WO2001081391A1

WO2001081391A1 - A novel polypeptide, a protein 29 similar to g beta and the polynucleotide encoding the polypeptide

Info

Publication number: WO2001081391A1
Application number: PCT/CN2001/000583
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Biowindow Gene Development Inc. Shanghai
Priority date: 2000-04-27
Filing date: 2001-04-23
Publication date: 2001-11-01
Also published as: AU7379201A; CN1320610A

Abstract

The present invention discloses a novel polypeptide, a protein 29 similar to G beta, the polynucleotide encoding the polypeptide and the method for producing the polypeptide by DNA recombinant technology. The invention also discloses the uses of the polypeptide in methods for treating various diseases, such as malignant tumour, hemopathy, development disorders, HIV infection, immunological disease, and various inflammations, etc. The invention also discloses the agonists against the polypeptide and the therapeutic action thereof. The invention also discloses the uses of the polynucleotide encoding the protein 29 similar to G beta.

Description

A new polypeptide, a G-beta-like protein 29, and a polynucleotide encoding the polypeptide TECHNICAL FIELD

The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a G-beta-like protein 29, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

Transducin is a G protein that activates cGMP-specific phosphodiesterase. β-transducin (G-β) is one of the three subunits of the G protein, and these three subunits are heterotrisomy of the GTP-binding protein. β-transductin plays an integral role in signal transduction produced by transmembrane receptors, that is, it acts as a transmembrane signal transduction intermediate in a variety of physiological responses. These physiological responses include visual and Hormonal response. The α subunit binds and hydrolyzes GTP and belongs to the GTPase superfamily. The β subunit and γ subunit are necessary for GTP to replace GDP and membrane anchoring and recognition of receptors. They also participate in the inactivation of the α subunit.

In higher eukaryotes, G-β belongs to a small polygene family that contains approximately 340 conserved amino acid residues. The G-β structure consists of eight repeaters of approximately 40 amino acids in series, each repeater containing a central Trp-Asp motif (WD-40). Proteins with the WD-40 motif are also commonly referred to as WD proteins or GH-WD proteins (GH stands for glycine-histidine). Such repeating structures are found in many proteins. The number of repeats varies from 5-8 in these proteins. In G-β and G-β-like proteins, repeat substructures are distributed throughout the entire sequence, while in other proteins, they are distributed only at the N-terminus, the central part, or the C-terminus. The conserved core structure of the WD repeat substructure is connected to a variety of different regions, which are likely to be located on the surface. The longest variable region is located in front of GH, but the other small variable regions are located in the conserved core region.

The core domain of the WD protein has a structural feature: [LIVMSTAC]-[LIVMFYWSTAGC]-[LIMXTAG]-[LIV MS TAGC]-X (2)-[DN] -X (2)-[LI VMWSTAC] -X- [LI VMFS TAG]-W- [DEN]-[LIVMFSTAGCN]. The continuous expression and hydrophobicity of this feature pattern indicate that the WD repeat substructure may fold into a variable loop located in front of GH, followed by a β-sheet-corner-β-sheet-corner sheet, and finally a WD repeater. It is likely that stable intramolecular dimers or tetramers are formed between the WD repeats. The core structure composed of WD repeats may interact with other cores, thus providing a platform for specific surface activity. The mobility of this platform provides a variable protein-protein reaction interface for WD family proteins, and different WD family proteins can have different functional reactions with different other proteins. GP-like protein is a eukaryotic subfamily protein of the WD protein family. This subfamily protein is characterized by a conservative conserved repeating structure consisting of 36-46 amino acids, ending with a tryptophan (W) and an asparagus Amino acid (D) [Simon, MI, StrathmanN, MP., Et al., 1991, Science252: 802-808] [Neer, EJ, Schmidt, CJ, et

al., 1994, Nature371: 297-300]. These WD proteins can inhibit the transcription of many genes. They do not directly bind to DNA, but function by reacting with DNA binding proteins [Kel Eher, CA, Redd, MJ, Schultz, J., et al., 1992 , Cel 168: 709-719].

The structure of GP-like proteins is very similar to that of G-β, and also has the above-mentioned structural characteristics. The polypeptides of the present inventors have 96% identity and 97% similarity at the protein level with similar proteins, have similar structural features to G β similar proteins, and belong to the similar protein family, so they are named GP similar proteins 29, and It is speculated that it has similar biological functions.

