摘要
利用PCR技术从猪细小病毒(PPV)SC1株基因组中扩增VP2全基因,并将其插入真核表达载体pPI-2.EG-FP中,构建转移载体pPI-2.EGFP.VP2。采用脂质体介导法将猪伪狂犬病病毒(PRV)SA215株DNA与pPI-2.EGFP.VP2DNA共转染Vero细胞,待出现细胞病变后收集病毒液。经空斑纯化,并同时采用检测PPV VP2基因的PCR方法筛选获得重组病毒PRV SA215/VP2株。以兔抗VP2的多克隆抗体建立的免疫荧光技术可检测到Vero细胞发出的特异性荧光,表明PPV VP2成功插入到PRV SA215基因组中,并获得表达。进一步电镜观察表明,感染PRV SA215/VP2的Vero细胞中可同时观察到PRV与PPV 2种类病毒样颗粒。结果表明,成功实现了利用PRV载体表达PPV VP2蛋白病毒样颗粒,为进一步研制PPV病毒样颗粒疫苗奠定了基础。
The present experiment was performed with the objective of studying the expression of porcine parvovirus-like particles formed by the VP2 protein in recombinant pseudorabies virus（PRV）.Amplified the VP2 gene from porcine parvovirus（PPV） SC1 strain by PCR,then inserted it into the eukaryotic expression vector pPI-2.EGFP,and the recombinant plasmid pPI-2.EGFP.VP2 was constructed.The recombinant plasmid and PRV SA215 strain genomic DNA were co-transfected into Vero cells with liposome,and the recombinant virus PRV SA215/VP2 was obtained with plaque purification and PCR method by examining the VP2 gene.Immunofluorescence was established to detect the Vero cells infected the recombinant virus by rabbit anti-VP2 IgG polyclonal antibody,and the specific fluorescence could be detected,indicating that PPV VP2 gene was inserted into PRV SA215 genome and expressed.The electron microscopy showed that PPV and PRV virus-like particles could be observed simultaneously in Vero cells infected PRV SA215/VP2.Above all,the results suggested that PPV virus-like particles formed by the VP2 protein could be expressed in recombinant PRV and laid a foundation of the further development of PPV virus-like particles vaccine.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2011年第10期1385-1389,共5页
Chinese Journal of Veterinary Science
基金
国家科技支撑计划资助项目(2006BAD06A18)
教育部长江学者和创新团队发展计划资助项目(IRT0848)
