CN1170850C - Human angiopoietin-like protein and coding sequence and use thereof - Google Patents

Abstract

The present invention discloses novel human angiogenin-like protein, polynucleotide for encoding the polypeptide, and a method for generating the polypeptide by recombination technology. The present invention also discloses a method by which the polypeptide is used for treating various diseases such as cancer, etc.; the present invention also discloses an antagonist for resisting the polypeptide, and a treating function thereof. The present invention also discloses the purpose of the polynucleotide for encoding the novel human angiogenin-like protein.

Description

Human angiopoietin-like protein and coding sequence and use thereof

technical field

The invention belongs to the field of biotechnology, in particular, the invention relates to a new polynucleotide encoding human angiopoietin-like protein and the polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation method of the polynucleotide and polypeptide.

Background technique

Angiogenesis is the key to the occurrence, evolution and metastasis of malignant tumors. It is known that vascular endothelial growth factor (VEGF) and its receptor (VEGFreceptor VEGFR) play an important role in tumors. VEGF and VEGFR are not only highly expressed in vascular endothelial cells, but also expressed in some malignant tumor cells, thus forming autocrine and adjacent secretory systems, supporting and accelerating the formation and development of malignant tumors.

Since 1996, it has been discovered that in addition to the VEGF/VEGFR system, there are still angiopoietin (Angiopoietin Ang) and its receptor TIE2 system, which plays an extremely important role in angiogenesis, and interacts with the VEGF/VEGFR system to jointly regulate blood vessels. Formation. Ang is a family, and the Ang family that has been discovered has Ang1-5. Among them, Ang1 has two sequences, ANGPT1 99.3.19 registration, registration number NM001146, Ang-1 97.3.26 registration, registration number U83508, Ang-3 has two sequences. ANGPT3 99.5.7 registration, NM_004673, Ang-1 99.5.24 registration, AF074332. That is a total of 7 members. In addition, South Korea registered an angiopoietin-related protein cDNA (Angiopoietin-related Protein, registration number AF153606) in Genbank in June 1999, which is an unpublished material.

Cancer is one of the major diseases that endanger human health. In order to effectively treat and prevent tumors, people have paid more and more attention to gene therapy of tumors. Therefore, there is an urgent need in this field to develop and study proteins with tumor suppressor effects and their agonists/inhibitors.

Contents of the invention

The object of the present invention is to provide a new class of angiopoietin-like protein polypeptides and fragments, analogs and derivatives thereof. The angiopoietin-like protein of the present invention is named as PP1158 protein.

Another object of the present invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide methods for producing these polypeptides and uses of the polypeptides and coding sequences.

In the first aspect of the present invention, a novel isolated angiopoietin-like protein polypeptide is provided, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an activity thereof derivative.

Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO:2.

In a second aspect of the present invention there is provided an isolated polynucleotide comprising a nucleotide sequence having at least 85% identity to a nucleotide sequence selected from the group consisting of: (a) a polynucleotide encoding the above-mentioned angiopoietin-like protein polypeptide; (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polypeptide encoded by the polynucleotide has the amino acid sequence of SEQ ID NO: 2. More preferably, the sequence of the polynucleotide is selected from the following group: the coding region sequence or full-length sequence of SEQ ID NO: 3.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned polynucleotides, and host cells transformed or transduced by the vectors or host cells directly transformed or transduced by the above-mentioned polynucleotides.

In the fourth aspect of the present invention, there is provided a method for preparing a polypeptide having human angiopoietin-like protein activity, the method comprising: (a) cultivating the above-mentioned transformed or transformed polypeptide under conditions suitable for expressing angiopoietin-like protein (b) isolating a polypeptide having human angiopoietin-like protein activity from the culture.

In the fifth aspect of the present invention, an antibody specifically binding to the above-mentioned angiopoietin-like protein polypeptide is provided. Also provided is a nucleic acid molecule useful for detection, which contains consecutive 10-800 nucleotides of the above-mentioned polynucleotides.

In the sixth aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of the angiopoietin-like protein polypeptide of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can treat diseases such as cancer and abnormal proliferation of cells.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Description of drawings

In the drawings, Fig. 1 shows the tumor suppressor effect of the angiopoietin-like protein gene PP1158 of the present invention. Among them, an empty vector control group (PCMV-Script) and a non-transformed cell control group were set up at the same time.

Figure 2 shows the expression profile of the human angiopoietin-like protein (PP1158) of the present invention.

Contents of the invention

The invention adopts large-scale cDNA clones to transfect cancer cells, and on the basis of obtaining tumor-suppressing effects, it is proved to be new genes by sequencing, and further obtains full-length cDNA clones. The DNA transfection test proves that the human angiopoietin-like protein (PP1158) of the present invention has the effect of inhibiting the colony formation of cancer cells (liver cancer cells).

In one embodiment of the present invention, the human angiopoietin-like protein gene is cDNA isolated from placenta, and its sequence is basically the same as that of Korean ARG after ATG, but the above-mentioned Korean ARG sequence, its complete cDNA, and No upstream termination codon sequence. The local sequences at the ATG end of the coding frame are slightly different between the two, and the ZZZ gene cloned by the inventor has an extra exon in the 3'-UTR region. The invention not only has the stop codon sequence of the 5' upstream region, but also proves to have inhibitory effect on the growth of malignant tumors, that is, liver cancer cells. Therefore, the gene has value in the treatment and diagnosis of malignant tumors.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated angiopoietin-like protein or polypeptide" means that the angiopoietin-like protein polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify angiopoietin-like proteins using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the angiopoietin-like protein polypeptide can be analyzed by amino acid sequence.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of human angiopoietin-like proteins. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the natural human angiopoietin-like protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, the term "human angiopoietin-like protein polypeptide" refers to a polypeptide having the sequence of SEQ ID NO.2 having human angiopoietin-like protein activity. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as human angiopoietin-like protein. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of human angiopoietin-like protein.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA that can hybridize to human angiopoietin-like protein DNA under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human angiopoietin-like protein polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human angiopoietin-like protein polypeptides or fragments thereof (such as fusion proteins comprising the sequence shown in SEQ ID NO: 2). In addition to substantially full-length polypeptides, the present invention also includes soluble fragments of human angiopoietin-like protein polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human angiopoietin-like protein polypeptide sequence. Preferably at least about 100 contiguous amino acids.

The invention also provides analogs of human angiopoietin-like protein or polypeptide. The difference between these analogues and the natural human angiopoietin-like protein polypeptide may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "human angiopoietin-like protein conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO: 2, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most Preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A initial residue representative replacement preferred substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence (position 173-1393) shown in SEQ ID NO: 3 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 3.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequence.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 60%, preferably at least 80%, more preferably at least 85%, and most preferably at least 90% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only the identity between the two sequences is at least 95%, more Preferably hybridization occurs above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate polynucleotides encoding human angiopoietin-like protein.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The DNA sequences of the present invention can be obtained in several ways. For example, DNA is isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous nucleotide sequences, and 2) antibody screening of expression libraries to detect cloned DNA fragments with common structural features .

The generation of specific DNA fragment sequence encoding human angiopoietin-like protein can also be obtained by the following methods: 1) isolation of double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of DNA sequence to obtain double-stranded DNA of the desired polypeptide.

Of the methods mentioned above, isolating genomic DNA is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice when the entire amino acid sequence of the desired polypeptide product is known. If the entire sequence of the desired amino acids is not known, direct chemical synthesis of the DNA sequence is not possible and the method of choice is isolation of the cDNA sequence. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature technologies for the method of extracting mRNA, and the kit is also available from commercial sources (Qiagene). And constructing a cDNA library is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as various cDNA libraries from the company Clontech. When combined with polymerase reaction technology, even minimal expression products can be cloned.

These cDNA libraries can be screened for the gene of the present invention by a conventional method. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) function appearance or loss of marker genes; (3) measuring the transcript level of human angiopoietin-like protein; (4) ) Detection of protein products expressed by genes by immunological techniques or assays of biological activity. The above methods can be used alone or in combination with multiple methods.

In the (1) method, the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2kb, preferably within 1kb. The probes used here are usually DNA sequences chemically synthesized based on the gene DNA sequence information of the present invention. The genes themselves or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, luciferin or enzymes (such as alkaline phosphatase) and the like.

In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the human angiopoietin-like protein gene.

A method of amplifying DNA/RNA using the PCR technique (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The nucleotide sequence of the gene of the present invention obtained as described above, or various DNA fragments, etc., can be determined by conventional methods such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such nucleotide sequence determination can also use commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or human angiopoietin-like protein coding sequences, and methods for producing the polypeptides of the present invention through recombinant techniques.

