含绿色荧光蛋白基因的猪细小病毒病-伪狂犬病重组病毒的构建

将扩增得到的PPV SC-1株VP2基因产物插入转移载体质粒pPI-2．EGFP中，构建了含PPV VP2基因及其EGFP报告基因的PRV基因缺失表达载体质粒pPI-2．EGFP．VP2。采用磷酸钙转染系统，将pPI-2．EGFP．VP2 DNA和PRV DNA共转染Vero细胞后通过直接荧光观察、Dot-blot核酸杂交筛选得到几株重组病毒，将其中一株（命名为SA215-B）DNA用BamHI酶切和Southern转印杂交进一步验证重组病毒构建成功，并通过SDS-PAGE电泳和Western免疫印迹检测表明PPV VP2基因在重组病毒内获得表达，产生大小约93ku的融合蛋白，同时融合蛋白中的PPV VP2结构蛋白仍然保持了原有的反应原性，表明成功构建了含绿色荧光蛋白基因的猪细小病毒病-伪狂犬病重组病毒。

Construction of a Recombinant Pseudorabies Virus Coexpressing Porcine Parvovirus VP2 Gene and a Marker Enhanced Green Fluorescence Protein Gene

The PPV VP2 gene obtained by PCR was inserted into pPI-2. EGFP vector, and the expression vector pPI-2. EGFP. VP2 was constructed. The results of Dot-blot hybridization and restriction enzymes digestion suggested that the construction of pPI-2. EGFP. VP2 was successful. PRV SA215 DNA and recombinant plasmid pPI-2. EGFP. VP2 DNA were co-transfected into Vero cells with calcium phosphate, and several recombinant viruses were screened out with fluorescence microscopy assay and Dot-blot hybridization. The recombinant virus named SA215-B was analyzed with BamHI restriction enzyme digestion, Southern blotting, SDS-PAGE and Western blotting, the result confirmed that PPV VP2 gene had been inserted into the PRV SA215 DNA and expressed about 93ku EGFP fusion proteins in recombinant virus. The successful construction of PRV-PPV recombinant virus would lay a foundation on the development of an genetically engineered PRV bivalent marker vaccine.