As mentioned above, G-beta-like protein 29 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more involved in these processes The G-beta-like protein 29 protein, in particular, identifies the amino acid sequence of this protein. The isolation of the new G-beta-like protein 29 protein-encoding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so isolating its coding DNA is important. Disclosure of invention

It is an object of the present invention to provide an isolated novel polypeptide, G-beta-like protein 29, as well as fragments, analogs and derivatives thereof.

Another object of the invention is to provide a polynucleotide encoding the polypeptide.

Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a G-beta-like protein 29.

It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a G-beta-like protein 29.

Another object of the present invention is to provide a method for producing G-beta-like protein 29.

Another object of the present invention is to provide an antibody against the polypeptide of the present invention, a G-beta-like protein 29.

Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, G-beta-like protein 29.

Another object of the present invention is to provide diagnosis and treatment of diseases related to abnormalities of G-beta-like protein 29. Methods.

The present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID D. 2;

(b) a polynucleotide complementary to polynucleotide (a);

(c) A polynucleotide having at least 97% identity to a polynucleotide sequence of (a) or (b).

More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID D NO: 1

A sequence of 400-1 1 82 bits; and (b) a sequence of 1 -1 698 bits in SEQ ID NO: 1.

The present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of G-be t a similar protein 29 protein, which comprises using the polypeptide of the invention. The invention also relates to compounds obtained by this method.

The invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a G-beta-like protein 29 protein, comprising detecting mutations in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.

The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.

The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of G-be t a similar protein 29.

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

The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof Minute. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

A "variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

"Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

"Insertion" or "addition" refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

"Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.

An "agonist" is a molecule that, when combined with a G-beta-like protein 29, causes the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to a G-beta-like protein 29.

An "antagonist" or "inhibitor" refers to a molecule that blocks or regulates the biological or immunological activity of G-beta-like protein 29 when bound to G-beta-like protein 29. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds G-beta-like protein 29.

"Regulation" refers to a change in the function of G-beta-like protein 29, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immunological changes in G-beta-like protein 29.

By "substantially pure" is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify G-beta-like protein 29 using standard protein purification techniques. The substantially pure G-beta-like protein 29 produces a single main band on a non-reducing polyacrylamide gel. The purity of G-beta-like protein 29 peptide can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-ACT". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands The efficiency and strength of hybridization between nucleic acid strands has a significant effect.

"Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of a completely homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences interact with each other specifically or selectively.

"Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences, such as sequence A and sequence B, is calculated by the following formula: Number of residues matching between sequence A and sequence X 100 Number of residues in sequence A-number of interval residues in sequence A B is a spacer sequence of residues may be measured as Jotun Hein percent identity between nucleic acid sequences or by using Cluster method known in the art (Hein J., (1990) methods in emzumology 183: 625-645) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

"Antisense" refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

"Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules. "Antibody" refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') ₂ and Fv, which can specifically bind to the epitope of G-beta-like protein 29.

A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

The term "isolated" refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .

As used herein, "isolated G-beta-like protein 29" means that G-beta-like protein 29 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify G-beta-like protein 29 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the G-beta-like protein 29 peptide can be analyzed by amino acid sequence.

The present invention provides a new polypeptide, G-beta-like protein 29, which is basically composed of SEQ ID

It consists of the amino acid sequence shown in NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives and analogs of G-beta-like protein 29. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the G-beta-like protein 29 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (in) such One of which mature peptide Fusion with another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence (such as a leader sequence or a secretion sequence) formed by fusing an additional amino acid sequence into a mature polypeptide Or the sequence used to purify this polypeptide or protease sequence) As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 1698 bases, and its open reading frame 400-1 1 82 encodes 260 amino acids. According to the amino acid sequence homology comparison, it was found that the polypeptide has 96% homology with the G-be t a similar protein, and it can be deduced that the G-be t a similar protein 29 has the similar structure and function of the G-be t a similar protein.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide can 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 that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only at Hybridization occurs when the identity between the two sequences is at least 95%, and more preferably 97%. In addition, 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 invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding G-beta-like protein 29.

The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the G-beta-like protein 29 of the present invention can be obtained by various methods. For example, polynucleotides are 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 polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.

Of the methods mentioned above, genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate niRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene). The construction of cDNA libraries 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 different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.

The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (DDNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of the transcript of G-beta-like protein 29; (4) by Immunological technology or determination of biological activity to detect protein products of gene expression. The above methods can be used alone or in combination.