By conventional recombinant DNA techniques (Science, 1984; 224:1431), the polynucleotide sequences of the present invention can be used to express or produce recombinant human angiopoietin-like protein polypeptides. Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding human angiopoietin-like protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In the present invention, the human angiopoietin-like protein polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include, but are not limited to: T7-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem.263:3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the human angiopoietin-like protein coding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage _PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS or Bowes melanoma cells, etc.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. An alternative is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be encapsulated inside the cell, outside the cell or expressed on the cell membrane or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The recombinant human angiopoietin-like protein or polypeptide has various uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by low or loss of human angiopoietin-like protein function (especially for inhibiting tumor growth), and screening for promoting or antagonizing human angiopoietin-like protein. Antibodies, peptides or other ligands for protein functions. For example, antibodies can be used to activate or inhibit the function of human angiopoietin-like protein. Screening the polypeptide library with expressed recombinant human angiopoietin-like protein can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of human angiopoietin-like protein.

The invention also provides methods of screening drugs to identify agents that increase (agonists) or repress (antagonists) human angiopoietin-like protein. Agonists enhance biological functions of human angiopoietin-like protein such as stimulating cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation such as various cancers. For example, mammalian cells or membrane preparations expressing human angiopoietin-like protein can be incubated with labeled human angiopoietin-like protein in the presence of the drug. The ability of the drug to enhance or repress this interaction is then determined.

Antagonists of human angiopoietin-like protein include screened antibodies, compounds, receptor deletions and analogs. Antagonists of human angiopoietin-like protein can bind to human angiopoietin-like protein and eliminate its function, or inhibit the production of human angiopoietin-like protein, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions Function. Antagonists of human angiopoietin-like protein are useful for therapeutic use.

In screening compounds for antagonists, human angiopoietin-like protein can be added to a bioanalytical assay to determine whether a compound is an antagonist by determining the compound's effect on the interaction between human angiopoietin-like protein and its receptor. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds.

The polypeptide of the present invention can be directly used in the treatment of diseases, for example, various malignant tumors, abnormal cell proliferation and the like.

The polypeptides of the present invention, and fragments, derivatives, analogs thereof or their cells can be used as antigens to produce antibodies. These antibodies can be polyclonal or monoclonal. Polyclonal antibodies can be obtained by injecting the polypeptide directly into animals. Techniques for preparing monoclonal antibodies include hybridoma technology, trioma technology, human B-cell hybridoma technology, EBV-hybridoma technology, and the like.

The polypeptides and antagonists of the present invention can be used in combination with suitable pharmaceutical carriers. These carriers can be water, dextrose, ethanol, salts, buffers, glycerol and combinations thereof. The composition contains safe and effective doses of polypeptides or antagonists as well as carriers and excipients that do not affect the drug effect. These compositions can be used as medicine for disease treatment.

The invention also provides kits or kits comprising one or more containers containing one or more ingredients of the pharmaceutical compositions of the invention. Along with these containers, there may be an indicative notice given by the governmental regulatory agency that manufactures, uses or sells the drug or biological product reflecting its approval for human administration by the governmental regulatory agency that manufactures, uses or sells the drug or biological product. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes of administration. Human angiopoietin-like protein is administered in an amount effective to treat and/or prevent the particular indication. The amount and dosage range of human angiopoietin-like protein administered to a patient will depend on many factors, such as the mode of administration, the health condition of the person to be treated, and the judgment of the diagnosing physician.

Polynucleotides of human angiopoietin-like protein may also be used for various therapeutic purposes. Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism due to non-expression of human angiopoietin-like protein or abnormal/inactive expression of human angiopoietin-like protein. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human angiopoietin-like protein to inhibit the activity of endogenous human angiopoietin-like protein. For example, a mutant human angiopoietin-like protein can be a shortened human angiopoietin-like protein that lacks a signal transduction domain, and although it can bind to a downstream substrate, it lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human angiopoietin-like protein. Virus-derived expression vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the human angiopoietin-like protein gene into cells. The method for constructing a recombinant viral vector carrying the human angiopoietin-like protein gene can be found in existing literature (Sambrook, et al.). In addition, the recombinant human angiopoietin-like protein gene can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human angiopoietin-like protein mRNA are also within the scope of the invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform an endonucleic cut. Antisense RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoamide chemical synthesis of oligonucleotides, which has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RNA. This DNA sequence has been integrated into the vector downstream of the RNA polymerase promoter. In order to increase the stability of nucleic acid molecules, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the connection between ribonucleosides should use phosphothioester bonds or peptide bonds instead of phosphodiester bonds.

The methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in the body; or first introducing polynucleotides into cells in vitro through vectors (such as viruses, phages, or plasmids, etc.) , and then transplant the cells into the body, etc.

The polypeptide of the present invention can also be used for peptide spectrum analysis, for example, the polypeptide can be specifically cleaved physically, chemically or enzymatically, and subjected to one-dimensional, two-dimensional or three-dimensional gel electrophoresis analysis.

The present invention also provides antibodies against epitopes of human angiopoietin-like protein. These antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

Antibodies against human angiopoietin-like protein are used in immunohistochemical techniques to detect human angiopoietin-like protein in biopsy specimens.

Monoclonal antibodies that bind to human angiopoietin-like proteins can also be labeled with radioactive isotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method for localization of tumor cells and judgment of metastasis.

The antibodies of the present invention can be used to treat or prevent diseases related to human angiopoietin-like protein. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human angiopoietin-like protein.

Antibodies can also be used to design immunotoxins that target a particular site in the body. For example, high-affinity monoclonal antibodies to human angiopoietin-like protein can be covalently combined with bacterial or plant toxins (such as diphtheria toxin, ricin, rhododine, etc.). A common method is to use a sulfhydryl cross-linking agent such as SPDP to attack the amino group of the antibody, and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill cells positive for human angiopoietin-like protein .

For the production of polyclonal antibodies, human angiopoietin-like protein or polypeptide can be used to immunize animals, such as rabbits, mice, rats, etc. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Human angiopoietin-like protein monoclonal antibody can be produced by hybridoma technology (Kohler and Milstein. Nature, 1975, 256:495-497). Chimeric antibodies combining human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81:6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against human angiopoietin-like protein.

The polypeptide molecule capable of binding to human angiopoietin-like protein can be obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to solid phases. For screening, the human angiopoietin-like protein molecule must be labeled.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human angiopoietin-like protein. These assays are well known in the art and include FISH assays and radioimmunoassays. The level of human angiopoietin-like protein detected in the test can be used to explain the importance of human angiopoietin-like protein in various diseases and to diagnose diseases in which human angiopoietin-like protein plays a role.

The polynucleotide of human angiopoietin-like protein can be used for the diagnosis and treatment of diseases related to human angiopoietin-like protein. In terms of diagnosis, the polynucleotide of human angiopoietin-like protein can be used to detect the expression of human angiopoietin-like protein or the abnormal expression of human angiopoietin-like protein in a disease state. For example, the human angiopoietin-like protein DNA sequence can be used for hybridization of biopsy specimens to determine the abnormal expression of human angiopoietin-like protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification with primers specific to human angiopoietin-like protein can also detect the transcript of human angiopoietin-like protein.

Detection of mutations in the human angiopoietin-like protein gene can also be used to diagnose diseases associated with the human angiopoietin-like protein. The mutated form of human angiopoietin-like protein includes point mutation, translocation, deletion, recombination and any other abnormalities compared with the normal wild-type human angiopoietin-like protein DNA sequence. Mutations can be detected using established techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene has a mutation.

The sequences of the invention are also valuable for chromosome identification. The sequence will be specific for a particular location on a human chromosome and can hybridize thereto. Currently, there is a need to identify the specific site of each gene on the chromosome. Currently, only a few chromosomal markers based on actual sequence data (repeat polymorphisms) are available to mark chromosomal positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on chromosomes.

In short, PCR primers (preferably 15-35bp) are prepared according to the cDNA, and the sequence can be positioned on the chromosome. These primers were then used for PCR screening of somatic heterozygous cells containing individual human chromosomes. Only those cells heterozygous for the human gene corresponding to the primer will produce an amplified fragment.

The PCR mapping method of somatic heterozygous cells is a quick method to locate DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, sublocalization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones by a similar method. Other similar strategies that can be used for chromosome mapping include in situ hybridization, chromosome prescreening by flow sorting with markers, and hybridization preselection to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosomes allows precise chromosomal mapping in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to an exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with gene map data. These data can be found, for example, in V. Mckusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and the disease that has been mapped to the chromosomal region.

Next, the cDNA or genome sequence differences between affected and non-affected individuals need to be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping techniques, the cDNA that is pinpointed to a disease-associated chromosomal region can be one of 50 to 500 potential disease-causing genes (assuming 1 megabase mapping resolution capacity and each 20kb corresponds to a gene).

