杆状病毒SpltMNPV gp41基因的克隆、表达及抗体制备  

Cloning and Expression of Spodoptera litura nucleopolyhedro virus gp41 Gene in Escherichia coil and Preparation of Its Antibody

作　　者：潘丽晶[1] 李朝飞[1] 尹崇[1] 吕雷[1] 庞义[1] 

机构地区：[1]中山大学生物防治国家重点实验室,广州510275

出　　处：《农业生物技术学报》2004年第6期676-679,共4页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究与发展规划(973)项目(No.G2000016209)资助

摘　　要：GP41是杆状病毒包埋型病毒粒子(Occlusion-derived virus, ODV)特有的糖蛋白,它介导芽殖型病毒粒子(Buddedvirus, BV)的核衣壳通过胞核。用PCR方法扩增得到斜纹夜蛾核多角体病毒(Spodoptera litura nucleopolyhedro virus, SpltMN-PV)gp41基因,并将其克隆至5′端有6xHis编码序列的原核表达载体pQE30上,转化大肠杆菌(Escherichia coli)M15[pREP4],在1 mmol/L异丙基-D-硫代半乳糖(IPTG)诱导下超量表达了与理论预测值相符的1个37.9kD融合蛋白。以纯化的融合蛋白为抗原,免疫新西兰大白兔制备了特异的抗GP41抗体。Western印迹分析表明,该抗体适合进一步分析SpltMNPVGP41蛋白。GP41, a major glycoprotein, has been identified in the Occlusion-derived virus (ODV) of baculoviruses, which was re- quired for the egress of nucleocapsids from the nucleus in the pathway of the Budded virus (BV)synthesis. The ORF of Spodoptera litura nucleopolyhedro virus(SpltMNPV)gp41 gene was obtained by PCR method from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). The gp41 gene was recombined in vitro with prokaryot- ic expression vector pQE30 and transformed into Escherichia. coli M15 [pREP4]. The M15 [pREP4] strain, containing gp41 recombi- nant plasmid, expressed a 37.9 kD 6×His-tag fusion protein after induction with 1 mmol/L IPTG (isopropylthio-β-D-galactoside). The fusion protein was purified with Ni-NTA resin column and used as the immunogen to raise a GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

关 键 词：GP41 表达 MNPV 基因 抗体制备 克隆 融合蛋白 病毒粒子 核衣壳 杆状病毒 

分 类 号：S433[农业科学—农业昆虫与害虫防治] R392[农业科学—植物保护]