牛病毒性腹泻-黏膜病病毒E_2基因重组卡介苗的构建  

Construction of Recombinant BCG for BVDV E2 gene

作　　者：时坤[1] 王文玉[2] 王慧慧 曾范利[1] 李健明[1] 杜锐[4] 

机构地区：[1]吉林农业大学中药材学院,吉林长春130118 [2]吉林农业大学动物科学技术学院,吉林长春130118 [3]长春西诺生物科技有限公司,吉林长春130012 [4]吉林农业大学研究生学院,教育部动物生产及产品质量安全重点实验室,吉林长春130118

出　　处：《中国兽医杂志》2014年第7期32-35,共4页Chinese Journal of Veterinary Medicine

基　　金：国家科技支撑计划(2011BAI03B02-1);国家自然科学基金(31272565);吉林省科技厅科技支撑计划(20120225);吉林农业大学博士启动基金(201209)

摘　　要：本试验将成功克隆的牛病毒性腹泻-黏膜病病毒Changchun184株E2基因与大肠杆菌-分枝杆菌整合表达载体pMV361连接,构建重组整合质粒pMV361-E2。并确定了电转化的最佳条件:2.0 kV/cm、25μF、1 000Ω,成功获得了E2基因重组卡介苗。在45℃下热诱导E2基因在重组卡介苗中的表达,通过SDS-PAGE电泳和免疫印迹检测,结果显示,在44 kD处出现目的条带,符合E2基因表达蛋白的大小,表明牛病毒性腹泻-黏膜病病毒E2基因在卡介苗中成功表达。E2 gene of BVDV Changchun 184 strain was cloned and inserted into the integration expression plasmid vector pMV361, and the recombinant integration plasmid pMV361-E2 was constructed.Then pMV361- E2 was transformed into BCG successfully and rec, ombiuant BCG resistive to kanamycin was obtained. After studying the main factors which can effect the result of electroporation, the optimal electroporation parameters were confirmed, which are 2.0 kv/cm, 25 μF, and 1000Ω .The resulting plasraids pMV361-E2 was electrotransfected into BCG that it was induced at 45 ℃, and then the SDS-PAGE and western blolting were used to analyze the expression product. Our results showed that the vector pMV361 containing E2 gene was successfully constructed, and Western blotting indicated that the E2 gene was successfully expressed in rBCG.

关 键 词：牛病毒性腹泻-黏膜病病毒 E2基因 重组卡介苗 构建 

分 类 号：S852.653[农业科学—基础兽医学]