Development Of PRRSV Specific Monoclonal Antibody Based On Single B Cell Antibody Technique

Porcine reproductive and respiratory syndrome(PRRS)is a highly contact infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).The clinical symptoms are mainly characterized by reproductive disorders of sows,respiratory symptoms of piglets and high mortality of suckling piglets.The persistent and secondary infection due to PRRSV infection will lead to a decline in herd performance,which causes great economic loss to pig farming and has become one of the important diseases affecting the healthy development of the pig industry.PRRSV is easy to mutate,and there are many epidemic strains.In addition,the use of commercially available attenuated vaccines will also lead to the occurrence of the recombination of epidemic virus and vaccine virus.All these factors make PRRS prevention and control extremely difficult.The structure of PRRSV antigens that induce protective immune responses is still not very clear.It is necessary to develop monoclonal antibodies to different structural proteins of PRRSV,analyze the antigen structure of PRRSV,and find the epitopes that induce neutralizing antibodies or even broadly neutralizing antibody,so as to provide new theoretical basis for the development of PRRSV prevention and control products.In this study,firstly,the pigs were sequentially immunized with commercially available vaccines and lab-made prevalent strain vaccines,and to induce the production of higher levels of neutralizing antibodies.Simultaneously,purify the whole virus antigen and label the virus particles with biotin;collect venous blood of 0922 # pig after the eighth immunization,EDTA anticoagulated blood sample was collected to isolate peripheral blood mononuclear cells(PBMCs),PRRSV-specific single B cells were sorted from PBMCs by flow cytometry using biotinylated PRRSV antigen as a bait and then were lysed.The pig IgG antibody heavy chain and light chain variable(VH and VL)gene sequences were amplified-independently by a nested PCR method using primers for porcine IgG.Then,the gene sequences were inserted into pcDNA3.4 eukaryotic vectors containing the constant region of porcine IgG antibody to construct complete antibody expression plasmids.The IgG antibody expression plasmids were transfected into Chinese hamster ovary suspension cells(CHO-S)for small amount of antibody expression,After that,the indirect immunofluorescence test(IFA)was used to verify the specificity of the small amount of expressed antibodies to recognize PRRSV,and the specific reaction antibodies were expressed and purified in large quantities.The antibodies were tested for biological activity and functionality by IFA,virus neutralization test(VNT),and Western blot(WB).The results showed that PRRSV virions were successfully purified by sucrose density gradient centrifugation and the virions were elliptic with a diameter of 50-80 nm by electron microscope,the highest levels of neutralizing antibodies against the attenuated vaccine VR2332,epidemic strains GSWW/15(HP-PRRSV-like strain)and GSWW/18(NADC30-like strian)after repeated immunization with different commercially available vaccines or lab-made virus vaccines could reach 1:45,1: 256 and 1:45,respectively.The results of IFA、VNT and WB showed that a total of 9 monoclonal antibodies were identified as PRRSV-specific non-neutralizing antibodies.These antibodies could only bind to the nucleoprotein(N Protein)of PRRSV and have good reactivity in both IFA and WB tests.In summary,9 strains of PRRSV-specific monoclonal antibodies were obtained based on single B-cell antibody technology,all of which mainly recognized the PRRSV N protein,indicating that N protein has a strong immune superiority.This study has accumulated experience for screening PRRSV neutralizing monoclonal antibodies,and also provided an antibody tool for the analysis of antigen epitopes in N protein.