Preparation Of Specific Monoclonal Antibody Against PCV2 Cap Protein Based On Single B Cell Antibody Technology

Porcine circovirus(PCV)has four genotypes(PCV1,PCV2,PCV3,PCV4),of which PCV2 is the main pathogen of weaned piglets with multiple system failure syndrome(PMWS).After infecting animals,PCV2 will inhibit the immune system of the body,cause reproductive disorders of pigs,and easily cause mixed infection of bacteria and viruses,causing great harm to the breeding industry.At present,there is no effective treatment.Therefore,it is necessary to develop monoclonal antibodies against different structural proteins of PCV2 and analyze their antigen structures to find out the antigen epitope information that can induce specific and even neutralizing responses,so as to provide a theoretical basis for the development of PCV2 prevention and control products.In order to obtain a specific monoclonal antibody against the capsid protein of circovirus type 2(PCV2 Cap)protein,this study used a commercial inactivated PCV2 vaccine to immunize pigs and induce them to produce a high level of specific antibody.At the same time,the plasmid containing PCV2-ORF2 gene stored in the laboratory was expressed and purified by BL21(DE3)prokaryotic method to obtain high-purity Cap protein,and denatured Cap protein was refolded by urea gradient method.After coating the refolded Cap protein,the content of specific antibody against Cap protein in immunized animals was detected by indirect ELISA.The results showed that Cap protein was successfully obtained by prokaryotic expression system,and the target band was about 25 k Da,which was consistent with the expected results.The indirect ELISA results showed that the specific antibody of Cap protein in the sera of immunized animals increased with time and immunization times.It provides a preliminary basis for the subsequent screening of PCV2 Cap protein specific antibodies.In order to obtain specific monoclonal antibody against PCV2 Cap protein by single B cell technology,biotin labeled Cap protein was used as capture antigen to collect anticoagulation of # 1516 pigs after the fourth immunization and separate their PBMCs.Flow cytometry was used to separate PCV2 Cap protein specific antibody secreting single B cells from PBMCs.The gene sequences of heavy chain variable region(VH)and light chain variable region(VL)of pig IgG antibody were amplified by nested PCR.Insert the paired IgG variable region genes(VH,VL)into the eukaryotic expression vector containing pig IgG antibody constant region,construct the complete antibody expression plasmid,and co transfect the antibody heavy chain and light chain plasmids into mammalian cells for expression and purification.The results showed that Cap protein was successfully labeled,and a single B cell secreting PCV2 Cap protein specific antibody was obtained by flow cytometry.Nine pairs of naturally paired VH and VL were amplified by nested PCR,and nine monoclonal antibodies were obtained by transfection with CHO-S.In order to verify the biological activity of the selected monoclonal antibody,enzyme linked immunosorbent assay(ELISA),Western blot(WB),indirect immunofluorescence assay(IFA)and virus neutralization test(VNT)were used to verify it.The results of indirect ELISA and WB experiments showed that 8 of the 9 monoclonal antibodies were specific for prokaryotic expression of PCV2 Cap protein,IFA results showed that 6 m Abs were specific for eukaryotic expression of PCV2 Cap protein,1 m Abs was specific for PCV2 d strain,3 m Abs(17B,21 B,23B)bound to linear epitopes,and 1 m Ab(2A)bound to conformational epitopes without neutralizing activity.To sum up,eight of the monoclonal antibodies screened by single B cell technology in this study recognize PCV2 Cap protein.This study provides an antibody tool for analyzing the antigen epitopes in PCV2 Cap protein and preparing detection kits,which is of great significance.