CN1065876C - Coded protein specific antibody having cytoskeleton gene - Google Patents

Abstract

The invention belongs to the field of human genome technology. The full-length cDNA sequence of the cytoskeleton-like gene (JWA) is 2107bp, its open reading frame sequence is 564bp, and encodes 188 amino acids in total. The gene number in GenBank is "BankIt 200994, Accession Number: AF070523". The invention utilizes partial sequences of 188 amino acids to synthesize polypeptides to immunize animals to prepare polyclonal and monoclonal antibodies.

Description

Antibodies specific for proteins encoded by cytoskeleton-like genes

The invention belongs to the technical field of human genome.

The cytoskeleton-like gene (JWA) of the present invention was obtained by using DD-PCR, phage screening method, RACE-PCR and other methods during the research period (1996.3～1998.3) of the inventor at the University of California, Davis (UCDavis). A novel gene related to the cytoskeleton cloned from a cDNA library constructed from human tracheal bronchial epithelial cells. The full-length cDNA sequence of the gene is 2107bp, and its open reading frame sequence is 564bp, encoding a total of 188 amino acids. Further studies showed that the open reading frame sequence of JWA gene contains three membrane translocation regions, each region contains 23 amino acids, and there are protein kinase C (PKC) phosphorylation sites at both ends of the three membrane translocation regions. Northern hybridization analysis showed that the gene had two mRNA transcripts of different sizes, and the exact termination positions of the two transcripts had been determined by reverse transcription, 3′-RACE-PCR, Southern hybridization analysis and Rnase Protection Assay. The results of immunofluorescence staining after transfection of S-6 and HBE cells with the expression vector carrying the open reading frame sequence of JWA gene (PCMV-2-Flag-JWA) showed that the protein encoded by JWA gene resembled cytoskeleton microtubules. The protein was clearly affected by the depolymerizing agents colchicine and nocodazole but not by cold treatment. In addition, the expression of this gene in several tumor cells was significantly affected by the demethylating drug 5-azacytidin.

