Cloning And Expression Of Porcine Antibody Gene Against Glycoprotein 5 Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS)has been one of the most economically significant swine diseases worldwide in the past 20 years.It is typically manifested by reproductive failures(e.g.late-term abortion,stillbirth and mummification)in sows and respiratory distress(e.g.interstitial pneumonia)in growing pigs.Despite billions of dollars spent over two decades on prevention and control of PRRS,it doesn't eliminate the prevalence and spread of PRRSV all over the world.Traditional methods for rapid diagnosis of PRRSV antigens or antibodies include reverse transcription-polymerase chain reaction(RT-PCR)and Enzymes linked immunosorbent assay(ELISA)and so on.Considering the false positive results caused by high sensitivity of RT-PCR and high cost of commercial ELISA kits,it is imperative to develop a highly specific and low-priced diagnostic method for PRRS.In recent years,with the development of recombinant DNA technique and protein engineering,a vast range of recombinant antibody-based reagents have been created for diagnosis of diseases.Due to its short producing time,low cost,easy transformation and specific binding to pathogen,recombinant antibody shows promising prospect in the field of identification and diagnosis of animal diseases(e.g.PRRS).To express prokaryotically the porcine recombinant antibody(PrAb)against PRRSV,the B cells from pigs immunized with PRRSV vaccine strain were sorted and isolated by Flow Cytometry with the utilization of conserved epitope of PRRSV and rabbit anti-pig IgG.Then,the encoding sequences of variable regions of heavy(VH)and light chains(VL)of pig antibody were amplified from positive B cells by PCR.The encoding sequences of constant regions of heavy(CH)and light chains(CL)of pig antibody were connected separately with the encoding sequences of VH and VL.To construct the whole gene sequence of H-Linker-L,a Linker was used in SOE-PCR.Then the recombinant H-Linker-L gene was cloned into pET-28 a vector to express protein in E.coli BL21.The Western blot result showed that the expressed protein was actually porcine recombinant antibody,and the ELISA result demonstrated that PrAb possessed specific binding activity to PRRSV.In this study,we establish a method to screen target B cells with utilization of conserved epitope and specific antibody binding surface antigen on B cells.After amplification of encoding sequence of VH and VL,we successfully constructed recombinant plasmid,pET-H-linker-L,and successfully expressed PrAb when transformed it into E.coli BL21.Experiment in vitro showed that PrAb can specifically recognize PRRSV.These results lay a foundation for further study on PrAb to diagnose PRRS.