CN1208733A - Coded protein specific antibody having cytoskeleton gene - Google Patents

Abstract

The present invention relates to human genome technology, said cell skeleton gene (JWA) whole length CDNA sequence is 2107 bp, its open-read frame sequence is 564 bp, altogether coding 188 amino acids. The GenBank number of said gene is "Bank It200994, Accession Number; AF070523". In the invention we use part of the sequence of the 188 amino acids to synthesize polypeptide immune animal to prepare polyclone and monoclone antibody.

Description

The coded protein specific antibody of cytoskeleton like gene

The invention belongs to the Human genome technical group field.

Cytoskeleton like gene of the present invention (JWA) is that contriver (1996.3～1998.3) during the Davis of California, USA university (UCDavis) visit research uses DD-PCR, the phage selection method, the brand-new gene relevant that methods such as RACE-PCR are cloned from the cDNA library that people's tracheae bronchial epithelial cell makes up with cytoskeleton.This full length gene cDNA sequence is 2107bp, and its open reading frame sequence is 564bp, 188 amino acid of encoding altogether.Further studies show that in the JWA gene open reading frame sequence and contain three film transit zones, 23 amino acid are contained in each district, and protein kinase C (PKC) phosphorylation site is arranged at the two ends in three film transit zones.The Northern hybridization analysis shows that this gene has two mRNA transcripts that vary in size, through reverse transcription, 3 '-RACE-PCR, the definite final position of two transcripts has been determined in research such as Southern hybridization analysis and Rnase Protection Assay.Show that with the immunofluorescence dyeing result behind expression vector (PCMV-2-Flag-JWA) transfection S-6 that carries JWA gene open reading frame sequence and the HBE cell albumen of JWA genes encoding exactly likes the cytoskeleton microtubule.This albumen is gone the influence of polymerizing agent colchicine and nocodazole significantly but is not subjected to the influence of deepfreeze.In addition, the expression of this gene in several tumour cells is subjected to the influence of demethylation medicine 5-azacytidin significantly.

