表达猪瘟病毒E2基因和gfp报道基因的伪狂犬病病毒双基因转移载体的构建

表达猪瘟病毒E2基因和gfp报道基因的伪狂犬病病毒双基因转移载体的构建 Construction of a Pseudorabies Virus Transfer Vector Coexpressing E2 Gene of Classical Swine Fever and a Maker Green Fluorescence Protein Gene 潘兹书 PAN Zi\|shu 1 2 first-author 张楚瑜 ZHANG Chu\|yu 1 2 陈玉栋 CHEN Yu\|dong 1 2 闵平 MIN Ping 1 2 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 武汉大学生命科学学院,湖北,武汉,430072 武汉大学生命科学学院,湖北,武汉,430072 将以PRVgG为启动子 ,SV4 0polyA为加尾信号的CSFVE2表达盒克隆于包含伪狂犬病病毒 （PRV）tk基因和gH基因片段的重组质粒pTK2 .5 ,替换掉tk基因的SalⅠ与XhoⅠ酶切位点之间的序列 ,构建了携带CSFVE2基因的转移载体pTKE2 免疫荧光检测证实 ,转染BHK2 1细胞的pTKE2在感染PRV的情况下 ,能表达E2蛋白 采用特异性引物PCR方法扩增gfp表达盒 ,对酶切位点进行改造 ,将其克隆到pTKE2的SalⅠ位点 ,构建了用PRVgG启动子和HCMVIE启动子分别控制E2基因和gfp基因表达、两基因取向相反的双基因转移载体pTKE2 .GFP 将双基因转移载体pTKE2 .GFP转染BHK2 1细胞中 ,12h后在荧光显微镜下观察到呈绿色荧光的GFP表达 ,表达的GFP不引起细胞毒性 The E2 gene of CSFV was inserted downstream of the promoter of PRV gG gene, and upstream of SV40 polyadenylation signal, and an E2 gene expression cassette was constructed. The tk gene sequence between Sal Ⅰ and Xho Ⅰ sites of the plasmid pTK2.5 containing the complete tk gene and partial gH gene of PRV was replaced by the E2 gene expression cassette, which resulted in a recombinant PRV transfer vector pTKE2. Transient expression of E2 from pTKE2 was identified by immuno\|fluroscence staining. A green flurocence protein gene （ gfp ） expression cassette was amplificated by PCR with the modified flanking sites to insert it in plasmid pTKE2. The gfp expression cassette was cloned into Sal Ⅰ site of pTKE2, and the resulted coexpression vector was named as pTKE2.GFP, and expression of E2 and gfp genes were controlled by the PRV gG and HCMV IE promoters, respectively. These two promoters were oriented in opposite directions to prevent the possibility of polarity effects. pTKE2.GFP was transfected into BHK21 cells, and GFP expression could be detected in the transfected cells as 12 h post\|transfection. These results might be contributive to the development of an genetically engineered PRV bivalent marker vaccine. 伪狂犬病毒 转移载体 绿色荧光蛋白 E2基因 pseudorabies virus transfer vector green flurosence protein E2 gene S852.651 湖北省科技攻关重点项目资助 2002-04-01 2021-04-01 4