中华蜜蜂囊状幼虫病毒RNA依赖性RNA聚合酶基因的原核表达及多克隆抗体制备  

Prokaryotic Expression of RNA-Dependent RNA Polymerase Gene of Chinese Sacbrood Virus and Preparation for Its Polyclonal Antibody

作　　者：张洁[1] 张淑钦 党晓群[1] 周泽扬[1] ZHANG Jie;ZHANG Shuqin;DANG Xiaoqun;ZHOU Zeyang(Chongqing Key Laboratory of Vector Control and Utilization,Key Laboratory of Conservation and Utilization of Pollinator Insect of the Upper Reaches of the Yangtze River(Co-Construction by Ministry and Province),Chongqing Normal University,Chongqing,401331,China)

机构地区：[1]重庆师范大学、农业农村部长江上游传粉昆虫资源保护与利用重点实验室(部省共建)媒介生物控制和利用重庆市重点实验室,重庆401331

出　　处：《重庆师范大学学报(自然科学版)》2024年第6期36-42,共7页Journal of Chongqing Normal University:Natural Science

基　　金：国家自然科学基金面上项目(No.32372944)。

摘　　要：为研究对中华蜜蜂(Apis cerana cerana)健康构成严重威胁的中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase,RdRp)基因RdRp的原核表达及制备对应的多克隆抗体,通过PCR扩增CSBV的RdRp全长序列,将它克隆到pET-28a原核表达载体,再将含有目的基因的载体转入到大肠杆菌(Escherichia coli)Rosetta(DE3)菌株中,经异丙基-β-D-硫代半乳糖苷诱导表达出大量目的蛋白,通过免疫小鼠(Mus musculus)制备对应的多克隆抗体,采用蛋白印迹法(Western blot)分析多克隆抗体的特异性。结果显示:成功构建了重组表达质粒pET28a-RdRp,在大肠杆菌Rosetta(DE3)菌株中成功表达出大量RdRp重组蛋白;Western blot分析结果表明制备的多克隆抗体能特异性地识别感染了CSBV的中华蜜蜂幼虫体内的RdRp。上述研究结果为今后研究RdRp的精确定位和掲示CSBV入侵宿主具体过程奠定了基础。To investigate the RNA-dependent RNA polymerase(RdRp)gene of Chinese sacbrood virus(CSBV),a serious threat to the health of Apis cerana cerana,its expression in prokaryotic cells and the preparation of corresponding polyclonal antibodies,the full-length sequence of RdRp gene was amplified by PCR,cloned into the pET-28a prokaryotic expression vector,and then introduced into Escherichia coli Rosetta(DE3)cells.The target protein was induced to express in large quantities by IPTG-β-Dthiogalactoside,and polyclonal antibodies were prepared by immunizing mice(Mus musculus).The specificity of the polyclonal antibody was analyzed by Western blot.The results showed that the recombinant expression plasmid pET28a-RdRp was successfully constructed,and large amounts of RdRp recombinant protein were expressed in E.coli Rosetta(DE3)cells.The Western blot analysis results showed that the polyclonal antibody prepared could specifically recognize RdRp in the larvae of Apis cerana cerana infected with CSBV.The above research results lay a foundation for further studying the precise location of RdRp and revealing the specific process of CSBV invading host cells.

关 键 词：中华蜜蜂囊状幼虫病毒 RNA依赖性RNA聚合酶 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]