CN1107072C - Apoptosis inducing molecule II - Google Patents

Abstract

The present invention relates to a new member of the TNF-ligand superfamily, apoptosis-inducing molecule II (AIM II). In particular, the invention provides isolated nucleic acid molecules encoding human AIM II protein. The present invention also provides AIM II polypeptides, as well as vectors, host cells and recombinant methods for producing the polypeptides. The invention also relates to screening methods for identifying agonists and antagonists of AIM II activity. The present invention also provides therapeutic methods for treating lymphadenopathy, autoimmune disease, graft-versus-host disease and inhibiting growth of cancer such as tumor cells.

Description

Apoptosis Inducing Molecule II

field of invention

The present invention relates to a new member of the TNF-ligand superfamily. Specifically, the present invention provides an isolated nucleic acid molecule encoding human apoptosis-inducing molecule II (Apoptosis Inducing Molecular II, AIM II). The invention also provides the AIM II polypeptide, as well as the vector, host cell and recombinant method for producing the polypeptide. The present invention also relates to screening methods for identifying agonists and antagonists of AIM II activity. The invention also provides therapeutic methods for treating lymphadenopathy, autoimmune disease, graft versus host disease, and inhibiting the growth of tumors, such as tumor cells.

Background technique

Human tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta, or lymphocytotoxins) are related members of a broad class of polypeptide-mediated molecules that includes interferons, interleukins, and growth Factors are collectively referred to as cytokines (Beutler B. and Cerami A., Annu. Ret. Immunol., 7: 625-655 (1989)).

Tumor necrosis factor (TNF-α and TNF-β) was originally discovered due to its antitumor activity effect, however it is now regarded as a pleiotropic cytokine with various biological activities including participation in some transformation Apoptotic cell death in cell lines, mediates cell activation and proliferation, and also plays an important role in immune regulation and inflammation.

So far known members of the TNF-ligand superfamily are: TNF-α, TNF-β (lymphotoxin-α), LT-β, OX40L, Fax ligand, CD30L, CD27L, CD40L and 4-IBBL. Ligands of the TNF ligand superfamily are acidic, TNF-like molecules that share about 20% (range, 12%-36%) sequence homology in the extracellular domain and exist primarily in a membrane-bound form. The biologically active type is the trimer/multimer complex. Only soluble forms of the TNF ligand superfamily have been identified so far from the TNF, LTα, and Fas ligands (for a general review, see Gruss H. and Dower S.K., Blood, 85(12):3378-3404 (1995)). This document is hereby incorporated by reference in its entirety.

These proteins are involved in the regulation of cell proliferation, activation, and differentiation processes, including the control of cell survival or death through apoptosis or cytotoxicity (Armitage R.J., Curr. Opin. Immunol., 6:407 (1994) and Smith C.A., Cell , 75:959 (1994)).

The development of mammals depends on the proliferation and differentiation of cells, and the programmed cell death (Walker et al., Methods Achiev. Exp. Pathol., 13: 18 (1988) that occurs in the programmed cell death process. Programmed cell death is in The destruction of immune thymocytes that recognize self-antigens plays a very important role. Failure of the normal elimination process may play a role in autoimmune diseases (Gammon et al., Immunology Today, 12:193 (1991)).

Itoh et al. (Cell, 66:233 (1991)) describe a cell surface antigen, Fas/CD95. It mediates apoptosis and is involved in the clonal deletion of T-cells. Fas is expressed in activated T-cells, B-cells and neutrophils, as well as thymus, liver, heart and lung. In addition to being expressed in activated T-cells, B-cells and neutrophils, Fas is also expressed in the adult mouse ovary (Watanate-Fukunaga et al., J. Immunol. 148:1274 (1992)). In experiments in which a monoclonal antibody to Fas was cross-linked to Fas, apoptosis was induced (Yonehara et al., J. Exp. Med. 169:1747 (1989); Trauth et al., Science, 245:301 (1989)). In addition, it has been demonstrated that binding of monoclonal antibodies to Fas has the potential to stimulate T-cells under certain conditions (Alderson et al., J. Exp. Med. 178:2231 (1993)).

Fas antigen is a cell surface protein with a relative molecular weight of 45kd. Human and mouse Fas genes have been cloned by Watanabe-Fukunaga et al. (Journal of Immunology, 148: 1274 (1992) and Itoh et al. (Cell, 66: 233 (1991). The proteins encoded by the two genes are transmembrane proteins, Structural homology to the nerve growth factor/tumor necrosis factor receptor superfamily, which includes the two TNF receptors, the low-affinity nerve growth factor receptor and the LTβ receptors CD40, CD27, CD30 and OX40.

The Fas ligand has been described recently (Suda et al., Cell 75:II69 (1993)). Its amino acid sequence shows that Fas ligand is a type II transmembrane protein belonging to the TNF family. Fas ligand is expressed in spleen cells and thymocytes, and the purified Fas ligand has a molecular weight of 40kd.

Recently, it has been shown that the interaction between Fas/Fas ligand is necessary for the apoptosis process after T cell activation (Ju et al., Nature, 373:444 (1995); Brunner et al., Nature, 373:441 (1995). )). Activation of T cells induces these two proteins on the cell surface, and subsequent interaction of the ligand with the receptor leads to apoptosis of the cell. This proves that in the normal immune response, the programmed cell death process induced by Fas/Fas ligand interaction may have a certain regulatory effect.

Based on structural and biological similarities, the polypeptide of the present invention has been identified as a new member of the TNF ligand superfamily.

Clearly, there should be factors that regulate the activation and differentiation of normal and abnormal cells. Therefore, there is a need to identify and characterize such factors that regulate the activation and differentiation of normal and abnormal cells in normal and disease states. In particular, there is a need to isolate and identify additional Fas ligands that control apoptosis for the treatment of autoimmune diseases, graft versus host disease, and lymphadenopathy.

Summary of the invention

The invention provides isolated nucleic acid molecules comprising a polynucleotide encoding an AIM II polypeptide having the amino acid sequence shown in Figure 1A-C (SEQ ID NO: 2) or derived from a cDNA clone deposited in a bacterial host The amino acid sequence encoding the bacterial host was deposited on August 22, 1996 under ATCC Accession No. 97689.

The present invention also relates to recombinant vectors containing the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, and methods for preparing the vectors and host cells by recombinant techniques and using them to produce AIM II polypeptides or peptides.

The invention also provides an isolated AIM II polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The term "AIM II" polypeptide as used herein includes membrane-bound proteins (containing a cytoplasmic domain, a transmembrane domain, and an extracellular domain) and truncated proteins thereof that retain the activity of the AIM II polypeptide. In one embodiment, the soluble AIM II polypeptide contains all or part of the extracellular domain of the AIM II protein, but lacks the transmembrane region that retains the polypeptide on the cell membrane. As long as the soluble AIM II protein can be secreted, it may also contain part of the transmembrane region or part of the cytoplasmic domain or other sequences. A heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide to allow secretion of the soluble AIM II polypeptide when expressed.

The present invention also provides AIM II polypeptides, especially human AIM polypeptides, which can be used to treat lymphadenopathy, autoimmune diseases and graft-versus-host diseases. The AIM II polypeptides can be used to stimulate peripheral tolerance, destroy some transformed cell lines, Mediates cell activation and proliferation, and functions as a primary mediator of immune regulation and inflammatory responses.

The present invention also provides compositions comprising AIM II polynucleotides or AIM II polypeptides that can be administered to cells in vitro, ex vivo and in vivo, or to multicellular organisms. In certain particularly preferred embodiments of this aspect of the invention, the composition contains an AIM II polynucleotide for expression in a host organism of an AIM II polypeptide for use in treating a disease. In this regard, expression in a patient is particularly preferred for the treatment of diseases associated with abnormal endogenous activity of AIMII.

The invention also provides a screening method for identifying compounds that enhance or inhibit a cellular response induced by AIM II, the screening method comprising contacting cells expressing AIM II with a candidate compound, testing the cellular response, and comparing the cellular response to a standard cell Responses are compared to the standard measured in the absence of exposure to the candidate compound; thus, an increase in the cellular response relative to the standard indicates that the compound is an agonist, and a decrease in the cellular response relative to the standard indicates that the compound is an antagonist.

In another aspect, the invention provides screening assays for AIM II agonists and antagonists. Antagonists are useful in the prevention of septic shock, inflammation, cerebral malaria, reactivation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis, and cachexia (wasting or malnutrition).

Another aspect of the invention relates to methods of treatment of individuals in need of higher levels of AIM II activity in vivo, said methods comprising administering to such individuals a composition comprising a therapeutically effective amount of an isolated AIM II polypeptide of the invention or an agonist thereof.

Another aspect of the invention also relates to methods of treatment of individuals in need of lower levels of AIM II activity in vivo, said methods comprising administering to such individuals a composition comprising a therapeutically effective amount of an AIM II antagonist.

Brief description of the drawings

Figure 1A-C shows the nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of AIM II. The deduced molecular weight of this protein is approximately 26.4 kDa. The putative transmembrane domain of the AIM II protein is underlined.

Figure 2A-F shows AIM II albumen and human TNF-alpha (SEQ ID NO: 3), human TNF-beta (SEQ ID NO: 4), human lymphocytotoxin (SEQ ID NO: 5) and human Fas ligand ( Similar regions between the amino acid sequences of SEQ ID NO: 6).

Figure 3A-F shows AIM II amino acid sequence analysis. The figure shows α, β, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic region, flexible region; antigenicity index and surface possibility. In the "Antigenicity Index - Jameson-Wolf" chart, approximately 13-20, 23-36, 69-79, 85-94, 167-178, 184-196, 221-233 amino acids in Figure 1A-C correspond to Shows a highly antigenic region in the AIM II protein.

Detailed description of the invention

The present invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding an AIM II polypeptide having the amino acid sequence shown in Figures 1A-C (SEQ ID NO: 2). AIM II protein of the present invention and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphocytotoxin (SEQ ID NO: 5) and human Fas ligand (SEQ ID NO: 6) has sequence homology (Fig. 2A-F). The nucleotide sequences shown in Figures 1A-C were obtained from the sequencing results of a cDNA clone deposited with the American Type Culture Collection on August 22, 1996, 12301 Park Lawn Drive, Rockville, Maryland 20852, The accession number is 97689. This deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).

Unless otherwise specified, all nucleotide sequences determined by DNA molecular sequencing herein are determined using an automated DNA sequencer (such as Model 373 of Applied Biosystems, Inc.), and all polypeptides encoded by the DNA molecules determined herein are The amino acid sequences were all estimated from the translation of the DNA sequences determined above. Accordingly, any nucleotide sequence determined herein may contain some errors, as is known in the art for any DNA sequence determined by this automated approach. Nucleotide sequences determined by automation are usually at least about 90% identical, more usually at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The true sequence can be more accurately determined by other means, including manual DNA sequencing well known in the art. It is also known in the art that a single insertion or deletion within a determined nucleotide sequence can cause a frameshift in translation of the nucleotide sequence compared to the actual sequence, thus resulting in a predicted amino acid sequence encoded by the determined nucleotide sequence. Starting at such insertion or deletion points is completely different from the amino acid sequence actually encoded by the sequenced DNA molecule.

Using the information provided herein, such as the nucleotide sequences in Figure 1A-C, it is possible to obtain polypeptides encoding AIM II by standard cloning and screening methods, such as those used when cloning cDNA using mRNA as starting material nucleic acid molecules of the invention. As described in the present invention, the nucleic acid molecule (SEQ ID NO: 1) described in Figure 1A-C was found in a cDNA library from human macrophage ox LDL (HMCCB64). This gene was also identified in a cDNA library from activated T cells (HT4CC72). The nucleotide sequence (SEQ ID NO: 1) measured by AIM II cDNA in Fig. 1A-C contains the open reading frame of the protein of coding 240 amino acid residues, and the initiation codon that it has is positioned at Fig. 1A-C (SEQ ID NO: 1) ID NO: 1) in the nucleotide sequence 49-51, the extracellular domain contains amino acid residues from about 60 to about 240 in Figure 1A-C (SEQ ID NO: 2), and the transmembrane domain contains Amino acid residues from about 37th to about 59th in Fig. 1A-C (SEQ ID NO: 2), the intracellular domain contains from about 1st to about 36th in Fig. 1A-C (SEQ ID NO: 2) Amino acid residues at the position, its molecular weight is deduced to be about 26.4kDa. The AIM II protein shown in Figure 1A-C (SEQ ID NO: 2) has about 27% identity and about 51% similarity with the amino acid sequence of human Fas ligand (Figure 2A-F), and human TNF-α (Figure 2A-F) The amino acid sequences of 2A-F) have about 26% identity and about 47% similarity.

One of ordinary skill will understand that due to the possibility of sequencing errors discussed above, the putative AIM II polypeptide encoded by the deposited cDNA contains approximately 240 amino acids, but the amino acid number may be 230-250. It is further understood that the exact positions of the extracellular, intracellular and transmembrane domains of AIM II polypeptides may vary slightly depending on the criteria used. For example, the exact position of the extracellular domain of AIM II in Figure 1A-C (SEQ ID NO: 2) may vary slightly due to the criteria used to define the region (e.g., the specific position may be between about "drift" between residues at position 5).

