Expression And Identification Of ORF1 Gene Of Porcine Circovirus Type 2 In Prokaryocyte And Baculovirus Expression System

Porcine circovirus (PCV) is one land of smallest viruses we have known by now, which has two gene types, PCV1 and PCV2. PCV1 can be detected in most PK15 cells and it doesn't cause cytopathic effect (CPE ). PCV2 has been implicated as the etiological agent of some diseases such as post weaning multisystemic wasting syndrome (PMWS), porcine dermatitis nephropathy syndrome (PDNS), reproductive failure and porcine respiratory disease complex (PRDC) etc. The wide spread of these diseases imposed serious threats on pig industry and caused huge loss in many nations and regions in the world, what is the worse is that the infection of PCV2 can depress the pig's immune system and lead to the second infection of other pathogens.Both PCV1 and PCV2 have two primary open reading frames (ORFs), ORF1 and ORF2.The Cap protein as main structural protein of PCV was encoded by ORF2,Rep and Rep' protein associated to replication were encoded by ORF1 of PCV1, while function of protein encoded by ORF1 of PCV2 is known little. The ORF1 gene of PCV2 were amplified and cloned into prokaryocyte and baculovirus expression system, recombinant protein were expressed as following:1. Expression of ORF1 gene of PCV2 in prokaryocyte system: In order to express the ORF1 gene of PCV2 , a pair of primers were designed according to the sequence of PCV2 SH strain. The ORF1 gene were amplified from recombinant plasmid pMD-18T-PCV2 SH by PCR using the primers that have EcoRâ… and Notâ… sites, and cloned into a prokaryotic expression plasmid pGEX-6p-1, then a recombinant plasmid named pGEX-6p-1-ORF1 was constructed and identified with restriction enzyme digestion and sequenced. The plasmid pGEX-6p-1-ORF1 was transformed into E.coli BLn and induced by IPTG. Result of SDS-PAGE indicated that ORF1 gene were expressed and the recombinant fusion protein GST-Rep was purified with a size of 62kDa. Finally, antibody against the nonstractural Rep protein was made by treating mice with purified recombinant fusion protein.2. Expression and identification of ORF1 gene of PCV2 in baculovirus expression system: According to the sequence of ORF1 gene of PCV2, a pair of primers were designed and ORF1 gene were amplified from recombinant plasmid pMD-18T-PCV2 SH. ORF1 gene were cloned into transfer vector pFastBac^TMDual using Xhoâ… and Kpnâ… digestion sites. E.coli DH₁₀Bac containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFastBacDual^TM-ORF1. The colonies of E.coli containing recombinant bacmid (rBacmid-ORF1) were collected by blue/white selection. The rBacmid-ORF1 was transfected into sf9 cells to yield Ac. NPV carrying the PCV2 ORF1 gene referred to as rAc-Rep. Expression of the ORF1 gene of PCV2 was confirmed by SDS-PAGE and Western-blotting. The Rep protein expressed by rAc-Rep had a molecular weight of 36kDa.In summary, in this experiment Rep protein gene of PCV2 was expressed in the prokaryotic expression system and Bac-to-Bac expression system respectively, and the specific antibody against Rep protein was made. The results makes stable groundwork for diagnosis reagent and gene engineering vaccine.