The full-length human angiopoietin-like protein nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be completely chemically synthesized. This DNA sequence can then be introduced into various DNA molecules (such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In one embodiment of the present invention, the full-length cDNA clone of angiopoietin-like protein was obtained from the human placenta cDNA library by in vitro DNA transfection screening and isolation of liver cancer cell 7721, and obtained by 5'-RACE and full-cloning technology . In addition, Northern hybridization proves that the angiopoietin-like protein gene of the present invention is expressed in brain and placenta, but not or weakly expressed in heart, lung, muscle, kidney and pancreas.

In view of the fact that PP1158 has the function of inhibiting the growth of cancer cells, it not only has the function of regulating cell division, but also has application value in the treatment and diagnosis of malignant tumors. In addition, since the human angiopoietin-like protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or be more active when administered to humans, compared to homologous proteins derived from other species. Low side effects (eg, less or no immunogenicity in humans).

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions specified by the manufacturer. suggested conditions.

Detailed ways

Example 1

Growth Inhibition of Human Hepatocellular Carcinoma Cells by Angiopoietin-like Protein Gene and Isolation of Full-length cDNA

Placenta tissues at 3, 6, and 10 months old were taken, and total RNA was extracted with Trizol reagent (GIBCO BRL Company) according to the manufacturer's instructions, and mRNA was extracted with an mRNA purification kit (Pharmacia Company). The cDNA library of the above mRNA was constructed with the pCMV-script TMXR cDNA library construction kit (Stratagene). The reverse transcriptase was changed to MMLV-RT-Superscript II (GIBCO BRL), and the reverse transcription reaction was carried out at 42°C. XL 10-Gold competent cells were transformed, and a cDNA library with a titer of 1×106cfu/μg cDNA was obtained. In the first round, cDNA clones were randomly selected, and then high-abundance cDNA clones and cDNA clones that had been proven to inhibit the growth of cancer cells were used as probes to hybridize and screen the cDNA library to pick weakly positive and negative clones. Plasmid DNA was extracted using Qiagen 96-well plate plasmid extraction kit according to the manufacturer's instructions. Plasmid DNA and empty vector were transfected into liver cancer cell line 7721 at the same time. After 100ng DNA was dried by alcohol precipitation, it was dissolved by adding 6μl H2O, and then transfected. Add 0.74 μl liposome and 9.3 μl serum-free medium to each DNA sample, mix well, and place at room temperature for 10 minutes. Add 150 μl of serum-free culture medium to each tube, add evenly to 7721 cells grown in 96-well plates in 3 wells, place at 37°C for 2 hours, add 50 μl of serum-free culture medium to each well, and keep at 37°C for 24 hours. Change 100 μl whole culture medium for each well, 37°C for 24 hours, change 100 μl whole culture medium containing G418, 37°C for 24 to 48 hours, change the culture medium with different concentrations of G418 while observing. After about 2 to 3 times, until the microscopic examination of the cells shows colony formation, count them. It was found that the PP1158 cDNA clone had the function of inhibiting the formation of clones (the number of empty vector clones was 42, 40, 56, and PP1158 was 0, 0, 0)

After analyzing the sequence of the PP1158 cDNA clone, it was found that the gene was not complete. The SMART RACE cDNA amplification kit (Cat.No.K1811-1) of Clontech Company was used, and the gene-specific primers were 5′-GTGGTCCC TCTGAGTCCCCAACTCC-3′ and 5′-CTCAGAAAGGGGGCTTCTCCAGTCG -3', operate according to the instructions to obtain full-length clones.

Embodiment 2: the sequence analysis of PP1158 cDNA

The full length of the cDNA obtained in Example 1 is 1943 nucleotides, including a reading frame of 1221 (positions 173-1393) nucleotides, and encodes a protein consisting of 406 amino acids (excluding stop codons).

A: Nucleotide sequence: (SEQ ID NO: 1) Length: 1943bp

1 GGAGAAGAAG CCGAGCTGAG CGGATCCTCA CACGACTGTG ATCCGATTCT

51 TTCCAGCGGC TTCTGCAACC AAGCGGGTCT TACCCCCGGT CCTCCGCGTC

101 TCCAGTCCTC GCACCTGGAA CCCCAACGTC CCCGAGAGTC CCCGAATCCC

151 CGCTCCCAGG CTACCTAAGA GGATGAGCGG TGCTCCGACG GCCGGGGCAG

201 CCCTGATGCT CTGCGCCGCC ACCGCCGTGC TACTGAGCGC TCAGGGCGGA

251 CCCGTGCAGT CCAAGTCGCC GCGCTTTGCG TCCTGGGACG AGATGAATGT

301 CCTGGCGCAC GGACTCCTGC AGCTCGGCCA GGGGCTGCGC GAACACGCGG

351 AGCGCACCCG CAGTCAGCTG AGCGCGCTGG AGCGGCGCCT GAGCGCGTGC

401 GGGTCCGCCT GTCAGGGAAC CGAGGGGTCC ACCGACCTCC CGTTAGCCCC

451 TGAGAGCCGG GTGGACCCTG AGGTCCTTCA CAGCCTGCAG ACACAACTCA

501 AGGCTCAGAA CAGCAGGATC CAGCAACTCT TCCACAAGGT GGCCCAGCAG

551 CAGCGGCACC TGGAGAAGCA GCACCTGCGA ATTCAGCATC TGCAAAGCCA

601 GTTTGGCCTC CTGGACCACA AGCACCTAGA CCATGAGGTG GCCAAAGCCTG

651 CCCGAAGAAA GAGGCTGCCC GAGATGGCCC AGCCAGTTGA CCCGGCTCAC

701 AATGTCAGCC GCCTGCACCG GCTGCCCAGG GATTGCCAGG AGCTGTTCCA

751 GGTTGGGGAG AGGCAGAGTG GACTATTTGA AATCCAGCT CAGGGGTCTC

801 CGCCATTTTT GGTGAACTGC AAGATGACCT CAGATGGAGG CTGGACAGTA

851 ATTCAGAGGC GCCACGATGG CTCAGTGGAC TTCAACCGGC CCTGGGAAGC

901 CTACAAGGCG GGGTTTGGGG ATCCCCACGG CGAGTTCTGG CTGGGTCTGG

951 AGAAGGTGCA TAGCATCACG GGGGACCGCA ACAGCCGCCT GGCCGTGCAG

1001 CTGCGGGACT GGGATGGCAA CGCCGAGTTG CTGCAGTTCT CCGTGCACCT

1051 GGGTGGCGAG GACACGGCCT ATAGCCTGCA GCTCACTGCA CCCGTGGCCG

1101 GCCAGCTGGG CGCCACCACC GTCCCACCCA GCGGCCTCTC CGTACCCTTC

1151 TCCACTTGGG ACCAGGATCA CGACCTCCGC AGGGACAAGA ACTGCGCCAA

1201 GAGCCTCTCT GGAGGCTGGT GGTTTGGCAC CTGCAGCCAT TCCAACCTCA

1251 ACGGCCAGTA CTTCCGCTCC ATCCCACAGC AGCGGCAGAA GCTTAAGAAG

1301 GGAATCTTCT GGAAGACCTG GCGGGGCCGC TACTACCCGC TGCAGGCCAC

1351 CACCATGTTG ATCCAGCCCA TGGCAGCAGA GGCAGCCTCC TAGCGTCCTG

1401 GCTGGGCCTG GTCCCAGGCC CACGAAAGAC GGTGACTCTT GGCTCTGCCC

1451 GAGGATGTGG CCGTTCCCTG CCTGGGCAGG GGCTCCAAGG AGGGGCCATC

1501 TGGAAACTTG TGGACAGAGA AGAAGACCAC GACTGGAGAA GCCCCCTTTC

1551 TGAGTGCAGG GGGGCTGCAT GCGTTGCCTC CTGAGATCGA GGCTGCAGGA

1601 TATGCTCAGA CTCTAGAGGC GTGGACCAAG GGGCATGGAG CTTCACTCCT

1651 TGCTGGCCAG GGAGTTGGGG ACTCAGAGGG ACCACTTGGG GCCAGCCAGA

1701 CTGGCCTCAA TGGCGGACTC AGTCACATTG ACTGACGGGG ACCAGGGCTT

1751 GTGTGGGTCG AGAGCGCCCT CATGGTGCTG GTGCTGTTGT GTGTAGGTCC

1801 CCTGGGGACA CAAGCAGGCG CCAATGGTAT CTGGGCGGCG TCACAGAGTT

1851 CTTGGAATAA AAGCAACCTC AGAACACTTA AAAAAAAAAA AAAAAAAAAA

1901 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA AAA

B: Amino acid sequence: (SEQ ID NO: 2) Length: 406 amino acids

1 MSGAPTAGAA LMLCAATAVL LSAQGGPVQS KSPRFASWDE MNVLAHGLLQ

51 LGQGLREHAE RTRSQLSALE RRLSACGSAC QGTEGSTDLP LAPESRVDPE

101 VLHSLQTQLK AQNSRIQQLF HKVAQQQRHL EKQHLRIQHL QSQFGLLDHK

151 HLDHEVAKPA RRKRLPEMAQ PVDPAHNVSR LHRLPRDCQE LFQVGERQSG

201 LFEIQPQGSP PFLVNCKMTS DGGWTVIQRR HDGSVDFNRP WEAYKAGFGD

251 PHGEFWLGLE KVHSITGDRN SRLAVQLRDW DGNAELLQFS VHLGGEDTAY

301 SLQLTAPVAG QLGATTPPPS GLSVPFSTWD QDHDLRRDKN CAKSLSGGWW

351 FGTCSHSNLN GQYFRSIPQQ RQKLKKGIFW KTWRGRYYPL QATTMLIQPM

401 AAEAAS

C: Clone number: PP1158 (SEQ ID NO: 3)