As noted above, nucleic acid molecules of the invention may be in the form of RNA, such as mRNA, or DNA, including cDNA and genomic DNA, such as produced by cloning or synthesis. DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be either the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).

An "isolated" nucleic acid molecule means a nucleic acid molecule - DNA or RNA - that is separated from its natural environment. For example, a recombinant DNA molecule contained in a vector is also considered "isolated" for the purposes of the present invention. Other examples of isolated DNA molecules include recombinant DNA molecules present in heterologous host cells or purified (partially or substantially) DNA molecules present in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules of the invention. According to the invention, isolated nucleic acid molecules also include such molecules produced synthetically.

The isolated nucleic acid molecules of the present invention include DNA molecules containing the open reading frame (ORF) shown in Figure 1A-C (SEQ ID NO: 1); containing the AIM II protein coding sequence shown in Figure 1A-C (SEQ ID NO: 2) and DNA molecules that contain sequences that are substantially different from the above sequences but still encode the AIM II protein due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Thus, methods for generating such degenerate variants are routine to those skilled in the art.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an AIM II polypeptide having the amino acid sequence encoded by a cNDA clone in a plasmid deposited on August 22, 1996 under ATCC Accession No. 97689 . Preferably, the nucleic acid molecule encodes the polypeptide encoded by the above-mentioned deposited cDNA clone. The present invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1A-C (SEQ ID NO: 1) or the nucleotide sequence of the AIM II cDNA contained in the above-mentioned preserved clone, or having one of the above-mentioned sequences Complementary sequences of nucleic acid molecules. Such isolated molecules, especially DNA molecules, are useful as probes for gene mapping by chromosomal in situ hybridization, and for detection of AIM II gene expression in human tissues by, for example, Northern blot analysis.

The present invention also relates to fragments of the isolated nucleic acid molecules described herein. Fragments having the nucleotide sequence of the deposited cDNA or of the isolated nucleic acid having the nucleotide sequence shown in Figure 1A-C (SEQ ID NO: 1) are meant to be effective as diagnostic probes and primers of a length of at least Fragments of about 15 nt, more preferably at least about 20 nt, more preferably at least about 30 nt, even more preferably at least about 40 nt. Of course, according to the present invention, larger fragments of 50-1500 nt in length are also useful, consistent with most (if not all) of the nucleotide sequence shown in the deposited cDNA or Figure 1A-C (SEQ ID NO: 1) Snippets are also useful. For example, a fragment of at least about 20 nt in length means a fragment containing 20 or more contiguous bases derived from the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figure 1A-C (SEQ ID NO: 1).

Preferably, the nucleic acid fragment of the present invention comprises a nucleic acid molecule encoding an epitope-containing region of the AIM II protein. Specifically, such nucleic acid fragments of the present invention include nucleic acid molecules encoding the following polypeptides: a polypeptide comprising amino acid residues from about 13th to about 20th in Figure 1A-C (SEQ ID NO: 2); A polypeptide of about 23 to about 36 amino acid residues in 1A-C (SEQ ID NO: 2); comprising about 69 to about 79 amino acid residues in Figure 1A-C (SEQ ID NO: 2) A polypeptide containing amino acid residues from about 85th to about 94th in Figure 1A-C (SEQ ID NO: 2); a polypeptide containing about 167th to about 94th amino acid residues in Figure 1A-C (SEQ ID NO: 2) A polypeptide comprising amino acid residue 178; a polypeptide comprising about amino acid residue 184 to about 196 in Figure 1A-C (SEQ ID NO: 2); and a polypeptide comprising Figure 1A-C (SEQ ID NO: 2) A polypeptide of amino acid residues from about 221 to about 233 in the . The inventors have determined that the above-mentioned polypeptide fragments are all antigenic regions of the AIM II protein. Methods for determining other epitope-containing regions of the AIM II protein are detailed below.

According to the present invention, AIM II polynucleotides can be used in a variety of applications, especially those that take advantage of the chemical and biological properties of AIM II. These applications involve autoimmune diseases and abnormal cell proliferation. Other applications involve the diagnosis and treatment of abnormalities in cells, tissues and organisms.

The invention also relates to the use of AIM II polynucleotides for the detection of complementary polynucleotide strands, for example as diagnostic reagents. Detection of mutated forms of AIM II associated with dysfunction would provide a diagnostic tool to enhance or limit disease or disease susceptibility resulting from underexpression, overexpression or aberrant expression of AIM II (e.g. autoimmune in the diagnosis of immune diseases). Since the AIM II gene exists in a variety of tumor cell lines, including pancreatic tumors, testicular tumors, endometrial tumors and T-cell lymph node tumors, the polynucleotide encoding AIM II can also be used as a diagnostic marker to detect the polypeptide of the present invention expression.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a polynucleotide which, under stringent hybridization conditions, is hybridized to the polynucleotide in the aforementioned nucleic acid molecule of the present invention (eg, the cDNA clone contained in ATCC Accession No. 97689). Acid segments are capable of hybridization. "Stringent hybridization conditions" means incubation overnight at 42°C in a solution containing the following components: 50% formamide, 5×SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 X Denhardt's solution, 10% dextran sulfate and 20 g/ml denatured sheared salmon sperm DNA, followed by washing the filter in 0.1 X SSC at approximately 65°C.

A polynucleotide capable of hybridizing to a "segment" of a polynucleotide means a polynucleotide that hybridizes to at least about 15 nucleotides (nt), more preferably at least about 20 nt, more preferably at least about 30 nt, even more preferably at least about 30 nt, or even More preferred are polynucleotides (DNA or RNA) that are hybridizable in segments of about 30-70 nt. As discussed in detail above and below, they are useful as diagnostic probes and primers.

For example, a segment of a polynucleotide of "at least 20 nt in length" means nucleosides derived from a standard polynucleotide such as a deposited cDNA or the nucleotide sequence shown in Figure 1A-C (SEQ ID NO: 1) 20 or more contiguous nucleotides in the acid sequence.

Of course, only the poly A sequence [the 3′ terminal poly (A) region of AIM II cDNA shown in Figure 1A-C (SEQ ID NO: 1)] or the complementary extension of T (or U) residues can hybridize to the multinuclear Nucleotides are not included in polynucleotides of the invention for use in hybridization to nucleic acid segments of the invention, since such polynucleotide molecules are compatible with any nucleic acid containing poly(A) extended strands or complementary segments thereof. Molecules (eg, virtually any double-stranded cDNA clone) can hybridize.

As described herein, nucleic acid molecules of the present invention that encode an AIM II polypeptide may include, but are not limited to, those nucleic acid molecules that encode the amino acid sequence of the polypeptide; polypeptide coding sequences and other sequences, such as coding leader sequences or A sequence of a secretory sequence (such as a preprotein or proprotein or preproprotein sequence); a polypeptide coding sequence that may or may not contain the aforementioned other coding sequences, the polypeptide coding sequence may also contain other non-coding sequences, including but not limited to Such as introns, 5' and 3' non-coding sequences (such as transcribed but not translated sequences that have functions such as binding ribosomes and stabilizing mRNA in transcription and mRNA splicing, including splicing and polyadenylation signals ); other coding sequences encoding other amino acids such as amino acids that serve other functions. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, eg, a sequence encoding the polypeptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the marker provided on the pQE vector (Qiagen, Inc.), many of which are commercially available. For example, the presence of hexa-histidine facilitates purification of fusion proteins as described by Gentz et al., Proc. Natl. Acad. Sci. USA, 86:821-824 (1989). The "HA" tag is another useful peptide for purification. This "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described in Wilson et al., Cell, 37:767 (1984) . Other such fusion proteins include AIM II fused to Fc at the N-terminus or C-terminus, as described below.

Nucleic acid molecules of the invention also include nucleic acid molecules encoding full-length AIM-II polypeptides lacking an N-terminal methionine.

The invention also relates to variants of the nucleic acid molecules of the invention, which variants encode segments, analogs or derivatives of the AIM II protein. Variants may be naturally occurring, such as natural allelic variants. An "allelic variant" means one of several variant forms of a gene located at a particular locus on a chromosome in an organism (Gene II, eds. Lewin B., John Wiley & Sons, New York ( 1985)). Non-naturally occurring variants can be generated using art-known mutagenesis techniques.

Such variants include nucleic acid molecules that may result from nucleotide substitutions, deletions, or additions that may involve one or more nucleotides. These variants can have changes in coding regions, noncoding regions, or both. Alterations within the coding region may result in conservative or non-conservative amino acid substitutions, deletions or additions. Silent substitutions, additions and deletions are particularly preferred among these changes, which do not change the properties and activities of the AIM II protein or segments thereof. In this context too, conservative substitutions are particularly preferred.

Another embodiment of the invention includes isolated nucleic acid molecules comprising a polynucleotide having at least 90%, more preferably at least 95%, 96% identity to one of the following nucleotide sequences , 97%, 98% or 99%: (a) the nucleotide sequence encoding the AIM II polypeptide having all the amino acid sequences in Figure 1A-C (SEQ ID NO: 2); The nucleotide sequence of the AIM II polypeptide of the amino acid sequence, the cDNA clone is included in the ATCC deposit number 97689; (c) the nucleotide sequence of the extracellular domain of the encoding AIM II polypeptide; (d) the transmembrane structure of the encoding AIM II polypeptide The nucleotide sequence of domain; (e) the nucleotide sequence of coding AIM II polypeptide intracellular domain; (f) the nucleotide sequence of coding soluble AIM II polypeptide, and this soluble AIM II polypeptide has extracellular and intracellular structure domain, but lacks a transmembrane domain; and (g) a nucleoside complementary to any of the nucleotide sequences of (a), (b), (c), (d), (e) or (f) above acid sequence.

For example, a polynucleotide comprising a nucleotide sequence that is at least 95% "identical" to a standard nucleotide sequence encoding an AIM II polypeptide means that the nucleotide sequence of the polynucleotide is substantially identical to the standard sequence Yes, but the polynucleotide sequence may contain no more than 5 point mutations within every 100 nucleotides in the standard nucleotide sequence encoding the AIM II polypeptide. In other words, in order to obtain a polynucleotide comprising a nucleotide sequence having at least 95% identity to the standard nucleotide sequence, no more than 5% of the nucleotides within the standard sequence may be deleted or replaced by other nucleotides or, the number of nucleotides accounting for no more than 5% of the total number of nucleic acids in the standard sequence can be inserted into the standard sequence. These mutations of the standard sequence can occur at the 5' or 3' terminal positions of the standard nucleotide sequence, and can also occur anywhere in between these terminal positions, and they are either scattered among the nucleotide sequences within the standard sequence, Or exist in the form of one or several continuous groups within the standard sequence.

As a practical matter, does any particular nucleic acid molecule share at least 90%, 95%, 96%, 97%, 98% Or 99% identity can be determined routinely using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, 575 Science Drive, Madison, WI 53711). The Bestfit program uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, 2: 482-489 (1981 ) to find the best segment of homology between two sequences. When utilizing Bestfit or any other sequence comparison program to determine whether a particular sequence has at least 95% identity with the standard sequence of the present invention, the parameters are of course set to ensure that the percentage of identity is relative to the standard nucleotide sequence The full length is calculated, and homology gaps accounting for no more than 5% of the total number of nucleotides in the standard sequence are also allowed.

This patent application relates to a nucleic acid having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequence shown in Figure 1A-C (SEQ ID NO: 1) or to the nucleic acid sequence of the deposited cDNA Molecules, and it is not required whether these nucleic acid molecules encode polypeptides with AIM II activity. This is because even if a specific nucleic acid molecule does not encode a polypeptide having AIM II activity, those skilled in the art will know how to use these nucleic acid molecules, such as as hybridization probes or polymerase chain reaction (PCR) primers. Nucleic acid molecules that do not encode polypeptides with AIM II activity in the present invention include the following applications: (1) isolate AIM II gene or its allelic variants from a cDNA library; (2) such as Verma et al., "Human Chromosome: Basic Technology Handbook, Pergamon Press, New York (1988), in situ hybridization (such as "FISH") with metaphase chromosomes for accurate chromosomal localization of the AIM II gene; and Northern blot analysis for detection of AIM mRNA expression in specific tissues .

However, preferred nucleic acid molecules have at least 90%, 95%, 96%, 97%, 98% or 99% identical sequence and indeed encodes a nucleic acid molecule of a polypeptide having AIM II protein activity. "Polypeptide having AIM II activity" means a polypeptide having an activity similar to, but not necessarily identical to, that of the AIM II protein of the present invention through specific biological tests. For example, AIM II protein cytotoxic activity can be measured by propidium iodide staining for apoptosis as described by Zarres et al., Cell, 70:31-46 (1992). Alternatively, AIM II-induced apoptosis can also be measured by TUNEL staining as described by Gavierli et al., J. Cell Biol., 119:493-501 (1992).

