摘要
为研究波形蛋白在病毒侵染过程中发挥的作用,原核表达猪波形蛋白与病毒互感后检测其复制能力。根据GenBank中已发表的波形蛋白序列,设计特异性引物,从PK-15细胞中扩增波形蛋白基因,并将其克隆至pET-28a(+)载体,转化大肠埃希菌BL21进行原核表达,表达产物经SDS-PAGE和Western blot鉴定后,进行目的蛋白的纯化及复性,并利用病毒抑制试验,检测其在病毒感染过程中的作用。结果表明,成功地克隆了猪波形蛋白基因,并克隆至pET-28a(+)载体,经诱导后高效表达,经纯化复性获得的波形蛋白抑制猪繁殖与呼吸综合征病毒(PRRSV)侵染Marc-145细胞,然而并不抑制口蹄疫(FMDV)侵染PK-15细胞。随后借助细胞流式术检测PK-15细胞中波形蛋白的分布,显示其主要存在于细胞膜内的基质中。猪源波形蛋白的克隆表达,为进一步研究猪源波形蛋白与口蹄疫病毒蛋白之间的相互作用奠定了基础。
The purpose of this study was to clone and express porcine vimentin and investigate the role of vimentin during virus infection.Specific primers were designed according to the published sequence of vimentin in GenBank.The vimentin gene was amplified from PK-15 cells and cloned into pET-28a (+) vector,and then it was transformed into E.coli BL21 for prokaryotic expression.The expressed products were identified by SDS-PAGE and Western blot.Purification and refolding of the target protein were then carried out.The role of vimentin in virus infection was tested by virus inhibiting assay.The porcine vimentin gene was successfully cloned into pET-28a (+) vector.After induction,the expressed vimentin was purified and renaturated,which inhibited the infection of PRRSV to Marc-145 cells,and did not inhibite FMDV infection to PK-15 cells.The successful cloning and expression vimentin of porcine source laid a foundation for further study of the interaction between vimentin and virus protein of FMDV.
作者
令瑛
马雪青
李平花
张婧
李坤
付元芳
冯若飞
马忠仁
卢曾军
刘在新
LING Ying;MA Xue-qing;LI Ping-hua;ZHANG Jing;LI Kun;FU Yuan-fang;FENG Ruo-fei;MA Zhong-ren;LU Zeng-jun;LIU Zai-xin(College of Life Science and Engineering,Northwest Minzu University,Lanzhou,Gansu,730030,China;State Key Laboratoryof Veterinary Etiological Biology,Key Laboratory of Animal Virology of Ministry of Agriculture,National Foot-and-Mouth DiseaseReference Laboratory,Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Lanzhou,Gansu,730046,China)
出处
《动物医学进展》
北大核心
2019年第1期12-19,共8页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31702266
31460665)
农业部中央级公益性科研院所基本科研业务费项目(1610312016016)
长江学者和创新团队发展计划项目(IRT-17R88)
关键词
猪波形蛋白
原核表达
口蹄疫病毒
PK-15细胞
porcine vimentin
prokaryotic expression
food-and-mouth disease virus
PK-15 cells