Start code: 173 ATG Stop code: 1393 TAG

Protein molecular weight: 45211.75

1 G GAG AAG AAG CCG AGC TGA GCG GAT CCT CAC ACG ACT GTG ATC CGA 46

47 TTC TTT CCA GCG GCT TCT GCA ACC AAG CGG GTC TTA CCC CCG GTC CTC 94

95 CGC GTC TCC AGT CCT CGC ACC TGG AAC CCC AAC GTC CCC GAG AGT CCC 142

143 CGA ATC CCC GCT CCC AGG CTA CCT AAG AGG ATG AGC GGT GCT CCG ACG 190

1 Met Ser Gly Ala Pro Thr 6

191 GCC GGG GCA GCC CTG ATG CTC TGC GCC GCC ACC GCC GTG CTA CTG AGC 238

7 Ala Gly Ala Ala Leu Met Leu Cys Ala Ala Thr Ala Val Leu Leu Ser 22

239 GCT CAG GGC GGA CCC GTG CAG TCC AAG TCG CCG CGC TTT GCG TCC TGG 286

23 Ala Gln Gly Gly Pro Val Gln Ser Lys Ser Pro Arg Phe Ala Ser Trp 38

287 GAC GAG ATG AAT GTC CTG GCG CAC GGA CTC CTG CAG CTC GGC CAG GGG 334

39 Asp Glu Met Asn Val Leu Ala His Gly Leu Leu Gln Leu Gly Gln Gly 54

335 CTG CGC GAA CAC GCG GAG CGC ACC CGC AGT CAG CTG AGC GCG CTG GAG 382

55 Leu Arg Glu His Ala Glu Arg Thr Arg Ser Gln Leu Ser Ala Leu Glu 70

383 CGG CGC CTG AGC GCG TGC GGG TCC GCC TGT CAG GGA ACC GAG GGG TCC 430

71 Arg Arg Leu Ser Ala Cys Gly Ser Ala Cys Gln Gly Thr Glu Gly Ser 86

431 ACC GAC CTC CCG TTA GCC CCT GAG AGC CGG GTG GAC CCT GAG GTC CTT 478

87 Thr Asp Leu Pro Leu Ala Pro Glu Ser Arg Val Asp Pro Glu Val Leu 102

479 CAC AGC CTG CAG ACA CAA CTC AAG GCT CAG AAC AGC AGG ATC CAG CAA 526

103 His Ser Leu Gln Thr Gln Leu Lys Ala Gln Asn Ser Arg Ile Gln Gln 118

527 CTC TTC CAC AAG GTG GCC CAG CAG CAG CGG CAC CTG GAG AAG CAG CAC 574

119 Leu Phe His Lys Val Ala Gln Gln Gln Arg His Leu Glu Lys Gln His 134

575 CTG CGA ATT CAG CAT CTG CAA AGC CAG TTT GGC CTC CTG GAC CAC AAG 622

135 Leu Arg Ile Gln His Leu Gln Ser Gln Phe Gly Leu Leu Asp His Lys 150

623 CAC CTA GAC CAT GAG GTG GCC AAG CCT GCC CGA AGA AAG AGG CTG CCC 670

151 His Leu Asp His Glu Val Ala Lys Pro Ala Arg Arg Lys Arg Leu Pro 166

671 GAG ATG GCC CAG CCA GTT GAC CCG GCT CAC AAT GTC AGC CGC CTG CAC 718

167 Glu Met Ala Gln Pro Val Asp Pro Ala His Asn Val Ser Arg Leu His 182

719 CGG CTG CCC AGG GAT TGC CAG GAG CTG TTC CAG GTT GGG GAG AGG CAG 766

183 Arg Leu Pro Arg Asp Cys Gln Glu Leu Phe Gln Val Gly Glu Arg Gln 198

767 AGT GGA CTA TTT GAA ATC CAG CCT CAG GGG TCT CCG CCA TTT TTG GTG 814

199 Ser Gly Leu Phe Glu Ile Gln Pro Gln Gly Ser Pro Pro Phe Leu Val 214

815 AAC TGC AAG ATG ACC TCA GAT GGA GGC TGG ACA GTA ATT CAG AGG CGC 862

215 Asn Cys Lys Met Thr Ser Asp Gly Gly Trp Thr Val Ile Gln Arg Arg 230

863 CAC GAT GGC TCA GTG GAC TTC AAC CGG CCC TGG GAA GCC TAC AAG GCG 910

231 His Asp Gly Ser Val Asp Phe Asn Arg Pro Trp Glu Ala Tyr Lys Ala 246

911 GGG TTT GGG GAT CCC CAC GGC GAG TTC TGG CTG GGT CTG GAG AAG GTG 958

247 Gly Phe Gly Asp Pro His Gly Glu Phe Trp Leu Gly Leu Glu Lys Val 262

959 CAT AGC ATC ACG GGG GAC CGC AAC AGC CGC CTG GCC GTG CAG CTG CGG 1006

263 His Ser Ile Thr Gly Asp Arg Asn Ser Arg Leu Ala Val Gln Leu Arg 278

1007 GAC TGG GAT GGC AAC GCC GAG TTG CTG CAG TTC TCC GTG CAC CTG GGT 1054

279 Asp Trp Asp Gly Asn Ala Glu Leu Leu Gln Phe Ser Val His Leu Gly 294

1055 GGC GAG GAC ACG GCC TAT AGC CTG CAG CTC ACT GCA CCC GTG GCC GGC 1102

295 Gly Glu Asp Thr Ala Tyr Ser Leu Gln Leu Thr Ala Pro Val Ala Gly 310

1103 CAG CTG GGC GCC ACC ACC GTC CCA CCC AGC GGC CTC TCC GTA CCC TTC 1150

311 Gln Leu Gly Ala Thr Thr Val Pro Pro Ser Gly Leu Ser Val Pro Phe 326

1151 TCC ACT TGG GAC CAG GAT CAC GAC CTC CGC AGG GAC AAG AAC TGC GCC 1198

327 Ser Thr Trp Asp Gln Asp His Asp Leu Arg Arg Asp Lys Asn Cys Ala 342

1199 AAG AGC CTC TCT GGA GGC TGG TGG TTT GGC ACC TGC AGC CAT TCC AAC 1246

343 Lys Ser Leu Ser Gly Gly Trp Trp Phe Gly Thr Cys Ser His Ser Asn 358

1247 CTC AAC GGC CAG TAC TTC CGC TCC ATC CCA CAG CAG CGG CAG AAG CTT 1294

359 Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro Gln Gln Arg Gln Lys Leu 374

1295 AAG AAG GGA ATC TTC TGG AAG ACC TGG CGG GGC CGC TAC TAC CCG CTG 1342

375 Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg Gly Arg Tyr Tyr Pro Leu 390

1343 CAG GCC ACC ACC ATG TTG ATC CAG CCC ATG GCA GCA GAG GCA GCC TCC 1390

391 Gln Ala Thr Thr Met Leu Ile Gln Pro Met Ala Ala Glu Ala Ala Ser 406

1391 TAG CGT CCT GGC TGG GCC TGG TCC CAG GCC CAC GAA AGA CGG TGA CTC 1438

407 *** 407

1439 TTG GCT CTG CCC GAG GAT GTG GCC GTT CCC TGC CTG GGC AGG GGC TCC 1486

1487 AAG GAG GGG CCA TCT GGA AAC TTG TGG ACA GAG AAG AAG ACC ACG ACT 1534

1535 GGA GAA GCC CCC TTT CTG AGT GCA GGG GGG CTG CAT GCG TTG CCT CCT 1582

1583 GAG ATC GAG GCT GCA GGA TAT GCT CAG ACT CTA GAG GCG TGG ACC AAG 1630

1631 GGG CAT GGA GCT TCA CTC CTT GCT GGC CAG GGA GTT GGG GAC TCA GAG 1678

1679 GGA CCA CTT GGG GCC AGC CAG ACT GGC CTC AAT GGC GGA CTC AGT CAC 1726

1727 ATT GAC TGA CGG GGA CCA GGG CTT GTG TGG GTC GAG AGC GCC CTC ATG 1774

1775 GTG CTG GTG CTG TTG TGT GTA GGT CCC CTG GGG ACA CAA GCA GGC GCC 1822

1823 AAT GGT ATC TGG GCG GCG TCA CAG AGT TCT TGG AAT AAA AGC AAC CTC 1870

1871 AGA ACA CTT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1918

1919 AAA AAA AAA AAA AAA AAA AAA AAA A 1943

D. Homology comparison

The PP1158 cDNA clone of the present invention contains a full-length cDNA sequence.