Since there is no homologous gene in the JWA gene sequence in the GenBank database, the inventors' research results on the structure and function of the JWA gene are all groundbreaking. The National Center for Biotechnology Information (NCBI) has recently officially accepted JWA gene as a new member of GenBank. The gene number is "BankIt 200994, Accession Number: AF070523". GAATTCGGCACGAGCCGAGACGGAGCCGCTGTCAACTCTCCAACTCAGCTCAGCTGATCGGTTGCCGCCGCCGCCGCCGCCAGATTCTGGAGGCGAAGAACGCAAAGCTGAGAACATGGACGTTAATATCGCCCCACTCCGCGCCTGGGACGATTTCTTCCCGGGTTCCGATCGCTTTGCCCGGCCGGACTTCAGGGACATTTCCAAATGGAACAACCGCGTAGTGAGCAACCTGCTCTATTACCAGACCAACTACCTGGTGGTGGCTGCCATGATGATTTCCATTGTGGGGTTTCTGAGTCCCTTCAACATGATCCTGGGAGGAATCGTGGTGGTGCTGGTGTTCACAGGGTTTGTGTGGGCAGCCCACAATAAAGACGTCCTTCGCCGGATGAAGAAGCGCTACCCCACGACGTTCGTTATGGTGGTCATGTTGGCGAGCTATTTCCTTATCTCCATGTTTGGAGGAGTCATGGTCTTTGTGTTTGGCATTACTTTTCCTTTGCTGTTGATGTTTATCCATGCATCGTTGAGACTTCGGAACCTCAAGAACAAACTGGAGAATAAAATGGAAGGAATAGGTTTGAAGAGGACACCGATGGGCATTGTCCTGGATGCCCTAGAACAGCAGGAAGAAGGCATCAACAGACTCACTGACTATATCAGCAAAGTGAAGGAATAAACATAACTTACCTGAGCTAGGGTTGCAGCAGAAATTGAGTTGCAGCTTGCCCTTGTCCAGACCTATGTTCTGCTTGCGTTTTTGAAACAGGAGGTGCACGTACCACCCAATTATCTATGGCAGCATGCATGTATAGGCCGAACTATTATCAGCTCTGATGTTTCAGAGAGAAGACCTCAGAAACCGAAAGAAAACCACCACCCTCCTATTGTGTCTGAAGTTTCACGTGTGTTTATGAAATCTAATGGGAAATGGATCACACGATTTCTTTAAGGGAATTAAAAAAAATAAAAGAATTACGGCTTTTACAGCAACAATACGATTATCTTATAGGAAAAAAAAAAATCATTGTAAAGTATCAAGACAATACGAGTAAATGAAAAGGCTGTTAAAGTAGATGACATCATGTGTTAGCCTGTTCCTAAATCCCTAGAATTGTAATGTGTGGGATATAAATTAGTTTTTATTATTCTCTTAAAAATCAAAGATGATCTCTATCACTTTGCCACCTGTTTGATGTGCAGTGGAAACTGGTTAAGCCAGTTGTTCATACTTCCTTTACAAATATAAAGATAGCTGTTTAGGATATTTTGTTACATTTTTGTAAATTTTTGAAATGCTAGTAATGTGTTTTCACCAGCAAGTATTTGTTGCAAACTTAATGTCATTTTCCTTAAGATGGTTACAGCTATGTAACCTGTATTATTCTGGACGGACTTATTAAAATACAAACAGACAAAAAATAAAACAAAACTTGAGTTCTATTTACCTTGCACATTTTTTGTTGTTACAGTGAAAAAAATGGTCCAAGAAAATGTTTGCCATTTTTGCATTGTTTCGTTTTTAACTGGAACATTTAGAAAGAAGGAAATGAATGTGCATTTTATTAATTCCTTAGGGGCACAAGGAGGACAATAATAGCTGATCTTTTGAAATTTGAAAAACGTCTTTAGATGACCAAGCAAAAAGACTTTAAAAAATGGTAATGAAAATGGAATGCAGCTACTGCAGCTAATAAAAAATTTTAGATAGCAATTGTTACAACCATATGCCTTTATAGCTAGACATTAGAATTATGATAGCATGAGTTTATACATTCTATTATTTTTCCTCCCTTTCTCATGTTTTTATAAATAGGTAATAAAAAATGTTTTGCCTGCCAATTGAATGATTTCGTAGCTGAAGTAGAAACATTTAGGTTTCTGTAGCATTAAATTGTGAAGACAACTGGAGTGGTACTTACTGAAGAAACTCTCTGTATGTCCTAGAATAAGAAGCAATGATGTGCTGCTTCTGATTTTTCTTGCATTTTAAATTCTCAGCCAACCTACAGCCATGATCTTTAGCACAGTGATATCACCATGACTTCACAGACATGGTCTAGAATCTGTACCCTTACCCACATATGAAGAATAAAATTGATTAAAGGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACTCGAGJWA基因cDNA序列MDVNIAPLRAWDDFFPGSDRFARPDFRDISKWNNRVVSNLLYYQTNYLVV           50AAMMISIV(1)GFLSPFNMILGGIVVVLVFTGFVWAAHN(2)KDVLRRMKKRYPTT    100FVMVVMLASYFLISMFGGVMVFVFGITFPL(3)LLMFIHASLRLRNLKNKLEN       150KMEGIGLKRTPMGIVLDALEQQEEGINRLTDYISKVKE                      188

Amino acid sequence of open reading frame of JWA gene

In the sequence (1), (2), (3) are three membrane translocation regions, SDR and SLR are PKC phosphorylation sites

