家蚕核型多角体病毒egt基因的原核表达及侵染BmE细胞中的EGT蛋白检测  

Prokaryotic Expression and Encoded Protein Detection in Infected BmE Cells ofegt Gene from Bombyx mori Nucleopolyhedrovirus

作　　者：刘红玲[1] 林英[1] 杨从文[1] 张梅[1] 朱传民[1] 孙晛 夏庆友[1] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学生物技术学院,重庆400716

出　　处：《蚕业科学》2013年第6期1115-1120,共6页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2011AA100306);国家自然科学基金青年基金项目(No.31101768);西南大学含弘杯竞赛专项(No.1131001)

摘　　要：杆状病毒的蜕皮甾体尿苷二磷酸葡萄糖基转移酶(ecdysteroid UDP glucosyltransferase,EGT)能使宿主昆虫体内的蜕皮激素失去活性而延迟蜕皮和化蛹,以利于病毒大量繁殖。从家蚕核型多角体病毒(BmNPV)基因组DNA中扩增获得egt基因全长序列片段,利用原核表达载体pET28a在大肠杆菌BL21(DE3)菌株中进行IPTG诱导表达。SDS-PAGE检测发现EGT蛋白以包涵体形式表达,并且在16℃条件下以0.4 mmol/L IPTG诱导20 h目的蛋白质的表达量最高。采用镍柱亲和层析和切胶回收的方法获得纯化的EGT蛋白,并制备兔源多克隆抗体,其效价>5.12×105。Western blotting检测发现BmNPV侵染BmE细胞后48 h开始有EGT蛋白合成,并一直持续到144 h细胞基本死亡时还存在大量的EGT蛋白。这些结果为进一步研究BmNPV与宿主的相互作用机制提供了一定的线索,也暗示可以利用EGT蛋白调控家蚕蜕皮激素含量,使开发"永久蛹"成为可能。Ecdysteroid UDP glucosyltransferase （EGT） of baculoviruses could trigger a delay of ecdysis and pupation by eliminating host insect ecdysone activity to facilitate viral multiplication. In this study, we cloned the full-length sequence fragment of egt gene from Bombxy mori nucleopolyhedrovirus （BmNPV） genome DNA, and expressed EGT protein in E. coil BL21 （DE3） after IPTG induction by using the prokaryotic expression vector pET28a. SDS-PAGE detection showed that EGT protein was expressed in the form of inclusion body and the expression level was the highest after in- duction with 0.4 mmol/L IPTG for 20 h at 16 ℃. We obtained the purified EGT protein by nickel column affinity chromatography and recovery from agarose gel and used it for preparation of rabbit polyclonal antibody whose titer was over 5.12 ×10^5. Western blotting detected the expression of EGT in BmE cells beginning at 48 h after BmNPV infection and being maintained at high level till 144 h at which the cells were almost dead. These results provide clues for further exploring the interaction between BmNPV and its host, and the possibility to utilize EGT for regulating ecdysone content in silkworm to develop ＂the permanent pupae＂.

关 键 词：家蚕核型多角体病毒 蜕皮甾体尿苷二磷酸葡萄糖基转移酶 原核表达 免疫印迹分析 

分 类 号：S884.51[农业科学—特种经济动物饲养] Q523.3[农业科学—畜牧兽医]