【摘要】 将包含有完整阅读框架的PPV VP2基因(1740 bp)和PCV2 ORF2基因(750 bp)插入真核表达载体pPI-2.EGFP中,构建了重组质粒pPI-2.EGFP.VP2.ORF2。采用脂质体介导法将重组质粒的DNA和PRV SA215株的DNA共转染Vero细胞,转染后20 h出现带荧光的空斑,挑斑纯化后繁殖,并收获病毒液。PCR和免疫荧光试验结果证实,成功构建了重组病毒,将其命名为PRV SA215(D1)株。细胞培养特性试验结果显示,重组病毒可以在Vero、MDBK、ST和IBRS-2等多种细胞上增殖,在Vero细胞上的增殖效价为1×106PFU。分别以1×105PFU/头、1×104PFU/头剂量接种28日龄仔猪,结果显示,免疫仔猪的PPV、PCV2抗体水平均逐渐升高,并分别从接种后第14 d和21 d开始,PPV、PCV2抗体转为阳性。此结果进一步佐证了重组PRV SA215(D1)株已构建成功。更多还原

【Abstract】 The recombinant plasmid pPI-2.EGFP.VP2.ORF2 was constructed by inserting VP2 gene of porcine parvovrius(PPV) and ORF2 gene of porcine circovirus type 2(PCV2) including complete open reading frame(ORF) into the eukaryotic expression vector pPI-2.EGFP.At hour 20 after co-transfection of the recombinant plasmid with the pseudorabies virus(PRV) strain SA215 genomic DNA into Vero cells with liposome,green fluorescent protein could be observed in some plaque using fluorescence microscope,and plaque purification was employed to obtain the virus,subsequently.The recombinant PRV named PRV SA215(D1) was collected through plaque purification repeatedly and proved to be successfully constructed by PCR amplification.The characterization of virus multiplication showed the recombinant virus could propagated on different cell lines such as Vero,MDBK,ST and IBRS-2 and PFU of the recombinant virus on Vero cell line was 1×106PFU.The animal test showed that 28 day-old piglets inoculated the recombinant virus(1×105PFU per piglet and 1×104PFU per piglet each) could generate specific PPV and PCV2 antibodies,which turned to positive on day 14 post-inoculation at 1×105PFU per piglet group,and on day 21 post-inoculation at 1×104PFU per piglet group.The result certificated that the recombinant virus PRV SA215(D1) was constructed successfully.更多还原