Construction And Screening Of Anti-PCV2 Cap Protein Phage Display ScFv Library

Porcine circovirus type 2(PCV2)is the main causative agent of porcine circovirus disease(PCVD),it damages the immune system of diseased piglets and caused mixed or secondary infection with other pathogenic,which resulted in a serious decline of pig reproductive capacity,and caused huge economic losses to the pig industry.Early diagnosis of PCV2 infection has a great signigicance to the comprehensive prevention and control of PCVD.At present,the main rapid detection methods of PCV2 pathogens are colloidal gold immunochromatographic test method and ELISA,the antibody used in those methods were mainly monoclonal antibody which have been prepared based on hybridoma cells.However traditional monoclonal antibody have shortcomings such as long production cycle,high cost and poor stability,which limit the rapid,efficient and inexpensive detection methods of PCV2.The single chain antibody(ScFv)prepared by genetic engineering has the advantages of short production cycle,low cost and simple operation,so it may be a potential candidates for the development of new immunological detectional methods of PCV2.The main contents and results of the experiment are as follows:1.Prokaryotic expression and identification of porcine circovirus type Ⅱ Cap proteinThe recombinant expression vector pET28a-ORF2 was transformed into E.coli BL21 and induced with IPTG,the recombinant protein was purified by Ni-NTA affinity chromatography and identified.The results showed that the recombinant protein was purified and had a molecular weight of about 26 kDa,which was soluble and could be recognized by His monoclonal antibody.BALB/C mice were immunized with recombinant protein,the murine antisear titers reached 1: 64000 by ELISA detection,the murine serum was also tested by PCV2 antibody PCV2 antibody test strips,the result was positive.The recombinant Cap protein was demonstrated to be of good solubility,high purity and good immunogenicity.Itprovides necessary experimental materials for the constructe of single chain antibody library of Cap protein.2.Construction and panning of ScFv phage display library against PCV2 Cap proteinTotal RNA was extracted from spleen of the BALB/C mice immunized with Cap protein.Complementary DNA fragments of variable heavy(VH)and variable light(VL)chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv with(G4S)3 by splice overlap extension PCR.The ScFv gene was inserted into the phagemid vector pCANTAB-5E and transformed into E.coli TG1 to obtain an antibody library,after infected by helper phage an ScFv phage display library of approximately 3.58 × 106 individual colonies was constructed.Three Cap protein-specific phage clones were isolated via phage display,in which ScFv-18 showed the highest specificity and reactivity to Cap protein.The crude extract of ScFv-P18 was identified by ELISA,and the result showed that it could be reacted with Cap protein.In this study,the soluble PCV2 Cap protein is expressed and purified,and the PCV2 Cap protein phage single chain antibody library is constructed.Furthermore,the specific single chain antibody with binding activity is isolated.The ScFv antibodies are expected to be used for the development of specific and rapid detection of PCV2.