伪狂犬病毒PK/gG/GFP重组转移载体的构建和表达

The Construction and Expression of PRV PK/gG/GFP Recombinant Transfer Vector

在构建了含伪狂犬病毒(pseudorabiesvirus,PRV)湖北株部分PK基因和gG基因转移载体的基础上,利用平端连接的方法将绿色荧光蛋白(GFP)的基因表达盒插入到缺失的部分,并在下游引入了1个多克隆位点,构建了重组转移载体KGDF.用限制性内切酶鉴定重组转移载体KGDF.根据质粒EGFP C1中的GFP基因序列设计1对引物鉴定GFP表达盒插入的正确性.用脂质体转染试剂盒将KGDF和PRVHB的基因组或病毒共转染BHK 21细胞,在荧光显微镜下将出现病变的荧光斑挑出得到重组病毒.将重组病毒扩大培养后提取基因组鉴定重组病毒中的GFP基因,并通过挑取病变的荧光斑的方法纯化重组病毒.

A transfer plasmid, KGDF was constructed from the plasmids containing part PK and gG genes.A report gene expression cassette ,GFP containing a multicloning sites ,was inserted bluntly into pPKgGD to construct a recombinant transfer vector,KGDF. That KGDF was constructed correctly was proven by restriction digestion and PCR analysis of GFP. KGDF was used to co-transfect BHK-21 cell together with the genome or the virus of PRV HB, and the recombinant virus with green fluorescence was picked using fluorescent microscope.The recombinant virus was further identified and purified using PCR of the gene GFP and pickup from the fluorescent cells.