In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 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 2000 nucleotides, preferably within 1000 nucleotides. The probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase). 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 of the G-beta-like protein 29 gene expression.

A method of applying a PCR technique to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-Rapid Amplification of cDNA Ends) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Selected and synthesized by conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

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

The present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a G-beta-like protein 29 coding sequence, and a recombinant technology for producing the polypeptide of the present invention method.

In the present invention, a polynucleotide sequence encoding a G-beta-like protein 29 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-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 baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a G-beta-like protein 29 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular CI on ing, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the _PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and the early and late SV40 promoters , Retroviral LTRs and other known controllable genes in prokaryotic cells or eukaryotic cells Promoters expressed in cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV4 0 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.

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, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

In the present invention, a polynucleotide encoding a G-be t a similar protein 29 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to 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: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS, or Bowes s melanoma cells, etc. .

Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l ₂ method used in steps well known in the art. The alternative is to use MgC l ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant G-be t a similar protein 29 (Scence, 1 984; 224: 1 4 31). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding human G-be t a similar protein 29 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) culturing host cells in a suitable medium;

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

In step (2), depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cell has grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is re- Cultivate for a while.

In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Brief description of the drawings

The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

Fig. 1 is a comparison diagram of amino acid sequence homology of G-beta-like protein 29 and G-beta-like protein according to the present invention. The upper sequence is G-beta-like protein 29, and the lower sequence is G-beta-like protein. Identical amino acids are represented by single character amino acids between the two sequences, and similar amino acids are represented by "+".

Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated G-beta-like protein 29.

29KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually performed according to conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.

Example 1: Cloning of G-beta-like protein 29

Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Using Quik mRNA Isolation Kit

(Qiegene product) Isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart 00 cDNA cloning kit (purchased from Clontech) was used to insert the 00 fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5oc. The bacteria formed a cDNA library. Dye terminate cycle reaction ion sequencing kit (Perkin Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with an existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones, 1393g01, was new DNA. The inserted cDNA fragment contained in the clone was synthesized by a series of primers Perform a two-way measurement. The results showed that the 1393g01 clone contained a full-length cDNA of 1698bp (as shown in Seq ID NO: 1), and a 783bp open reading frame (0RF) from 400bp to 1182bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-1393g01 and the encoded protein was named G-beta-like protein 29. Example 2: Homologous search of cDNA clones

The sequence of the G-beta-like protein 29 of the present invention and the protein sequence encoded by the same were used by the Blast program (Basiclocal Alignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] Perform homologous searches in databases such as Genbank and Swissport. The gene with the highest homology to the G-beta-like protein 29 of the present invention is a known G-beta-like protein, and the accession number of the encoded protein in Genbank is AF051155. The protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 96% and a similarity of 97%. Example 3: Cloning of a gene encoding G-beta-like protein 29 by RT-PCR

CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction.

After purification of Qiagene's kit, PCR amplification was performed with the following primers:

Primerl: 5'- GGCCATCGATAACTCTACGCTCGG —3, (SEQ ID NO: 3)

Primer 2: 5,-TGTCGGTCTGCTTTTATTACCTCC -3 '(SEQ ID NO: 4)

Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;

Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.

Amplification conditions: 50 μl KC1, 10 μl / L Tris-Cl, (pH8.5), 1.5mmol / L MgCl ₂ , 200 μmol / L dNTP in a 50 μ 1 reaction volume , lOpmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. The DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as that of 1-1698bp shown in SEQ ID NO: 1. Example 4: Analysis of the expression of G-beta-like protein 29 gene by Northern blot:

Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25 μτιM sodium citrate, 0.2M sodium acetate (ρΗ4.0), and 1 volume of phenol and 1Z5 volume of chloroform-isoamyl alcohol (49: 1) are added, After mixing Heart. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. With a ^"2 P dATP Preparation ^{32 Ρ-} DNA probe labeled by the random primer method. The DNA probe used was the PCR-amplified G-beta-like protein 29 coding region sequence (400bp to 1182bp) shown in FIG. 1. The 32P- labeled probe (approximately 2 X 10 ⁶ cpm / ml) and nitrocellulose transferred RNA in 42 "C overnight in a hybridization solution, the solution comprising 50% formamide -25mM KH ₂ P0, (pH7.4) -5 χ SSC-5 χ Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 χ SSC-0.1% SDS at 55 ° C for 30 minutes. Then, Phosphor Imager Analysis and quantification Example 5: In vitro expression, isolation and purification of recombinant G-beta-like protein 29

