新型鹅细小病毒VP2蛋白的原核可溶性表达及优化  

Prokaryotic soluble expression and optimization of novel goose parvovirus VP2 protein

作　　者：向凤君 刘琳敏 雷白时 杨颖 郭笑然 路文斌 田甜 张武超 赵款 袁万哲 XIANG Feng-jun;LIU Lin-min;LEI Bai-shi;YANG Ying;GUO Xiao-ran;LU Wen-bin;TIAN Tian;ZHANG Wu-chao;ZHAO Kuan;YUAN Wan-zhe(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding 071001,China;North China Observation and Experimental Station of Animal Disease Pathogen Biology,Ministry of Agriculture and Rural Affairs,Baoding 071001,China)

机构地区：[1]河北农业大学动物医学院 [2]河北省兽医生物技术创新中心 [3]农业农村部动物疫病病原生物学华北科学观测实验站

出　　处：《中国兽医科学》2023年第10期1285-1291,共7页Chinese Veterinary Science

基　　金：河北省自然基金面上项目(C2021204028);河北农业大学科研发展基金计划项目(JY2022026)。

摘　　要：本研究利用基因工程技术将VP2编码基因克隆至载体pET-32a(+)中,成功构建重组质粒pET-32a-VP2,通过表达条件摸索发现VP2为包涵体表达。为了实现VP2蛋白的可溶性表达,制备含有能表达分子伴侣蛋白groES-groEL-tig的质粒pG-Tf2和重组质粒pET-32a-VP2双质粒表达系统。通过对双质粒表达系统表达条件的探索,发现在IPTG为0.25 mmol/L、四环素碱为250μg/L、诱导时间12 h、诱导温度20℃时可溶性表达效果最佳,可溶性蛋白占总表达量80%。经过Western-blot分析,证实重组蛋白具有良好反应原性。结果表明,利用分子伴侣与NGPV VP2蛋白的共表达系统,实现了新型鹅细小病毒(NGPV)VP2在大肠杆菌中的可溶性表达,为检测方法的建立和亚单位疫苗的研发奠定基础。The VP2 encoding gene was cloned into the vector pET-32a(+)by genetic engineering technology to obtain the recombinant plasmid pET-32a-VP2.However,the expression of these recombinant protein using a variety of expression induction conditions invariably resulted in inclusion bodies.To achieve the soluble expression of VP2 protein,the chaperone co-expression system of plasmid p G-Tf2 to express the molecular chaperone gro ES-gro EL-tig and recombinant plasmid pET-32a-VP2 were prepared.The optimal conditions for the soluble expression of the VP2 protein were confirmed,inducing for 12 h at 20℃with 0.25 mmol/L IPTG and 250μg/L tetracycline when the soluble VP2 protein accounted for 80%of the total expression.The target protein was identified by Western-blot as having good antigenicity.This study successfully obtained the soluble expression of NGPV VP2 in Escherichia coli,which laid the foundation for the establishment of detection methods and the development of subunit vaccines.

关 键 词：新型鹅细小病毒 分子伴侣 可溶性表达 原核表达 

分 类 号：S852.659.2[农业科学—基础兽医学]