摘要
目的利用原核表达系统,表达了GⅡ.2型诺如病毒VP1蛋白,优化诱导条件并进行蛋白的纯化。方法扩增诺如病毒的VP1基因,克隆至pET32a(+)载体,构建重组质粒pET32a-VP1。将pET32a-VP1转化至大肠埃希菌BL21(DE3),加入IPTG进行诱导表达并优化诱导条件。利用His标签镍离子蛋白纯化柱纯化表达蛋白,SDS-PAGE电泳鉴定蛋白纯度。结果成功扩增大小为1659 bp的诺如病毒VP1基因。PCR检测重组质粒pET32a-VP1构建成功。将重组质粒转化至BL21(DE3),IPTG诱导表达70×10^3的VP1重组蛋白,优化的最佳诱导表达条件为37℃,0.6 mmol/L IPTG诱导5 h。重组蛋白以包涵体的形式表达,并SDS-PAGE鉴定镍柱纯化产物为单一电泳条带的目的蛋白。结论利用原核表达系统成功构建了重组质粒pET32a-VP1,并表达纯化了诺如病毒VP1重组蛋白,为单克隆及多克隆抗体的制备、检测试剂盒以及新型疫苗的研发奠定了基础。
Objective To use a prokaryotic expression system to express the VP1 protein of GⅡ.2 Norovirus,to optimize the conditions for induction of its expression,and to purify the expressed protein.Methods The VP1 gene of Norovirus was amplified and cloned into a pET32 a(+)vector to construct the recombinant plasmid pET32 a-VP1.PET32 a-VP1 was transformed into Escherichia coli BL21(DE3),IPTG was added to induce expression of the protein,and conditions for induction of its expression were optimized.The expressed protein bearing a His tag was purified on a nickel ion affinity column,and protein purity was determined using SDS-PAGE electrophoresis.Results The Norovirus VP1 gene,1,659 bp in length,was successfully amplified.PCR results indicated that the recombinant plasmid pET32 a-VP1 was successfully constructed.The recombinant plasmid was transformed into BL21(DE3),and expression of 70×10^3 of VP1 recombinant protein was induced with IPTG.The optimal expression conditions were 37℃and 0.6 mmol/L of IPTG for 5 h.The recombinant protein was expressed in the form of inclusion bodies,and SDS-PAGE revealed that the purified product from the nickel ion affinity column was the target protein,which produced a single electrophoretic band.Conclusion The recombinant plasmid pET32 a-VP1 was successfully constructed via a prokaryotic expression system,and the recombinant protein Norovirus VP1 was successfully expressed and purified.These findings have laid the foundation for the preparation of monoclonal and polyclonal antibodies,detection kits,and the development of new vaccines.
作者
李秋璇
韩继成
付婷婷
张金勇
王茂鹏
李金凤
解长占
李卓昕
肖朋朋
鲁会军
金宁一
LI Qiu-xuan;HAN Ji-cheng;FU Ting-ting;ZHANG Jin-yong;WANG Mao-peng;LI Jin-feng;XIE Chang-zhan;LI Zhuo-xin;XIAO Peng-peng;LU hui-jun;JIN Ning-yi(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130000,China;Military Veterinary Institute,Academy of Military Sciences;Medical College,YanBian University;Institute of Virology,Wenzhou University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第7期745-748,754,共5页
Journal of Pathogen Biology
基金
国家科技重大专项"艾滋病和病毒性肝炎等重大传染病防治"课题资助项目(No.2018ZX10102001)。
