猪瘟病毒E2蛋白A/D抗原区基因在酵母中的分泌表达与鉴定  

Secretion Expression of the Gene Encoding Classical Swine Fever Virus E2 A/D Antigenic Domain in Pichia pastoris and Identification of the Protein

作　　者：徐学清[1] 张素芳[1] 郑其升[1] 苏小运[1] 任雪枫[1] 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,江苏南京210095

出　　处：《中国病毒学》2004年第6期598-601,共4页Virologica Sinica

基　　金：国家 863 高技术发展计划资助项目(2001AA249012)。

摘　　要：基于猪瘟病毒主要保护性抗原 E2 囊膜糖蛋白有两个相对独立的抗原结构单位--B/C 抗原区和 A/D 抗原区,设计一对特异性的引物扩增猪瘟病毒 E2 蛋白的 A/D 抗原区基因, 并将 PCR 产物克隆入含有强启动子 PAOX1和α-MF 信号肽序列的巴斯德毕赤酵母表达载体 pPICZαC 中,构建成重组质粒 pPICZα-AD,酶切线性化后电穿孔导入巴斯德毕赤酵母 X33菌中,经 ZeocinTM筛选得到 5 株高拷贝转化子,甲醇诱导表达。SDS-PAGE 和 Western blot试验表明酵母培养上清液中含有具有良好反应原性的 E2 蛋白,蛋白表达量达 175.8μg/mL。N-糖基化分析显示该表达蛋白在分泌过程中发生糖基化。该研究为研制防治猪瘟的亚单位疫苗与诊断试剂盒奠定基础。Based on the fact that the envelope glycoprotein E2 which can protect swine from virulent attack of Classical swine fever virus(CSFV) has two structural antigenic domains B/C and A/D, a pair of specific primers was designed to amplify the gene fragment encoding A/D antigenic domain of E2 protein. The 373 bp PCR product was directionally cloned into Pichia pastoris secretory expression vector pPICZαC under the control of the AOX1 promoter and α-factor secretion signal sequence. After being linearized with restriction endonuclease Dra I, the recombinant plasmid was transformed into Pichia pastoris by electroporation. Five transformants with high copies were acquired when selected under ZeocinTM and were induced with methanol. SDS-PAGE indicated that the supernatant of the induced P. pastoris culture contained the recombinant protein E2 (175.8ug/mL). Western-blot analysis proved that the recombinant protein had good reactimmunity against positive CSFV serum. N-glycosylation analysis of expressed products showed that the recombinant protein was glycosylated in the process of secretion. Our research provided a basis to develop sub-unit vaccine and diagnostic antigen against CSFV.

关 键 词：猪瘟病毒 A/D抗原区 毕赤酵母 表达 

分 类 号：S852.65[农业科学—基础兽医学]