杆状病毒AcMNPV p35基因的克隆表达及多抗制备  

Cloning and Expression of Autographa californica Nucleopolyhedrovirus p35 Gene in E. coli and Preparation of Multiclonal Antibody

作　　者：李玲玲[1] 李朝飞[2] 庞义[2] 

机构地区：[1]华南师范大学激光生命科学教育部重点实验室,广东广州510631 [2]中山大学生物防治国家重点实验室,广东广州510275

出　　处：《微生物学杂志》2007年第5期1-4,共4页Journal of Microbiology

基　　金：国家重大基础研究项目(973项目;G2000016209)

摘　　要：用PCR方法扩增得到苜蓿丫纹夜蛾核多角体病毒(Autographa californic anucleopolyhedrovirus,AcM N-PV)p35基因,将其克隆至质粒pET-32a(+)上,构建得到重组质粒pET-p35,转化大肠杆菌BL21(DE3),经IPTG诱导,表达了1条约为55 ku的蛋白带。以Ni2+-NTA偶连抗体检测证明所表达的蛋白为带有组氨酸的融合蛋白。采用割胶回收的方法纯化融合蛋白,以纯化的融合蛋白制备多克隆抗体,效价为1/1 024。免疫印迹分析表明,该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。Autographa californica nucleopolyhedrovirus （AcMNPV） p35 gene was amplified from AcMNPV genomic DNA via PCR. The PCR product was cloned into the expression vector pET-32a（ ＋ ） and transformed into Escherichia coli BL21 （DE3） , and a protein around 55 ku was expressed after induction with IPTG （ isopropylthio-β-D-galaetoside）. Western blot using Ni-NTA Conjugate indicated that the protein contained 6 x His tag. The fusion protein was separated on SDS-PAGE and recovered by gel extraction, and used as the immunogen to raise multielonal antibody, which had a titer of 1/1 024. Western blotting analysis indicated that the antibody could react with the corresponding protein existed in AeMNPV-infeeted cells.

关 键 词：苜蓿丫纹夜蛾核多角体病毒p35基因基因表达多克隆抗体 

分 类 号：Q93[生物学—微生物学]