WO2001072819A1 - A new polypeptide- human gap connexin 9.35 and the polynucleotide encoding it - Google Patents

Abstract

The present invention discloses a new polypeptide- human gap connexin 9.35, the polynucleotde encoding it and a method producing the polypeptide by DNA recombinant technology. The present invention further discloses a method treating various disorders, e.g., malignant neoplasm, hematopathy, HIV infection and immunological disease and various inflammation etc. The present invention also discloses an antagonist of the polypeptide and its therapeutic action. The present invention further discloses the use of the polynucleotide encoding the new human gap connexin 9.35.

Description

 A new polypeptide-human gap junction protein 9.35 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-human gap junction protein 9.35, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

 In tissues, cells can share ions, second messengers, and small metabolites through specific intercellular channels, called gap junctions. The linker that accomplishes this function consists of a complete hexamer of membrane proteins. This membrane protein is often called gap junction protein (Cx). A channel contains two groups of hexamers located on two cells. Assembly of connexins. The gap connexin family is a multi-gene family that is interconnected. Although structurally similar within a particular species, there are still many differences and tissue specificities.

 Structurally, a single connexin consists of a short cytoplasmic N-terminal region, the intermediate peptide is bent and folded into four transmembrane segments, two cells, and an intracellular loop structure. The C-terminus is also located in the cytoplasm. In addition, there is a very large difference in length between different connexins, ranging from 20 amino acid residues (Cx 26) to 260 amino acid residues (Cx 56). The entire conformation is irregularly W-shaped.

 The gap between connexin proteins is about 50% to 80%. The difference is mainly located in the C-terminal region of the cytoplasm. There are great differences in amino acid length and amino acid sequence. The two loops outside the cell membrane are very conserved. Each loop contains three Cy s residues, and the cell-to-cell connection is achieved through the formation of sulfur dioxide bonds. The similarity of the extracellular position facilitates the tight adhesion between cells, and the specificity of the intracellular part helps to make the necessary selection of small molecular substances that pass through the channel to avoid the invasion of unwanted or harmful substances .

The transmembrane channels formed by the gap connection realize the diffusion of small molecules from one cell to neighboring cells, and achieve the most reasonable material distribution, each taking what is needed. This exchange of intercellular substances helps to regulate cell activity, including regulation of cell growth and regulation of cell differentiation. Metabolic capacity also changes after cell adhesion, and connexin can also act as an inhibitor of somatic mutations involving enzymes in certain metabolic pathways. The activation of tyrosine kinase receptors on the cell membrane can inhibit interstitial intercellular communication, such as hepatocyte growth factor, whose surface contains tyrosine kinase receptors, which can inhibit keratinocyte connectivity. PTA, a factor that promotes tumor growth, can also inhibit intercellular communication. In the human body, the material exchange between cells is very closely related to various tissues and organ tumors, such as breast cancer, epithelial cell carcinoma, hepatocellular carcinoma, artificial bone cell carcinoma, and so on. Cx43 and Cx 32 are abundantly expressed in normal hepatocytes, but at the site of hepatocellular carcinoma, the expression of Cx 32 is significantly weaker than the surrounding non-tumor areas, proving that the connections between cells are affected and the activity of carcinogens is affected Significantly decreased. It has also been found that Cx4 3 expression increases during pregnancy and during delivery.

 Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv 304 cell line, PMA-Ecv 304 cell line, unstarved L 02 cell line, arsenic stimulation 1 L02 cell line for hours, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, fetal lung, and In the fetal heart, the expression profile of the polypeptide of the present invention is very similar to the expression profile of human gap junction protein 9.35, so the functions of the two may also be similar. The invention is named human gap junction protein 9.35.

 As mentioned above, the human gap junction protein 9.35 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 it has been necessary to identify more involved in these The process of human gap junction protein 9.35 protein, especially the amino acid sequence of this protein is identified. Newcomer gap junction protein 9.35 protein gene isolation also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for disease 1 and it is important to isolate its coding DNA. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-human gap junction protein 9.35 and 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 human gap junction protein 9.35.

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human gap junction protein 9.35.

 Another object of the present invention is to provide a method for producing human gap junction protein 9.35.

 Another object of the present invention is to provide an antibody against the polypeptide-to-human gap junction protein 9.35 of the present invention.

Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the human gap junction protein 9.35 of the polypeptide of the present invention. Another object of the present invention is to provide a method for diagnosing and treating diseases related to human gap junction protein 9.35 abnormalities.

 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 D0: 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 70% 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) a sequence having positions 31 to 571 in SEQ ID NO: 1; and (b) a sequence having positions 1 to 3 in SEQ ID NO: 1 167 0-bit sequence.

 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said 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 human gap junction protein 9.35 protein, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of human gap junction protein 9.35 protein in vitro, comprising detecting a mutation 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 present invention also relates to a pharmaceutical composition comprising a polypeptide of the present 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 human gap junction protein 9.35.

 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 otherwise:

"Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, and may also refer to Depending on the group or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense zinc or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. 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 the same. The changes may include amino acid sequences or amino acid or Deletion, insertion or replacement of nucleotides. Variants can have "conservative" changes, where the substituted amino acid has similar structural or chemical properties as the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as using Tryptophan replaces glycine.

 "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 specific antibodies in a suitable animal or cell.

 An "agonist" refers to a molecule that, when combined with human gap junction protein 9.35, 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 human gap junction protein 9.35.

 An "antagonist" or "inhibitor" refers to a molecule that, when combined with human gap junction protein 9.35, can block or regulate the biological or immunological activity of human gap junction protein 9.35. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human gap junction protein 9.35.

 "Regulation" refers to a change in the function of human gap junction protein 9.35, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human gap junction protein 9.35. change.

"Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. 35. Those skilled in the art can purify human gap junction protein 9.35 using standard protein purification techniques. Substantially pure human gap junction protein 9.35 produces a single main band on a non-reducing polyacrylamide gel. Interstitial The purity of the catenin 9.35 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by 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-A-C-T". The complementarity between two single-stranded molecules can be subdivided or all. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that can partially inhibit the hybridization of a completely complementary sequence to a target nucleic acid. 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 binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences be combined with each other as a specific or selective interaction.

 "Percent identity" refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through 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, D. G. and P.M. Sharp (1988) Gene 73: 237-244). The Cluster 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: The number of matching residues between sequence A and sequence B

 100

The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun Hein. , (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. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? ^ It can specifically bind to the epitope of human gap junction protein 9.35.

 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 a substance from its original environment (for example, its natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, 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 part 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 existing in the natural state. .

 As used herein, "isolated human gap junction protein 9. 35" refers to human gap junction protein 9. 35 which is essentially free of other proteins, lipids, carbohydrates or other substances that are naturally associated with it. Those skilled in the art can purify human gap junction protein 9.35 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human gap junction protein 9.35 peptide can be analyzed by amino acid sequences.

 The present invention provides a novel polypeptide-human gap junction protein 9.35, which is basically composed of the amino acid sequence shown in SEQ ID 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 polypeptides of the invention may be glycosylated or 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 human gap junction protein 9.35. As the present invention As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of human gap junction protein 9.35 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 the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (in) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained 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 a 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 of 1670 bases in length and its open reading frame 31 3-571 encodes 85 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to human gap junction protein 9.35, and it can be deduced that the human gap junction protein 9.35 has similar functions to human gap junction protein 9.35.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DM. DNA can be single-stranded or double-stranded. DNA can be a coding or non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or it may be a degenerate variant. As used herein, the "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 that includes the polypeptide and a polynucleotide that includes 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 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 Formula, it 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 (with at least two sequences between

50%, preferably with 70 »/. The same). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present 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) added during hybridization Iy。 Using denaturants, such as 50% (v / v) formamide, 0.1% calf serum / 0. iy. Ficoll, 42 ° C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97¾ or more. 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 invention also relates to a nucleic acid fragment that hybridizes to the sequence 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 human gap junction protein 9.35.

 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 human gap junction protein 9.35 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 the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting 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.

These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of human gap junction protein 9.35 transcripts; (4) By immunological techniques or by measuring biological activity Detects protein products expressed by genes. The above methods can be used singly 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 2,000 nucleotides, preferably within 1000 nucleotides. The probe used here 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 method (4), the protein products of the human gap junction protein 9.35 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

 A method for amplifying DNA / RNA by PCR (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 based on the polynucleotide sequence information of the invention disclosed herein It is appropriately selected and synthesized by a conventional method. 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. In order to obtain the full-length cDNA sequence, 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 produced by genetic engineering using the vector of the present invention or directly using a human gap junction protein 9.35 coding sequence, and a recombinant technology to produce a polypeptide of the present invention. method.