The purpose of the present invention is: in view of the fact that the JWA gene is a brand-new gene discovered by the inventor, the current human knowledge and understanding of this gene is limited to the scientific research work done by the inventor. The present inventors use methods such as Northern Blot, Western Blot, gene transfection and fluorescent immunohistochemical staining to confirm that JWA gene expresses a new cytoskeleton protein. This gene widely exists in various tissues and cells studied, and its mRNA expression is significantly affected by the differentiation-inducing agent retinoic acid (Retinoid acids), the oxidizing agent hydrogen peroxide (H2O2), and the demethylation drug (5-Azacytidin). Influence; the expression of the encoded protein of this gene is obviously affected by the depolymerization agent colchicine (Colchicine) and Nocodazole and heat shock treatment. The evolutionary conservation of the JWA gene and its active expression suggest that the gene has very active biological functions. Especially as a new cytoskeleton protein, it plays a very important role in cell proliferation and differentiation. Further research on the exact location of the protein encoded by the JWA gene in cells will help to understand the biological function of the gene. Gene-encoded protein-specific antibodies are an indispensable and important tool for studying gene structure and function. JWA gene encodes protein polyclonal and monoclonal specific antibodies, which can be used as immunohistochemical staining, ELISA, immunoelectron microscopy, immunoprecipitation and other detection methods designed according to immunological principles. It can be used for the localization and quantitative research of the protein encoded by the gene in tissue cells, the research of content changes in various biological samples (such as blood, urine, etc.), and the research of protein-protein interaction.

The technical solution of the present invention is: the amino acid sequence of the entire open reading frame of the JWA gene (a total of 188 amino acids, as listed in this application), especially the C-terminal 15 amino acids containing the hydrophilic amino acid region, the N-terminal 30 amino acids, and the 80th amino acid sequence. ~100 amino acids and amino acid sequences 135-155. Select a partial sequence of the above sequence to synthesize a polypeptide, combine with KLH carrier protein, mix with complete and incomplete adjuvant, immunize New Zealand rabbits (200ug/rabbit/time, stimulate injection once a week), and prepare polyclonal antibody (anti- JWA IgG); immunize Balb C mice (100ug/mouse/time, challenge injection once a month), separate spleen B lymphocytes and fuse with myeloma cells to generate hybridoma cells, and further use subcloning methods, ELISA methods and antibody typing Methods The monoclonal antibody secreting IgG was screened to prepare the monoclonal antibody (anti-JWA IgG).

Embodiments of the invention:

1. Cytoskeleton-like gene-encoded protein-specific polyclonal antibody takes 10 mg of synthetic JWA gene-encoded amino acid C-terminal 15 amino acid polypeptide (sequence: DALEQQEEGINRLTD, molecular weight: 1731), combined with carrier protein KLH (Keyhole LimpetHymocyanin), and then dialyzed Mix with Complete/Incomplete Freund's Adjuvant, immunize New Zealand rabbits (subcutaneous injection on the back), challenge and inject once every 3 to 4 weeks, and collect blood to separate serum after three months. Antibody titers in each immunized rabbit serum were determined by ELISA with pre-immunized animal serum as a control. Screen high-titer sera, and further use the antigen as a competitive inhibition immunoassay to determine the specificity of the antigen. According to the results of ELISA, the dilution doublings for immunohistochemical staining were determined to be 1:200-500.