Because the JWA gene order still do not have homologous genes in the GenBank database, so the contriver all is initiative for the 26S Proteasome Structure and Function result of study of JWA gene.(National Center for Biotechnology Information NCBI) has formally accepted the newcomer of JWA gene as GenBank in recently in American National biotechnology information center.This gene is numbered that " BankIt 200994, Accession Number:AF070523 ＂.GAATTCGGCACGAGCCGAGACAGAGCCGCTGTCAACTCTCCAACTCAGCTCAGCTGATCGGTTGCCGCCGCCGCCGCCGCCAGATTCTGGAGGCGAAGAACGCAAAGCTGAGAACATGGACGTTAATATCGCCCCACTCCGCGCCTGGGACGATTTCTTCCCGGGTTCCGATCGCTTTGCCCGGCCGGACTTCAGGGACATTTCCAAATGGAACAACCGCGTAGTGAGCAACCTGCTCTATTACCAGACCAACTACCTGGTGGTGGCTGCCATGATGATTTCCATTGTGGGGTTTCTGAGTCCCTTCAACATGATCCTGGGAGGAATCGTGGTGGTGCTGGTGTTCACAGGGTTTGTGTGGGCAGCCCACAATAAAGACGTCCTTCGCCGGATGAAGAAGCGCTACCCCACGACGTTCGTTATGGTGGTCATGTTGGCGAGCTATTTCCTTATCTCCATGTTTGGAGGAGTCATGGTCTTTGTGTTTGGCATTACTTTTCCTTTGCTGTTGATGTTTATCCATGCATCGTTGAGACTTCGGAACCTCAAGAACAAACTGGAGAATAAAATGGAAGGAATAGGTTTGAAGAGGACACCGATGGGCATTGTCCTGGATGCCCTAGAACAGCAGGAAGAAGGCATCAACAGACTCACTGACTATATCAGCAAAGTGAAGGAATAAACATAACTTACCTGAGCTAGGGTTGCAGCAGAAATTGAGTTGCAGCTTGCCCTTGTCCAGACCTATGTTCTGCTTGCGTTTTTGAAACAGGAGGTGCACGTACCACCCAATTATCTATGGCAGCATGCATGTATAGGCCGAACTATTATCAGCTCTGATGTTTCAGAGAGAAGACCTCAGAAACCGAAAGAAAACCACCACCCTCCTATTGTGTCTGAAGTTTCACGTGTGTTTATGAAATCTAATGGGAAATGGATCACACGATTTCTTTAAGGGAATTAAAAAAAATAAAAGAATTACGGCTTTTACAGCAACAATACGATTATCTTATAGGAAAAAAAAAAATCATTGTAAAGTATCAAGACAATACGAGTAAATGAAAAGGCTGTTAAAGTAGATGACATCATGTGTTAGCCTGTTCCTAAATCCCTAGAATTGTAATGTGTGGGATATAAATTAGTTTTTATTATTCTCTTAAAAATCAAAGATGATCTCTATCACTTTGCCACCTGTTTGATGTGCAGTGGAAACTGGTTAAGCCAGTTGTTCATACTTCCTTTACAAATATAAAGATAGCTGTTTAGGATATTTTGTTACATTTTTGTAAATTTTTGAAATGCTAGTAATGTGTTTTCACCAGCAAGTATTTGTTGCAAACTTAATGTCATTTTCCTTAAGATGGTTACAGCTATGTAACCTGTATTATTCTGGACGGACTTATTAAAATACAAACAGACAAAAAATAAAACAAAACTTGAGTTCTATTTACCTTGCACATTTTTTGTTGTTACAGTGAAAAAAATGGTCCAAGAAAATGTTTGCCATTTTTGCATTGTTTCGTTTTTAACTGGAACATTTAGAAAGAAGGAAATGAATGTGCATTTTATTAATTCCTTAGGGGCACAAGGAGGACAATAATAGCTGATCTTTTGAAATTTGAAAAACGTCTTTAGATGACCAAGCAAAAAGACTTTAAAAAATGGTAATGAAAATGGAATGCAGCTACTGCAGCTAATAAAAAATTTTAGATAGCAATTGTTACAACCATATGCCTTTATAGCTAGACATTAGAATTATGATAGCATGAGTTTATACATTCTATTATTTTTCCTCCCTTTCTCATGTTTTTATAAATAGGTAATAAAAAATGTTTTGCCTGCCAATTGAATGATTTCGTAGCTGAAGTAGAAACATTTAGGTTTCTGTAGCATTAAATTGTGAAGACAACTGGAGTGGTACTTACTGAAGAAACTCTCTGTATGTCCTAGAATAAGAAGCAATGATGTGCTGCTTCTGATTTTTCTTGCATTTTAAATTCTCAGCCAACCTACAGCCATGATCTTTAGCACAGTGATATCACCATGACTTCACAGACATGGTCTAGAATCTGTACCCTTACCCACATATGAAGAATAAAATTGATTAAAGGTTAAAAAAAAAAAAAAAAAAAAAAACTCGAG

JWA gene cDNA sequence MDVNIAPLRAWDDFFPGSDRFARPDFRDISKWNNRVVSNLLYYQTNYLVV 50AAMMISIV (1) GFLSPFNMILGGIVVVLVFTGFVWAAHN (2) KDVLRRMKKRYPTT 100FVMVVMLASYFLISMFGGVMVFVFGITFPL (3) LLMFIHASLRLRNLKNKLEN 150KMEGIGLKRTPMGIVLDALEQQEEGINRLTDYISKVKE 188

In the JWA gene open reading frame aminoacid sequence sequence (1), (2), (3) are three film transit zones, and SDR and SLR are the PKC phosphorylation site