 In the present invention, a polynucleotide sequence encoding human gap junction protein 9.35 can 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 the 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 replication origins, promoters, marker genes, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct DNA containing human gap junction protein 9.35 An expression vector of sequences and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, 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 promoters that control the expression of genes in prokaryotic or eukaryotic 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 SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.

 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 human gap junction protein 9.35 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 Sf9; animal cells such as CH0, COS or Bowes melanoma cells.

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 absorbing DNA can be harvested after the exponential growth phase and treated with CaCl. The steps used are well known in the art. Alternatively, MgCl ₂ is used. 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.

Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human gap junction protein 9.35 (Science, 1984; 224: 1431). Generally there are the following steps: (1) using the polynucleotide (or variant) encoding human human gap junction protein 9.35 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), the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, 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, ionization 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 gene chip expression profiles of human gap junction protein 9.35 and human gap junction protein 9.35 of the present invention. The upper graph is a graph of the expression profile of human gap junction protein 9. 35, and the lower graph is the graph of the expression profile of human gap junction protein 9. 35. Among them, 1 indicates fetal kidney, 2 indicates fetal large intestine, 3 indicates fetal small intestine, 4 indicates fetal muscle, 5 indicates fetal brain, 6 indicates fetal bladder, 7 indicates unstarved LG2, 8 indicates L02 +, l hr, As ³⁺ , 9 ECV304 PMA-, 10 ECV304 PMA +, 11 fetal liver, 12 normal liver, 13 thyroid, 14 skin, 15 fetal lung, 16 lung, 17 lung cancer, 18 fetal spleen, 19 Indicates the spleen, 20 indicates the prostate, 21 indicates the fetal heart, 22 indicates the heart, 23 indicates muscle, 24 indicates testes, 25 indicates fetal thymus, and 26 indicates thymus.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human gap junction protein 9.3. 9kDa 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. The specific experimental methods are not indicated in the following examples, and generally follow 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 manufacturer Suggested conditions.

 Example 1: Cloning of human gap junction protein 9.35

 Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. The Quik mRNA Isolation Kit (product of Qiegene) was used to isolate poly (A) mRNA and 2ug poly (A) niRNA from total RNA to form cDNA by reverse transcription. Using the Smart cDNA Cloning Kit (purchased from (ontech)) the c.DNA fragment Directly inserted into the multicloning site of pBSK (+) vector (Clontech), transformed DH5cx, and bacteria formed a cDNA library. Dye terminate cycle reaction ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkhi-Elmer) Determined the sequences of the 5 'and 3' ends of all clones. Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one clone 0129A01 was new A series of primers were used to synthesize the inserted cDNA sequence in this clone. The results showed that the full-length cDNA contained in clone 0129A01 was 1670bp (as shown in Seq ID NO: 1), from 313bp to 571bp has a 258bp open reading frame (ORF), which encodes a new protein (as shown in Seq ID NO: 2). We named this clone PBS-0129 A01, the encoded protein is named human gap junction protein 9.35. Example 2: Cloning the gene encoding human gap junction protein 9.35 by RT-PCR method

 CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:

 Primerl: 5-GATCTCTCATTATGGTTTTGATTT-3 '(SEQ ID NO: 3)

 Primer2: 5'- TCAACAGACATTTATAGATGTCTA-3- (SEQ ID NO: 4)

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

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

Amplification conditions: 50 μl of KC1, 10 μl / L Tris-Cl, (ρΗ8.5 ·), 1.5 μl / L MgCl ₂ , 200 μmol / L dNTP in a reaction volume of 50 μ 1 , lOpniol primer, 1U dnaq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perk in-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 5 ° C 30sec; 72 '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 using a TA cloning kit (Invitrogen product). DNA sequence analysis showed that the DNA sequence of the PCR product The column is exactly the same as l-1670bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human gap junction protein 9.35 gene expression:

