人7型腺病毒Hexon蛋白原核表达及蛋白纯化  

Prokaryotic expression and purification of Hexon gene of Human Adenovirus-7

作　　者：方瑞康 肖妍 李菁菁 金宁一 鲁会军 李一权 高旭[1] 韩继成[2,3] FANG Ruikang;XIAO Yan;LI Jingjing;JIN Ningyi;LU Huijun;LI Yiquan;GAO Xu;HAN Jicheng(Department of Veterinary Medicine,Agricultural College,Yanbian University,Yanbian 133000,Jilin,China;Academician Workstation,Changchun University of Chinese Medicine;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences)

机构地区：[1]延边大学农学院动物医学系,吉林延边133000 [2]长春中医药大学科研处院士工作室 [3]中国农业科学院长春兽医研究所

出　　处：《中国病原生物学杂志》2024年第9期1004-1008,共5页Journal of Pathogen Biology

基　　金：吉林省青年科技人才托举工程项目(No.QT202228);吉林省教育厅科学技术研究项目(JJKH20241039KJ)。

摘　　要：目的将人7型腺病毒(Human Adenovirus-7,HAdV-7)六邻体蛋白(Hexon)通过原核表达系统进行表达,筛选不同时间、浓度、温度诱导下的最佳表达条件,并进行蛋白纯化研究。方法通过PCR扩增获得Hexon目的基因,并将目的基因克隆至原核表达载体pET30a(+),构建pET30a-Hexon重组质粒,转化至宿主菌BL21(DE3)。经IPTG诱导表达,筛选蛋白表达条件,利用His标签镍离子蛋白纯化柱纯化蛋白。结果RT-PCR结果显示可扩增出2805 bp Hexon目的片段,将Hexon目的基因成功克隆至pET30a(+)载体,转化至BL21(DE3),成功构建pET30a-Hexon重组蛋白;筛选出pET30a-Hexon重组蛋白的最佳表达条件为34℃、1 mmol/L诱导剂诱导8 h,重组蛋白以包涵体的形式进行表达,通过His标签镍离子纯化柱可纯化出目的蛋白。结论成功表达pET30a-Hexon重组蛋白,筛选出最佳诱导条件并纯化出Hexon目的蛋白,为研究制备单克隆抗体及新型疫苗的开发提供了理论依据。Objective To express the six-neighbourhood protein(Hexon)of Human Adenovirus-7(HAdV-7)through a prokaryotic expression system,to screen the optimal expression conditions under different time,concentration,and temperature induction,and to study the protein purification.Methods The target gene of Hexon was obtained by PCR amplification,and the target gene was cloned into the prokaryotic expression vector pET30a(+),constructed the pET30a-Hexon recombinant plasmid,and transformed into the host bacterium BL21(DE3).The expression was induced by IPTG,the protein expression conditions were screened,and the protein was purified using His-tagged nickel ion protein purification column.Results RT-PCR results showed that 2805 bp Hexon target fragment could be amplified,and the Hexon target gene was successfully cloned into pET30a(+)vector,transformed into BL21(DE3),and pET30a-Hexon recombinant protein was successfully constructed;the optimal expression conditions for the pET30a-Hexon recombinant protein were screened to be 34℃,1 mmol/L inducer induced for 8 h,and the recombinant protein was expressed as inclusion bodies,and the target protein could bepurified by His-tagged nickel ion purification column.Conclusion In this experiment,pET30a-Hexon recombinant protein was successfully expressed,and the optimal induction conditions were screened and the target protein of Hexon was purified,which provided a theoretical basis for the study of preparing monoclonal antibodies and the development of novel vaccines.

关 键 词：人7型腺病毒 HEXON 原核表达 蛋白纯化 

分 类 号：R392-33[医药卫生—免疫学]