The objective of the invention is: in view of the JWA gene is the brand-new gene that the inventor finds, human at present understanding and the understanding to this gene only limits to the scientific research that the inventor does.The inventor uses NorthernBlot, Western Blot, and methods such as gene transfection and immunohistochemistry's dyeing studies confirm that a kind of new cytoskeletal protein of JWA genetic expression.This gene extensively is present in multiple tissue and the cell of being studied, and differentiation agent vitamin A acid (Retinoid acids), oxidants hydrogen peroxide (H are induced in the expression of its mRNA ₂O ₂), the obvious influence of demethylation medicine (5-Azacytidin) etc.: the influence of the influence of polymerizing agent colchicine (Colchicine) and Nocodazole and heat-shocked processing etc. is gone in the expression of this gene coded protein significantly.The evolution conservative of JWA gene is expressed active feature and is pointed out this gene to have very active biological function.Especially it is as a kind of new cytoskeletal protein, and the propagation of pair cell and differentiation all have important effect.Further research JWA gene coded protein helps the understanding to this biology of gene function in intracellular definite location.The gene coded protein specific antibody then is the indispensable important tool of research gene structure and function.JWA gene coded protein polyclone and mono-clonal specific antibody can be used as immunohistochemical staining, ELISA, immuno-electron microscope, multiple detection method according to the immunology principle design such as immunoprecipitation.Can be used for location and the quantitative examination of this gene coded protein in histocyte, content research in various biological specimens (as blood, urine etc.) and protein-protein interaction research etc.

Technical scheme of the present invention is: the whole open reading frame aminoacid sequences of JWA gene (totally 188 amino acid, see that the application is listed), 15 amino acid of C-terminal that particularly contain the hydrophilic amino acid district, 30 amino acid of N-terminal, the 80th～100 amino acid and the 135th～155 aminoacid sequence.Select the synthetic polypeptide of partial sequence in the above-mentioned sequence, after the KLH carrier proteins combines, mix with complete and Freund, immune new zealand rabbit (the 200ug/ rabbit/time, booster injection is once weekly), preparation polyclonal antibody (anti-JWA IgG); Immunity Balb C mouse (the 100ug/ mouse/time, booster injection in every month once), the separating spleen bone-marrow-derived lymphocyte and and the myeloma cell merge the generation hybridoma, further use the subclone method, the mono-clonal of ELISA method and antibody typing method screening IgG secretion antibody, preparation monoclonal antibody (anti-JWA IgG).

The embodiment of the invention: 1. cytoskeleton like gene coded protein specific polyclonal antibody is got 15 amino acid polypeptides of synthetic JWA gene coding amino acid C-terminal (sequence is: DALEQQEEGINRLTD, molecular weight: 1731) 10mg, combine with carrier proteins KLH (Keyhole LimpetHymocyanin), after dialysis again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) mixes, immunity new zealand rabbit (back subcutaneous injection), every 3～4 all booster injections once, after three months, get blood system from serum.Be ELISA in contrast with animal serum before the immunity and measure antibody titers in each immunize rabbit serum.Screen high titre serum, further make competition inhibition immunity test and determine antigenic specificity with antigen.Determine that according to ELISA result its dilution that is used for immunohistochemical staining multiplication number is 1: 200～500.Cytoskeleton like gene coded protein specific polyclonal antibody is used for westem blot test: (1)., use then/H that need not 1～3mM in 60mm culture dish to 100% fraction of coverage (common 5～7 days) with RPMI-1640 (containing 10% foetal calf serum) cultivation CHL/SMMC7723 cell ₂O ₂The colchicine of the 5-Azacytin/10ng/ml of vitamin A acid/5～10ng/ml of/10 (6) M handled cell 3～24 hours; (2). wash cell 3 times with PBS, PBS is removed in suction, SDS dissolving and collecting cell with 2 times of 100ul, the β mercaptoethanol of adding 5%, 95 ℃ were heated 10 minutes, add after the tetrabromophenol sulfonphthalein polyacrylamide gel electrophoresis protein isolate (70V 30min), then albumen is transferred on the nylon membrane with 14.5%.(3). seal nylon membrane (putting 30min on the shaking table) earlier with the PBST that contains 5% skim-milk and 1/500 normal sheep serum.One anti-(the anti-JWA IgG of rabbit) that adds 1: 200 then, room temperature 1 hour.(4). with PBST washing five times, each five minutes.Add biotin labeled goat anti-rabbit igg (1: 1000) then, room temperature 1 hour.(5). with PBST washing five times, each five minutes.Add ABC reagent, room temperature 30 minutes.(6). with PBST washing five times, each five minutes.Develop the color with DAB.The variation of cell JWA gene coded protein under the different treatment condition more on the same group.Thereby analyzing influence JWA protein expression factor.2. cytoskeleton like gene coded protein specific monoclonal antibody is got 15 amino acid polypeptides of synthetic JWA gene coding amino acid C-terminal (sequence is: DALEQQEEGINRLTD, molecular weight: 1731) 10mg, combine with carrier proteins KLH (Keyhole LimpetHymocyanin), after dialysis again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) mixes, immunity Balb/C mouse (abdominal part hypodermic), booster injection in every month once, continuous immunity is got blood system from serum after four months, get spleen and separate bone-marrow-derived lymphocyte, merge the generation hybridoma with the myeloma cell then.Through subclone repeatedly with obtain the hybridoma cell strain of the synthetic polypeptide IgG of the anti-JWA of secretion with antigen (synthetic polypeptide) and IgG screening.Determine the titre of antibody with ELISA.