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-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA was precipitated at 70 ° / °. Wash with ethanol, dry and dissolve in water. Using 20 μg 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. A- "PdATP was used to prepare a labeled DNA probe by a random primer method. The DNA probe used was the PCR amplified human gap junction protein 9.35 coding region sequence (313bp to 571bp) shown in Figure 1. The 32P-labeled probes (approximately 2 X 10 ⁶ cpm / ml) and transferred to a nitrocellulose membrane RNA is hybridized overnight at 42 ° C in a solution, the solution comprising 50% formamide _{- 25mM KH 2 P0 (pH7.4)} - 5 χ SSC-5 Denhardt's solution and 200 yg / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1 »/. SDS for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human gap junction protein 9.35

 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:

 Primer3: 5'- CATGCTAGCATGCTTTTCGTGTCATATCTGAAA-3 '(Seq ID No: 5)

 Primer4: 5'-CATGGATCCCTAATCTTTGATGAGGCACCAAGA_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 target gene, respectively. The Nde I and BamH I restriction sites correspond to the expression vector plasmid pET- 28 b (+) (Nova gen, Cat. No. 69865.3). PCR was performed using the pBS-0129A01 plasmid containing the full-length target gene as a template. PCR reaction conditions were: 1 in a total volume of 50μ containing plasmid pBS-Ol AOl 10pg, primer Primer- 3 and the other points Pr imer- 4 ¹ 1 Opmol, Advantage polymerase Mix (Clontech Products) 1 μ 1. 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 ligated product was transformed into E. coli DH5cc using the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 g / ml), positive colonies were screened by colony PCR and sequenced. A positive clone with the correct sequence was selected (pET-0129A01). The recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (Novagen) using the calcium chloride method. Products). In LB liquid medium containing kanamycin (final concentration 30 g / ml), the host bacteria BL21 (pET-0129A01) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and continued Incubate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidines (6His-Tag). Purified human gap junction protein 9.35. After SDS-PAGE electrophoresis, a single band was obtained at 9 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Production of anti-human gap junction protein 9.35 antibodies

 The following peptides specific to human gap junction protein 9.35 were synthesized using a peptide synthesizer (product of PE):

NH2-Me t-Leu-Phe-Va 1-Ser-Tyr-Leu-Lys-Asn-H is—Cy s— G hi—G 1 y-G 1 n- Cy s— C00H (SEQ ID NO: 7) . The polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Avrameas, et a 1. Immmochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned cyanin peptide 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. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to human gap junction protein 9.35. Example 6: Application of the 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 embodiment 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, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter. These same steps are: The sample-fixed filter is first The hybridization buffer is pre-hybridized so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic 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 the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

First, the selection of the probe

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

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

2. The GC content is 30% -70%, and the 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 1 (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 -TGCTTTTCGTGTCATATCTGAAAAATCATTGCCAAGGCCAA-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 D NO: 1 or its complementary fragment:

 5 -TGCTTTTCGTGTCATATCTGCAAAATCATTGCCAAGGCCAA-3 '(SEQ ID NO: 9)

Please refer to the literature for other unlisted common reagents and their preparation methods related to the following specific experimental steps: DNA PROBES GH Keller; MM Manak; Stockton Pres s, 1989 (USA) and more commonly used molecular cloning experiment manual books such as (Molecular Cloning Experiment Guide, U 998, 2nd Edition) [US] Sam Brook, et al., Science Press. Sample Preparation:

 1. Extract DNA 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; 2 5mmol / L Tris-HCl} , pH7.5; 25mmol / LnaCl; 25mmol / L MgCl 2) was suspended precipitate (about lOml / g) ₀ 4) 4 ° C Homogenize the tissue suspension 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 (1 ml of the initial tissue sample per 0.1 gram), and then follow the phenol extraction method below.

 2, DNA phenol extraction method

Steps: 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (1 x 10 ⁸ cells / ml) and apply a minimum of 100 ul of lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly 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 200 _Ug / 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. Purification of DNA and ethanol precipitation

Steps: 1) Add 10 volumes of 2mol / L sodium acetate and 1 volume of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C 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 allow the surface ethanol to evaporate. 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. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, every 1- 5 χ 10 ⁶ cells extracted about plus lul.