(1) Nucleotide sequence homology comparison

Query＝PP1158 (1943 nucleotides)

>GB_PR4: AF153606 AF153606 human angiopoietin-related protein

mRNA, complete cds.6/1999 length = 1860

Score=2813bits(1419), expected value=0.0

Identity = 1440/1443 (99%), Similarity = 1440/1443 (99%), Gap = 3/1443 (0%)

Query: 20 gcggatcctcacacgactgtgatccgattctttccagcggcttctgcaaccaagcgggtc 79

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1 gcggatcctcacacgactgtgatccgattctttccagcggcttctgcaaccaagcgggtc 60

Query: 80 ttacccccggtcctccgcgtctccagtcctcgcacctggaaccccaacgtccccgagagt 139

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 61 ttacccccggtcctccgcgtctccagtcctcgcacctggaaccccaacgtccccgagagt 120

Query: 140 ccccgaatccccgctcccaggctacctaagaggatgagcggtgctccgacggccggggca 199

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 121 ccccgaatccccgctcccaggctacctaagaggatgagcggtgctccgacggccggggca 180

Query: 200 gccctgatgctctgcgccgccaccgccgtgctactgagcgctcagggcggacccgtgcag 259

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 181 gccctgatgctctgcgccgccaccgccgtgctactgagcgctcagggcggacccgtgcag 240

Query: 260 tccaagtcgccgcgctttgcgtcctgggacgagatgaatgtcctggcgcacggactcctg 319

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 241 tccaagtcgccgcgctttgcgtcctgggacgagatgaatgtcctggcgcacggactcctg 300

Query: 320 cagctcggccaggggctgcgcgaacacgcggagcgcacccgcagtcagctgagcgcgctg 379

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 301 cagctcggccagggg-tgcgcgaacac-cggagcgcacccgcagtcagctgagcgcgctg 358

Query: 380 gagcggcgcctgagcgcgtgcgggtccgcctgtcagggaaccgaggggtccaccgacctc 439

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 359 gagcg-cgcctgagcgcgtgcgggtccgcctgtcagggaaccgaggggtccaccgacctc 417

Query: 440 ccgttagcccctgagagccgggtggaccctgaggtccttcacagcctgcagacacaactc 499

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 418 ccgttagcccctgagagccgggtggaccctgaggtccttcacagcctgcagacacaactc 477

Query: 500 aaggctcagaacagcaggatccagcaactcttccacaaggtggcccagcagcagcggcac 559

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 478 aaggctcagaacagcaggatccagcaactcttccacaaggtggcccagcagcagcggcac 537

Query: 560 ctggagaagcagcacctgcgaattcagcatctgcaaagccagtttggcctcctggaccac 619

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 538 ctggagaagcagcacctgcgaattcagcatctgcaaagccagtttggcctcctggaccac 597

Query: 620 aagcacctagaccatgaggtggccaagcctgcccgaagaaagaggctgcccgagatggcc 679

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 598 aagcacctagaccatgaggtggccaagcctgcccgaagaaagaggctgcccgagatggcc 657

Query: 680 cagccagttgacccggctcacaatgtcagccgcctgcaccggctgcccagggattgccag 739

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 658 cagccagttgacccggctcacaatgtcagccgcctgcaccggctgcccagggattgccag 717

Query: 740 gagctgttccaggttggggagaggcagagtggactatttgaaatccagcctcaggggtct 799

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 718 gagctgttccaggttggggagaggcagagtggactatttgaaatccagcctcaggggtct 777

Query: 800 ccgccatttttggtgaactgcaagatgacctcagatggaggctggacagtaattcagagg 859

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 778 ccgccatttttggtgaactgcaagatgacctcagatggaggctggacagtaattcagagg 837

Query: 860 cgccacgatggctcagtggacttcaaccggccctgggaagcctacaaggcggggtttggg 919

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 838 cgccacgatggctcagtggacttcaaccggccctgggaagcctacaaggcggggtttggg 897

Query: 920 gatccccacggcgagttctggctgggtctggagaaggtgcatagcatcacgggggaccgc 979

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 898 gatccccacggcgagttctggctgggtctggagaaggtgcatagcatcacgggggaccgc 957

Query: 980 aacagccgcctggccgtgcagctgcgggactgggatggcaacgccgagttgctgcagttc 1039

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 958 aacagccgcctggccgtgcagctgcgggactgggatggcaacgccgagttgctgcagttc 1017

Query: 1040 tccgtgcacctgggtggcgaggacacggcctatagcctgcagctcactgcacccgtggcc 1099

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1018 tccgtgcacctgggtggcgaggacacggcctatagcctgcagctcactgcacccgtggcc 1077

Query: 1100 ggccagctgggcgccaccaccgtcccaccccagcggcctctccgtacccttctccacttgg 1159

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1078 ggccagctgggcgccaccaccgtcccaccccagcggcctctccgtacccttctccacttgg 1137

Query: 1160 gaccaggatcacgacctccgcagggacaagaactgcgccaagagcctctctggaggctgg 1219

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1138 gaccaggatcacgacctccgcagggacaagaactgcgccaagagcctctctggaggctgg 1197

Query: 1220 tggtttggcacctgcagccattccaacctcaacggccagtacttccgctccatccccacag 1279

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1198 tggtttggcacctgcagccattccaacctcaacggccagtacttccgctccatccccacag 1257

Query: 1280 cagcggcagaagcttaagaagggaatcttctggaagacctggcggggccgctactacccg 1339

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1258 cagcggcagaagcttaagaagggaatcttctggaagacctggcggggccgctactacccg 1317

Query: 1340 ctgcaggccaccaccatgttgatccagcccatggcagcagaggcagcctcctagcgtcct 1399

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1318 ctgcaggccaccaccacatgttgatccagcccatggcagcagaggcagcctcctagcgtcct 1377

Query: 1400 ggctgggcctggtcccaggcccacgaaagacggtgactcttggctctgcccgaggatgtg 1459

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1378 ggctgggcctggtcccaggcccacgaaagacggtgactcttggctctgcccgaggatgtg 1437

Query: 1460 gcc 1462

|||

Sbjct: 1438 gcc 1440

Score = 680bits (343), expected value = 0.0

Identity = 353/355 (99%), Similarity = 353/355 (99%), Gap = 1/355 (0%)

Query: 1523 aagaccacgactggagaagccccctttctgagtgcaggggggctgcatgcgttgcctcct 1582

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1441 aagaccacgactggagaagccccctttctgagtgcaggggggctgcatgcgttgcctcct 1500

Query: 1583 gagatcgaggctgcaggatatgctcagactctagaggcgtggaccaaggggcatggagct 1642

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1501 gagatcgaggctgcaggatatgctcagactctagaggcgtggaccaaggggcatggagct 1560

Query: 1643 tcactccttgctggccagggagttggggactcagagggaccacttggggccagccagact 1702

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1561 tcactccttgctggccaggggagttggggactcagagggaccacttggggccagccagact 1620

Query: 1703 ggcctcaatggcggactcagtcacattgactgacggggaccagggcttgtgtgggtcgag 1762

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1621 ggcctcaatggcggactcagtcacattgactgacggggaccagggcttgtgtgggtcgag 1680

Query: 1763 agcgccctcatggtgctggtgctgttgtgtgtaggtcccctggggacacaagcaggcgcc 1822

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||

Sbjct: 1681 agcgccctcatggtgctggtgctgttgtgtgtaggtcccctggggacacaagcaggcgcc 1740

Query: 1823 aatggtatctgggcggcg-tcacagagttcttggaataaaagcaacctcagaaca 1876

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||

Sbjct: 1741 aatggtatctgggcggagctcacagagttcttggaataaaagcaacctcagaaca 1795

Score = 71.9bits(36), predicted value = 7e-10

Identity = 36/36 (100%), Similarity = 36/36 (100%)

