Construction And Identification Of Recombinant Adenovirus Expressing Fusion Protein Of PCV2 Cap And Porcine IFNa

Porcine circovirus 2 (PCV2) is causal agent of postweaning multisystemic wasting syndrome (PMWS). PCV2 genome contains at least two large open reading frames named ORF1 and ORF2, which encode the Rep protein involved in replication and main structure Cap protein, respectively. It was found that the main epitopes exist on Cap protein which could induce immunoprotection against PCV2 infection in swine.Interferons (IFN) belong to the family of antivirus proteins, which can inhibit some viruses proliferation. IFNa has a broad-spectrum antivirus effect, which display on inhibiting virus replication, degrading conveying and decreasing the lesion of cells.1. The expression and purification and immunogenicity of PCV2 Cap protein in E.coliThe ORF2 gene of PCV2 was amplified by PCR, and the C-terminal gene was digested with EcoRI/NotI which was cloned into a prokaryotic expression plasmid pGEX-6p-1. After identification with enzyme digestion and sequence analysis, the recombinant vector was transformed into E.coli BL21 strain. By optimization for induce condition, the fusion protein of about 40kD was expressed correctly with IPTG at concentration of 0.3mmol/L and induction at 30℃for 6 hours. The results of westernblot showed that the expression protein has a good immunity of PCV2,and it could exist as inclusion-body and soluble protein. Thus the protein wwas purified with GST-affinity column and washing after carbamide denaturation, respectly, and the immunogenicity was detected in ELISA with PCV2 positive serum. The results indicated that there were no obviously differences between the soluble protein and inclusion-body. It could be used as antigen in ELISA for detecty antigen to PCV2.2. Construction and identification of recombinant adenovirus expressing Cap and IFNa proteinIn this study, Cap protein gene in recombinant plasmid rShuttle-ORF2-ORF5 was digested with KpnⅠand cloned into a transfer vector pShuttle-CMV-IFNa that contein IFNa gene. A recombinant plasmid, called pShuttle-ORF2-IFNa, was constructed and identified by PCR and enzyme digestion with KpnⅠ. After co-transformation of PmeⅠ-linearized recombinant plasmid pShuttle-ORF2-IFNa and the bone vector pAdEasy-1 into E.coli bacteria strain BJ5183, recombinant plasmid containing both Cap protein gene and IFNa gene (rAd-ORF2-IFNa) was obtained, and identified with PCR. Upon transfection of PacⅠ-linearized plasmid pAd-ORF2-IFNa in 293 cell line, a recombinant adenovirus was obtained, and named as rAd-Cap-IFNa. The fusion product of about 49kD was expressed correctly which is confirmed by RT-PCR and westernblot. In order to examine the immunity responses of the recombinant adenovirus expressing the Cap and IFNa proteins of PCV2, the mice were separated for 4 groups: rAd-IFNa, rAd-Cap, rAd-Cap-IFNa and control. After different time after inoculation, the titer of serum antibody to PCV2 was tested by ELISA with the antigen of recombinant Cap protein. The results showed that antibody to Cap protein cann't be detected except the group of rAd-Cap-IFNa, two weeks after inoculating. At 2 weeks after booster, the titer was up to 1:800 in the group of rAd-Cap-IFNa, while the group of rAd-Cap to 1:50. It demonstrated that the expression of the fusion protein could enhance the humoral immunity to PCV2 in mice comparing to the group of rAd-Cap. It laid the foundation of development of the recombinant vaccine against PCV2.