【作者】 孙京臣； 戴伟君； 谭佩婵； 谭玉蓉； 张景强； 徐兴耀；

【Author】 SUN Jingchen~(1,2) DAI Weijun~2 TAN Peichan~1 TAN Yurong~2 ZHANG Jingqiang~2 XU Xing-yao~1 (1 College of Animal Science,South China Agricultural University,Guangzhou,510642;2 China State Key Laboratory for Biocontrol,Zhongshan University,Guangzhou 510275,China)

【机构】 华南农业大学动物科学学院； 中山大学生命科学院生物防治国家重点实验室；

【摘要】 应用分段RT-PCR 的方法从家蚕质多角体病毒(Bombyx mori cytoplasmic Polyhedrosis Virus,BmCPV)中国株的dsRNA 中成功地克隆了RDRP 基因(RNA-dependent RNA polymerase),基因序列全长3691bp,并登录GenBank,序列号为AY496445。利用质粒载体pET-28b 成功构建了表达质粒pET28b-RDRP,并用IPTG 诱导的方法在大肠杆菌BL21(DE3)中获得表达,分子量约为138kDa。以重组蛋白的兔抗为一抗,15nm 山羊抗兔IgG-胶体金为二抗,进行免疫标记,电镜定位,结果显示在病蚕中肠的柱状细胞的游离病毒粒子和多角体内的病毒粒子上均能结合胶体金颗粒,标记率为30%,左右.证明BmCPV 病毒的RDRP 复合物确实位于病毒衣壳上。更多还原

【Abstract】 RDRP gene of the BmCPV,(China strain) were cloned respectively by usingstep-by-step RT-PCR.The whole RDRP gene of 3691bp was sequenced (GenBank accessionnumber:AY496445).Vector pET-28b (+) was applied to construct the expression vector pET28b-RDRP which was expressed in the E.coli BL21 (DE3) bacteria after induced with IPTG.The molecular weight of the expression product was about 138kDa.The first antibody was rabbitantibody against BmCPV-RDRP recombinant protein and the second antibody was 15nmimmunogol labeled goat anti rabbit IgG.Immunogold bound mostly to the virion thatdispersedly located in virus generation matrix and polyhedron in the columnar cells of the midgutof silkworms,and the average marking ration was about 30%.This result demonstrated thatBmCPV-RDRP complex does locate at the capsid of BmCPV.更多还原