Query: 1841 tcacagagttcttggaataaaagcaacctcagaaca 1876

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 1820 tcacagagttcttggaataaaagcaacctcagaaca 1855

(2) Comparison of amino acid sequence and related genes

Query＝PP1158AA (406 amino acids)

Sequences that yield significant alignments: (bits) Value

NG27 585 e-171

gi|4557315|ref|NP_001138.1||Angiopoietin 2 167 3e-45

gi|4378598|gb|AAD19608.1|Angiopoietin Y1 162 7e-44

gi|4502087|ref|NP_001137.1||Angiopoietin 1 149 6e-40

>NG27 Length=410

Score = 585bits (1493), predicted value = e-171

Identity = 286/390 (73%), Similarity = 318/390 (81%), Gap = 6/390 (1%)

Query: 22 SAQGGPVQSKSPRFASWDEMNVLAHXXXXXXXXXREHAERTRSQLSALERRLSACGSACQ 81

SAQG P Q + PRFASWDEMN+LAH REH ERTR QL ALERR++ACG+ACQ

Sbjct: 22 SAQGRPAQPEPPRFASWDEMNLLAHGLLQLGHGLREHVERTRGQLGALERRMAACGNACQ 81

Query: 82 GTEGSTDLPLAP-ESRVD----PEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLR 136

G +G D P E RV PE L SLQTQLKAQNS+IQQLF KVAQQQR+L KQ+LR

Sbjct: 82 GPKGK-DAPFKDSEDRVPEGQTPETLQSLQTQLKAQNSKIQQLFQKVAQQQRYLSKQNLR 140

Query: 137 IQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGE 196

IQ+LQSQ LL HLD+ V K +R KRLP+M Q + N + LHR PRDCQELFQ GE

Sbjct: 141 IQNLQSQIDLLAPTHLDNGVDKTSRGKRLPKMTQLIGLTPNATHLHRPPRDCQELFQEGE 200

Query: 197 RQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFW 256

R SGLF+IQP GSPPFLVNC+MTSDGGWTVIQRR +GSVDFN+ WEAYK GFGDP GEFW

Sbjct: 201 RHSGLFQIQPLGSPPFLVNCEMTSDGGWTVIQRRLNGSVDFNQSWEAYKDGFGDPQGEFW 260

Query: 257 LGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATT 316

    LGLEK+HSITG+R S+LAVQL+DWDGNA+LLQF +HLGGEDTAYSLQLT P A +LGAT

Sbjct: 261 LGLEKMHSITGNRGSQLAVQLQDWDGNAKLLQFPIHLGGEDTAYSLQLTEPTANELGATN 320

Query: 317 VPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKK 376

V P+GLS+PFSTWDQDHDLR D NCAKSLSGGWWFGTCSHSNLNGQYF SIP+QRQ+ KK

Sbjct: 321 VSPNGLSLPFSTWDQDHDLRGDLNCAKSLSGGWWFGTCSHSNLNGQYFHSIPRQRQERKK 380

Query: 377 GIFWKTWRGRYYPLQATTMLIQPMAAEAAS 406

GIFWKTW+GRYYPLQATT+LIQPM A AAS

Sbjct: 381 GIFWKTWKGRYYPLQATTLLIQPMEATAAS 410

>gi|4557315|ref|NP_001138.1||Angiopoietin 2 Length＝496

Score = 167bits (418), expected value = 3e-45

Identity = 93/221 (42%), Similarity = 127/221 (57%), Gap = 16/221 (7%)

Query: 186 RDCQELFQVGERQSGLFEIQ-PQGSPPFLVNCKMTSDGG-WTVIQRRHDGSVDFNRPWEA 243

RDC E+F+ G +G++ + P + C M + GG WT+IQRR DGSVDF R W+

Sbjct: 282 RDCAEVFKSGHTTNGIYTLTFPNSTEEIKAYCDMEAGGGGWTIIQRREDGSVDFQRTWKE 341

Query: 244 YKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGN-AELLQFSVHLGGEDTAYSL 302

                                              

Sbjct: 342 YKVGFGNPSGEYWLGNEFVSQLTNQQRYVLKIHLKDWEGNEAYSLYEHFYLSSEELNYRI 401

Query: 303 QL--TAVAGQLGATTPPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLN 360

AG++ + + P + FST D D+D K C++ L+GGWWF C SNLN

Sbjct: 402 HLKGLTGTAGKISSISQPGN----DFSTKDGDNDKCICK-CSQMLTGGWWFDACGPSNLN 456

Query: 361 GQYFRSIPQQRQKLKK--GIFWKTWRGRYYPLQATTMLIQP 399

G Y+ QRQ K GI W W+G Y L+ATTM+I+P

Sbjct: 457 GMYY----PQRQNTNKFNGIKWYYWKGSGYSLKATTMMIRP 493

Score = 22.3bits (46), predicted value = 0.11

Identity = 10/34 (29%), Similarity = 18/34 (52%)

Query: 122 KVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHE 155

++ ++ QQR++ K HL+ + L +H +L E

Sbjct: 362 QLTNQQRYVLKIHLKDWEGNEAYSLYEHFYLSSE 395

>gi|4378598|gb|AAD19608.1|Angiopoietin Y1 Length=491

Score = 162bits (406), expected value = 7e-44

Identity = 88/218 (40%), Similarity = 128/218 (58%), Gap = 8/218 (3%)

Query: 186 RDCQELFQVGERQSGLFEIQPQGSP-PFLVNCKMTSD-GGWTVIQRRHDGSVDFNRPWEA 243

+DCQ+ + G SG++ I+P+ S P + C+ + D GGWTVIQ+R DGSV+F R WE

Sbjct: 278 KDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTDGSVNFFRNWEN 337

Query: 244 YKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQF-SVHLGGEDTAYSL 302

                                   

Sbjct: 338 YKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAEYSSFRLEPESEFYRL 397

Query: 303 QLTAPVAGQLGATTPPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQ 362

+L G G G + + +G F+T D+D D+ NCA GGWW+ C+HSNLNG

Sbjct: 398 RL-GTYQGNAGDSMMWHNGKQ--FTTLDRDKDMYAG-NCAHFHKGGWWYNACAHSNLNGV 483

Query: 363 YFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPM 400

        ++R   R K + GIFW +RG Y L+A M+I+P+

Sbjct: 454 WYRG-GHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPI 490

Score = 21.2bits(43), predicted value = 0.24

Identity = 26/111 (23%), Similarity = 47/111 (41%), Gap = 10/111 (9%)

Query: 105 LQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKR 164

L+ + + NSR+ QL+ ++ + + L + L+++ ++ E+ K A R R

Sbjct: 119 LRKESRNMNSRVTQLYMQLLHEIIRKRDNSLELSQLENKI-----LNVTTEMLKMATRYR 173

Query: 165 LPEM--AQPVDPAHNVS-RLHRLPRDCQELF--QVGERQSGLFEIQPQGSP 210

E+ A D +N S + L C +F Q L ++ PQ P

Sbjct: 174 ELEVKYASLTDLVNNQSVMITLLEEQCLRIFSRQDTHVSPPLVQVVPQHIP 224

>gi|4502087|ref|NP_001137.1||Angiopoietin 1 Length＝192

Score = 149bits (372), expected value = 6e-40

Identity = 85/194 (43%), Similarity = 110/194 (55%), Gap = 11/194 (5%)

Query: 211 PFLVNCKM-TSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDR 269

P V C M + GGWTVIQ R DGS+DF R W+ YK GFG+P GE+WLG E + +IT R

Sbjct: 4 PKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQR 63

Query: 270 NSRLAVQLRDWDGNAELLQFS-VHLGGEDTAYSLQLTAPVAGQLGATTPPPSGL--SVPF 326

L ++L DW+GN Q+ H+G E Y L L G G S + F

Sbjct: 64 QYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYL----KGHTGTAGKQSSLILHGADF 119

Query: 327 STWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGR 386

ST D D+D K CA L+GGWWF C SNLNG ++ + Q KL GI W ++G

Sbjct: 120 STKDADNDNCMCK-CALMLTGGWWFDACGPSNLNGMFY-TAGQNHGKL-NGIKWHYFKGP 176

Query: 387 YYPLQATTMLIQPM 400

Y L++TTM+I+P+

Sbjct: 177 SYSLRSTTMMIRPL 190

Example 3: Angiopoietin-like protein gene-transformed cells (L929) tumorigenesis experiment in vivo

Eight-week-old male nude mice were randomly divided into three groups: experimental group (Elpp1158), empty vector control group (PCMV-Script) and non-transformed cell control group (Control), with 4, 6 and 6 nude mice in each group .

