A Preliminary Study On The New Vaccine Of Porcine Circovirus By Gene Regulation

According to the published genomic sequence of porcine circovirus type2, twopairs of specific primers were designed and synthesized.One pair of primers were designed to amplify the complete genome of porcinecircovirus type2,the complete genome was cloned into pET30a.The sequencingresult showed the genome of porcine circovirus type2was1767(bp).This sequencewas designated as sh0901(GenBank accession no. GU124593.1).The preferencecodons on the sequence of the porcine circovirus type2were changed into nonpreference codons with the rare codon. The nucleotides sequence encoding amino acidsof PCV2genomic from685bp to843bp were changed into the non rare codon, andnamed pET30a-PCV2-C.The pET30a-PCV2and the pET30a-PCV2-C weredigested with the EcoRâ… and purified (1767bp),respectively the circular PCV2genomic DNA was generated by self-ligating with T4DNA ligase. Then, theproducts were transfected into PK-15cells by using LipofectamineTM2000Reagentand cultured for72-96hours.The PCV2and PCV2-C virus were collectedrespectively. After six continuous passages, the PCV2and PCV2-C virus wereidentified by PCR and indirect immunofluorescence assay (IFA).The results showedwe have rescued the PCV2and the PCV2-C virus, which can replicate and proliferatein PK-15cells.The proliferation rate of the PCV2-C virus was lower than that ofPCV2.In a word, the genetic modified strain of PCV2-C was successfully obtainedand the attenuated strain of PCV2-C had the infection ability.The other pair specific primer to the Cap gene of porcine circovirus type2(PCV2) were designed and synthesized.The Cap gene was amplified by PolymeraseChain Reaction (PCR).The PCR products were cloned into pMD18-T vector andconfirmed by restrict enzyme analysis and DNA sequencing.The prokaryoticexpression plasmid pET-30a-Cap was constructed and identified.The recombinantplasmid pET30a-Cap was transformed into E.coli BL21(DE3) strain,The recombinantprotein had been expressed and the molecular weight of the Cap protein was28.0kDby SDS-PAGE analysis.The expression products existed as a soluble fusionprotein.Western blot assay showed that the recombinant protein reacted with thepolyclonal antibodies against PCV2,which implied that the Cap protein might besuitable as potential antigen for further development of immunoreagents,The proteinwas expected to lay foundation for further studies on the subunit vaccine of PCV2.