猪脑心肌炎病毒2A蛋白的原核表达与纯化  

Prokaryotic Expression and Purification of 2A Protein of Porcine Encephalomyocarditis Virus

作　　者：韩若婵 梁瑞英[1] 左玉柱 萧飒[2] 崔尚金[1] HAN Ruochan;LIANG Ruiying;ZUO Yuzhu;XIAO Sa;CUI Shangjin(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Department of Animal Husbandry and Veterinary Medicine,Baoding Vocational and Technical College,Baoding 071001,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]西北农林科技大学动物医学院,杨凌712100 [3]保定职业技术学院畜牧兽医系,保定071001 [4]河北农业大学动物医学院,保定071001

出　　处：《中国畜牧兽医》2020年第11期3454-3459,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金面上项目“猪脑心肌炎病毒2A蛋白调控宿主细胞凋亡分子机制研究”(31873019);河北省重点研发计划项目(19226622D)。

摘　　要：本试验对猪脑心肌炎病毒(encephalomyocarditis virus,EMCV)2A蛋白进行原核表达和纯化。采用大肠杆菌原核表达系统,将EMCV 2A基因插入原核表达载体pET-28a-sumo中构建重组原核表达质粒pET28a-EMCV-2A,经PCR和测序鉴定无误。将重组原核表达质粒转化至大肠杆菌BL21(DE3)感受态细胞中,超声破碎后,2A蛋白的表达主要以包涵体形式存在。通过优化蛋白表达条件,使目的蛋白能够实现可溶性表达。利用磁珠纯化方法对重组蛋白进行纯化,并以SDS-PAGE和Western blotting进行双重鉴定。结果显示,本试验成功构建2A的重组表达质粒;重组蛋白分子质量约为25 ku,与预期大小一致;IPTG浓度1.0 mmol/L、16℃低温诱导16 h为最佳诱导条件,约有50%的蛋白呈现可溶性表达;纯化后获得2A重组蛋白。本试验通过对EMCV 2A蛋白的原核表达及纯化,成功获得纯度较高的EMCV 2A蛋白,为进一步阐明EMCV的分子致病机制以及研发基因疫苗和抗病毒药物奠定基础。In this study,encephalomyocarditis virus 2A protein was prokaryotic expressed and purified.E.coli was used to express foreign proteins,the EMCV 2A gene was inserted into the prokaryotic expression vector pET-28a-sumo to obtain the recombinant plasmid pET28a-EMCV-2A and confirmed by PCR and sequencing.The plasmid was transformed into E.coli BL21(DE3).After ultrasound fragmentation,the expression of 2A protein was mainly in the form of inclusion bodies.By optimizing the protein expression conditions,the soluble expression of the target protein could be achieved.The recombinant protein was purified by beads and identified by SDS-PAGE and Western blotting.The results showed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant 2A protein was about 25 ku.The best condition was 16℃with 1.0 mmol/L IPTG,about 50%of the proteins were soluble.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and indicated that the purified protein was the recombinant protein.The study provided the basic theoretical basis for elucidating the molecular pathogenesis of EMCV and preparation of gene vaccines and antiviral drugs.

关 键 词：脑心肌炎病毒(EMCV) 2A蛋白 原核表达 纯化 

分 类 号：S855.3[农业科学—临床兽医学]