Each nude mouse was subcutaneously inoculated with 2× ¹⁰⁶ transformed cells on the ventral side of the right nape of the nude mouse. The experimental observation period was 4 weeks. ) compared with the empty vector control group (PCMV-Script) had a very significant difference (p<0.01), that is, it had the effect of significantly inhibiting the growth of transformed cells (see Table 1 and Figure 1).

Table 1: Comparison table of tumor size in transformed cell (L929) tumor formation experiment serial number Elpp1158 PCMV-Script vector No treatment cell group 1 0.3 3.9 0.9 2 0.7 3.7 0.6 3 0.7 3.3 0.2 4 0.3 3 0 5 2.8 0 6 2.1 0 Mean ± SD 0.5* 3.133+0.653 0.283

^* : p<0.01

Example 4: mRNA expression of angiopoietin-like protein cDNA in various tissues

Using the angiopoietin-like protein gene as a probe, hybridization was performed on various tissue mRNA patches (Clontech). The results are shown in the figure, and it was found that the angiopoietin-like protein gene was expressed in the brain and placenta, but not or weakly expressed in the heart, lung, muscle, kidney, and pancreas (see Figure 2).

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

(1) General information:

(i) Applicant: Shanghai Cancer Institute

(ii) Title of the invention: human angiopoietin-like protein and its coding sequence and its use

(iii) Number of sequences: 3

(2) Information on SEQ ID NO.1

(i) Sequence features:

(A) Length: 1943bp

(B) Type: Nucleotide

(C) chain: double chain

(D) Topology: Linear

(ii) Molecular type: Nucleotide

(ix) Sequence description: SEQ ID NO.1:

GGAGAAGAAG CCGAGCTGAG CGGATCCTCA CACGACTGTG ATCCGATTCT TTCCAGCGGC 60

TTCTGCAACC AAGCGGGTCT TACCCCCGGT CCTCCGCGTC TCCAGTCCTC GCACCTGGAA 120

CCCCAACGTC CCCGAGAGTC CCCGAATCCC CGCTCCCAGG CTACCTAAGA GGATGAGCGG 180

TGCTCCGACG GCCGGGGCAG CCCTGATGCT CTGCGCCGCC ACCGCCGTGC TACTGAGCGC 240

TCAGGGCGGA CCCGTGCAGT CCAAGTCGCC GCGCTTTGCG TCCTGGGACG AGATGAATGT 300

CCTGGCGCAC GGACTCCTGC AGCTCGGCCA GGGGCTGCGC GAACACGCGG AGCGCACCCG 360

CAGTCAGCTG AGCGCGCTGG AGCGGCGCCT GAGCGCGTGC GGGTCCGCCT GTCAGGGAAC 420

CGAGGGGTCC ACCGACCTCC CGTTAGCCCC TGAGAGCCGG GTGGACCCTG AGGTCCTTCA 480

CAGCCTGCAG ACACAACTCA AGGCTCAGAA CAGCAGGATC CAGCAACTCT TCCACAAGGT 540

GGCCCAGCAG CAGCGGCACC TGGAGAAGCA GCACCTGCGA ATTCAGCATC TGCAAAGCCA 600

GTTTGGCCTC CTGGACCCA AGCACCTAGA CCATGAGGTG GCCAAAGCCTG CCCGAAGAAA 660

GAGGCTGCCC GAGATGGCCC AGCCAGTTGA CCCGGCTCAC AATGTCAGCC GCCTGCACCG 720

GCTGCCCAGG GATTGCCAGG AGCTGTTCCA GGTTGGGGAG AGGCAGAGTG GACTATTTGA 780

AATCCAGCT CAGGGGTCTC CGCCATTTTT GGTGAACTGC AAGATGACCT CAGATGGAGG 840

CTGGACAGTA ATTCAGAGGC GCCACGATGG CTCAGTGGAC TTCAACCGGC CCTGGGAAGC 900

CTACAAGGCG GGGTTTGGGG ATCCCCACGG CGAGTTCTGG CTGGGTCTGG AGAAGGTGCA 960

TAGCATCACG GGGGACCGCA ACAGCCGCCT GGCCGTGCAG CTGCGGGACT GGGATGGCAA 1020

CGCCGAGTTG CTGCAGTTCT CCGTGCACCT GGGTGGCGAG GACACGGCCT ATAGCCTGCA 1080

GCTCACTGCA CCCGTGGCCG GCCAGCTGGG CGCCACCACC GTCCCACCCA GCGGCCTCTC 1140

CGTACCCTTC TCCACTTGGG ACCAGGATCA CGACCTCCGC AGGGACAAGA ACTGCGCCAA 1200

GAGCCTCTCT GGAGGCTGGT GGTTTGGCAC CTGCAGCCAT TCCAACCTCA ACGGCCAGTA 1260

CTTCCGCTCC ATCCCACAGC AGCGGCAGAA GCTTAAGAAG GGAATCTTCT GGAAGACCTG 1320

GCGGGGCCGC TACTACCCGC TGCAGGCCAC CACCATGTTG ATCCAGCCCA TGGCAGCAGA 1380

GGCAGCCTCC TAGCGTCCTG GCTGGGCCTG GTCCCAGGCC CACGAAAGAC GGTGACTCTT 1440

GGCTCTGCCC GAGGATGTGG CCGTTCCCTG CCTGGGCAGG GGCTCCAAGG AGGGGCCATC 1500

TGGAAACTTG TGGACAGAGA AGAAGACCAC GACTGGAGAA GCCCCCTTTC TGAGTGCAGG 1560

GGGGCTGCAT GCGTTGCCTC CTGAGATCGA GGCTGCAGGA TATGCTCAGA CTCTAGAGGC 1620

GTGGACCAAG GGGCATGGAG CTTCACTCCT TGCTGGCCAG GGAGTTGGGG ACTCAGAGGG 1680

ACCACTTGGG GCCAGCCAGA CTGGCCTCAA TGGCGGACTC AGTCACATTG ACTGACGGGG 1740

ACCAGGGCTT GTGTGGGTCG AGAGCGCCCT CATGGTGCTG GTGCTGTTGT GTGTAGGTCC 1800

CCTGGGGACA CAAGCAGGCG CCAATGGTAT CTGGGCGGCG TCACAGAGTT CTTGGAATAA 1860

AAGCAACCTC AGAACACTTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1920

AAAAAAAAAAA AAAAAAAAA AAA 1943

(2) Information of SEQ ID NO.2

(i) Sequence features:

(A) Length: 406 amino acids

(B) type: amino acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: polypeptide

(ix) Sequence description: SEQ ID NO.2:

MSGAPTAGAA LMLCAATAVL LSAQGGPVQS KSPRFASWDE MNVLAHGLLQ 50

LGQGLREHAE RTRSQLSALE RRLSACGSAC QGTEGSTDLP LAPESRVDPE 100

VLHSLQTQLK AQNSRIQQLF HKVAQQQRHL EKQHLRIQHL QSQFGLLDHK 150

HLDHEVAKPA RRKRLPEMAQ PVDPAHNVSR LHRLPRDCQE LFQVGERQSG 200

LFEIQPQGSP PFLVNCKMTS DGGWTVIQRR HDGSVDFNRP WEAYKAGFGD 250

PHGEFWLGLE KVHSITGDRN SRLAVQLRDW DGNAELLQFS VHLGGEDTAY 300

SLQLTAPVAG QLGATTPPPS GLSVPFSTWD QDHDLRRDKN CAKSLSGGWW 350

FGTCSHSNLN GQYFRSIPQQ RQKLKKGIFW KTWRGRYYPL QATTMLIQPM 400

AAEAAS 406

(2) Information of SEQ ID NO.3

(i) Sequence features:

(A) Length: 1943bp

(B) Type: Nucleotide

(C) chain: double chain

(D) Topology: Linear

(ii) Molecular type: Nucleotide

(ix) Sequence description: SEQ ID NO.3:

