【摘要】 目的:构建甜菜夜蛾核多角体病毒(Spodoptera exigua nucleopolyhedrovirus,SeMNPV)ORF100(Se100)和ORF101(Se101)基因的原核表达载体,表达并纯化两种蛋白。方法:用PCR方法扩增Se100和Se101基因,分别将它们克隆至原核表达载体pQE-30上,转化宿主菌M15[pREP-4],用IPTG进行诱导表达,表达产物用Ni-NTA金属螯合层析法进行纯化,SDS-PAGE检测表达的目的蛋白。结果:构建了分别含有Se100和Se101基因的原核表达质粒pQE100和pQE101;SDS-PAGE检测显示,表达的两个融合蛋白的分子量分别为15kDa和31kDa,比预期分子量稍大;Ni-NTA亲和层析结果显示:6×His-Se100和6×His-Se101融合蛋白主要存在于pH值为4.5的缓冲液中。结论:成功克隆并高效表达了Se100和Se101两个基因,有效纯化了两种蛋白,为深入进行基因的功能研究奠定了基础。更多还原

【Abstract】 Objective:To construct Spodoptera exigua nucleopolyhedrovirus(SeMNPV) open reading frame 100 and 101 prokaryotic expression vectors,and to express and purify fusion proteins.Methods:Se100 and Se101 genes were amplified by PCR,and then PCR products were cloned into the expression vector pQE-30 and transformed into Escherichia coli M15[pREP-4].The target proteins were expressed with induction of IPTG and purified by Ni-NTA metal chelation chromatography.The expressed fusion protein was separated on SDS-PAGE.Results: The expression vectors pQE100 and pQE101 were constructed.SDS-PAGE showed that these two fusion proteins had a molecular weight 15 kDa and 31 kDa respectively,which were a little larger than expected size.Additionally,Ni-NTA affinity chromatography analysis showed that both 6×His-Se100 and 6×His-Se101 existed mainly in the elution buffer at pH 4.5.Conclusion:In this study,Se100 and Se101 were cloned and expressed in Escherichia coli and fusion proteins were successfully purified under denaturing conditions.This lays a foundation for the further research of the gene functions.更多还原