Briefly, propidium iodide staining is performed as follows: tissue or culture-derived cells are fixed in formaldehyde, cryosectioned, and stained with propidium iodide. Nuclei can be visualized by propidium iodide using confocal fluorescence microscopy. Cell death is indicated by pyknotic nuclei (chromosomal condensation, shrinkage, and/or fragmentation of the nucleus).

Of course, due to the degeneracy of the genetic code, those of ordinary skill in the art will quickly understand that the nucleic acid sequence containing the deposited cDNA or the nucleic acid sequence shown in Figure 1A-C (SEQ ID NO: 1) has at least 90%, 95% %, 96%, 97%, 98% or 99% identical sequences of a large number of nucleic acid molecules also encode polypeptides "with AIM II protein activity". In fact, since the degenerate variants of these nucleotide sequences all encode the same protein, it will be clear to those skilled in the art even without performing the above comparison test. Those skilled in the art also understand that for those nucleic acid molecules that are not degenerate variants, a considerable portion can also encode polypeptides with AIM II protein activity. This is because those amino acid substitutions that are less likely or unlikely to significantly affect protein activity are well understood by those skilled in the art (eg, substitution of one aliphatic amino acid for another aliphatic amino acid).

For example, Bowie J.U. et al., "Deciphering Information Within Protein Sequences: Tolerance of Amino Acid Substitutions", Science, 247:1306-1310 (1990) describe rules for how to make amino acid substitutions without phenotypic changes, where The authors point out that the protein is surprisingly tolerant of amino acid substitutions. Vectors and host cells

The present invention also relates to vectors containing the isolated DNA molecules of the present invention, host cells genetically manipulated using the recombinant vectors, and the production of AIM II polypeptides or fragments thereof by recombinant techniques.

The polynucleotide can be linked to a vector containing a selectable marker for propagation in the host. Usually plasmid vectors can be introduced into the host via precipitates such as calcium phosphate precipitates or complexes with charged lipids. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and transduced into host cells.

The DNA insert should be operably linked to an appropriate promoter, such as the bacteriophage λPL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, retrovirus LTR promoter, etc. Suitable other promoters are also well known to those skilled in the art. The expression construct also contains a transcription initiation point, termination point, and ribosome binding site for translation within the transcribed region. The coding segment of the mature transcript expressed by this construct preferably contains a translation initiation sequence at the beginning and a stop codon (UAA, UGA or UAG) at the appropriate position at the end of the polypeptide to be translated.

As noted above, expression vectors preferably contain at least one selectable marker. Such markers include the neomycin resistance gene for eukaryotic cell culture, the dihydrofolate reductase gene, and the tetracycline or ampicillin resistance gene for E. coli and other bacterial culture. Representative examples of suitable hosts are bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanin tumor cells; and plant cells, but not limited to the above cells. Appropriate culture media and culture conditions for the above-mentioned host cells are well known in the art.

Preferred vectors for use in bacteria include pQE70, pQE60 and pEQ-9 available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A and pNH46A available from Stratagene; and ptrc99a, pKK223 available from Pharmacia -3, pKK233-3, pDR540, and pRIT5; and the like. Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and the like. Suitable other vectors will also be apparent to those skilled in the art.

Constructs can be introduced into host cells by calcium phosphate precipitation transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. These methods are described in various standard laboratory manuals, such as Davis et al., Fundamental Methods in Molecular Biology (1986).

Polypeptides can be expressed in modified forms, such as fusion proteins, and can contain not only secretion signals but also other heterologous functional domains. For example, regions containing other amino acids, particularly charged amino acids, may be added at the N-terminus of the polypeptide to provide stability and persistence within the host cell, during purification or during subsequent use and storage. In addition, small peptides can also be added to the polypeptides to facilitate purification, and such regions can be removed prior to final preparation of the polypeptides. Small peptides are added to polypeptides to secrete away such regions. The techniques of adding small peptides to polypeptides to secrete and excrete polypeptides, improve the stability of polypeptides, and facilitate the purification of polypeptides are skilled and routine techniques in the art. Preferred fusion proteins contain heterologous domains derived from immunoglobulins that facilitate protein solubilization. For example, EP-A-0464533 (Canadian counterpart No. 2045869) discloses fusion proteins comprising various segments of the constant regions of immunoglobulin molecules and other human proteins or parts thereof. In many cases, the Fc part of fusion proteins is very useful in therapy and diagnosis, and can improve eg pharmacokinetic properties (EP-A0232262). On the other hand, in some applications it is desirable to remove the Fc portion of the fusion protein after expression, detection and purification of the fusion protein by the preferred methods already described. This is the case when the Fc segment proves to have adverse effects on the application of the fusion protein in therapy and diagnosis, such as the use of the fusion protein as an immunizing antigen. For example, in drug discovery, human proteins such as hIL-5 are fused to the Fc region for the purpose of high-capacity screening assays to identify hIL-5 antagonists. See D. Bennett et al., J. Molecular Recognition, Vol. 8: 52-58 (1995) and K. Johanson et al., J. Biochemistry, Vol. 270, No. 16: 9459-9471 (1995).

By well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and exogenous agglutination AIM II polypeptides can be recovered and purified from recombinant cell cultures by methods such as plain chromatography. During purification, high performance liquid chromatography ("HPLC") is most preferably used. Polypeptides of the present invention include natural purified products, products produced by chemical synthesis, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including bacteria, yeast, higher plants, insects, and mammalian cells. Depending on the type of host used in the recombinant production method, the polypeptides of the invention may or may not be glycosylated. In addition, polypeptides of the invention may also contain modified initial methionine residues, in some cases as a result of host-mediated processes. AIM II peptides and fragments

The present invention also provides an isolated AIM II polypeptide having the amino acid sequence encoded by the deposited cDNA or the amino acid sequence in Figure 1A-C (SEQ ID NO: 2), or a peptide or polypeptide comprising a segment of the above polypeptide.

It is well known in the art that some amino acid sequences of AIM II polypeptides can be altered without significantly affecting the structure or function of the protein. Careful analysis of such sequence differences should reveal the presence of important regions of the protein that determine its activity.

Accordingly, the present invention also includes variants of AIM II polypeptides that have significant AIM II polypeptide activity or that contain regions of the AIM II protein (such as protein segments described below). Such mutants include deletions, insertions, transitions, duplications and type substitutions. As noted above, rules regarding which amino acid changes are likely to be non-phenotypic can be found in Bowie J.U. et al., "Interpreting information in protein sequences: tolerance to amino acid substitutions", Science, 247:1306-1310 (1990 ).

Thus, fragments, derivatives or analogs of the polypeptide of Figure 1A-C (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA may be: (i) wherein one or more amino acid residues are conserved or non- Conserved amino acid residues (preferably conserved amino acid residues), which may or may not be amino acids encoded by the genetic code; or (ii) one or more of the amino acid residues Contains substituents; or (iii) wherein the mature polypeptide is fused to another compound such as a compound that increases the half-life of the polypeptide (e.g. polyethylene glycol); or (iv) wherein the mature polypeptide is fused to other amino acids such as IgG Fc fusion region peptide, leader sequence, secretory sequence or sequence that can be used for purification of mature polypeptide, or proprotein sequence. Such fragments, derivatives and analogs are also within the purview of those skilled in the art as taught herein.

Particularly striking is the substitution of a charged amino acid by another charged amino acid, a neutral or negatively charged amino acid, which results in a less positively charged protein and thus improves the properties of the AIM II protein. It is extremely necessary to avoid agglutination. Aggregation of proteins not only destroys activity, but can also cause major problems in the preparation of pharmaceutical formulations due to their immunogenicity (Pinckard et al., Clin. Experimental Immunology, 2: 331-340 (1967); Robbins et al., Diabetes, 36 : 838-845 (1987)); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems, 10: 307-377 (1993)).

Amino acid substitutions can also alter the selective binding of a polypeptide to cell surface receptors. Ostade et al., Nature, 361:266-268 (1993) describe certain mutations that result in selective binding of TNF-[alpha] to only one of the two known types of TNF receptors. Therefore, the AIM II receptor of the present invention may contain one or more amino acid substitutions, deletions or additions, and such changes may be the result of natural mutations or artificial manipulations.

As noted, less impactful changes are preferred, such as conservative amino acid substitutions that do not significantly affect protein folding or activity (see Table 1).

Table 1 Conservative amino acid substitutions aromatic amino acid Phenylalanine Tryptophan Tyrosine hydrophobic amino acid Leucine Isoleucine Valine polar amino acids glutamine asparagine basic amino acid arginine lysine histidine acidic amino acid aspartic acid glutamic acid small amino acid Alanine Serine Threonine Methionine Glycine

The AIM II proteins of the present invention can be identified by methods known in the art, such as site-specific mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science, 244:1081-1085 (1989)). An amino acid that is essential for its function. The latter method is to introduce a single alanine mutation at any residue in the molecule, and then measure the biological activity of the mutant molecule thus obtained, such as receptor binding ability, or proliferative activity in vivo or in vitro. It can also be determined by structural analysis, such as crystallization processes, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Molecular Biology, 224:899-904 (1992) and deVos et al., Science, 255:306-312 (1992)). Sites critical for ligand-receptor binding.

The polypeptides of the invention are preferably provided in isolated form. "Isolated polypeptide" means a polypeptide that has been removed from its natural environment. Thus, a polypeptide produced and contained within a recombinant host cell is also considered "isolated" for the purposes of the present invention. "Isolated polypeptide" also refers to a polypeptide that has been partially or substantially purified from within a recombinant host. For example, recombinant producer AIM II polypeptides can be substantially purified by the one-step procedure described by Smith and Hohnson, Gene, 67:31-40 (1988).

Polypeptides of the present invention include polypeptides encoded by deposited cDNAs, polypeptides of Figures 1A-C (SEQ ID NO: 2), polypeptides of Figures 1A-C (SEQ ID NO: 2) lacking an N-terminal methionine, cellular Extracellular domains, transmembrane domains, intracellular domains, soluble polypeptides containing all or part of the extracellular and intracellular domains but lacking transmembrane domains, and polypeptides encoded by deposited cDNA or Figure 1A-C (SEQ ID NO 2): The identity between the polypeptides of 2) is at least 80%, more preferably at least 90% or 95%, more preferably at least 96%, 97%, 98% or 99%, and also includes polypeptides containing at least 30 amino acids , more preferably segments of such polypeptides of at least 50 amino acids.

A polypeptide containing an amino acid sequence having at least 95% "identity" with the standard amino acid sequence of an AIM II polypeptide means that the amino acid sequence of the polypeptide is substantially identical to the standard sequence, except that the polypeptide sequence is in every 100 of the standard amino acids of the AIM II polypeptide amino acids may contain no more than 5 amino acid changes. In other words, in order to obtain a polypeptide having an amino acid sequence having at least 95% identity with the standard amino acid sequence, no more than 5% of the amino acid residues in the standard sequence may be deleted or substituted by other amino acids; or, Amino acids accounting for no more than 5% of the total amino acid residues in the standard sequence are inserted into the standard sequence. These alterations to the standard sequence may be at the amino- or carboxy-terminal positions of the standard amino acid sequence, or anywhere in between these terminal positions, either individually dispersed among the residues of the standard sequence or in one or more contiguous groups Exists within the standard sequence.

As a practical matter, known computer programs such as the Bestfit program (Wisconsin Sequence analysis Package, 575 Science Drive, Madison, WI 53711) can be used to routinely determine the relationship between any given polypeptide and that shown in Figures 1A-C (SEQ ID NO: 2). Whether the indicated amino acid sequence or the amino acid sequence encoded by the deposited cDNA clone is at least 90%, 95%, 96%, 97%, 98% or 99% identical. When using Bestfit or any other sequence comparison program to analyze whether there is at least 95% identity between a specific sequence and the standard sequence of the present invention, the parameters are of course set to ensure that the percentage of identity is the full range relative to the standard amino acid sequence. Homology gaps of up to 5% of the total number of amino acids in the standard sequence are also allowed.

The term "AIM II" polypeptide as used herein includes membrane-bound proteins (comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain), as well as truncated proteins that retain AIM II polypeptide activity. In one embodiment, the soluble AIM II polypeptide contains all or part of the extracellular domain of the AIM II protein, but lacks the transmembrane segment that retains the polypeptide at the cell membrane. Soluble AIM II may also contain part of the transmembrane domain or part of the cytoplasmic domain or other sequences as long as it is secreted. A heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide to allow secretion of the soluble AIM II polypeptide after expression.

Polypeptides of the invention can be used as molecular weight markers on SDS-PAGE gels or molecular sieve gel filtration columns using methods well known to those skilled in the art.

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-containing segment of a polypeptide of the invention. The epitope of this polypeptide segment is an immunogenic or antigenic epitope of a polypeptide described herein. "Immunogenic epitope" refers to the whole protein as the part of the protein that can trigger the antibody response during immunization, while the region in the protein molecule that can bind to the antibody is called "antigenic epitope". The number of immunogenic epitopes within a protein is generally smaller than that of antigenic epitopes. See, eg, Geysen et al., Proceedings of the National Academy of Sciences of USA, 81:3998-4002 (1983).