Cytoskeleton-like gene-encoded protein-specific polyclonal antibody was used in western blot test: (1). Culture CHL/SMMC7723 cells in 1640 culture medium (containing 10% fetal bovine serum) in 60mm culture dishes to 100% coverage (usually 5-7 days), and then use/not use 1-3mM H ₂ O ₂ /10( -6) M retinoic acid/5-l0ng/ml 5-Azacytin/10ng/ml colchicine treated cells for 3-24 hours; (2). Wash the cells with PBS for 3 times, suck off the PBS, dissolve and collect the cells with 100ul of 2 times SDS, add 5% β-mercaptoethanol, heat at 95°C for 10 minutes, add bromophenol blue, gel with 14.5% polyacrylamide The protein was separated by gel electrophoresis (70V 30min), and then the protein was transferred to a nylon membrane. (3). The nylon membrane was first blocked with PBST containing 5% skimmed milk powder and 1/500 normal sheep serum (on a shaker for 30 min). Then a 1:200 primary antibody (rabbit anti-JWA IgG) was added for 1 hour at room temperature. (4). Wash five times with PBST for five minutes each. Then biotin-labeled goat anti-rabbit IgG (1:1000) was added for 1 hour at room temperature. (5). Wash five times with PBST for five minutes each. Add ABC reagent, room temperature for 30 minutes. (6). Wash five times with PBST for five minutes each. Color was developed with DAB. Compare the changes of JWA gene encoded protein in different groups of cells under different treatment conditions. In order to analyze the factors affecting the expression of JWA protein.

2. Cytoskeleton-like gene-encoded protein-specific monoclonal antibody takes 10 mg of synthetic JWA gene-encoded amino acid C-terminal 15 amino acid polypeptide (sequence: DALEQQEEGINRLTD, molecular weight: 1731), combined with carrier protein KLH (Keyhole LimpetHymocyanin), and then dialyzed Mix with Complete/Incomplete Freund's Adjuvant, immunize Balb/C mice (abdominal subcutaneous injection), challenge injection once a month, after four months of continuous immunization, take blood to separate serum, and take spleen to separate B The lymphocytes are then fused with myeloma cells to produce hybridoma cells. A hybridoma cell line secreting anti-JWA synthetic polypeptide IgG was obtained through multiple subcloning and screening with antigen (synthetic polypeptide) and IgG. Antibody titers were determined by ELISA.

Cytoskeleton-like gene-encoded protein-specific monoclonal antibody used in fluorescent immunohistochemical staining test of cultured cells: (1). Cultivate CHL/SMMC7723 cells in 1640 culture medium (containing 10% fetal bovine serum) to 100% coverage (usually 5-7 days) in a 35mm culture dish, and then use/not use 1-3mm of H ₂ O ₂ /10( -6) Treat the cells with M retinoic acid/5-10 ng/ml 5-Azacytin/10 ng/ml colchicine for 3-24 hours; fix the cells with methanol for 10 minutes. (2) Wash the cells 2-3 times with PBS, add 1:500 normal goat serum to block for 30 min. Then wash the cells with PBS for 2-3 times, add 1:1000 mouse anti-JWA gene-encoded protein monoclonal antibody, and incubate at 37°C for 1 hour. (3). Wash the cells 5 times with PBS, add 1:200 fluorescein-labeled goat anti-mouse IgG and incubate at 37°C for 1 hour. (4) The cells were washed 5 times with PBS, and the distribution characteristics of the JWA gene protein in the cells were observed with a fluorescence microscope after preparation.

Claims (3)

1.一种细胞骨架样基因(JWA)编码蛋白特异性抗体，其特征是JWA基因全部开放读框氨基酸序列为：MDVNIAPLRAWDDFFPGSDRFARPDFRDISKWNNRVVSNLLYYQTNYLVVAAMMISIV(1)GFLSPFNMILGGIVVVLVFTGFVWAAHN(2)KDVLRRMKKRYPTTFVMVVMLASYFLISMFGGVMVFVFGITFPL(3)LLMFIHASLRLRNLKNKLENKMEGIGLKRTPMGIVLDALEQQEEGINRLTDYISKVKE 2. Any polyclonal antibody prepared against the amino acid sequence of the JWA open reading frame according to claim 1 is characterized in that the polyclonal antibody can recognize the encoded protein corresponding to the amino acid sequence of the JWA open reading frame by using immunohistological techniques. 3. Any monoclonal antibody prepared against the amino acid sequence of the JWA open reading frame according to claim 1 is characterized in that the monoclonal antibody can recognize the encoded protein corresponding to the amino acid sequence of the JWA open reading frame by immunological techniques.