利用BrdU在CEF(TK^+)细胞中筛选TK^-重组鸡痘病毒  

Selection of TK^- Recombinant Fowlpox Virus in CEF Cell (TK^+) in the Presence of BrdU

作　　者：郭志儒[1] 金宁一[1] 方厚华[1] 罗坤[1] 夏志平[1] 殷震[1] 

机构地区：[1]解放军军需大学军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2001年第6期543-545,共3页Chinese Journal of Veterinary Science

基　　金：国家"8 6 3"计划资助项目 ( 10 1-0 5 -0 3-0 1)

摘　　要：将新城疫病毒 (NDV)四平株 HN基因 ,插入不含报告基因、以 TK为侧翼的鸡痘病毒表达载体 p UTA- 2中的复合启动子下游 ,获得重组表达质粒 p UTA- 2 HN。将重组表达质粒转染鸡痘病毒 (FPV)感染的鸡胚成纤维细胞 (CEF) ,培养、收获病毒后 ,用含 40 m g/ L 5 -溴 - 2 -脱氧尿嘧啶 (Brd U )的培养液 ,在 CEF(TK+)细胞中筛选培养 2代 ,然后用不含 Brd U的培养液进行病毒蚀斑纯化 ,成功筛选出表达 NDV HN蛋白的A recombinant expression plasmid pUTA-2HN was constructed by inserting the HN gene of Newcastle disease virus(NDV) strain Siping, into the SmaⅠ site downstream of a hybrid poxvirus promoter which is flanked by the TK gene of fowlpox virus(FPV) strain 282E4. For production and selection recombinant fowlpox virus expressing the HN gene of NDV and without containing additional reporter gene, the constructed pUTA-2HN was firstly transfected into chicken embryo fibroblast(CEF) cells preinfected with FPV strain 282E4, then the viruses resulted from the transfection were selected for 2 passages by culturing in CEF cells with MEM medium containing 40 mg/L 5-bromo-2′deoxy-uridine(BrdU). The selected viruses were plaque-purified in CEF cells cultured with MEM media without BrdU. After Western blot analysis the lysate of CEF cells which were infected by the purified FPV, recombinant FPVs (rFPVs) expressing the HN protein were obtained. This indicated that rFPVs containing a foreign gene interrupted TK gene (TK -) could be selected in the CEF(TK +) cell in the presence of BrdU.

关 键 词：BRDU 鸡痘病毒 TK基因 重组病毒 

分 类 号：S858.31[农业科学—临床兽医学] S852.65[农业科学—兽医学]