Discovery of candidate site in virus-like particles(VLPs) of porcine parvovirus(PPV) for inserting foreign protein and the construction of the recombinant VLPs contained the ORF2 protein of porcine circovirus type 2(PCV2)
Abstract：Discovery of candidate site in virus-like particles(VLPs) of porcine parvovirus(PPV) for inserting foreign protein and the construction of the recombinant VLPs contained the ORF2 protein of porcine circovirus type 2(PCV2)
ZHU Ling,GUO Wan-zhu**,JIANG Qing-rong,XU Zhi-wen,XIONG Ding-jie,ZHANG Bo,WANG Yin,ZHAO LING
(Animal Biotechnology Center, Sichuan Agricultural University, YA’an,625014,China)
Abstract:The plasmid pMD-VP2 was constructed by cloning the VP2 gene fragment gained by PCR from the PPV(SC-1)strain template into pMD18-T vector. The ORF2 gene fragment of PCV2(SC)strain was amplified using two pairs of primers ,each pair of which added identical restrict site at the 5’end of the forward and the downward (one pair added HindⅢ,the other added SacI),then the two PCR product were cloned into pMD18-T vector to construct pMD-ORF2.A and pMD-ORF2.B,respectively. Through digesting the two plasmid with the corresponding restrict endonuclease HindⅢ and SacI respectively, the expected fragments could be reclaim and were inserted into the restrict cloning sites, which were corresponding to the 1/3 position of the N-terminus and the C-terminus of the VP2 gene fragment in the pMD-VP2 ,and then the pPVP2－ORF2.A and pPVP2－ORF2.B were constructed. After verification, the correct directional cloning pPVP2－ORF2.A and pPVP2－ORF2.B were digested with restrict endonuclease KpnI、BamHI and ApaLI to get the expected gene fragments which contained VP2 and ORF2 gene, and then the two gene fragments were cloned into enkaryotic expression vector pEGFP-C1 to construct the two recombinant plasmid pEGFP.VO.A and pEGFP.VO.B. Employed fluorescence microscope and electron microscope to confirm the fusion gene expression after transfected the two recombinant plasmids into the COS-7 cell line with liposome, we found that VLPs form in the cell transfected the pEGFP.VO.A only but not in the group of pEGFP.VO.B. In animal, the BALB/C mice after been immunized the purified VLPs could produce comparatively high-level cytoimmunity and special humoral immunity against PPV and PCV2. The result indicated that the recombinant VLPs be composed of PPV-VP2 and PCV2-ORF2 is available in this study and revealed the 1/3 position of the N-terminus of the VP2 gene is suitable for inserting foreign protein to construct recombinant VLPs.
Key Words: Porcine Parvovirus, Porcine Circovirus type 2, Virus-like particles, recombinant
