GST pull-down验证新城疫病毒基质蛋白与鸡importinβ1蛋白的相互作用  

Characterization of the Interaction between Newcastle Disease Virus Matrix Protein and Chicken Importin β1 Protein by GST Pull-down Assay

作　　者：胡焱[1] 段志强[1,2] 嵇辛勤[1,2] 赵佳福[1,2] 邓珊珊 李世静 熊建民[1] HU Yan;DUAN Zhiqiang;JI Xinqin;ZHAO Jiafu;DENG Shanshan;LI Shijing;XIONG Jianmin(College of Animal Science,Guizhou University,Guiyang 550025,China;Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountainous Region,Ministry of Education,Guizhou University,Guiyang 550025,China;Bureau of Agriculture,Huaxi District,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [3]贵阳市花溪区农业局,贵阳550025

出　　处：《畜牧兽医学报》2019年第1期126-133,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31502074;31760732);贵州省科技计划项目(黔科合平台人才[2017]5788号);贵州省农业攻关项目(黔科合支撑[2016]2588号);贵州省科学技术基金(黔科合J字[2015]2054号)

摘　　要：旨在利用GST pull-down技术验证新城疫病毒(Newcastle disease virus,NDV)基质(matrix,M)蛋白与鸡importinβ1蛋白在体外的相互作用。根据NDV ZJ1株M基因和鸡importinβ1基因序列设计引物,通过PCR扩增获得NDV M基因和鸡importinβ1基因,将其分别定向插入到原核表达载体pGEX-6p-1和pET-32a(+)构建重组原核表达载体pGEX-6p-M、pET-32a-importinβ1,然后转化至大肠杆菌BL21(DE3)后进行IPTG诱导表达,SDSPAGE检测重组蛋白的表达情况,并对包涵体重组蛋白进行变性和复性处理。然后以GST-M重组蛋白为诱饵蛋白,His-importinβ1重组蛋白为猎物蛋白,利用GST pull-down技术验证M蛋白与importinβ1蛋白的相互作用。结果表明,成功构建了重组原核表达载体pGEX-6p-M和pET-32a-importinβ1,将其转化至大肠杆菌BL21(DE3)获得了正确表达。SDS-PAGE电泳检测显示GST-M重组蛋白主要以包涵体形式存在,而His-importinβ1重组蛋白以可溶性和包涵体两种形式存在。利用蛋白重折叠试剂盒获得了有活性的GST-M重组蛋白,将其作为诱饵蛋白,可以捕获并检测到His-importinβ1重组蛋白,但是GST标签蛋白不能结合His-importinβ1重组蛋白。利用GST pull-down技术证实了NDV M蛋白与鸡importinβ1蛋白在体外具有直接的相互作用,这为深入研究importinβ1蛋白在NDV M蛋白细胞核定位的分子机制以及在NDV复制和致病中的作用奠定了基础。The purpose of this study was to verify the in vitro interaction between Newcastle disease virus(NDV)matrix(M)protein and chicken importinβ1 protein.The products of NDV M gene and chicken importinβ1 gene were obtained by PCR amplification,and then subcloned into the prokaryotic expression vectors pGEX-6p-1 and pET-32a(+)to construct plasmids pGEX-6p-M and pET-32a-importinβ1,respectively.The recombinant proteins were expressed by transforming the plasmids into BL21(DE3)under the condition of IPTG and then analyzed by SDS-PAGE.The obtained inclusion body weight histones were dealt with through denaturation and renaturation to recover activity.Then the interaction between GST-M(bait protein)and His-importinβ1(prey protein)was examined by GST pull-down assay.The results showed that the recombinant prokaryotic expression vectors pGEX-6p-M and pET-32a-importinβ1 were successfully constructed,and the recombinant proteins were correctly expressed in BL21(DE3).However,GST-M existed in the form of inclusion bodies,but His-importinβ1 existed in the form of both solubility and inclusion bodies.The activated GST-M recombinant protein was recovered by protein refolding kit and could capture the His-importinβ1 protein when used as bait protein,while GST alone could not.The in vitro interaction between NDV M protein and chicken importinβ1 protein was verified by GST pull-down assay,which will provide foundation for further studying the role of importinβ1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.

关 键 词：新城疫病毒 基质蛋白 importinβ1蛋白 GSTpull-down 蛋白相互作用 

分 类 号：S855.3[农业科学—临床兽医学]