As for the selection of peptides or polypeptides containing antigenic epitopes (that is, peptides or polypeptides containing regions of protein molecules that can bind to antibodies), it is well known in the art that relatively short synthetic peptides that closely resemble the sequence of a part of a protein generally stimulate Antisera are raised that react with proteins similar to this part. See, eg, Sutcliffe H.G., Shinnick T.M., Green N. and Learner R.A. (1983), "Antibodies Reactive with Predetermined Sites in Proteins", Science 219:660-666. Peptides that elicit protein-reactive sera are mostly represented by the primary sequence of the protein, can be determined by a simple set of chemical rules, and are not limited to immunodominant regions of the intact protein (i.e., immunogenic epitopes) or other amino or carboxyl terminus.

Therefore, the peptides and polypeptides containing antigenic epitopes of the present invention can be effectively used to prepare antibodies, including monoclonal antibodies, that specifically bind to the polypeptides of the present invention. See, eg, Wilson et al., Cell, 37:767-778 (1984), at page 777.

A sequence contained in the peptides and polypeptides carrying antigenic epitopes of the present invention is preferably at least 7, more preferably at least 9, and most preferably at least 15 to about 30 of the amino acid sequences of the polypeptides of the present invention. of amino acids.

Examples of antigenic polypeptides or peptides that can be used to prepare AIM II-specific antibodies include, but are not limited to, the following: A polypeptide; a polypeptide comprising amino acid residues from about 23rd to about 36th in Figure 1A-C (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about 69th to about 36th in Figure 1A-C (SEQ ID NO: 2) A polypeptide at amino acid residue 79; a polypeptide comprising about amino acid residue 85 to about 94 in Figure 1A-C (SEQ ID NO: 2); a polypeptide comprising about amino acid residue 94 in Figure 1A-C (SEQ ID NO: 2); A polypeptide comprising amino acid residues 167 to about 178; a polypeptide comprising amino acid residues 184 to about 196 in Figure 1A-C (SEQ ID NO: 2); and a polypeptide comprising amino acid residues 1A-C (SEQ ID NO: 2) NO: a polypeptide at about 221 to about 233 amino acid residues in 2). As mentioned above, the present inventors have determined that the above polypeptide fragments are antigenic regions of the AIM II protein.

The epitope-bearing peptides and polypeptides of the invention can be produced by any conventional means. Houghten R.A. (1985), "A general method for rapid solid-phase synthesis of large numbers of polypeptides: Specificity of antigen-antibody reactions at the level of single amino acids", Proceedings of the National Academy of Sciences of USA, 82: 5131-5135. This "simultaneous multiple polypeptide synthesis (SMPS)" pathway is further described in US Patent No. 4,631,211 to Houghten et al.

The AIM II polypeptides of the invention can be used to treat lymphoproliferative disorders that induce lymphadenopathy. Since AIM II can mediate apoptosis by stimulating clonal deletion of T-cells, AIM II can be applied to the treatment of autoimmune diseases, stimulation of peripheral tolerance, and T-cell-mediated programming of cytotoxicity sex cell death process. AIM II can also be used as a research tool to elucidate the biological mechanisms of autoimmune disorders, including systemic lupus erythematosus (SLE), immunoproliferative diseases, Lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (AIL), lymphoblastic lymphadenopathy (IBL), rheumatoid arthritis, diabetes and multiple sclerosis.

The AIM II polypeptides of the invention can also be used to inhibit tumors, such as tumor cell growth. AIM II polypeptide may play a certain role in the destruction of tumors through programmed cell death and cytotoxicity to certain cells. Since AIM II can stimulate the proliferation of endothelial-derived cells, AIM II can also be used to treat some diseases that require growth-promoting activity, such as restenotic diseases. Moreover, AIM II can also be used to regulate hematopoiesis during endothelial cell development.

The invention also provides methods of identifying molecules, such as receptor molecules, that bind to AIM II. Genes encoding proteins capable of binding AIM II, such as receptor proteins, can be identified by numerous methods known to those skilled in the art, such as ligand selection and FACS sorting. Such methods are described in many laboratory manuals such as Coligan et al., Current Methods in Immunology, 1(2):Chapter 5 (1991).

For example, expression cloning methods can be used for this purpose. To achieve this purpose, first prepare polyadenylated RNA from cells that respond to AIM II, construct a cDNA library from this RNA, divide the library into several groups, and then transform cells that do not respond to AIM II. Transformed cells were then mixed with labeled AIM II. (AIM II can be labeled by a variety of well-known techniques, including radioactive iodine treatment or inclusion of site-specific protein kinase recognition sites and other standard methods.) After treatment, cells are fixed and the rate of AIM II incorporation is determined. This method can be easily performed on glass slides.

Groups containing cDNA capable of producing AIM II binding cells were identified. These positive groups were divided into subgroups, and then the host cells were transformed and screened according to the above method. Through this iterative sub-pooling and re-screening process, one or more single clones encoding putative binding molecules such as receptor molecules can be isolated.

Alternatively, the labeled ligand is photoaffinity linked to a cell extract, such as a membrane, or a membrane extract prepared from cells expressing a molecule, such as a receptor molecule, that binds the ligand of. The cross-linked material is separated by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film. The tagged complex containing the ligand-receptor is excised, cleaved into peptide fragments, and subjected to protein microsequencing. Using the amino acid sequences obtained from microsequencing results, specific or degenerate oligonucleotide probes can be designed to screen cDNA libraries to identify genes encoding putative receptor molecules.

The receptor peptides of the invention can also be used to demonstrate the ability of AIMII-binding molecules, such as receptor molecules, to bind AIMII in cellular or cell-free preparations.

Those skilled in the art are well aware that the AIM II polypeptide of the present invention or its epitope-containing fragment described above can be combined with a part of the constant domain of an immunoglobulin (IgG) to generate a chimeric polypeptide. Such fusion proteins can be easily purified and have a longer half-life in vivo. For example, the chimeric protein containing the first two domains of human CD4 polypeptide and the constant domains of mammalian immunoglobulin heavy chain or light chain reflects the above characteristics (EPA394,827; Traunecker et al., Nature, 331:84 -86(1988)). Fusion proteins with a disulfide-linked diabody structure due to the IgG portion also bind and neutralize other molecules more efficiently than monomeric AIMII proteins or protein fragments alone (Fountoulakis et al., J. Biochem., 270: 3958-3964 (1995)).

The present inventors have found that AIM II is expressed in spleen, thymus and bone marrow tissues. For a range of disorders such as septic shock, inflammation, cerebral malaria, reactivation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis, and cachexia, in some individuals with such disorders AIM II that is significantly higher or lower than "standard" AIM II gene expression levels can be detected in tissues (such as spleen, thymus, and bone marrow tissue) or body fluids (such as serum, cytosol, urine, synovial fluid, or spinal fluid) Gene expression, where the "standard" AIM II gene expression level is the expression level of AIM II in the tissues or body fluids of individuals who do not have such disorders. Therefore, the present invention provides a very useful diagnostic method in the process of disorder diagnosis, which method comprises: (a) measuring the expression level of AIM II gene in cells or body fluids of individuals; (b) converting the measured AIM II gene Expression levels are compared to standard AIM II gene expression levels, where a measured AIM II gene expression level above or below the standard expression level is indicative of a disorder. AIM II agonists and antagonists

The invention also provides methods of screening compounds to identify compounds that enhance or inhibit the effect of AIM II on a cell, such as the interaction with an AIM II binding molecule such as a receptor molecule. Agonists are compounds that enhance the natural biological functions of AIM II or act in a manner similar to AIM II; antagonists reduce or eliminate such functions.

For example, cellular components, such as membrane preparations, can be prepared from cells expressing molecules that bind AIM II, such as molecules that are regulated by AIM II through signaling or regulatory pathways. The preparation is incubated with labeled AIM II in the absence or presence of a test molecule, which may be an agonist or antagonist of AIM II. The reduced level of labeled ligand binding reflects the ability of the test molecule to bind to the binding molecule or to AIM II itself. Molecules that bind "gratis," ie, do not induce an AIM II response when bound to an AIM II-binding molecule, are most likely to be good antagonists. Molecules that bind well and act the same or closely related to AIM II are good agonists.

For example, the activity of a second messenger system measured by the interaction of a potential agonist or antagonist with a cell or an appropriate cell preparation and compared to the activity of AIM II or a molecule with the same function as AIM II can be measured. The AIM II similarity activity of the molecule to be tested is obtained. In this regard, useful second messenger systems include, but are not limited to, AMP guanylate cyclase, ion channels, or phosphoinositide hydrolysis second messenger systems.

Another example of an AIM II antagonist assay is the competition assay, in which AIM II and potential antagonists are combined with membrane-bound AIM II receptor molecules or recombinant AIM II receptor molecules combined. AIM II can be labeled, e.g., by radioactivity, so that the number of AIM II molecules bound to the receptor molecule can be accurately determined to estimate the efficacy of this potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptides of the present invention to inhibit or eliminate their activity. Potential antagonists may also be small organic molecules that bind to the same site of a binding molecule such as a receptor molecule without inducing AIM II-inducing activity, thereby inhibiting the action of AIM II by preventing the binding of AIM II, Peptides or polypeptides, such as closely related proteins or antibodies.

Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression by antisense DNA or antisense RNA or by triplex helix formation. Antisense technology has been described, eg, in Okano, Journal of Neurochemistry, 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, RL (1988). Triplet helix formation is described, eg, in Lee et al., Nucleic Acids Res. 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1360 (1991). These methods are all based on the binding of polynucleotides to complementary DNA or RNA. For example, antisense RN oligonucleotides of about 10 to 40 base pairs in length can be designed using the 5' coding region of the polynucleotide encoding the mature polypeptide of the present invention. DNA oligonucleotides are designed to be complementary to gene regions involved in transcription, thereby inhibiting the transcription and production of AIM II. Antisense RNA oligonucleotides hybridize to mRNA in vivo, thereby blocking the translation of mRNA molecules into AIM II polypeptides. The above-mentioned oligonucleotides can also be introduced into cells so as to express the antisense RNA or DNA in vivo, thereby inhibiting the production of AIM II.

The antagonist can be formulated into a composition together with a pharmaceutical carrier (as described later) for application.

For example, antagonists could be used to treat cachexia, a disorder in which lipid clearance is defective due to a systemic deficiency of lipoprotein lipase, which is said to be suppressed by AIM II. inhibition. Antagonists of AIM II may also be used in the treatment of cerebral malaria, in which AIM II may have a pathological role. This antagonist can also be used to treat rheumatoid arthritis by inhibiting the production of AIM II-induced inflammatory factors such as IL1 in synoviocytes. In the treatment of arthritis, the AIM II antagonist is preferably injected into the joint.

AIM II antagonists can also be used to prevent graft-host rejection in the presence of a graft by preventing stimulation of the immune system.

Antagonists of AIM II may also be used to inhibit bone resorption, thereby treating and/or preventing osteoporosis.

Antagonists may also be used as anti-inflammatory agents and to treat endotoxic shock. This dangerous state results from an extraordinary response to bacterial or other types of infection. cancer prediction

Mammals with cancer express significantly reduced levels of AIM II protein and mRNA encoding AIM II protein in certain tissues compared to corresponding "normal" mammals. A normal mammal refers to an individual that does not suffer from cancer and belongs to the same species as the cancer-affected mammal. In addition, AIM II levels measured in certain body fluids (such as serum, cytosol, urine, and spinal fluid) of mammals with cancer were significantly higher than those of mammals of the same species but without cancer reduce. Therefore, the present invention provides a diagnostic method that can be effectively applied in tumor diagnosis. This method includes measuring the expression level of the gene encoding AIM II protein in mammalian cells or body fluids, and comparing the measured gene expression level with the standard AIM II protein. Compared with the gene expression level, if the measured gene expression level is lower than the standard, it indicates that there is a certain tumor.

The present invention can be effectively used as a predictive indicator when tumor diagnosis has been performed according to conventional methods, wherein patients with enhanced AIM II gene expression may have better clinical outcomes than patients with lower gene expression levels.

"Determining the expression level of a gene encoding an AIM II protein" means directly (such as by determining or estimating the absolute amount of protein levels or mRNA levels) or relatively (such as by comparing the AIM II protein levels or mRNA levels of a second biological sample) Level comparison) qualitatively or quantitatively measure or estimate the level of AIM II protein or the level of mRNA encoding AIM II protein in the first sample.

Preferably, the AIM II protein level or mRNA level in the first biological sample is determined or estimated and compared to a standard AIM II protein level or mRNA level obtained from a second biological sample from an individual without cancer measured in. It is well known in the art that once a standard AIM II protein level or mRNA level has been measured, that value can be repeated as a comparison standard.

"Biological sample" means any biological sample obtained from an individual, cell line, tissue culture, or other source that contains AIM II protein or mRNA. Biological samples include mammalian body fluids (such as serum, cytoplasm, urine, synovial fluid, and spinal fluid) that contain secreted mature AIM II protein, as well as ovary, prostate, heart, placenta, pancreas, liver, spleen, lung, breast and umbilical tissue.

