Characterization Of Two Broad Neutralizing Mouse Monoclonal Antibodies (mAbs) Against PRRSV And Construction Of Mouse-Swine Chimeric Recombinant Antibody

Porcine reproductive and respiratory syndrome(Porcine reproductive and respiratory syndrome,PRRS)is an infectious disease induced by porcine reproductive and respiratory syndrome virus(Porcine reproductive and respiratory syndrome virus,PRRSV),which causes sow miscarriage,piglet respiratory disturbance and severe immunosuppressive response.The virus is widely spread in the global pig population,large-scale vaccine immunization has not been able to eliminate its epidemic,the disease has caused huge economic losses to the swine industry in the world and has become one of the major epidemic diseases in large-scale pig farms around the world.It is also a major problem in global pig disease control.In our previous study,we have obtained two mouse monoclonal antibodies against PRRSV.They have broadspectrum reactivity and can neutralize the infection of PRRSV.By using these two special monoclonal antibodies,this study intends to sequence the light chain and heavy chain of the antibodies and get coding genes in its hybridoma.Then the chimeric genetic engineering monoclonal antibody against the host source of PRRSV virus has developed by constant region substitution,then identified its biological function identified.The work and results of this study are as follows:1.Purification and reactivity determination of Mab-M and Mab-GP3 antibodies.Hybridoma cells secreting monoclonal antibodies against Mab-M and Mab-GP3 injected into the abdominal cavity of BALB / c mice by in vivo preparation method to prepare ascites,and high purity monoclonal antibodies purified from ascites by ammonium sulfate precipitation method and Protein G Resin chromatography.It verified by SDS-PAGE.Then the subtypes of antibodies were determined and using IFA to identify Marc-145 cells which infected with different strains of PRRSV-1 and type 2.It was proved that all infected cells could be recognized by the antibodies.The broad-spectrum recognition and reactivity of the two monoclonal antibodies to PRRSV were verified.2.Mab-M and Mab-GP3 have broad-spectrum neutralizing activity against PRRSV virus.PAM cells were incubated with two different concentrations of antibodies at 37 ℃ for 1h.Then infected with PAM cells for 1 h,then cultured in a new culture medium for 36 h,and then the lysates were collected for Western blot and q PCR tests.It proved that the two monoclonal antibodies could neutralize the virus and inhibit the proliferation of PRRSV in cells.At the same time,it detected that the virus titer in the corresponding cell culture supernatant.The gradient decrease of virus titer further explained the neutralization effect of the antibody on the virus.All cells which infected by PRRSV-1 and PRRSV-2 can be identified by Mab-M and Mab-GP3.It indicated that they have broad-spectrum neutralizing activity with PRRSV.3.Preparation of Chimeric Antibody against PRRSV.According to the two monoclonal antibodies Mab-M and Mab-GP3,the total RNA of hybridoma cells were extracted and the variable regions of heavy chain and light chain of mouse monoclonal antibody were amplified and sequenced.After that,Mab-GP3,was selected to synthesize the chimeric antibody by connecting the variable region of the heavy chain light chain of the antibody with the constant region of the heavy chain light chain of the pig antibody and cloning the chimeric antibody.It demonstrated that the synthesized chimeric antibody could still bind to Marc-145 cells infected with PRRSV by IFA test,so it verified that the chimeric antibody can still bind to Marc145 cells which infected with PRRSV.In conclusion,this study proved that Mab-M and Mab-GP3 had broad-spectrum neutralizing activity against PRRSV virus,could inhibit the replication of different genotypes of PRRSV virus,and sequenced the heavy chain light chain variable region of the two Mabs.The chimeric antibody Mab-GP3 was prepared.The results showed that the monoclonal antibody had broad-spectrum neutralizing activity against PRRSV,and the mouse-pig chimeric antibody was prepared,which provided a new direction for PRRSV antiviral therapy.