 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 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentration is 0.5% and 100ug / ml. Incubate at 37 ° C for 30 minutes. 10) Use an equal volume of benzene Phenol: chloroform: isoamyl alcohol (25: 24: 1) The reaction solution was extracted and centrifuged for 10 minutes. 11) Carefully remove the aqueous phase and 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 let stand at -20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Determination _Α 26β and Α _28. To detect the purity and yield of DNA. 15) Stored in -20 ^u (.

Preparation of sample film:

 1) Take 4 x 2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number lightly with a pencil. Two NC membranes are needed for each probe for subsequent experiments. In the step, the film is washed with high-strength conditions and strength conditions, respectively.

 2) Pipette 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 at 60-80 "C for 2 hours.

 Labeling of probes

1) 3 μ IProbe (0.1 IOD / Ιθμ 1), add 2 μ IK inase 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 (moni tor can be used for monitoring).

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

 7) Monitor the amount of isotope with a liquid scintillator

8) The combined solution after the first peak was collected as ^{32 P_Probe} (the second peak to prepare the desired free ^{γ-: '2} Ρ- dATP). Pre-hybridization

 The sample membrane was placed in a plastic bag, and 3-lOmg pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml) was added.

CT DNA (calf thymus DNA). ), After sealing the bag, shake in a 68 C water bath for 2 hours.

 Cross

 Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake in a water bath overnight.

 Wash film:

 High-intensity washing film:

1) Take out the hybridized sample membrane. 2) 2xSSC, 0.1% SDS, 40 ^Q C for 15 minutes (twice).

 3) Wash in 0.1xSSC, 0.1% SDS for 15 minutes at 40 ° C (twice).

 4) In 0.1xSSC, 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) 2xSSC, 0.1% SDS, wash at 37 ° C for 1 minute (twice).

 3) 0.1xSSC, 0.1% SDS, 37. C Wash for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice), and dry at room temperature. X-ray auto-development:

 -70 ° C, X-ray autoradiography (pressing 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 radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than that of hybridization spots. The radioactive intensity of the hybridization spot of the other probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Example 7 DNA Microarray

 Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature, for example, see the literature DeRisi, J. L., Lyer, V. & Brown, P.0.

 (1997) Science 278, 680-686. And Helle, R. A., Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

A total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between points is 280 μm. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

3. Wash twice with ddH ₂ 0 for 1 minute each time;

4. NaBH _{4 is} blocked for 5 minutes;

 5. 95 ° C water for 2 minutes;

 6. Wash with 0.2% SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry and store at 25 ° C in the dark for future use.

 (Two) probe marking

Were extracted from the body with a one-step mixing with body tissue specific tissue (or cell line after stimulation) total mRNA, and treated with Oligotex mRNA Midi Kit (available from QiaGen Inc.) purified mRNA, the other points will be ¹ J burned by reverse transcription Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and ¹ J Cy5dUTP ( 5-Amino-propargyl-2--deoxyur idine 5--tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the specific tissue (or stimulated cell line) mRNA of the body, and purified the probe to prepare the probe . For specific steps and methods, see:

 Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dari., Davis, RW ( 1995) Science. 270. (20): 467-480.

(Three) cross

 The probes from the two types of tissues and the chips were hybridized in a UniHyb ™ Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000. The scanner (purchased from General Scanning Company, USA) was used for scanning. The scanned image was analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.

The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Draw a fold based on these 13 Cy3 / Cy5 ratios Party illustration. (figure 1 ) . It can be seen from the figure that the expression of human gap junction protein 9.35 and human gap junction protein 9.35 according to the present invention are very similar. Industrial applicability

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

 In tissues, cells can share ions, second messengers, and small metabolites through specific intercellular channels, called gap junctions. Gap junction protein helps to achieve the connection function. Specific intercellular channels are assembled by gap connexins. The transmembrane channels formed by gap junctions realize intercellular communication, and help to regulate cell activity, including regulation of cell growth and regulation of cell differentiation. Metabolic capacity also changes after cell adhesion, and connexin can also be used as an inhibitor of enzymes and cell mutations involved in certain metabolic pathways.

