Prokaryotic Expression Of Porcine Circovirus Type 2 Cap Protein And Preparation Of Rabbit ScFv

Porcine circovirus type 2(PCV2)is a small,single-stranded circular DNA virus that causes immunosuppression in piglets,leading to various complications.The Cap protein of PCV2 is the only structural protein of the virus and has multiple epitopes that can induce neutralizing antibodies.Single-chain antibody(sc Fv)is an important material in the fields of immunological diagnosis and treatment.Screening out the single-chain antibody of PCV2can play an important role in the detection and diagnosis of the disease.In this study,Novopro protein structure prediction software was used to analyze the secondary structure and corresponding epitopes of PCV2-Cap protein(Gen Bank accession number:MF981845.1).Sequence,the 50-234 amino acid sequence of Cap protein was used as the target fragment.The Cap gene was amplified by PCR,and the recombinant plasmid p ET-28a-PCV2-Cap was constructed and expressed in prokaryotic cells.The soluble protein with a size of 37 k Da was successfully expressed.After immunizing rabbits with the successfully expressed protein,the antibody titer reached 8×10~4.RNA was extracted from the spleen of the immunized rabbit,and then the RNA was reverse transcribed to obtain the c DNA sequence.The antibody heavy chain VH and light chain VL genes were amplified by PCR,and then the VH and VL were spliced into the sc Fv gene using SOE-PCR.The sc Fv gene fragment was ligated with the phage vector p Comb3XSS,electroporated into ER2738 competent cells,and then infected with M13K07helper phage to construct a phage display library.Then the specific single-chain antibody was panned with PCV2-Cap as the target protein,and finally the sc Fv was successfully panned.The successfully panned sc Fv was cloned into the eukaryotic vector p TT5-Fc for eukaryotic expression,and the recombinant single-chain antibody PCV2-sc Fv was obtained.The EC50 of the recombinant antibody was determined to be 10.68 ng/m L.Through phage display technology,the corresponding sc Fv antibodies of PCV2 were screened out,which provided a certain possibility for the establishment of a rapid,accurate and timely detection method,and also laid a foundation for the immunological detection and diagnosis of the virus.