1 G G GAG AAG AAG CCG AGC TGA GCG GAT CCT CAC ACG ACT GTG ATC CGA 46

47 TTC TTT CCA GCG GCT TCT GCA ACC AAG CGG GTC TTA CCC CCG GTC CTC 94

95 CGC GTC TCC AGT CCT CGC ACC TGG AAC CCC AAC GTC CCC GAG AGT CCC 142

143 CGA ATC CCC GCT CCC AGG CTA CCT AAG AGG ATG AGC GGT GCT CCG ACG 190

1 Met Ser Gly Ala Pro Thr 6

191 GCC GGG GCA GCC CTG ATG CTC TGC GCC GCC ACC GCC GTG CTA CTG AGC 238

7 Ala Gly Ala Ala Leu Met Leu Cys Ala Ala Thr Ala Val Leu Leu Ser 22

239 GCT CAG GGC GGA CCC GTG CAG TCC AAG TCG CCG CGC TTT GCG TCC TGG 286

23 Ala Gln Gly Gly Pro Val Gln Ser Lys Ser Pro Arg Phe Ala Ser Trp 38

287 GAC GAG ATG AAT GTC CTG GCG CAC GGA CTC CTG CAG CTC GGC CAG GGG 334

39 Asp Glu Met Asn Val Leu Ala His Gly Leu Leu Gln Leu Gly Gln Gly 54

335 CTG CGC GAA CAC GCG GAG CGC ACC CGC AGT CAG CTG AGC GCG CTG GAG 382

55 Leu Arg Glu His Ala Glu Arg Thr Arg Ser Gln Leu Ser Ala Leu Glu 70

383 CGG CGC CTG AGC GCG TGC GGG TCC GCC TGT CAG GGA ACC GAG GGG TCC 430

71 Arg Arg Leu Ser Ala Cys Gly Ser Ala Cys Gln Gly Thr Glu Gly Ser 86

431 ACC GAC CTC CCG TTA GCC CCT GAG AGC CGG GTG GAC CCT GAG GTC CTT 478

87 Thr Asp Leu Pro Leu Ala Pro Glu Ser Arg Val Asp Pro Glu Val Leu 102

479 CAC AGC CTG CAG ACA CAA CTC AAG GCT CAG AAC AGC AGG ATC CAG CAA 526

103 His Ser Leu Gln Thr Gln Leu Lys Ala Gln Asn Ser Arg Ile Gln Gln 118

527 CTC TTC CAC AAG GTG GCC CAG CAG CAG CGG CAC CTG GAG AAG CAG CAC 574

119 Leu Phe His Lys Val Ala Gln Gln Gln Arg His Leu Glu Lys Gln His 134

575 CTG CGA ATT CAG CAT CTG CAA AGC CAG TTT GGC CTC CTG GAC CAC AAG 622

135 Leu Arg Ile Gln His Leu Gln Ser Gln Phe Gly Leu Leu Asp His Lys 150

623 CAC CTA GAC CAT GAG GTG GCC AAG CCT GCC CGA AGA AAG AGG CTG CCC 670

151 His Leu Asp His Glu Val Ala Lys Pro Ala Arg Arg Lys Arg Leu Pro 166

671 GAG ATG GCC CAG CCA GTT GAC CCG GCT CAC AAT GTC AGC CGC CTG CAC 718

167 Glu Met Ala Gln Pro Val Asp Pro Ala His Asn Val Ser Arg Leu His 182

719 CGG CTG CCC AGG GAT TGC CAG GAG CTG TTC CAG GTT GGG GAG AGG CAG 766

183 Arg Leu Pro Arg Asp Cys Gln Glu Leu Phe Gln Val Gly Glu Arg Gln 198

767 AGT GGA CTA TTT GAA ATC CAG CCT CAG GGG TCT CCG CCA TTT TTG GTG 814

199 Ser Gly Leu Phe Glu Ile Gln Pro Gln Gly Ser Pro Pro Phe Leu Val 214

815 AAC TGC AAG ATG ACC TCA GAT GGA GGC TGG ACA GTA ATT CAG AGG CGC 862

215 Asn Cys Lys Met Thr Ser Asp Gly Gly Trp Thr Val Ile Gln Arg Arg 230

863 CAC GAT GGC TCA GTG GAC TTC AAC CGG CCC TGG GAA GCC TAC AAG GCG 910

231 His Asp Gly Ser Val Asp Phe Asn Arg Pro Trp Glu Ala Tyr Lys Ala 246

911 GGG TTT GGG GAT CCC CAC GGC GAG TTC TGG CTG GGT CTG GAG AAG GTG 958

247 Gly Phe Gly Asp Pro His Gly Glu Phe Trp Leu Gly Leu Glu Lys Val 262

959 CAT AGC ATC ACG GGG GAC CGC AAC AGC CGC CTG GCC GTG CAG CTG CGG 1006

263 His Ser Ile Thr Gly Asp Arg Asn Ser Arg Leu Ala Val Gln Leu Arg 278

1007 GAC TGG GAT GGC AAC GCC GAG TTG CTG CAG TTC TCC GTG CAC CTG GGT 1054

279 Asp Trp Asp Gly Asn Ala Glu Leu Leu Gln Phe Ser Val His Leu Gly 294

1055 GGC GAG GAC ACG GCC TAT AGC CTG CAG CTC ACT GCA CCC GTG GCC GGC 1102

295 Gly Glu Asp Thr Ala Tyr Ser Leu Gln Leu Thr Ala Pro Val Ala Gly 310

1103 CAG CTG GGC GCC ACC ACC GTC CCA CCC AGC GGC CTC TCC GTA CCC TTC 1150

311 Gln Leu Gly Ala Thr Thr Val Pro Pro Ser Gly Leu Ser Val Pro Phe 326

1151 TCC ACT TGG GAC CAG GAT CAC GAC CTC CGC AGG GAC AAG AAC TGC GCC 1198

327 Ser Thr Trp Asp Gln Asp His Asp Leu Arg Arg Asp Lys Asn Cys Ala 342

1199 AAG AGC CTC TCT GGA GGC TGG TGG TTT GGC ACC TGC AGC CAT TCC AAC 1246

343 Lys Ser Leu Ser Gly Gly Trp Trp Phe Gly Thr Cys Ser His Ser Asn 358

1247 CTC AAC GGC CAG TAC TTC CGC TCC ATC CCA CAG CAG CGG CAG AAG CTT 1294

359 Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro Gln Gln Arg Gln Lys Leu 374

1295 AAG AAG GGA ATC TTC TGG AAG ACC TGG CGG GGC CGC TAC TAC CCG CTG 1342

375 Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg Gly Arg Tyr Tyr Pro Leu 390

1343 CAG GCC ACC ACC ATG TTG ATC CAG CCC ATG GCA GCA GAG GCA GCC TCC 1390

391 Gln Ala Thr Thr Met Leu Ile Gln Pro Met Ala Ala Glu Ala Ala Ser 406

1391 TAG CGT CCT GGC TGG GCC TGG TCC CAG GCC CAC GAA AGA CGG TGA CTC 1438

407 *** 407

1439 TTG GCT CTG CCC GAG GAT GTG GCC GTT CCC TGC CTG GGC AGG GGC TCC 1486

1487 AAG GAG GGG CCA TCT GGA AAC TTG TGG ACA GAG AAG AAG ACC ACG ACT 1534

1535 GGA GAA GCC CCC TTT CTG AGT GCA GGG GGG CTG CAT GCG TTG CCT CCT 1582

1583 GAG ATC GAG GCT GCA GGA TAT GCT CAG ACT CTA GAG GCG TGG ACC AAG 1630

1631 GGG CAT GGA GCT TCA CTC CTT GCT GGC CAG GGA GTT GGG GAC TCA GAG 1678

1679 GGA CCA CTT GGG GCC AGC CAG ACT GGC CTC AAT GGC GGA CTC AGT CAC 1726

1727 ATT GAC TGA CGG GGA CCA GGG CTT GTG TGG GTC GAG AGC GCC CTC ATG 1774

1775 GTG CTG GTG CTG TTG TGT GTA GGT CCC CTG GGG ACA CAA GCA GGC GCC 1822

1823 AAT GGT ATC TGG GCG GCG TCA CAG AGT TCT TGG AAT AAA AGC AAC CTC 1870

1871 AGA ACA CTT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1918

1919 AAA AAA AAA AAA AAA AAA AAA AAA A 1943

Claims (9)

1. isolating PP1158 polypeptide is characterized in that this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through disappearance, insertion or the replacement of 1-10 amino-acid residue, and have suppress hepatoma cell line 7721 clone's formation functions by (a) polypeptides derived.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with aminoacid sequence of SEQ ID NO:2.

3. isolating polynucleotide is characterized in that, are selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of SEQID NO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:

Coding region sequence or the full length sequence of SEQ ID NO:3.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it is a kind of host cell that is selected from down group:

(a) host cell that transforms or transduce with the described carrier of claim 6;

(b) host cell that transforms or transduce with the described polynucleotide of claim 3.

8. the preparation method with the active polypeptide of PP1158 is characterized in that, this method comprises:

(a) being fit to express under the condition of PP1158, cultivate the described host cell of claim 7;

(b) from culture, isolate and have the active polypeptide of PP1158.

9. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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