Preparation Of Monoclonal Antibodies Against Porcine Reproductive And Respiratory Syndrome Virus And Establishment Of Indirect ELISA Method

Objective: PRRSV NSP7 protein was expressed by Prokaryotic expression and indirect ELISA method was established which can be used to monitor wild-ear virus infection in pigs without vaccination.Monoclonal antibodies against five highly pathogenic PRRSV and traditional PRRSV(CH-1a)were prepared based on surface fluorescence method,which provides a material basis for the establishment of serological methods to differentiate high pathogenic PRRSV from traditional PRRSV(CH-1a).Methods: 1?The expression,purification and immunological activity of NSP7 protein were identified by means of gene engineering,protein purification and ELISA.The optimal encapsulation quantity,encapsulation conditions,serum dilution multiple and incubation time of NSP7 protein was detect by indirect ELISA.The established indirect ELISA method was used to detect actual samples and the coincidence rate was calculated with IDEXX kit.2?Hybridoma cell lines secreting stable antibodies were prepared by immunization of mice,cell fusion and hybridoma cell surface fluorescence screening.Cell lines capable of differentiating highly pathogenic PRRSV(TP,JN,QJ,LQ,JN)from traditional PRRSV(CH-1a)were selected.The purity,specificity,subtype and titer of the obtained antibody were analyzed and identified by SDS-PAGE and indirect ELISA.3?Molecular biological techniques were used to extract and sequence the variable region genes of light and heavy chain of monoclonal antibody 4D5;Discovery studio 4.5 Software was used to modeling and docking of Antibody 4D5;Indirect ELISA was used to identify the reaction between 4D5 and N protein.Results: 1?After codon optimization,NSP7 protein can be expressed in soluble form;The optimal expression temperature of NSP7 protein is 34 C,the optimal induction concentration of IPTG is 0.8 mmol/L,and the optimal induction time is 7 h;The purified NSP7 protein has immunological activity;2?An indirect ELISA method based on NSP7 protein was successfully established;Compared with IDEXX kit,the coincidence rate was 88.3%.3?A total of 36 monoclonal antibody cell lines against PRRSV were screened,and 10 of them secreted antibody with good binding ability.;4D5 can distinguish five highly pathogenic PRRSV viruses(TP,QJ,LQ,TY,JN)from one traditional PRRSV(CH-1a).4?The light and heavy chain variable region gene of antibody 4D5 was successfully obtained;The antibody model of 4D5 and the molecular docking model with N protein were established;Indirect ELISA proved that there was a positive reaction between 4D5 and N protein.Conclusion:An indirect ELISA method based on NSP7 protein was established,which could use to monitor wild virus infection in pigs without vaccination;A monoclonal antibody can differentiate five highly pathogenic PRRSV(TP,QJ,LQ,TY,JN)from one traditional PRRSV(CH-1a)was obtained.It provides a material basis for the serological detection of highly pathogenic PRRSV(TP,QJ,LQ,TY,JN)and traditional PRRSV(CH-1a).