The present invention can be effectively used in the diagnosis of cancer in mammals. The present invention is particularly useful in the diagnosis of cancer in mammals of the following organ types: breast, ovary, prostate, bone, liver, lung, pancreas and spleen. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Humans are especially preferred.

Total cellular RNA can be isolated from biological samples using the one-step guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi, Anal. Biochem., 162:156-159 (1987). The level of mRNA encoding the AIM II protein is then determined using an appropriate method. These methods include Northern blot analysis (Harada et al., Cell, 63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell, 49:357-367 (1987)), polymerase chain reaction (PCR) , a combination of reverse transcription and polymerase chain reaction (RT-PCR) (Makino et al., Technology, 2:295-301 (1990)), and a combination of reverse transcription and ligase chain reaction (RT-LCR).

Antibody-based techniques can be used to determine the level of AIM II protein in biological samples. For example, by conventional immunohistological methods (Jalkanen M. et al., J. Cell Biology, 101:976-985 (1985); Jalkanen M. et al., J. Cell Biology, 105:3087-3096 (1987)), can To study the expression of AIM II protein in tissues.

Other antibody-based methods that can be effectively used for the determination of AIM II protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).

Various suitable labels are well known in the art and include enzyme labels such as glucose oxidase, radioactive isotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ C), tritium ( ³ H), indium ( ¹¹² In) and technetium ( ^99m Tc), and fluorescent labels such as fluorescein, rhodamine and biotin. therapy

AIM II polypeptides, especially human AIM II polypeptides, can be used for the treatment of neoplasia, lymphadenopathy, autoimmune diseases, graft versus host disease. In addition, the AIMII polypeptides of the present invention can also be used to inhibit neoplasia, such as the growth of tumor cells. AIM II polypeptides may participate in the destruction of tumors through apoptosis and cytotoxic effects on certain cells. Since AIM II has a proliferative effect on endothelial-derived cells, AIM II can also be used in the treatment of diseases that require growth-promoting activity, such as restenosis. Therefore, AIM II can also be used to regulate hematopoiesis during endothelial cell development. Method of administration

It should be understood that the above-mentioned conditions can be treated by administering AIM II protein. Accordingly, the present invention also provides methods of treating individuals in need of increased levels of AIM II activity, the method comprising administering to such individuals a pharmaceutical composition comprising an effective amount of an isolated AIM II polypeptide of the invention, particularly a mature form of AIM II. Compositions to effectively increase the level of AIM II activity in such individuals.

Although the dosage can be flexibly controlled according to the needs of the treatment as mentioned above, as a general recommendation, the total therapeutically effective dose of AIM II polypeptide administered parenterally is about 1 μg/kg patient body weight/day to 10mg/kg patient body weight/day per dose . More preferably, the dose is at least 0.01 mg/kg/day, and most preferably the hormone is administered at a dose of about 0.01-1 mg/kg/day for humans. If administered continuously, it can usually be accomplished by 1 to 4 injections per day or continuous subcutaneous infusion (such as using a mini pump), wherein the dose rate of AIM II polypeptide is about 1 μg/kg/hour to 50 μg/kg/hour. Intravenous pack solutions may also be used.

The pharmaceutical composition containing AIM II of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powder, ointment, drops, or epidermal patch), bucally, or by oral or nasal spray. "Pharmaceutical carrier" means a non-toxic solid, semi-solid or liquid formulation, diluent, encapsulating substance or any type of formulation auxiliary. The term "parenteral" as used herein is meant to include modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

In addition to soluble AIM II polypeptides, AIM II polypeptides containing transmembrane regions suitably solubilized by the addition of detergents such as Triton X-100 and buffer can also be used. chromosome detection

The nucleic acid molecules of the invention are also valuable in chromosome identification work. This sequence can be specifically directed to and hybridizes to a specific site on a human individual chromosome. According to the present invention, mapping DNA to chromosomes is a very important first step in linking sequences to disease-associated genes.

In certain preferred embodiments in this regard, the genomic DNA of the AIM II protein can be cloned using the cDNA disclosed herein. This can be done using a variety of techniques and libraries well known in the art. These libraries are generally commercially available. This genomic DNA is then used for in situ chromosome mapping by techniques well known in the art that allow for in situ chromosome mapping.

Additionally, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from cDNA. By computer analysis of the 3' untranslated region of the gene, primers that do not span more than one exon in the genomic DNA can be quickly selected, thereby complicating the amplification process. Then, using these primers, PCR screening was performed on somatic cell hybrids containing human individual chromosomes.

Fluorescence in situ hybridization ("FISH") of metaphase chromosomes using cDNA clones provides precise chromosomal localization in one step. Probes as short as 50 or 60 bp from cDNA can be used in this technique. For a review of this technique see Verma et al., The Human Chromosome: A Handbook of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise location on a chromosome, the physical location of the sequence on the chromosome can be linked to genetic map data. Such data can be found, eg, in V. McKusick, Mendelian Inheritance in Humans, available online through Johns Hopkins University, Welch Medical Library. Linkage analysis (co-inheritance of physically adjacent genes) is then used to identify the relationship between genes that have been located in the same chromosomal region and the disease.

Next, it is necessary to assay for differences between the cDNA or genome sequences of infected and uninfected individuals. If a mutation is observed in some or all infected individuals but not in any normal individuals, then the mutation is likely to be the cause of the disease.

Having generally described the present invention, the following examples will help to better understand the present invention, and these examples are provided by way of illustration, but not intended to limit the present invention. Example Example 1: Expression and purification of AIM II in Escherichia coli

A. Expression of AIM II with an N-terminal HA tag

Using PCR oligonucleotide primers specific to the amino terminal sequence of the AIM II protein and the carrier sequence on the 3' side of the gene, the DNA sequence encoding the AIM II protein in the preserved cDNA clone was amplified. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5' and 3' sequences, respectively.

A 22kDa fragment of the AIM II protein (lacking the N-terminal and transmembrane regions) was expressed using the following primers:

The sequence of the 5' oligonucleotide primer is 5' GCG GGATCC GGAGAGATGGTCACC3' (SEQ ID NO: 3), which corresponds to positions 244-258 in the AIM II protein coding sequence in Fig. 1A-C (SEQ ID NO: 1) nucleotides, and contains an underlined BamHI restriction site therein;

The sequence of the 3' primer is 5'CGC AAGCTT CCTTCACACCATGAAAGC3' (SEQ ID NO: 8), which contains an underlined Hind III restriction site followed by the 756- 783 nucleotides.

The complete AIM II protein can be expressed with the following primers:

The sequence of the 5' oligonucleotide primer is 5' GACC GGATCC ATG GAG GAGAGT GTC GTA CGG C3' (SEQ ID NO: 9), which corresponds to the AIM II protein coding sequence in Fig. 1A-C (SEQ ID NO: 1) 22 nucleotides within and contains a BamHI restriction site marked by the following underline;

The sequence of the 3' primer is 5'CGC AAGCTT CCT TCA CAC CAT GAAAGC 3' (SEQ ID NO: 10), which contains an underlined Hind III restriction site followed by the 756-783 nucleotides are shown.

These restriction sites match the restriction sites in the bacterial expression vector pQE9, which can be used for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE9 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS"), a 6-His tag and some restriction enzyme sites.

The amplified AIM II DNA and vector pQE9 were digested with BamHI and Hind III, and the digested DNA was ligated together. After inserting the AIM II protein DNA in the pQE9 vector that has been restricted, the AIM II protein coding region is placed downstream of the IPTG-inducible promoter in the vector and is effectively connected to it, and the insertion position of the AIM II protein coding region makes It is in-frame with the initiation AUG codon, which is properly placed to facilitate translation of the AIM II protein. B. Expression of AIM II with a C-terminal HA tag

In this example, the bacterial expression vector pQE60 was used for bacterial expression. (QIAGEN, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS"), encoding histidine residues The six codons of the six codons, and an appropriate single restriction enzyme site, wherein the histidine residues can be obtained by using the nickel-nitrilotriacetic acid ("Ni -NTA") affinity resin for affinity purification. These elements are arranged in such a way that the expressed polypeptide can be covalently linked with 6 histidine residues at its carboxyl terminus after insertion of the DNA fragment encoding the polypeptide (ie "6x His tag").

Using PCR oligonucleotide primers that anneal to the amino-terminal sequence of the desired AIM II protein segment and to the sequence 3′ to the cDNA coding sequence in the deposited construct, respectively, amplify the desired protein from the deposited cDNA clone. The DNA sequence of the AIM II protein segment. Additional nucleotides containing restriction sites to facilitate cloning into the pQE60 vector were added to the 5' and 3' sequences, respectively.

In order to clone the protein, the sequence of the 5' primer used is 5'GACGC CCATGG AG GAG GAG AGT GTC GTA CGC C3' (SEQ ID NO: 17), which contains the NcoI restriction site marked by underline, followed by Nucleotides that may be complementary to the amino-terminal coding sequence of the AIM II sequence in Figures 1A-C. Of course, it is well known to those of ordinary skill in the art that the 5' primer start site within the protein coding sequence can be altered to amplify a DNA fragment (shorter or longer) encoding any desired segment of the complete protein. The sequence of the 3' primer is 5'GACC GGATCC CAC CAT GAA AGCCCC GAA GTA AG3' (SEQ ID NO: 18), which contains the underlined BamHI restriction site, followed by the same sequence as shown in Figures 1A-C The 3'-terminal complementary nucleotides of the coding sequence before the stop codon in the AIM II DNA sequence, the coding sequence and the restriction site are arranged in a way that ensures that its reading frame is consistent with the reading frame of the 6 His codons in the pQE60 vector .

The amplified AIM II DNA fragment and vector pQE60 were digested with BamHI and NcoI, and then the digested DNA was ligated together. Insert the AIM II DNA into the restriction-digested pQE60 vector so that the AIM II protein coding region is placed downstream of the IPTG-inducible promoter, and is consistent with the start codon AUG and the six histidine codons in the reading frame .

The ligation mixture containing the HA-tagged expression construct prepared in A or B above was transformed into competent E. coli cells using standard methods. Such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The E. coli M15/rep4 strain containing multiple copies of plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan ^r "), was used to perform the Examples described herein. This strain is only one of many strains suitable for expressing AIM II protein, and it is available from Qiagen.

Transformants were identified by their ability to grow on LB plates containing ampicillin and kanamycin. Plasmid DNA was extracted from resistant colonies, and the identity of the cloned DNA was confirmed by restriction analysis.

Clones containing the desired construct were grown overnight ("O/N") in liquid LB medium supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml).

O/N cultures were inoculated into bulk cultures at an equilibration of about 1:100 to 1:250. Culture the cells until the optical density value at 600 nm ("OD600") is 0.4-0.6, and then add isopropyl-β-D-thiogalactoside ("IPT G") to a final concentration of 1 mM to inactivate The lacI repressor induces transcription from a lac repressor-sensitive promoter. Cells were then further cultured for 3 to 4 hours. Cells are then harvested by centrifugation and lysed by standard methods. Inclusion bodies were purified from lysed cells using conventional harvesting techniques, and the proteins in the inclusion bodies were dissolved in 8M urea. A solution of 8M urea containing solubilized protein in 2X phosphate buffered saline ("PBS") was passed through a PD-10 column, thereby removing the urea, exchanging the buffer, and allowing the protein to refold. The protein is further purified to remove endotoxin by another step - chromatography. Then, perform sterile filtration. Sterile-filtered protein preparations were stored in 2X PBS at a concentration of 95 [mu]/ml. C. Expression and purification of AIM II protein without HA tag

In this example, the bacterial expression vector pQE60 was used for bacterial expression. (QIAGEN, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS"), encoding histidine residues The six codons of the six codons, and an appropriate single restriction enzyme site, wherein the histidine residues can be obtained by using the nickel-nitrilotriacetic acid ("Ni -NTA") affinity resin for affinity purification. These elements are arranged in such a way that the expressed polypeptide can be covalently linked with 6 histidine residues at its carboxyl terminus after insertion of the DNA fragment encoding the polypeptide (ie "6x His tag"). However, in this example, the polypeptide coding sequence was inserted in such a way that the six histidine codons could not be translated, and thus the resulting polypeptide did not contain a 6×His tag.

Using PCR oligonucleotide primers that anneal to the amino-terminal sequence of the desired AIM II protein segment and to the sequence 3' to the cDNA coding sequence in the deposited construct, respectively, amplify the DNA encoding the desired protein from the deposited cDNA clone. DNA sequence of the AIM II protein segment lacking a hydrophobic leader sequence. Additional nucleotides containing restriction sites to facilitate cloning into the pQE60 vector were added to the 5' and 3' sequences, respectively.

In order to clone the protein, the sequence of the 5' primer used is 5'GACGC CCATGG AG GAG GAG AGT GTC GTA CGC C3' (SEQ ID NO: 17), which contains the NcoI restriction site marked by underline, followed by Nucleotides that may be complementary to the amino-terminal coding sequence of the AIM II sequence in Figures 1A-C. Of course, it is well known to those of ordinary skill in the art that the 5' primer start site within the protein coding sequence can be altered to amplify a DNA fragment (shorter or longer) encoding any desired segment of the complete protein. The sequence of the 3' primer is 5'CGC AAGCTT CCTT CAC ACC ATGAAA GC3' (SEQ ID NO: 19), which contains the underlined Hind III restriction site, followed by Nucleotides complementary to the 3' end non-coding sequence within the AIM II DNA sequence.

