Prokaryotic Expression And Purification Of ASFV CP204L Gene And Its Mabs Preparation

African swine fever(ASF)is a major new animal epidemic with short onset time and high mortality in China in recent years.There is no effective vaccine for the prevention and control of ASF,which seriously threatens the development of the global pig breeding industry.Therefore,the development of detection methods and reagents with strong specificity,high sensitivity,and excellent safety is of great significance to the early diagnosis and monitoring of the virus.In this study,the p ET-28a-CP204 L prokaryotic expression vector and pc DNA3.1-CP204 L eukaryotic expression vector were constructed thro?gh the bioinformatics analysis of the CP204 L gene.Prokaryotic expression and purification of p30 protein and preparation of its monoclonal antibody.The results showed that the prokaryotic expression vector p ET-28a-CP20 L successfully expressed the p30 fusion protein containing 6×His tag.SDS-PAGE showed that a single purpose band was obtained,which could be combined with His tag antibody.The purified p30 protein was immunized to mice,and the antibody titer reached 1:102,400.The prepared hybridoma cells were screened by ELISA method,and finally 5 positive cell lines stably secreting p30 protein antibody were obtained,and the monoclonal antibody subclass was identified as Ig G1 and Ig G2 a.The results of Western Blotting showed that the selected monoclonal antibody can specifically bind to the p30 protein.The results of IFA showed that the selected monoclonal antibody binds to the p30 protein expressed by the eukaryotic plasmid pc DNA3.1-CP204 L,and can bind to the ASF whole virus antigen,and has good reactogenicity.