 Studies have found that activation of tyrosine kinase receptors on cell membranes can inhibit interstitial intercellular communication, such as hepatocyte growth factor, whose surface contains tyrosine kinase receptors, which can inhibit the connection of keratinocytes. PTA, a factor that promotes tumor growth, can also inhibit intercellular communication. In tissue cells such as breast cancer, epithelial cell carcinoma, hepatocellular carcinoma, and artificial osteoblastoma, abnormalities in the material communication between the cells occur. For example, Cx43 and Cx 32 are abundantly expressed in normal liver cells, but the expression of Cx 32 in hepatocellular carcinoma sites is significantly weaker than the surrounding non-tumor areas, which significantly reduces the activity of carcinogens. It has also been found that Cx43 expression increases during pregnancy and during childbirth

 The connexin family is an interconnected multi-gene family, but its specific conserved sequences are necessary to form its active mot if. It can be seen that the abnormal expression of the specific gap junction protein mo tif will cause the function of the polypeptide containing the mo tif of the present invention to be abnormal, resulting in abnormal cell-to-cell material communication, which will affect signal transmission and cell metabolism, and produce correlation Diseases such as tumors, embryonic developmental disorders, growth and development disorders, infectious inflammation, biliary cirrhosis, breast cysts, thyroid cysts, etc.

 It can be seen that the abnormal expression of human gap junction protein 9.35 of the present invention will produce various diseases, especially tumors, embryonic development disorders, growth and development disorders, infectious inflammation, biliary cirrhosis, breast cysts, thyroid cysts, These diseases include, but are not limited to:

Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, malignant histiocytosis, melanoma, teratoma, sarcoma, adrenal cancer, bladder cancer, bone cancer, Osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymus tumor, nasal cavity and sinus tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, pleural mesothelioma, fiber Tumor, fibrosarcoma, lipoma, liposarcoma, leiomyoma

 Fetal developmental disorders: congenital abortion, cleft palate, facial oblique fissure, limb absentness, limb differentiation disorder, gastrointestinal atresia or stenosis, hyaline membrane disease, pulmonary insufficiency, polycystic kidney disease, ectopic kidney, double ureter, crypto, Congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract , Congenital glaucoma or cataract, congenital deafness

 Growth and development disorders: mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation

 Infectious inflammation: bacterial infection, viral infection, mycoplasma infection

 The abnormal expression of the human interstitial protein 10 of the present invention will also cause certain hereditary, hematological and immune system diseases.

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human gap junction protein 9.35. Agonists enhance human gap junction protein 9.35 to stimulate biological functions such as 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 gap junction protein 9.35 can be cultured with labeled human gap junction protein 9.35 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human gap junction protein 9.35 include antibodies, compounds, receptor deletions and analogs that have been screened. Antagonists of human gap junction protein 9.35 can bind to human gap junction protein 9.35 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 that act as antagonists, human gap junction protein 9.35 can be added to the bioanalytical assay to determine whether the compound is a compound by measuring its effect on the interaction between human gap junction protein 9.35 and its receptor. 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 human gap junction protein 9.35 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 9.35 molecules of human gap junction protein should 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 human gap junction protein 9.35 epitopes. 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 human gap junction protein 9.35 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 Agent. Techniques for preparing monoclonal antibodies against human gap junction protein 9.35 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridization Tumor technology, etc. Chimeric antibodies that bind human constant regions to non-human-derived 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. 4945718) can also be used to produce single-chain antibodies against human gap junction protein 9.35.

 Antibodies against human gap junction protein 9.35 can be used in immunohistochemistry to detect human gap junction protein 9.35 in biopsy specimens.

 Monoclonal antibodies that bind to human gap junction protein 9.35 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, human gap junction protein 9. 35 High affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of the antibody with a thiol crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human connexin 9.35 positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to human gap junction protein 9.35. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human gap junction protein 9.35.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human gap junction protein 9.35 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human gap junction protein 9.35 detected in the test can be used to explain the importance of human gap junction protein 9.35 in various diseases and to diagnose diseases where human gap junction protein 9.3 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.

The polynucleotide encoding human gap junction protein 9.35 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 human gap junction protein 9.35. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated 35. Human gap junction protein 9. 35 to inhibit endogenous human gap junction protein 9. 35 activity. Example τ:. A variant of human connexin 9.35 can be shortened and lacks the signal transduction domain. Human connexin 9.35, although it can bind to downstream substrates, but lacks signal transduction activity . Therefore, the recombinant base 3 therapeutic vector can be used to treat diseases caused by abnormal expression or activity of human connexin 9.35. Expression vectors of source two viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human gap junction protein 9.35 into cells. A method for constructing a recombinant viral vector carrying a polynucleotide encoding human gap junction protein 9.35 can be found in existing literatures (Sambook, etal.). In addition, a recombinant polynucleotide encoding human gap junction protein 9.35 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 body tissue; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid). Into the body and so on.