Based on the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed. The sequences are as follows:

Primer 3: 5'- CCCCATATGATGTATGATCTCAACTCCAATAAC -3 '(Seq ID No: 5) Primer4: 5'- CATGGATCCCTAGCCCAGCACACTGTCATTGAA -3' (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively , Followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively. The Ndel and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site. The PCR reaction was performed using the pBS-1393g01 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μΐ containing 10 pg of pBS-1393g01 plasmid, primers Primer-3 and Primer-4 were lOpmol and Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into coliform bacteria DH5C by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (PET-I393g01) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In containing kanamycin (final concentration of 30 μ g / ml) of LB liquid medium, host strain BL21 _(P ET-1393g01) incubated at 37 ° C to logarithmic phase, IPTG was added to a final concentration of 1 ol implicit / L, continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by ultrasonication. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). The purified G-beta-like protein 29 was purified. After SDS-PAGE electrophoresis, a single band was obtained at 29 KDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, 15 amino acids at the N-terminus and SEQ ID NO: 2 The N-terminal 15 amino acid residues shown are identical. Example 6 Production of Anti-G-beta Similar Protein 29 Antibody

A peptide synthesizer (product of PE company) was used to synthesize the following G-beta-like protein 29-specific peptides: NH2-Me t-Tyr-As p-Leu-Asn-Ser-Asn-Asn-Pro-Asn-Pro- I l eI l e-Ser-Tyr-C00 H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemi stry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polymorphic complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Seph _{ar 0 S} e4B column, and the anti-peptide antibody was separated from the total I gG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to G-beta-like protein 29. Example 7: Use of a polynucleotide fragment of the present invention as a hybridization probe

Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

The purpose of this example is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this embodiment, the dot blot method is used to fix the sample on the filter membrane. Under high-intensity washing conditions, the first type of probe hybridizes with the sample. The strongest specificity is retained.

First, the selection of the probe

The selection of oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:

1. The preferred range of probe size is 18-50 nucleotides;

2, GC content is 30% -70%, non-specific hybridization increases when it exceeds;

3. There should be no complementary regions inside the probe;

4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;

5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

After completing the above analysis, select and synthesize the following two probes:

Probe l (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

5'- TGTATGATCTCAACTCCAATAACCCTAACCCCATCATCAGC -3 '(SEQ ID NO: 8)

Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

5-TGTATGATCTCAACTCCAATCACCCTAACCCCATCATCAGC -3 '(SEQ ID NO: 9) For other common reagents and their preparation methods not listed in the following specific experimental steps, please refer to the literature:

DNA PROBES GH Keller; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experiment manuals such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambruck et al., Science Press .

Sample Preparation:

1. Extract DM from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) cold homogenization buffer (0.25mol / L sucrose; 25mmol / L Tris-HCl, pH7.5; 25 awake ol / LnaCl; 25mmol / L MgCl 2) was suspended precipitate (approximately 10ml / g). 4) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (add 1-5 ml per 0.1 g of the original tissue sample) and centrifuge at 1000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (add 1ml per 0.1 g of the original tissue sample), then connect the following Phenol extraction method.

2, DNA phenol extraction method

Steps: 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1,000 g for 10 minutes. 2) Resuspend the pelleted cells (1 × 10 ⁸ cells / ml) with cold cell lysate and apply a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing DNA to a new tube. The DNA was then purified and ethanol precipitated.

3, DNA purification and ethanol precipitation

Steps: 1) Add 10 volume of 2mol / L sodium acetate and 1 volume of cold 100% ethanol to the DNA solution and mix. Centrifuge at -2 (TC for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air-dry for 10-15 minutes to evaporate the surface ethanol. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Vortex at low speed or pipette with suction while gradually increasing TE, mix until the DNA is fully lysed, add approximately 1 ul per 1-5 x lO ⁶ cells extracted.

The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100 ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5% and 100ug / ml. Incubate at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volume of cold ethanol, mix and set-2 (TC for 1 hour. 13) wash the precipitate with 70% ethanol and 100% ethanol, air dry, and weigh Suspension of nucleic acids, the process is the same as steps 3-6. 14) Measure A ₂₆ . And A _2S . To detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing.

Preparation of sample film:

1) Take 4 x 2 nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number with a pencil. Two NC membranes are required for each probe for subsequent experiments. In the step, the film is washed with high-strength conditions and strength conditions, respectively. 2) Aspirate and control 15 microliters each, spot on the sample film, and dry at room temperature.

