Construction Of Prokaryotic Expression Plasmid Of Porcine Circovirus Type 2 Cap Protein Carrying Molecular Markers

Porcine circovirus type 2(PCV2)belong to the Circovirus family of Circovirus and is a non-enveloped single-stranded circular negative-stranded DNA virus.The mixed infection of PCV2 and other pathogens is a great threat to pig farming in China.Vaccine immunity is a routine method for the prevention and control of PCV2.The PCV2 vaccine on the market is mainly inactivated vaccine,which plays an important role in the prevention and control of PCV2.However,there are some defects in these kinds of vaccines,such as the inability to induce the host to produce cellular immunity,and the antibodies can not be distinguished from the wild-types,and they are expensive.Therefore,it is important to study the subunit vaccine of the PCV2,because subunit vaccines are not only safe,efficient and inexpensive,but also the antibodies produced by the subunit vaccine and the antibody produced by the wild poison attack can be can be distinguished by the corresponding ELISA kit.In this study,prokaryotic expression system was used to express PCV2 Cap protein with V5 tag.Genetic engineering technology was used to introduce V5 epitope tags to the ends of PCV2 ORF2 gene and construct corresponding prokaryotic expression vectors.1.Construction of tagged pET32a(+)-PCV2-ORF2m-V5 recombinant plasmid.In this experiment,the online software was used to compare the ORF2 of the PCV2 GZ-RH1 strain with the codon of Escherichia coli.The results showed that the frequency of the rare codons was higher when the ORF2 of the PCV2 GZ-RH1 strain was selected for the expression of the Escherichia coli expression system,and the optimization of the codon could improve the expression efficiency.Accordingly,the PCV2 ORF2m-V5 gene was synthesized by introducing V5 tags at 3 ends and replacing some rare codons.The synthetic gene was cloned by T and was correctly identified by enzyme digestion and plasmid PCR.The positive plasmid of T clone was extracted by double enzyme,and the gene was subcloned into pET32a(+)prokaryotic expression vector,pET32a(+)-PCV2-ORF2m-V5 Recombinant Prokaryotic expression plasmid was successfully constructed.2.Prokaryotic expression of PCV2 Cap-V5 protein carrying molecular markersIn this study,we constructed the pET32a(+)-PCV2-ORF2m-V5 plasmid and transformed it into E.coli BL21(DE3).Positive colonies after transformation by Amp screening for proliferation culture,and 1 mmol/L IPTG was used as an inducer to induce the expression of the bacteria solution at 37? and 16? for 3h.Then,the transformed strains not induced by IPTG and the induced by IPTG transformants were processed,and analyzed by SDS-PAGE and Western-blotting.The results showed that the size of the recombinant marker protein was consistent with the expected result,which was approximately 47.6kDa,and was mainly expressed in inclusion bodies.Both His and V5 monoclonal antibodies can bind to Cap-V5 protein and have good reaction properties;under the same conditions,the expression of Cap-V5 protein was induced at 37°C higher than 16°C.This study lays an experimental foundation for the pre-study of PCV2 subunit marker vaccines and the establishment of differential vaccine antibodies and wild-type antibody ELISA methods.