The amplified AIM II DNA fragment and vector pQE60 were digested with Hind III and NcoI, and then the digested DNA was ligated together. The AIM II DNA was inserted into the pQE60 vector that had been restricted, so that the AIM II protein coding region and its stop codon were placed downstream of the IPTG-inducible promoter and in the same reading frame as the start codon AUG. This stop codon prevents translation of the 6 histidine codons downstream of the insertion site.

The ligation mixture was transformed into competent large intestine using standard methods, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). in bacillus cells. The E. coli M15/rep4 strain containing multiple copies of plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan ^r "), was used to perform the Examples described herein. This strain is only one of many strains suitable for expressing AIM II protein, and it can be purchased from Qiagen (same address as above). Transformants were identified by their ability to grow on LB plates containing ampicillin and kanamycin. Plasmid DNA was extracted from resistant colonies, and the identity of the cloned DNA was confirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired construct were grown overnight ("O/N") in liquid LB medium supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). The O/N culture was then inoculated into a bulk culture at a dilution of approximately 1:25 to 1:250. Culture the cells until the optical density at 600 nm ("OD600") is 0.4-0.6, and then add isopropyl-β-D-thiogalactopyranoside ("IPTG") to a final concentration of 1 mM to inactivate lacI repressor to induce transcription from the lac repressor-sensitive promoter. Cells were then cultured for a further 3 to 4 hours before harvesting by centrifugation.

Cells were then homogenized in 6M guanidine hydrochloride, pH 8, for 3-4 hours at 4°C. Cell debris was removed by centrifugation and the supernatant containing AIM II was dialyzed against 50 mM sodium acetate buffer pH 6 supplemented with 200 mM NaCl. Alternatively, proteins can be successfully refolded by dialyzing the supernatant against 500 mM NaCl, 20% glycerol, 25 mM Tris HCl, pH 7.4, containing protease inhibitors. After renaturation, proteins can be purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. Alternatively, pure AIM II protein can be obtained by an affinity chromatography step, such as using an antibody column. Store purified protein at 4°C, or freeze at -80°C. Embodiment 2: Cloning and expressing AIM II protein in the baculovirus expression system

The cDNA sequence encoding the full-length AIM II protein in the deposited clone was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the AIM II gene:

The sequence of the 5' primer is 5'GCT CCA GGA TCC GCC ATC ATG GAGGAG AGT GTC GTA CGG C3' (SEQ ID NO: 1), which contains the underlined BamHI restriction site and the subsequent Figure 1A- 22 bases within the AIMII protein sequence in C. The 5' end of the amplified fragment encoding AIM II provides an efficient signal peptide upon insertion into an expression vector as described below. Efficient signals for translation initiation in eukaryotic cells are placed at appropriate positions within the vector segment in the construct as described by Kozak M., J. Molecular Biology, 196: 947-950 (1987);

The sequence of the 3' primer is 5'GA CGC GGT ACC GTC CAA TGC ACCACG CTC CTT CCT TC3' (SEQ ID NO: 12), which contains the underlined Asp718 restriction site followed by the same sequence as in Figures 1A-C Nucleotides complementary to nucleotides 769-795 within AIM II.

Amplified fragments were separated from 1% agarose gels using a commercially available kit ("Geneclean", BIO101 Inc., La Jolla, Ca.). This fragment was then digested with BamHI and Asp718 and purified on a 1% agarose gel. The fragment is named F2 here.

The vector pA2-GP was used to express the AIM II protein in a baculovirus expression vector by standard methods as described by Summers et al., Baculovirus Vector Methods and Insect Cell Culture Technique Manual, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) . This expression vector contains the strong polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by convenient restriction sites. The signal peptide of AcMNPVgp67 including the N-terminal methionine is located just upstream of the BamHI site. The polyadenylation site of Simian Virus 40 ("SV40") was also used in this vector to efficiently accomplish polyadenylation. In order to facilitate the screening of recombinant viruses, the vector also inserts the β-galactosidase gene derived from E. coli in the same direction as the polyhedrin gene promoter, followed by the polyadenylation signal of the polyhedrin gene . The polyhedrin gene sequence is flanked by viral sequences to allow cell-mediated homologous recombination with wild-type viral DNA to generate active virus expressing the cloned polynucleotide.

As known to those skilled in the art, as long as the construction process can provide the required signals for transcription, translation, transport, etc. in proper arrangement, such as AUG and signal peptide with the same reading frame, other baculovirus vectors can be used instead pA2-GP. Such vectors are described, among others, in Luckow et al., Virology, 170:31-39.

By conventional techniques in the art, the plasmid was digested with restriction enzymes BamHI and Asp718, and dephosphorylated with calf intestinal phosphatase. This DNA was then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO101 Inc., La Jolla, Ca). The vector DNA is named "V" here.

Fragment F2 and dephosphorylated plasmid V2 were ligated together by T4 DNA ligase. Escherichia coli HB101 cells were transformed with the ligation mixture, and spread on culture plates. The DNA from each colony was digested with XbaI, and the digested products were analyzed by gel electrophoresis to identify the bacteria containing the plasmid carrying the human AIM II gene. The sequence of the cloned fragments was confirmed by DNA sequencing. The plasmid is named pBac AIM II here.

5 μg of plasmid pBac AIM II was mixed with 1.0 μg of commercially available linearized baculovirus DNA (“Baculo Gold ^TM baculovirus DNA", Pharmingen, San Diego, CA.) together for co-transfection. In a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD), mix 1 μg of Baculo Gold ^TM virus DNA and 5 μg of plasmid pBac AIM II, then add 10 μl of Lipofectin and 90 μl of Grace's culture base, mix and let stand at room temperature for 15 minutes. The transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL1711 ) seeded on 35 mm tissue culture plates containing 1 ml of serum-free Grace's medium. The plate was shaken back and forth to mix the newly added solution, and the plate was incubated at 27°C for 5 hours. After 5 hours, the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal bovine serum was added. The plates were returned to the incubator and incubated for an additional 4 days at 27°C.

After 4 days, supernatants were collected and plaque assays were performed as described by Summers and Smith, cited above. The use of agarose gels containing "Blue Gal" (Life technologies Inc., Gaithersburg) for easy identification and isolation of galactosidase-expressing clones that produce blue-stained Plaque (a detailed description of this "plaque assay" can also be found in Insect Cell Culture and Baculovirology Instructions for Use, Life Technologies Inc., Gaithersburg, pages 9-10).

After 4 days of serial dilution, the virus was added to the cells. After proper incubation, the blue-stained plaques were picked out with the tip of an Eppendorf pipette, and then the agar containing the recombinant virus was resuspended in an Eppendorf tube containing 200 μl of Grace’s medium. The agar was removed by slight centrifugation, and then the supernatant containing the recombinant baculovirus was used to infect the Sf9 cells inoculated on a 35 mm plate. After 4 days, the supernatants from these plates were collected and they were stored at 4°C. Clones containing the correctly inserted hESSBI, II and III were identified by DNA analysis methods such as restriction enzyme mapping and sequencing. This clone is named V-AIM II in this paper.

Sf9 cells were cultured in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected at a multiplicity of infection ("MOI") of around 2 (around 1 to around 3) with recombinant baculovirus V-AIM II. After 6 hours, the medium was removed and replaced with methionine- and cysteine-free SF900 II medium (available from LifeTechnologies Inc., Gaithersburg). After 42 hours, 5 μCi ³⁵ S-methionine and 5 μCi ³⁵ S-cysteine (available from Amersham) were added. The cells were cultured for 16 hours, and then the cells were collected by centrifugation, lysed, and the labeled proteins were observed by SDS-PAGE and autoradiography. Example 3: Cloning and Expression in Mammalian Cells

Most of the vectors used for transient expression of AIM II protein in mammalian cells should carry the SV40 origin of replication. This helps to ensure that the vector replicates to high copy numbers in cells expressing the T antigen required for initiation of viral DNA synthesis, such as COS cells. Any other mammalian cell line may also be used for this purpose.

A typical mammalian expression vector contains a promoter element that mediates transcription initiation of mRNA, protein coding sequence, and signals required for transcription termination and transcript polyadenylation. Additional elements include enhancers, Kozak sequences, and intervening sequences flanked by donor and acceptor sites required for RNA splicing. High-efficiency transcription can be obtained by using the early and late promoters of SV40, the long terminal repeat regions (LTRs) of retroviruses such as RSV, HTLVI, and HIVI, and the early promoters of cytomegalovirus (CMV). However, cellular signals (such as the human actin promoter) can also be used. Suitable expression vectors that can be used to carry out the present invention include vectors such as pSVL, pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC67109). Available mammalian cells include human Hela cells, 283 cells, H9 cells and Jurkart cells, mouse NIH3T3 cells and C127 cells, Cosl cells, Cos7 cells and CV1 cells, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.

Alternatively, the gene can also be expressed in a stable cell line in which the gene is integrated chromosomally. Transfected cells can be identified and isolated by co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin genes.

A transfected gene can also be amplified to express the protein it encodes in large quantities. DHFR (dihydrofolate reductase) is a useful marker when developing cell lines carrying hundreds or even thousands of copies of a gene of interest. Another useful selectable marker is glutamine synthetase (GS) (Murphy et al., J. Biol. Chem., 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, these mammalian cells are grown in selective media and the most resistant cells are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells are often used to produce proteins.

Expression vectors pC1 and pC4 contain the Rous sarcoma virus strong promoter (LTR) (Cullen et al., Molecular and Cellular Biology, 438- 447 (March 1985)). Multiple cloning sites, such as BamHI, XbaI and Asp718 restriction enzyme sites, can facilitate the cloning of genes of interest. The vector also contains the 3' intron, polyadenylation and termination signal for the rat preproinsulin gene. Example 3(a): Cloning and expression in COS cells

The expression plasmid pAIM II HA was made by cloning the cNDA encoding AIM II into the expression vector pcDNA I/Amp (available from Invitrogen Inc.).

This expression vector pcDNA I/amp contains: (1) the Escherichia coli origin of replication that can be effectively used for amplification in Escherichia coli and other prokaryotic cells; (2) the ampicillin resistance gene that can be used for the selection of prokaryotic cells containing plasmids; ( 3) SV40 origin of replication that can be used for eukaryotic intracellular amplification; (4) CMV promoter, multi-site linker, SV40 intron and polyadenylation signal, the arrangement of these sequences can ensure the passage of multi-site linker Restriction sites within the cDNA conveniently place the cDNA under the expression control of the CMV promoter and are operatively linked to the SV40 intron and polyadenylation signal.

The DNA fragment encoding AIM II protein and HA tag fused at its 3′ end was cloned into the multi-site linker region of the vector, so as to express recombinant protein under the guidance of CMV promoter. This HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell, 37:767 (1984). Fusion of the HA tag within the target protein allows for easy detection of the recombinant protein by antibodies recognizing HA epitopes.

The strategy for plasmid construction is as follows. Similar to the above process of constructing an expression vector expressing AIM II in E. coli, the AIM II cDNA in the deposited clone was amplified using primers containing convenient restriction sites. To facilitate detection, purification and identification of expressed AIM II, one of the primers contained the above-mentioned hemagglutinin tag ("HA tag").

Suitable primers are the following primers used in this example: a 5' primer containing the underlined BamHI site and the AUG initiation codon, the sequence of which is as follows:

5'G CTC GGA TCC GCC ATC ATG 3' (SEQ ID NO: 13);

The 3' primer containing the underlined XbaI site, stop codon, 9 codons that can form the hemagglutinin HA tag, and the 3' coding sequence (at its 3' end) of 31 bp has the following sequence:

5'GAT GT T CTA GA A AGC GTA GTC TGG GAC GTC GTATGG GTA CAC CAT GAA AGC CCC GAA GTA AGA CCG GGTAC 3' (SEQ ID NO: 14).

The PCR amplified DNA fragment and the carrier pcDNAI/Amp were digested by HindIII and XhoI, and then they were connected. The ligation mixture was transformed into E. coli SURE strain (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA92037), the transformed culture was plated on plates containing ampicillin medium, and the plates were grown to ensure the growth of ampicillin-resistant colonies. Plasmid DNA was isolated from resistant colonies, and the presence of the AIM II coding fragment in the plasmid DNA was detected by restriction analysis and gel electrophoresis.

For expression of recombinant AIM II, DEAE-DEXTRAN as described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) can be used to transfect COS with the above-mentioned expression vector. cell. Cells were cultured under conditions suitable for vector expression of AIM II.

