伪狂犬病毒TK基因转移载体构建及LacZ基因表达

伪狂犬病毒TK基因转移载体构建及LacZ基因表达 Construction of the Pseudorabies Virus Transfer Vector and the Expression of E. coli β-Galactosidase 潘兹书 PAN Zi\|shu 1 first-author 张楚瑜 ZHANG Chu\|yu 1 赵伟光 ZHAO Wei\|guang 1 郑从义 ZHENG Cong\|yi 1 丁建华 DING Jiang\|hua 1 武汉大学生命科学院,湖北,武汉,430072 武汉大学生命科学院,湖北,武汉,430072 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 在扩增、克隆 PRV tk、g H基因的基础上 ,构建了包含 tk和 g H基因片段的转移载体质粒 p TK2 .5 .以PRV糖蛋白 g G启动子 （ Pg G）为控制外源基因表达的启动子 ,以 E.coli lac Z为报道基因 ,用其替换 p TK2 .5质粒tk区的 Sal I与 Xho I之间的序列 ,构建了转移载体质粒 p TK- L ac Z.瞬时表达证实 ,转染细胞的 p TK - lac Z质粒在野生型 PRV感染的情况下 ,能有效表达β- Gal酶活性 .将 p TK- lac Z转染 BHK 2 1细胞后再以 PRV感染进行同源重组 ,在 143TK- 细胞上经 5 -溴脱氧尿苷选择 ,Vero细胞纯化 ,X- Gal染色 ,蓝斑筛选 ,分离到重组体 PRV （ r PRV） .r PRV与野生型 PRV在细胞上具有类似的生长特性 ,且经过连续传代的 r PRV仍能稳定的表达β- Gal酶活性 A recombinant PRV transfer vector （pTK\|lacZ） expressing E.coli LacZ gene contolled by PRV glycoprotein gG promotor（PgG） was constructed. The LacZ expression cassette was inserted at the thymidine kinase（tk） locus of PRV. The transient expression of β\|galactosidase gene on the vector could be easily tested in the transfected BHK21 cells when infected with the wild type PRV. The recombinant PRV （rPRV） could be yielded by homologous recombination with pTK\|lacZ and PRV DNA, and selected by 5\|bromodeoxyuridine when grew on 143TK - cells and isolated by plaque\|staining with X\|gal on Vero cells. Recombinant PRV could stably express the β\|galactosidase activity and it’s biological characteristics were similar to that of wild type PRV when they grew on the cells. These results will not only be contributive to the developement of an genetically engineered PRV vaccine but should also help for analyzing behaviour of rPRV in the infected animal. 伪狂犬病毒 胸苷激酶基因 转移载体 LacZ基因 pseudorabies virus thymidine kinase gene transfer vector LacZ gene 10.14188/j.1671-8836.2000.06.017 S852.65 武汉市科技攻关重点项目资助!（ 962 0 0 10 18） 2000-06-01 2021-04-01 6