法氏囊病病毒vp2基因在酵母中的分泌表达及鉴定  

Secretive Expression and identification of vp2, the Major Antigenic Protein of Infectious Bursal Disease Virus in Yeast

作　　者：贾赟[1,2] 张素芳[1] 周斌[1] 徐学清[1] 曹瑞兵[1] 赵玉军[3] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室 [2]沈阳农业大学畜牧兽医学院辽宁沈阳110161 [3]沈阳农业大学畜牧兽医学院

出　　处：《中国病毒学》2004年第6期582-586,共5页Virologica Sinica

基　　金：国家 863 课题资助(项目号:2002AA245051)

摘　　要：应用 Pichia 酵母表达系统高效分泌表达了传染性传染性法氏囊病病毒 vp2 基因片段。首先用 Primer5.0 设计 1 对引物 P1、P2,以插入 IBDV vp2 基因的 PMD18-T-VP2 载体为模板,扩增出带有终止密码子的 1.4 kb 片段,将此片段正向亚克隆到酵母表达载体pPICZα-A上,将构建好的重组载体pPICZα-A-VP2用SacⅠ内切酶线性化后,电击转化整合入 Pichia PastorisX-33 酵母菌,经高浓度 ZeocinTM 筛选、表型鉴定、诱导表达及表达产物的鉴定,得到高效表达 vp2 基因的酵母工程菌 X-33/ pPICZα-A-VP2。工程菌 72h 培养上清的 SDS-PAGE 电泳与免疫印迹结果显示,vp2 基因表达产物大小约为 55 kDa,比预期的 41kDa 大。凝胶薄层扫描结合 Bradford 蛋白质总含量测定结果表明,表达产物占工程菌培养上清总蛋白的 28%,表达量可达 23 mg/ L。间接 ELISA 结果表明重组表达产物能够有效地区分法氏囊病病毒标准阳性与阴性血清。Here we report that the major antigenic protein VP2 of very virulent Infectious bursal disease virus (vvIBDV) was secretively expressed with high level in yeast (Pichia pastoris) expression system. Primers were designed to amplify the previously cloned vvIBDV-vp2 gene from the plasmid pMD18-T- VP2. The amplified 1.4kb vp2 gene fragment was sub-cloned into the yeast-expressing plasmid pPICZα-A. The recombinant pPICZα-A-VP2 was analyzed by restriction enzymes and sequenced, which was then linearized by SacⅠand transfected into Pichia pastoris X-33. After selection with ZeocinTM, the phenotype identity, the inductive expression, the SDS-PAGE and the western blot analysis of culture supernatant, an engineering Pichia pastoris strain X-33/pPICZαA-VP2 with which the VP2 protein could be expressed with high level was obtained. SDS-PAGE and Western blot analysis indicated that the expressed product of the VP2 in culture supernatant of X-33/pPICZαA-VP2 was about 55kDa, a bit larger than expected. Gel scanning and Bradford protein analysis showed that the expressed product was as high as 23mg/ L, or 28% of total proteins in culture supernatant of X-33/ pPICZαA-VP2.

关 键 词：传染性法氏囊病病毒 VP2基因片段 PICHIA pastoris表达系统 

分 类 号：S852.65[农业科学—基础兽医学]