The AIM II HA fusion protein was determined by radiolabeling and immunoprecipitation as described in Harlow et al., "Antibodies: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). Express. For this purpose, 2 days after transfection, the cells were labeled by culturing them in medium containing ³⁵ S-cysteine. Cells and culture medium were collected, cells were washed, and then according to the method described by Wilson et al. 40, 0.5% DOC, 50 mM Tris, pH 7.5 to lyse the cells. Proteins were precipitated from cell lysates and tissue culture media using HA-specific monoclonal antibodies. Precipitated proteins were then analyzed by SDS-PAGE gels and radiography. An expression product of the expected size was observed in the cell lysate, which was absent in the negative control. Example 3(b): Cloning and expression in CHO cells

The AIM II protein was expressed using the vector pC4. Plasmid pCl is a derivative of plasmid pSV2-dhfr [ATCC Deposit No. 37146]. Both plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. Dihydrofolate-free Chinese hamster ovary cells or other cells transfected with these plasmids can be selected by culturing the cells in selective medium (alpyha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) has been well documented (see e.g., Alt F.W., Kellems R.M., Bertino J.R. and Schimke R.T., 1978, Journal of Biochemistry, 253:1357-1370; Hamlin J.L. and Ma C., 1990, Biochem et Biophys. Acta, 1097: 107-143; Page M.J. and Sydenham M.A., 1991, Biotechnology, Vol. 9: 64-68). Cells grown at higher concentrations of MTX were able to enhance their resistance to this drug through amplification of the DHFR gene and overexpression of the target enzyme DHFR. If the DHFR gene is linked to another gene, it is usually coamplified and overexpressed as well. The development of cell lines containing more than 1000 copies of genes is an important work in the field. Thus, after methotrexate was withdrawn, the cell line still contained the amplified gene integrated into the chromosome.

Plasmid pC4 contains a strong promoter of the Rous sarcoma virus long terminal repeat (LTR) for expression of the gene of interest (Cullen et al., Mol. (CMV) a fragment isolated from the enhancer of the immediate early gene (Boshart et al., Cell, 41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI and Asp718 restriction sites that allow gene insertion. This plasmid also contains the 3' intron and polyadenylation site of the rat preproinsulin gene after these cloning sites. Other high-efficiency promoters can also be used for expression, such as the human β-actin gene promoter, the early or late promoter of SV40, or the long terminal repeat regions of other retroviruses such as HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems can be used to express AIM II in a regulated manner in mammalian cells (Gossen M. and Bujard H., 1992, Proceedings of the National Academy of Sciences of the United States of America, 89: 5547-5551). As for the polyadenylation of the mRNA, other signals such as those derived from the human growth hormone or globin genes can also be used. Stable cell lines carrying the gene of interest integrated into the chromosome can also be selected by co-transformation with selectable markers such as gpt, G418 or hygromycin. It is best to start with multiple selectable markers, such as G418 and methotrexate at the same time.

Plasmid pC4 was digested with restriction enzymes BamHI and Asp718, and then dephosphorylated with calf intestinal phosphatase by methods known in the art. The vector was then separated by 1% agarose gel.

The DNA sequence encoding the complete AIM II protein and its leader sequence was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene, respectively. The sequence of the 5' primer is: 5'GCT CCA GGA TCC GCC ATC ATG GAG GAG AGTGTC GTA CGG C3' (SEQ ID NO: 15), which contains the underlined BamHI restriction enzyme site followed by Kozak M., Journal of Molecular Biology, 196: 947-950 (1987) describes efficient signals for translation initiation in eukaryotes, and the AIM II code shown in Figure 1A-C (SEQ ID NO: 1) 22 bases within the sequence. The sequence of the 3' primer is: 5' GA CGC GGT ACC GTC CAA TGC ACC ACG CTC CTT CCT TC3' (SEQ ID NO: 16), which contains the Asp718 restriction site represented by the underline and the same as in Fig. 1A-C ( SEQ ID NO: 1) Complementary nucleotides to nucleotides 769-795 of the AIM II gene.

The amplified fragment was digested with endonucleases BamHI and Asp718, and repurified on 1% agarose gel. Then connect the isolated fragment with the dephosphorylated vector by T4 DNA ligase, and then transform Escherichia coli HB101 or XL1-Blue cells, and identify the bacteria containing the fragment inserted into the plasmid pC4 by analysis such as restriction enzymes .

Chinese hamster ovary cells lacking an active DHFR gene were used for transformation. 5 μg of expression plasmid pC4 was co-transformed with 0.5 μg of plasmid pSV2-neo using Lipofectin (Felgner et al., supra). Plasmid pSV2 neo contains a dominant selectable marker, the Tn5-derived neo gene, which encodes enzymes that confer resistance to a group of antibiotics including G418. The cells were seeded in alpha minus MEM medium supplemented with 1 mg/ml G418. After 2 days, the cells were trypsinized and plated on hybridoma cloning plates (Greiner, Germany) containing alpha minus MEM medium supplemented simultaneously with 10.25 or 50 ng/ml methotrexate and 1 mg/ml G418. After approximately 10-14 days, individual clones were trypsinized and seeded in 6-well dishes or 10 ml Erlenmeyer flasks with different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones grown in the highest concentration of methotrexate were transferred to new 6-well plates containing higher concentrations of methotrexate (1 μΜ, 2 μΜ, 5 μΜ, 10 mM, 20 mM). This step was repeated until a clone capable of growing at a concentration of 100-200 μM was obtained. The expression of the gene product of interest is analyzed by methods such as SDS-PAGE, Western blot, or reverse-phase HPLC analysis. Example 4: Tissue distribution of AIM II protein expression

Northern blot analysis was performed to study AIM II gene expression in human tissues using the method described by Sambrook et al. (supra) and elsewhere. The cDNA probe containing the entire nucleotide sequence of AIM II protein (SEQ ID NO: 1) was labeled with ³² P using the rediprime ^™ DNA labeling system (Amersham LifeScience) according to the manufacturer's instructions. After labeling, the probe was purified using a CHROMA SPIN-100 ^™ column (Clontech Laboratories) according to the manufacturer's No. PT1200-1 technique. The purified labeled probes were then used to analyze the levels of AIM II mRNA in various human tissues.

A multi-tissue Northern (Multiple Tissue Northern, MTN) blot containing multiple human tissues (H) or human immune system tissues (IM) was obtained from Clontech, and according to the manufacturer's PT1190-1 technical protocol, using ExpressHyb ^™ hybridization solution ( Clontech), these blots were analyzed by labeled probes. After hybridization and washing, the blots were fixed and exposed to film overnight at -70°C, and the film was developed according to standard methods.

Obviously, the invention may be practiced otherwise than as detailed in the specification and examples herein.

Many modifications and variations of the present invention may be made in light of the above teachings and they are therefore also within the scope of the appended claims.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are incorporated herein by reference.

Sequence Listing (1) General information:

(i) Applicant: Human Genome Sciences Corporation

                  9410 Key West Avenue

Rockville, MD 20850

U.S

Applicant/Inventor Ebner, Reinhard

Person: Yu, Guo-Liang

Ruben, Steven M.

(ii) Invention name: Apoptotic Cell Death Inducing Molecule II

(iii) Number of sequences: 19

(iv) Correspondence address:

(A) Recipient: Sterne, Kessler, Goldstein & Fox, P.L.L.C

(B) Street: 1100 New York Ave., Suite 600

(C) City: Washington

(D) State: DC

(E) Country: United States

(F) Zip code: 20005-3934

(v) in computer readable form:

(A) Media type: Floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: Patent In Release#1.0, Version#1.30

(vi) Current application information

(A) Application number: TBA

(B) Filing date: Attached hereto

(C) Classification:

(vii) Prior application information:

(A) Application number: 60/013,923

(B) Application date: March 22, 1996

(C) Classification:

(viii) Agent/Agency Information:

(A) Name: Goldstein, Jorge A.

(B) Registration number: 29,021

(C) Material/document number: 1488.065 PC 01/JAG/EKS/KMT(ix) Communication information:

(A) Tel: 202-371-2600

(B) Telex: 202-371-2540 (2) SEQ ID NO: 1 Information: (i) Sequence Characteristics:

(A) Length: 1169 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: linear (ii) Molecular type: DNA (genome) (ix) Properties:

(A) Name/keyword: CDS

(B) Position: 49..768 (xi) Sequence description: SEQ ID NO: 1: GAGGTTGAAG GACCCAGGCG TGTCAGCCCT GCTCCAGAGA CCTTGGGC ATG GAG GAG 57

Met Glu Glu

1AGT GTC GTA CGG CCC TCA GTG TTT GTG GGA CAG GAC GAC GAC ATC 105ser Val Val PHE Val Val Val Val Vln Thr ASP Ile

5 10 15cca TTC ACG ACG ACG GGA CGA AGC CAC CAC CAC AGA CAG TCG TGC AGT GTG 153PRO PHR ARG Leu Gly ARG ARG GLN SER Val Val Val Val Val 20 30 35GCC CGG GGT CTG CTG CTG CTG TTG CTG TTG CTG CTG CTG CTG CTG GGG CTG 201Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly Ala Gly Leu

40 45 50GCC GTC CGC TGG TGG TTC CTG CTG CAG CAC CAC CAC CGG CGT CTA GGA 249ALA Val Gln Gly Tru Leu Gln Leu His Tru GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY

55 60 65ATG GTC ACC CGC CGC CTG CCT GGA CCT GCA GGC TCC TGG CAG CAG CGG 297MET Val THR ARG Leu Pro ASLA GLY Serp Gln Leu Gln Leu Gln Leu

70 75 80ATA CAA GAA GAG TCT CAG GAG GAG GAG GAG GCA GCA GCA GCG Cat CTC ACA 345ile Gln Gln Gln Glu ARG Serg Serg Serg Serg

85                  90                  95GGG GCC AAC TCC AGC TTG ACC GGC AGC GGG GGG CCG CTG TTA TGG GAG         393Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu100                 105                 110                 115ACT CAG CTG GGC CTG GCC TTC CTG AGG GGC CTC AGC TAC CAC GAT GGG 441Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly

120 125 130GCC CTT GTG GTC ACC AAA GCT GGC TAC TAC TAC TAC TCC TCC AAG GTG 489ALA Leu Val Val THR LA GLY Tyr Tyr Tyr Lyser Tyr Lyser Tyr Lys Lys that was Lysle Tyr -I Lysle Tyr's Tyr that Tyr that T that?

135 140 145CAG CTG GGC GGT GGT GGC TGC CCG CTG GCC CTG GCC AGC ACC ACC 537GLN Leu GLY GLY VAL VAL GLY CYS PRL Leu Ala Serte Thr Ile Thr

150 155 160CAC GGC CTC TAC AAG CGC ACA CCC CGC CGC CCC GAG GAG GAG GAG GAG GAG 585HIS GLY Leu Tys ARG THR PRU Glu Glu Glu Glu Glu Glu Glu Glu G

165                 170                 175TTG GTC AGC CAG CAG TCA CCC TGC GGA CGG GCC ACC AGC AGC TCC CGG         633Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg180                 185                 190                 195GTC TGG TGG GAC AGC AGC TTC CTG GGT GGT GTG GTA CAC CTG GAG GCT 681Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala

200 205 20GGG GAG GAG GAG GAG GTG GTC GTC CGT GTG CTG GAA CGC CGC CTG GTT CGA CGA 729GLY GLU Val Val ARG Val Leu ARG Leu Val ARG Leu

215 220 225CGT GGT ACC CGG TCT TCTC GGG GGG GCT TG GTG GTG TGAAGGG 778ARG ASP GLY THR AR TYR ALA PHE MET Val Val Val Val Val Val Val Val Val Val

230                 235                 240AGCGTGGTGC ATTGGACATG GGTCTGACAC GTGGAGAACT CAGAGGGTGC CTCAGGGGAA       838AGAAAACTCA CGAAGCAGAG GCTGGGCGTG GTGGCTCTCG CCTGTAATCC CAGCACTTTG       898GGAGGCCAAG GCAGGCGGAT CACCTGAGGT CAGGAGTTCG AGACCAGCCT GGCTAACATG       958GCAAAACCCC ATCTCTACTA AAAATACAAA AATTAGCCGG ACGTGGTGGT GCCTGCCTGT       1018AATCCAGCTA CTCAGGAGGC TGAGGCAGGA TAATTTTGCT TAAACCCGGG AGGCGGAGGT       1078TGCAGTGAGC CGAGATCACA CCACTGCACT CCAACCTGGG AAACGCAGTG AGACTGTGCC       1138TCAAAAAAAA AAAAAAAAAA AAAAAAAAAA A                                      1169(2)SEQ ID NO： 2 Information: (i) Sequence Features:

(A) Length: 240 amino acids

(B) Type: amino acid

(D) Topological structure: linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 2: Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Gln1 5 5 10 T le P hr sp hr 1 Arg Leu Gly Arg Ser His Arg Arg Gln Ser

20 25 25 30Cys Ser Val Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly

35 40 45Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg

50 55 60leu GLY GLU MET VAL THR ARG Leu Pro ASP GLY Pro Ala Gly Serp65 70 75 80Glu Gln Leu Ile Gln Glu ARG Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg's Glu Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala AR -ALU that

85 90 95His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu

100 105 110Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr

115 120 125His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr

130                 135                 140Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser145                 150                 155                 160Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu

165 170 175Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser

180 185 190Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His

195 200 205 Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu

210                 215                 220Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val225                 230                 235                  240(2)SEQ ID NO：3信息：(i)序列特征：

