In Vitro Expression Of ORF3-,ORF4-and ORF5-encoding Products For Type Ⅱ Of Porcine Circovirus

Porcine circovirus type 2 (PCV-2) is a member of Circoviridae, which causes a disease named as postweaning multisystemic wasting syndrome (PMWS). It has made a great loss in world's swine industry. Up to now, we don't understand the pathogenisis of PMWS thoroughly. PCV-2 contains 11 open reading frames (ORFs). ORF1 encodes a putative protein involved in viral replication (Rep protein), and ORP2 becomes a hot spot because ORF2-coding product was the major structural protein for PCV-2, while other ORF-coding products in genome of PCV-2 have not been reported. In this study, we successfully cloned ORF3, ORF4 and ORF5 genes based on PCV-2, constructed the prokaryotic expression vectors of ORF3, ORF4, ORF5, purified ORF3-, ORF4- and ORF5-encoding products expressed in E.coli, and also investigated the expressions of ORF3-, ORF4- and ORF5-encoding products in eukaryotic cell utilizing enhanced green fluorescent protein (EGFP). These research results would provide a basis for futher studying these proteins. Main results as below:1. ORF3 of PCV-2 were re-constructed by designing and synthesizing specific primers according to PCV-2 nucleotide sequence, for removing the rare codons disturbing gene expression in E.coli, using fusion PCR techniques. pGEX-4T-l-ORF3 expression system was further built for expression of ORF3-encoding products in prokaryotic cells. Futhermore, the prokaryotic expression system of ORF3-encoding product was also constructed.2. The expression of ORF3-, ORF4- and ORF5-encoding products was confirmed by SDS-PAGE analysis and western blot. Western blot analysis showed that ORF3, ORF4- and ORF5-encoding products expressed in E.coli were specifically recongnized by the polyclonal antibody to GST. The molecular weights of ORF3, ORF4- and ORP5-encoding products are 37.7, 32.5 and 32 kDa, respectively. These results proved that ORF-3, ORF4- and ORF5-encoding products were expressed in E.coli. Meanwhile, the expression of ORF3 indicated that the rare codons of foreign gene play an important role in interfereing the expression of the foreign gene in E.coli.3. Analysesed by SDS-PAGE electrophoresis, PCV-2 ORF3 and ORF4 recombinat protein expressed in E.coli mainly existed as inclusion body. The denatured or refolded proteins could not bind to the Hitrap column via the GST tag, suggesting the fusion of ORF3 or ORF4 changes the configuration of GST significantly and interfere its binding ability. The expressed proteins were then recovered by a cutting gel method successfully for monoclonal antibody production.4. In order to explore the expression and location of PCV-2 ORF3- ORF4- and ORF5-encoding products in eukaryote, we constructed eukaryotic expression vectors of ORF3, ORF4 and ORF5 using the vector of pEGFP, respectively. The results displayed that the PCV-2 ORF3- ORF4- and ORF5-encoding products were expressed in PK-15 cells. The expressed proteins mainly were shown to appear in cellular nucleus and cytoplasma, and have some toxicity to PK-15 cells.