3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

4) Clamp in clean filter paper, wrap with aluminum foil, and dry under vacuum for 60-80 for 2 hours.

Labeling of probes

1) 3 μ IProbe (0.1 OD / 10 μ 1), add 2 μ IKinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

2) Incubate at 37 ° C for 1 hour.

3) Add 1/5 volume of bromophenol blue indicator (BPB).

4) Pass Sephadex G-50 column.

5) Before the ³² P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).

6) 5 drops / tube, collect 10-15 tubes.

7) Monitor the amount of isotope with a liquid scintillator

8) After combining the collection liquids of the first peak, the ³² P-Probe (the second peak is free γ- ³² P-dATP) is prepared. Pre-hybridization

Place the sample film in a plastic bag, add 3-10 mg of pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).), Seal the bag, and shake at 68 ° C in water for 2 hour.

Cross

Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake in a 42 C water bath overnight. Wash film:

High-intensity washing film:

1) Take out the hybridized sample membrane.

2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

3) 0.1xSSC, 0.1% SDS, wash at 40 C for 15 minutes (2 times;).

4) 0. IxSSC, 0.1% SDS, wash at 55 "C for 30 minutes (twice), and dry at room temperature. Low-intensity washing film:

1) Take out the hybridized sample membrane.

2) Wash in 2xSSC, 0.1% SDS, 37 "C for 15 minutes (twice).

3) 0. IxSSC, 0.1¾SDS, wash at 37 ° C for 15 minutes (twice).

4) 0. IxSSC, 0.1% SDS, wash at 40 "C for 15 minutes (twice), and dry at room temperature. X-ray autoradiography:

-70 ° C, X-ray autoradiography (compression time depends on the radioactivity of the hybrid spot).

Experimental results:

The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactivity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than that of hybrid spots. The radioactive intensity of the hybridization spot of the other probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1. Industrial applicability

The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.

Transducin is a G protein that activates cGMP-specific phosphodiesterase, and β-transducin is a subunit of G protein. β-transductin plays an integral role in signal transduction produced by transmembrane receptors, that is, it acts as a transmembrane signal transduction intermediate in a variety of physiological responses. These physiological responses include visual and Hormonal response. The α subunit binds and hydrolyzes GTP and belongs to the GTPase superfamily. The β subunit and γ subunit are necessary for GTP to replace GDP and membrane anchoring and recognition of receptors. They also participate in the inactivation of the α subunit.

G-β contains a conserved structure of WD repeats. WD repeats participate in a variable protein-protein reaction. G-β-like proteins are eukaryotic subfamily proteins of the WD protein family. They can inhibit the transcription of many genes. It functions by reacting with DNA-binding proteins.

The polypeptide of the present invention and the G-β-like protein are homologous proteins and contain characteristic sequences of the G-β-like protein family, and both have similar biological functions. It participates in the transmission of information in the body, can inhibit the transcription of many genes, and is necessary for the normal physiological function of cells. Its abnormal expression is usually closely related to growth and development, immune system formation, tumorigenesis, and related diseases.

It can be seen that the abnormal expression of G-be ta-like protein 29 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth and development disorders, inflammation, and immune diseases. These diseases include but are not Limited to:

Tumors of various tissues: stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, chest Adenocarcinoma, Nasopharyngeal Carcinoma, Laryngeal Carcinoma, Trachea Tumor, Fibroma, Fibrosarcoma, Lipoma, Liposarcoma Embryonic Disorders: Congenital Abortion, Cleft Palate, Limb Absence, Limb Differentiation Disorder, Atrial Septal Defect, Neural Tube Defect , Congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness

Growth and development disorders: mental retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome Sexual retardation

Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations

Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome

Abnormal expression of the G-be t a similar protein 29 of the present invention will also cause certain hereditary, hematological diseases and the like. The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth and development disorders, inflammation, immunity Sexual diseases, certain hereditary, blood diseases, etc.

The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) G-be t a similar protein 29. Agonists enhance biological functions such as G-beta-like protein 29 to stimulate 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 G-be t a similar protein 29 can be cultured with labeled G-be t a similar protein 29 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

Antagonists of G-beta-like protein 29 include antibodies, compounds, receptor deletions, and analogs that have been screened. An antagonist of G-be ta-like protein 29 can bind to G-be ta-like protein 29 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot exert its biology Features.

