凋亡素原核表达载体的构建和表达  

Study on construction and expression of a prokaryotic vector for expression of apoptin

作　　者：朱惠明 杨俊文 蔡筱彦 李银鹏 顾红祥 黄勋 

机构地区：[1]暨南大学附属第二医院,广东省深圳市人民医院消化内科,518020

出　　处：《广东医学》2003年第10期1058-1060,共3页Guangdong Medical Journal

摘　　要：目的　构建凋亡素原核表达系统 ,以制备抗原物质凋亡素融合蛋白。方法　通过PCR方法 ,以pcDNA -VP3质粒为模板 ,扩增出凋亡素VP3基因。将其克隆到原核表达载体pET -DsbA的多克隆位点 ,构建成凋亡素的高效原核表达载体pET -DsbA -VP3 ,将该质粒转化到大肠杆菌E .coliBL2 1(DE3 )plysS中 ,以异丙基硫代 -β -D -半乳糖苷(isopropylthio -β -D -galactoside ,IPTG)对其进行诱导表达 ,聚丙烯酰胺凝胶电泳分析目的蛋白。 结果　转化有凋亡素原核表达载体pET -DsbA -VP3的大肠杆菌E .coliBL2 1(DE3 )plysS经IPTG诱导后 ,聚丙烯酰胺凝胶电泳出现 3 83 0 0的目的蛋白条带。结论　凋亡素原核表达载体pET -DsbA -VP3能高效表达出凋亡素融合蛋白。Objective To construct an apoptin expression system to produce an antigen, apoptin fusion protein. Methods The apoptin gene(VP3) was amplified from the template of plasmid pcDNA-VP3 by means of PCR. The VP3 was subcloned into the multiple clone site of plasmid pET-DsbA to get the plasmid pET-DsbA-VP3, which was transformed into E.coliBL21(DE3)plysS. Expression of E.coliBL21(DE3)plysS was induced by isopropylthio-β-D-galactoside (IPTG). The protein was analyzed by polyacrylamide gel electrophoresis.Results E.coliBL21(DE3)plysS with the vector for expression of apoptin, pET-DsbA-VP3, was induced by IPTG and several classes of proteins were partitioned by polyacrylamide gel electrophoresis. The protein of 38?300 , a fusion protein of DsbA and VP3, was separated.Conclusion The apoptin expression system with pET-DsbA-VP3 can effectively express apoptin fusion protein.

关 键 词：凋亡素 原核表达载体 融合蛋白 基因表达 克隆 

分 类 号：R394[医药卫生—医学遗传学]