家蚕核型多角体病毒IE1的多克隆抗体制备及鉴定  

Preparation and Verification of Polyclonal Antibody of Bombyx mori nucleopolyhedrovirus IE1

作　　者：董战旗 张军[1] 胡楠[1] 鲁成[1,2] 潘敏慧[1,2] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学,重庆400716 [2]农业部家蚕功能基因组和生物学重点实验室,西南大学,重庆400716

出　　处：《西南大学学报（自然科学版）》2014年第10期76-81,共6页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(31272505;31172269)

摘　　要：家蚕核型多角体病毒(BmNPV)ie1基因是BmNPV DNA复制的必需基因,其编码的IE1蛋白能够反式转录激活杆状病毒早期基因的表达.为了进一步研究IE1蛋白在家蚕核型多角体病毒感染宿主过程中具体的功能,研究通过PCR扩增BmNPVie1基因片段,亚克隆到原核表达载体pET32,获得重组质粒pET32-IE1,经测序正确后,转化至大肠杆菌BL21(DE3)表达菌株.通过IPTG诱导融合蛋白原核表达后,初步纯化收集抗原,免疫新西兰大白兔,制备IE1多克隆抗体.应用制备的免疫兔血清进行Western-blot分析,结果显示多克隆抗体能特异识别BmNPV的IE1和IE0蛋白.免疫荧光结果同样显示IE1蛋白定位于宿主细胞核病毒复制中心.IE1抗体制备的成功为进一步研究IE1在病毒感染家蚕中的作用机理奠定了基础.Bombyx mori nucleopolyhedrovirus（BmNPV）. ie1 gene is essential for viral DNA replication, and the IE1 protein encoded by it can transcriptionally transactivate the expression of the early genes of baculovirus. In order to further study the specific function of IE1 protein in the process of BmNPV infec-tion, we amplified the BmNPV ie1 gene fragment by PCR and subcloned it into the prokaryotic expression vector pET32, thus obtaining the recombinant plasmid pET32-IE1. After pET32-IE1 nucleotide was cor-rectly sequenced, it was transformed into E. coli strain BL21 （DE3）. After the prokaryotic expression of fusion protein with IPTG induction, the antigen was preliminarily purified and was then used to immunize New Zealand white rabbits to prepare the polyclonal antibody. Western-blot analysis of the immune rabbit serum showed that the polyclonal antibody specifically identified the expression of IE0 and IE1 protein. Im-munofluorescence results also showed that the IE1 protein was located at the viral replication center of the infected cell. The successful preparation of the IE1 antibody has laid a foundation for the further study of the mechanism of IE1 gene during viral infection of the host.

关 键 词：家蚕 家蚕核型多角体病毒 IE1 原核表达 多克隆抗体 

分 类 号：S851.347.31[农业科学—预防兽医学]