杆状病毒SeMNPV Se29基因的克隆、表达与抗体制备  

Cloning and Expression of Spodoptera exigua Multicapsid Nucleopolyhedrovirus Se29 Gene in Escherichia.coli and Preparation of Antibody

作　　者：李赛男[1,2] 李朝飞[2] 庞义[2] 杨凯[2] 

机构地区：[1]肇庆学院生命科学学院,中国肇庆526061 [2]中山大学有害生物控制与资源利用国家重点实验室,中国广州510275

出　　处：《湖南师范大学自然科学学报》2011年第4期78-81,87,共5页Journal of Natural Science of Hunan Normal University

基　　金：国家重大基础研究资助项目(G2000016209);广东省教育厅自然科学基金重点资助项目(06Z026);肇庆市科技创新资助项目(203301);中山大学生物防治国家重点实验室开放课题(2004)

摘　　要：目的:克隆和表达甜菜夜蛾核多角体病毒(Spodoptera exigua multicapsid nucleopolyhedrovirus,SeMN-PV)ORF29全长基因(Se29),制备SE29蛋白的多克隆抗体.方法:用PCR的方法扩增得到Se29基因,将其克隆至原核表达载体pET-28a(+)上,转化大肠杆菌BL21(DE3),在IPTG诱导下超量表达了SE29蛋白.采用割胶回收的方法纯化SE29蛋白,以纯化的SE29蛋白作为抗原,免疫新西兰大白兔制备了特异的抗SE29蛋白的抗体.结果:构建了Se29基因原核表达质粒,在大肠杆菌中实现了SE29蛋白的原核表达,获得了SE29蛋白的多克隆抗体,West-ern blot分析表明该抗体能与SeMNPV感染的细胞蛋白样品发生特异性反应.结论:SE29蛋白的多克隆抗体的获得为进一步研究SE29蛋白的功能奠定了基础.Objective： To clone and express Spodoptera exigua multicapsid nucleopolyhedrovirus （SeMNPV） Se29 gene, and to prepare its antibody for further function analysis of Se29 gene. Methods： Se29 gene was obtained by PCR method from SeMNPV genomic DNA. The PCR product was cloned into the expression vector pET-28a （ ＋ ） and transformed into Escherichia coli BL21 （DE3）, and SE29 protein was expressed after the induction with IPTG （Isopropylthio-β-D-galactoside）. The SE29 protein was separated on SDS-PAGE and recovered by gel extraction. New Zealand white rabbits were immunized with the purified protein to harvest polyclonal antibodies. Resuits： The expression plasmid of Se29 gene was constructed and expressed successfuUy in E. coli, and its polyclonal antibody was obtained from the rabbit. Western blotting analysis indicated that the antibody could react with the corresponding protein existed in SeMNPV-infected Se301 cells. Conclusion： SE29-specific antibody is suitable to be used for further analysis of SE29 protein.

关 键 词：甜菜夜蛾核多角体病毒 Se29 克隆 原核表达 抗体 

分 类 号：Q786[生物学—分子生物学]