猪瘟病毒E2蛋白B/C抗原区基因在毕赤酵母中的表达与鉴定  

Secretion Expression of the Gene Encoding Classical Swine Fever Virus E2 B/C Antigenic Domain in Pichia Pastoris and Identification of the Protein

作　　者：徐学清[1] 郑其升[1] 曹瑞兵[1] 苏小运[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,南京210095

出　　处：《中国生物工程杂志》2004年第9期53-57,共5页China Biotechnology

基　　金：国家"8 63"计划资助项目 ( 2 0 0 1AA2 490 12 )

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 ,设计引物扩增编码猪瘟病毒E2蛋白B C抗原区的基因 ,将大小为2 61bp的PCR产物插入含有强启动子PAOX1和α MF信号肽序列的巴斯德毕赤酵母 (Pichiapastoris)表达载体pPICZαC中 ,构建成重组质粒pPICZα BC ,酶切线性化后电穿孔导入巴斯德毕赤酵母菌X3 3 中 ,经ZeocinTM 筛选得到 3株高拷贝转化子 ,甲醇诱导表达 ,SDS PAGE和Westernblot及ELISA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。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 primer was designed to amplify the gene fragment encoding B/C antigenic domain of E2 protein.The 261 bp PCR product was directionally inserted into Pichia pastoris secretory expression vector pPICZ αC under the control of AOX1 promoter and α-factor secretion signal sequence.After being linearized with restrict enzyme SacⅠ,The recombinant plasmid was transformed into Pichia pastoris by electroporation to integrate with the genome.three transformants with high copies were acquired when selected under Zeocin TM and were induced with methonl.SDS-PAGE indicated that the supernatant of the induced P.pastoris culture contained the recombinant protein E2 (175.8μg/ml).Western-blot analysis proved that the recombinant protein had good reactimmunity against positive CSFV serum.A basis to develop sub-unit vaccine and diagnostic antigen against CSFV was provided.

关 键 词：C抗原 E2蛋白 B/C 表达 基因 膜糖蛋白 D抗原 猪瘟病毒 株高 诊断抗原 

分 类 号：Q786[生物学—分子生物学] R457[医药卫生—治疗学]