Cytoskeleton like gene coded protein specific monoclonal antibody is used for immunohistochemistry's stain test of culturing cell: (1). and cultivate the CHL/SMMC7723 cell in 35mm culture dish to 100% fraction of coverage (common 5～7 days) with RPMI-1640 (containing 10% foetal calf serum), use then/H that need not 1～3mm ₂O ₂The colchicine of the 5-Azacytin/10ng/ml of vitamin A acid/5～10ng/ml of/10 (6) M handled cell 3～24 hours; With methyl alcohol fixed cell 10min.(2) use PBS washed cell 2～3 times, add 1: 500 normal sheep serum sealing 30min.Again with the mouse-anti JWA gene coded protein monoclonal antibody that adds 1: 1000 behind the PBS washed cell 2～3 times, 37 ℃ of incubations 1 hour.(3). with PBS washed cell 5 times, add 37 ℃ of incubations of fluorescein-labeled sheep anti-mouse igg 1 hour of 1: 200.(4) use PBS washed cell 5 times, use fluorescence microscope JWA gene protein after the film-making in intracellular distribution characteristics.

Claims (3)

1, a kind of cytoskeleton like gene (JWA) coded protein specific antibody, it is characterized in that referring to the whole open reading frame amino acid series of JWA gene (totally 188 amino acid, see that the application is listed), 15 amino acid of C-terminal that particularly contain the hydrophilic amino acid district, 30 amino acid of N-terminal, the 80th～100 amino acid and the 135th～155 aminoacid sequence.

2, aminoacid sequence according to claim 1, it is characterized in that selecting the synthetic polypeptide of partial sequence in 188 amino acid, after the KLH carrier proteins combines, mix with complete and Freund, the immunity new zealand rabbit (the 200ug/ rabbit/time, booster injection is once weekly), preparation polyclonal antibody (anti-JWA IgG).

3, aminoacid sequence according to claim 1, it is characterized in that selecting the synthetic polypeptide of partial sequence in 188 amino acid, after the KLH carrier proteins combines, mix with complete and Freund, immunity Balb C mouse (the 100ug/ mouse/time, booster injection in every month once), the separating spleen bone-marrow-derived lymphocyte and and the myeloma cell merge the generation hybridoma, further use the subclone method, the mono-clonal of ELISA method and antibody typing method screening IgG secretion antibody, preparation monoclonal antibody (anti-JWA IgG).