When screening compounds as antagonists, G-be ta-like protein 29 can be added to bioanalytical assays to determine whether a compound is Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to G-be t a similar protein 29 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the G-be t a similar protein 29 molecule should generally be labeled.

The present invention provides the use of polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. Methods of producing antibodies. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the G-beta similar protein 29 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

Polyclonal antibodies can be produced by injecting G-beta-like protein 29 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait. Techniques for preparing monoclonal antibodies to G-beta-like protein 29 include, but are not limited to, hybridoma technology (ohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV- Hybridoma technology, etc. Chimeric antibodies that bind human constant regions to 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 G-beta-like protein 29.

Antibodies against G-beta-like protein 29 can be used in immunohistochemical techniques to detect G-beta-like protein 29 in biopsy specimens.

Monoclonal antibodies that bind to G-beta-like protein 29 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, G-beta-like protein 29 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill G-beta-like protein 29 positive cells .

The antibodies of the present invention can be used to treat or prevent diseases related to G-beta-like protein 29. Administration of an appropriate dose of antibody can stimulate or block G-beta-like protein 29 production or activity.

The invention also relates to a diagnostic test method for quantitative and localized detection of G-beta-like protein 29 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The levels of G-beta-like protein 29 detected in the test can be used to explain the importance of G-beta-like protein 29 in various diseases and to diagnose diseases in which G-beta-like protein 29 plays a role.

The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.

Polynucleotides encoding G-beta-like protein 29 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of G-beta-like protein 29. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated G-beta-like protein 29 to inhibit endogenous G-beta-like protein 29 activity. For example, a variant G-beta-like protein 29 may be a shortened G-beta-like protein 29 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of G-beta-like protein 29. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a G-beta-like protein 29 into a cell. Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a G-beta-like protein 29 can be found in the literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding G-beta-like protein 29 can be packaged into liposomes and transferred into cells.

Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit G-beta-like protein 29 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidation synthesis of oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.

A polynucleotide encoding G-beta-like protein 29 can be used for the diagnosis of diseases related to G-beta-like protein 29. Polynucleotides encoding G-beta-like protein 29 can be used to detect the expression of G-beta-like protein 29 or the abnormal expression of G-beta-like protein 29 in disease states. For example, the DNA sequence encoding G-beta-like protein 29 can be used to hybridize biopsy specimens to determine the expression of G-beta-like protein 29. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissue. G-beta-like protein 29 specific primers can also be used to detect G-beta-like protein 29 transcripts by in vitro amplification of RNA-polymerase chain reaction (RT-PCR).

Detecting mutations in the G-beta-like protein 29 gene can also be used to diagnose G-beta-like protein 29-related diseases. G-beta-like protein 29 mutant forms include a normal wild-type G-beta-like protein 29 DNA sequence compared to point mutations, translocations, deletions, recombinations and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome 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 a chromosome.

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

PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

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

Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene). The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

The present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them. In addition, the polypeptide of the present invention can be used in combination with other therapeutic compounds.

The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. G-be t a similar protein 2 9 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of G-beta-like protein 2 9 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Claims

Claim

1. An isolated polypeptide-G-beta-like protein 29, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative thereof.

2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;

(b) a polynucleotide complementary to the polynucleotide (a); or

(c) A polynucleotide that is at least 97% identical to (a) or (b).

5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.

6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises a sequence of positions 400-1182 in SEQ ID NO: 1 or a sequence of positions 1-1698 in SEQ ID NO: 1. .

7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant constructed from the polynucleotide according to any one of claims 4 to 6 and a plasmid, virus or vector expression vector Carrier.

8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

(a) a host cell transformed or transduced with the recombinant vector of claim 7; or

(b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having G-beta-like protein 29 activity, characterized in that the method comprises:

(a) culturing the engineered host cell of claim 8 under the condition of expressing G-beta-like protein 29;

(b) Isolating a polypeptide with G-beta-like protein 29 activity from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to a G-beta-like protein 29.

11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they mimic, promote, Compounds that antagonize or inhibit the activity of G-beta-like protein 29.

12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

13. Use of a compound according to claim 11, characterized in that said compound is used for a method for regulating the activity of G-beta-like protein 29 in vitro and in vivo.

14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

15. Use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of G-beta-like protein 29; or for peptides Fingerprint identification.

16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.

17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with G-beta-like protein 29 abnormality.

18. The use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HIV infection and immune diseases and drugs of various inflammations.