凋亡素基因的原核表达构建与提纯

Study on expression and purification of apoptin from prokaryotic vector

目的构建凋亡素原核表达系统,制备提纯抗原物质凋亡素融合蛋白。方法用PCR的技术,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.coliBL21(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,固化Ni离子金属鏊合纯化带6X组氨酸标签的凋亡素融合蛋白,聚丙烯酰胺凝胶电泳分析目的蛋白。结果转化有凋亡素基因的原核表达载体pET-DsbA-VP3的大肠杆菌E.coliBL21(DE3)plysS经IPTG诱导,固化Ni离子金属鏊合亲和层析纯化凋亡素融合蛋白,聚丙烯酰胺凝胶电泳获得分子量为38.3KD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）. and the protein was analysized by polyacrylamidedel electrophoresis. The fusion protein with apoptin and DsbA expressed in E.coli BL21 （DE3）plysS was purified through Ni-NTA His Bind Resins and the several classes of protein were partitioned by polyacrylamide gel electrophoresis. [Results] The cracked bacteria E.coli BL21 （DE3）plysS which induced by IPTG was purified and partitioned by polyacrylamide gel electrophoresis. The protein with 38.3 KD, a fusion protein of DsbA and VP3, was separated. [Conclussion] The apoptin expression system with pET-DsbA-VP3 can effectively express apoptin fusion protein.