 Oligonucleotides (including antisense RNA and DNA) that inhibit human gap junction protein 9.35 mRNA and ribozymes are also within the scope of the present invention. A ribozyme is an enzyme-like RNA that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. The sensed RNA, DNA and ribozyme can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoryl chemistry synthesis of oligonucleotides has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription by encoding the DNA sequence of the RNA. This DNA sequence has been integrated downstream of the RM 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 the side, and the phosphorothioate or peptide bond is used instead of the phosphodiester bond to link the ribonucleosides.

 The polynucleotide encoding human gap junction protein 9.35 can be used for the diagnosis of diseases related to human gap junction protein 9.35. A polynucleotide encoding human gap junction protein 9.35 can be used to detect the expression of human gap junction protein 9.35 or the abnormal expression of human gap junction protein 9.35 in a disease state. For example, the DNA sequence of human gap connexin 9. 35 can be used to hybridize biopsy specimens to determine the expression of human gap connexin 9. 35. Hybridization techniques include Sout hern blotting, Nor t hern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. Part or all of the polynucleotides of the invention can be used as probes to be fixed on a microarray (Mic Roa ray) or DNA chip (also known as "gene chip"), used to analyze the differential expression analysis of genes in tissues and Genetic diagnosis. Human interstitial connexin 9. 35 specific primers were used for in vitro amplification of RNA-polymerase chain reaction (RT-PCR). Transcripts of human interstitial junction 9.35 were also detected.

Detection of mutations in the human gap junction 9.35 gene can also be used to diagnose human gap junction 9.35 phase Related diseases. The form of human gap junction 9.55 mutations includes point mutations, mutations, deletions, recombinations, and any other variants compared to the normal wild-type human gap junction 9.35 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequencing, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target the specific position of a human chromosome and can hybridize with it. Currently, the specific site of each gene on the chromosome needs to be identified. Currently, only few chromosome markers based on actual sequence and data (repeating polymorphisms) can be used to mark chromosome positions. According to the present invention, in order for these sequences to be associated with disease-related genes. The important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared based on the cDNA, and the sequence can be mapped on the chromosome. These primers were then used for PCR to select somatic promiscuous cells containing individual human chromosomes. Only those synaptic cells containing the human genes corresponding to the primers 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, by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. "≡: Other similar strategies for chromosomal localization include in situ hybridization, chromosome pre-selection using labeled flow sorting, and hybrid pre-selection 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, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the base and the disease that has been mapped to the chromosomal region.

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 is available at the chromosomal level or is it based on a cDNA sequence? ^^ Can detect 'missing or translocation'. 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. Human gap junction protein 9. 35 is administered in an amount effective to treat and / or prevent specific indications. The amount and range of human connexin 9.35 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

Request for Rights I. An isolated polypeptide-human gap junction protein 9.35, 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 an amino acid sequence represented by SEQ ID D NO: 2 of at least 95 »/. The same.  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; or  (c) A polynucleotide that is at least 70% 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 the sequence of positions 31 to 571 in S EQ ID NO: 1 or 1 to 1 67 in SEQ ID NO: 1 A sequence of zero bits.  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 human gap junction protein 9.35 activity, characterized in that the method comprises:  (a) culturing the engineered host cell of claim 8 under the condition of expressing human gap junction protein 9.35;  (b) Isolating a polypeptide having human gap junction protein 9.35 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 human gap junction protein 9.35. II. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they mimic, promote, A compound that antagonizes or inhibits the activity of human gap junction protein 9.35.  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 the compound is used for a method for regulating the activity of human gap junction connexin 9.35 in vivo and in vitro.  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. The use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening a mimetic, agonist, antagonist or inhibitor of human gap junction protein 9.35; or using a dipeptide fingerprint Atlas 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 used 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 abnormalities of human gap junction protein 9.35.  18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said 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.