Preparation And Characterization Of Rabbit Monoclonal Antibody To H4K8 Crotonylation

Background : Crotonoylation is one of the highly conserved post-translation modfication in progess.Specifically,histone kcr is main concentration in human somatic cells,gene transcription activity of the promoter region and inhancr in male mice.However,it's still very few research about histone kcr mechanism and fuction at present due to limitation of catalytic of crotonlation.Compared with mouse monoclonal antibodies,rabbit monoclonal antibodies(RabMAbs)had the advantages of higher binding affinity and specificity,and was easier to be humanized.It's inportmant to develop PTM antibody which is further reveal new epigentec markers in tumorigenesis,development and drug treatment.And it's more suitable to develop PTM antibody fot RABMAB's high affinity and strong spcifity.There's an urgent need to develop H4K8 cr rabbit monoclonal antibody,in order to speed up its biological function of exploration research.Objective:Using the rabbit monoclonal hybridoma technology and Recombinant Antibody technology to develop anti-H4K8 cr RABMAB which are applied to Western Blot,immunohistochemistry and immunocytochemistry.Methods: In our experiment,we synthesized two peptides of H4K8 CR,mix 2 peptide and immunize four experimental rabbits for each two peptide.After preliminary screening by serum titer and blot experiment,the splenocytes were fusioned with rabbit myeloma-like cell line of 240E-W2(Epitomics Inc.,USA),then we acquired rabbit-rabbit hybridoma cells which secreted H4K8 CR antibodies.The fusioned cells were maintained in HAT selection medium.To acquire positive clones,ELISA? WB?Peptide array ? ICC assays were used for screening.The one with the strongest signal of WB and specific Peptide array was chosen to plasmid recombinant phase.After further screening,the best plasmid pair was used for large-scale transfection(production).Results : In this expriment,we get 114 ELISA positive clones at primrary confirm stage,20 Western Blot positive clones,and only 1 clone show no reaction with acetylation in the same modified site.So we choose this clone to IgG cloning,and finally we get 2 mL,IgG concentration is 1.562 mg/ml antibody.Western Blot result show 11 KDa band in Hela?3T3?C6 and PC-12 cell lysates.IHC and ICC are all show nucleus subcellular location.Peptide array show no cross reaction with all crotonylation and actylation modified site about H2A?H2B ?H3 and H4 which are all find in current research.Conclusion : In this experiment,we get H4k8 cr rabbit monoclone antibody by hybridoma and recombinant technology.It can be used in ELISA?WB?IHC?ICC?Peptide array immunoassay,and it's also can be react with mouse and rat in Western blot,it has potential application prospect for it is suitable for scientific research,fill in the market without H4K8 cr monoclonal antibody shortcomings,it can promote the crotonylation research.