Cloning And Sequence Analysis Of The Complete Genomes Of PCV2 And The Fused Expression Of The Nucleocapsid Protein

Porcine circovirus 2 (PCV2) is the causative agent of postweaningmultisystemic wasting syndrome (PMWS) ,porcine dermatitis and nephropathysyndrome (PDNS),reproductive failure, porcine respiratory disease complex,granulomatous enteritis, necrotizing lymphadenitiscongenital tremor andexudative epidermitis. Since no PCV2 vaccine was available so far, we havedeveloped a specific PCV2 vaccine candidate, the work were done through threeassay below:1. PCV2 complete genomes were amplified by PCR from total DNA extractedfrom PMWS samples. The PCR products were cloned into pMD18-T easy vectorand the recombinant plasmids were obtained. The sequences of the cloned PCRproducts were sequenced and analysed. The complete genomes is 1768bp inlength. The sequences was Submited to GenBank, the accession number isAY943819. and was compared with the other PCV2 isolates in GenBank.2. Porcine circovirus type 2 (PCV2) genome contains two major open readingframes(ORFs), ORF1 encoding the viral replication-associated protein (Rep)and ORF2 encoding the viral capsid protein (Cap). Cap protein was found to bea major immunogen。To express the Cap protein in prokaryote system,PCV2-ORF2 gene was amplified by PCR and inserted into pET28a(+) plasmidvector. The 3' terminus 396 bp sequence of Cap(Δcap) were cutdown byEcoRⅠand HindⅢand purified, and then cloned to pETLTB vector. Thus, therecombinant plasmid pETORF2Δcap has been constructed. The recombinantplasmid pETLTBΔcap was high level expressed in BL21(DE3) pLysS of E.coliby induced with IPTG. The molecular weight of the recombinant proteinapproximately equal 29 kD by SDS-PAGE with a expression amount ofapproximately 36.67% of total bacterial protein. The fused protein could berecognized by antiserum against PCV2 in Western blot. The study laid thefoundation for development of the recombinant vaccine against PCV2.3. The recombinant LTBΔcap protein was isolated and purified. Kunmin/c mice aged 8 weeks old were randomly assigned to 2 groups. The 8 mice in group 1were each immunized with The recombinant LTBΔcap protein, named immunegroup; another 8 mice in group 2 were each immunized with phosphate-bufferedsaline buffer as as control. 20 days later, all the mice were inoculated with tissuehomogenates from mice which infected by PCV2. As evaluated byHistopathologic analysis, PCV2 genome copys by PCR。the mice were protectedagainst a PCV2 challenge after vaccination.