(A) Length: 455 amino acids

(B) Type: amino acid

(C) Chain type: irrelevant

(D) Topological structure: irrelevant (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 3: Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu 1 5 5 u u Val lu G 10 Le u Val lu G 1 Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

20 25 25 30His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

35 40 45Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

50 55 60Gly Thr Tyr Leu Tyr Asn ASP CYS Pro Gly Pro GLN ASP THR ASP65 70 75 80CY GLU CYS GLY Serge Thr Ala Sern His Leu

85 90 95Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

100 105 110Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

115 120 125Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

130                 135                 140Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu145                 150                 155                 160Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

165 170 175 Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

180 185 190Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser

195 200 205Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu

210 215 220leu Seru Leu PHE Ile Gly Leu Met Tyr ARG TYR GLN ARG TRP LYS225 235 240SER LEU TYR Serle Val Cys GLY LYS SER Pro Glu LYS GLU

245 250 255Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser

260 265 270Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val

275 280 285Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys

290 295 300Pro Asn Phe Ala Ala Pro ARG Glu Val Ala Pro Tyr Gln GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN Gln

325 330 335Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp

340 345 350Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val Glu Asn Val Pro Pro

355 360 365Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu

370                 375                 380Ile Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln385                 390                 395                 400Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg Glu Ala

405 410 415Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly

420 425 430Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro

435 440 445Pro Ala Pro Ser Leu Leu Arg

450 455(2) SEQ ID NO: 4 Information: (i) Sequence Characteristics:

(A) Length: 205 amino acids

(B) Type: amino acid

(C) Chain type: irrelevant

(D) Topological structure: irrelevant (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 4: Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr1 5 5 is u Le u Le u Le u H 1 Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

20 25 30Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

35 40 45Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala

50 55 60ALA His Leu Ile Gly ASP Pro Ser Lysn Gln Seru TRP ARG65 75 80ALA Asn Thr ARA PHE Leu Gln ASP GLY PHE Seru Seru Seru Seru Seru Seru Seru Seru Seru Ser Asr

85 90 95Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

100 105 110Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro

115 120 125 Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

130 135 140His Val Pro Leu Leu Ser Sergs Met Val Tyr Pro GLY Leu Gln145 150 155 160GLU TRP Leu His SER HIS GLY Ala Phe Gln Leu Thr.

165 170 175Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val

180 185 190Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

195 200 205 (2) SEQ ID NO: 5 information: (i) Sequence characteristics:

(A) Length: 205 amino acids

(B) Type: amino acid

(C) Chain type: irrelevant

(D) Topological structure: irrelevant (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 5: Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr1 5 5 is u Le u Le u H 1 5 Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

20 25 30Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

35 40 45Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala

50 55 60ALA His Leu Ile Gly ASP Pro Ser Lysn Gln Seru TRP ARG65 75 80ALA Asn Thr ARA PHE Leu Gln ASP GLY PHE Seru Seru Seru Seru Seru Seru Seru Seru Seru Ser Asr

85 90 95Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

100 105 110Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro

115 120 125 Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

130 135 140His Val Pro Leu Leu Ser Sergs Met Val Tyr Pro GLY Leu Gln145 150 155 160GLU TRP Leu His SER HIS GLY Ala Phe Gln Leu Thr.

165 170 175Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val

180 185 190Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

195 200 205 (2) SEQ ID NO: 6 information: (i) sequence characteristics:

(A) Length: 281 amino acids

(B) Type: amino acid

(C) Chain type: irrelevant

(D) Topology: Unrelated (II) Molecular type: Protein (xi) sequence description: SEQ ID NO: 6: Met Gln Gln Pro PHE Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Ala Ser Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys

20 25 25 30Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro Pro

35 40 45Pro Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

50 55pro leu project

85 90 95Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

100 105 110 Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Ser Leu Glu

115 120 125Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg

135 135140lys Val Ala His Leu Thr Gly Lysn Serg Serg Serg Serg Serite Proo Leu145 150 155 160GLU TRU ASP THR GLY ILE Val Leu Sering Val Lys Tyr

165 170 175Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr

180 185 190Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser

195 200 205 His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met

210 215 220MET GLY LYS MET MET MET MET SER CYS Thr THR GLN MET TRA225 230 235 240ARG Ser Tyr Leu Gly Ala Val Phr Sera Ala aser

245 250 255Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser

260 265 270Gln Thr Phe Phe Gly Leu Tyr Lys Leu

275 280(2) SEQ ID NO: 7 information: (i) Sequence characteristics:

(A) Length: 24 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 7: GCGGGATCCG GAGAGATGGT CACC 24 (2) SEQ ID NO: 8 Information: (i) Sequence features:

(A) Length: 27 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 8: CGCAAGCTTC CTTCACACCA TGAAAGC 27 (2) SEQ ID NO: 9 Information: (i) Sequence features:

(A) Length: 32 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 9: GACCGGATCC ATGGAGGAGA GTGTCGTACG GC 32 (2) SEQ ID NO: 10 Information: (i) Sequence features:

(A) Length: 27 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 10: CGCAAGCTTC CTTCACACCA TGAAAGC 27 (2) SEQ ID NO: 11 information: (i) Sequence characteristics:

(A) Length: 40 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 11: GCTCCAGGAT CCGCCATCAT GGAGGAGAGTGTCGTACGGC 40 (2) SEQ ID NO: 12 information:

(i) Sequential features:

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 12: GACGCGGTAC CGTCCAATGC ACCACGCTCC TTCCTTC 37 (2) SEQ ID NO: 13 information: (i) Sequence features:

(A) Length: 19 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 13: GCTCGGATCC GCCATCATG 19 (2) SEQ ID NO: 14 information: (i) Sequence features:

(A) Length: 71 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear (II) Molecular type: CDNA (XI) sequence description: SEQ ID NO: 14: Gatgttctag AAAGCGTAGT CTGGGGTC GTACAAAG CCCCCGGGGTA C 71 (2) sequence: (2) information: (I): (2) :

(A) Length: 40 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 15: GCTCCAGGAT CCGCCATCAT GGAGGAGAGTGTCGTACGGC 40 (2) SEQ ID NO: 16 information: (i) Sequence features:

(A) Length: 37 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 16: GACGCGGTAC CGTCCAATGC ACCACGCTCC TTCCTTC 37 (2) SEQ ID NO: 17 information: (i) Sequence features:

(A) Length: 32 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 17: GACGCCCATG GAGGAGGAGA GTGTCGTACG GC 32 (2) SEQ ID NO: 18 information: (i) Sequence features:

(A) Length: 33 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 18: GACCGGATCC CACCATGAAA GCCCCGAAGT AAG 33 (2) SEQ ID NO: 19 information: (i) Sequence features:

(A) Length: 27 base pairs

(B) Type: nucleic acid

(C) Chain type: single chain

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 19: CGCAAGCTTC CTTCACACCA TGAAAGC 27

Claims (19)

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence of at least 95% identity to a sequence selected from the group consisting of: (a) coding has the nucleotide sequence of the AIM II polypeptide of complete aminoacid sequence (SEQ ID NO:2) among Fig. 1A-C; (b) a nucleotide sequence encoding an AIM II polypeptide having the entire amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 97689; (c) a nucleotide sequence encoding the extracellular domain of the AIM II polypeptide; (d) the nucleotide sequence of coding AIM II polypeptide transmembrane domain; (e) a nucleotide sequence encoding the intracellular domain of the AIM II polypeptide; (f) a nucleotide sequence encoding a soluble AIM II polypeptide that contains extracellular and intracellular domains, but lacks a transmembrane domain; and (g) A nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above. 2. The nucleic acid molecule of claim 1, wherein said polynucleotide has the entire nucleotide sequence (SEQ ID NO: 1) in Figures 1A-C. 3. The nucleic acid molecule of claim 1, wherein said polynucleotide has a nucleotide sequence encoding an AIM II polypeptide among Fig. 1A-C (SEQ ID NO: 1), and said AIM II polypeptide has among Fig. 1A-C Full amino acid sequence (SEQ ID NO: 2). 4. The nucleic acid molecule of claim 1, wherein said polynucleotide has the entire nucleotide sequence of the cDNA clone contained in ATCC Accession No. 97689. 5. The nucleic acid molecule of claim 1, wherein said polynucleotide has a nucleotide sequence encoding an AIM II polypeptide having the entire amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 97689. 6. An isolated nucleic acid molecule comprising a section of polynucleotide that can contain (a), (b), (c), (d), (e), ( f) or (g) a polynucleotide of the same nucleotide sequence as in (g), wherein said polynucleotide hybridizes under stringent hybridization conditions to a nucleus containing only A residues or only T residues Nucleotide sequences cannot hybridize. 7. An isolated nucleic acid molecule, which contains the polynucleotide of the amino acid sequence of the segment having an epitope in the encoding AIM II polypeptide, and the AIM II polypeptide has (a), (b), (c) of claim 1 ), (d, ) the amino acid sequence in (e) or (f). 8. The nucleic acid molecule of the separation of claim 7, its coding AIM II polypeptide has the segment of epitope, and described segment is selected from in following group: containing about the first in Fig. 1A-C (SEQ ID NO:2) A polypeptide comprising amino acid residues at position 13 to approximately 20; a polypeptide comprising amino acid residues at approximately position 23 to approximately 36 in Figure 1A-C (SEQ ID NO: 2); a polypeptide comprising amino acid residues at approximately position 23 to approximately 36 in Figure 1A-C (SEQ ID NO: 2); 2) A polypeptide comprising amino acid residues at about 69 to about 79; a polypeptide comprising amino acid residues at about 85 to about 94 in Figure 1A-C (SEQ ID NO: 2); comprising Figure 1A-C (SEQ ID NO: 2); A polypeptide comprising about amino acid residues 167 to about 178 of C (SEQ ID NO: 2); a polypeptide comprising amino acid residues about 184 to about 196 of Figures 1A-C (SEQ ID NO: 2) Polypeptide; a polypeptide comprising about amino acid residues 221 to about 233 of Figures 1A-C (SEQ ID NO: 2). 9. A method of preparing a recombinant vector, said method comprising inserting the isolated nucleic acid molecule of claim 1 into the vector. 10. A recombinant vector prepared by the method of claim 9. 11. A method for preparing a recombinant host cell, said method comprising introducing the recombinant vector of claim 10 into the host cell. 12. A recombinant host cell prepared by the method of claim 11. 13. A method for recombinantly producing an AIM II polypeptide, said method comprising cultivating the recombinant host cell of claim 12 under conditions in which said polypeptide can be expressed; and reclaiming said polypeptide. 14. An isolated AIM II polypeptide comprising an amino acid sequence having at least 95% identity to a sequence selected from the group consisting of: (a) the amino acid sequence of the AIM II polypeptide having the entire amino acid sequence in Figures 1A-C (SEQ ID NO: 2); (b) the amino acid sequence of the AIM II polypeptide having the entire amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 97689; (c) the amino acid sequence of the extracellular domain of the AIM II polypeptide; (d) the amino acid sequence of the transmembrane domain of the AIM II peptide; (e) the amino acid sequence of the intracellular domain of the AIM II polypeptide; (f) the amino acid sequence of a soluble AIM II polypeptide having all or part of the extracellular and intracellular domains, but lacking a transmembrane domain; and (g) the amino acid sequence of the epitope-containing segment of any one of (a), (b), (c), (d), (e) or (f). 15. An isolated polypeptide comprising a segment of an AIM II protein having an epitope, wherein said segment is selected from the group consisting of about position 13 in Figure 1A-C (SEQ ID NO: 2) A polypeptide to about amino acid residues 20; a polypeptide comprising amino acid residues from about 23 to about 36 in Figure 1A-C (SEQ ID NO: 2); a polypeptide comprising Figure 1A-C (SEQ ID NO: 2) A polypeptide comprising about 69th to about 79th amino acid residue in Figure 1A-C (SEQ ID NO: 2); a polypeptide comprising about 85th to about 94th amino acid residue in Figure 1A-C (SEQ ID NO: 2); comprising Figure 1A-C ( A polypeptide comprising amino acid residues from about 167 to about 178 of SEQ ID NO: 2); a polypeptide comprising amino acid residues from about 184 to about 196 in Figures 1A-C (SEQ ID NO: 2); and polypeptides comprising amino acid residues from about 221 to about 233 in Figures 1A-C (SEQ ID NO: 2). 16. An isolated antibody capable of specifically binding to the AIM II polypeptide of claim 14. 17. The polypeptide in (a), (b), (c), (d), (e) or (f) of claim 14 is used in the preparation of a medicament for the treatment of a state or disease selected from the group consisting of Use in the composition: lymphadenopathy, autoimmune disease and graft versus host disease. 18. Use of the polypeptide in (a), (b), (c), (d), (e) or (f) of claim 14 in the preparation of a pharmaceutical composition useful for inhibiting neoplasia. 19. Use of an antagonist of a polypeptide according to claim 14 for the preparation of a pharmaceutical composition useful for the prevention of a state or disease selected from the group consisting of: septic shock, infection, cerebral malaria, HIV-viral activation , bone resorption, rheumatoid arthritis and cachexia.

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