摘要
目的利用毕赤酵母系统表达口蹄疫病毒(FMDV)非结构蛋白3AB,用于FMDV感染与疫苗免疫动物的鉴别诊断和3AB蛋白功能研究。方法采用PCR方法从pGEM-T-Easy-3ABC质粒中扩增3AB基因,将其插入pPICZαA酵母表达载体中,构建的重组表达质粒线性化后电转入毕赤酵母GS115中,在0.5%甲醇诱导下表达3AB蛋白,运用SDS-PAGE和ELISA方法鉴定表达蛋白。结果成功构建了pPICZαA-3AB重组酵母表达质粒,并转化入毕赤酵母GS115中,SDS-PAGE和ELISA鉴定结果表明,重组酵母菌株表达出约19kD的3AB蛋白,与针对FMDV的3AB蛋白单克隆抗体有特异性反应。结论在毕赤酵母中成功表达了FMDV3AB蛋白,与抗FMDV3AB蛋白单克隆抗体具有反应性。
To express nonstructural protein 3AB of foot-and-mouth disease virus（FMDV） in Pichia pastoris expression system in order to differentiate the infected and vaccinated animals and for the further study of 3AB protein function,the gene 3AB was amplified from the recombinant plasmid of pGEM-T-Easy-3ABC by PCR and inserted into the Pichia pastoris expression vector pPICZαA. The recombinant expression plasmid was then linearized and transformed into Pichia pastoris GS115 by electroporation method. The expressed protein with the induction 0.5 % methanol was analysed by SDS-PAGE and ELISA. It was demonstrated that the recombinant Pichia pastoris expression plasmid pPICZαA-3AB was successfully constructed and transformed into Pichia pastoris GS115. SDS-PAGE and ELISA analysis of the culture supernatants showed that the 3AB protein of 19 kD was expressed in Pichia pastoris. The expressed 3AB protein was reactive with the 3AB protein McAb against FMDV. It was concluded that the FMDV nonstructural protein 3AB was expressed in Pichia pastoris successfully. The expressed 3AB protein shared reaction with the 3AB protein McAb against FMDV.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第12期1100-1103,1134,共5页
Chinese Journal of Zoonoses
基金
国家"十五"科技攻关计划"口蹄疫防治科技攻关"项目资助(2004BA519A-40)
关键词
口蹄疫病毒
非结构蛋白
毕赤酵母
真核表达
Foot-and-mouth disease virus
nonstructural protein
Pichia pastoris
eukaryofic expression
作者简介
通讯作者：田克恭，Email：tiankegong@nvdc．cn
