Preparation Of Polyclonal Antibody And Construction Of Expressing Stable Cell Line Of Porcine Circovirus Type 2 Cap Protein

Porcine circovirus 2(PCV2)is the main pathogen causing the postweaning multisystemic wasting syndrome.The main capsid protein Cap of the virus can be self-assembled into virus-like particles in vitro.The study showed that Cap protein interacted with the protein such as complement protein C1 q membrane receptor gC1 qR and Dynein protein light chain DYNLL1,which affected the virus replication and cell function.The previous study of Cap protein interaction protein is helpful in understanding the molecular mechanism of PCV2 infection.Therefore,it is necessary to study the interaction protein of Cap protein of PCV2.According to the PCV2 gene sequence in GenBank,the cap gene specific primers were designed to clone the gene.The prokaryotic expression plasmid pET-28a-Cap was constructed.The recombinant Cap protein expression was induced and purified to immunize New Zealand white rabbits to obtain the polyclonal antibody of Cap protein.A cap gene specific primer containing att B recombination site was designed.The cap gene fragment with attB1 and attB2 sequence was obtained by PCR.The eukaryotic stable expression plasmid pDEST-SFB-Cap was constructed by Gateway technique.pDEST-SFB-Cap was transfected into porcine kidney cells PK-15 with puromycin selection to obtain stable expression of recombinant Cap protein cell lines.The recombinant Cap protein was detected by CoImmunoprecipitation and Western blot.Indirect immunofluorescence assay showed that the recombinant Cap protein was distributed in the dot in some cells and form virus-like particles.Flow cytometry showed that Cap protein expression induced apoptosis of PK-15 cells.In this study,we successfully obtained the polyclonal antibody of Cap protein and established a stable expression cell line of expressing Cap protein carrying SFB tag.This study laid the foundation for the further study of Cap protein interaction protein by tandem affinity chromatography/mass spectrometry.