牛传染性鼻气管炎病毒gB基因的截短表达与抗原反应性分析  

Expression and identification of truncated GB protein of bovine rhinotracheitis virus

作　　者：石建平[1,2] 孟日增[2] 马海利[3] 牛得料[3] 肖成蕊[2] 刘晶[2] 丁壮[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林出入境检验检疫局,吉林长春130062 [3]山西农业大学动物科技学院,山西太谷030801

出　　处：《中国兽医学报》2010年第10期1313-1317,共5页Chinese Journal of Veterinary Science

基　　金：国家质检总局科研项目(2006IK009)

摘　　要：根据牛传染性鼻气管炎病毒(IBRV)全基因组序列设计合成gB基因的特异性引物,采用PCR方法扩增IBRgB基因,并将其克隆到pMD18-T载体。用DNA Star分析IBR gB基因编码的吸附蛋白抗原性、亲水性和表面展示概率,将gB基因截短成4个片段,用Oligo 6设计4对引物,并以pMD18-T-gB为模板,用PCR方法分别扩增出gB1、gB2、gB3和gB44个基因片段,其大小分别为402、312、306和318 bp,分别定向插入到pGEX-6p-1表达载体中,转入表达菌BL21中,经IPTG诱导表达。SDS-PAGE电泳表明,表达产物以包涵体形式存在,融合蛋白大小约为40 300、39 000、38 700和37 800,与预测值相符。利用GSTrap HP层析柱纯化重组蛋白,经Western-blot和ELISA鉴定4种融合蛋白中gB3和gB4有较好的抗原反应性和特异性。The total IBRV gB gene was amplified by PCR and connected to the cloning vector pMD18-T.It was named pMD18-T-gB.Four pairs of primers for amplification antigenic sites were designed and synthesized in accordance with DNAstar biology software based on the IBRV Bartha Nu/67 gB gene published on GenBank.EcoRⅠand SalⅠwere inse ted in primers.Four antigenic sites were amplified using recombinant pMD18-T-gB as a template,fragments of four antigenic sites gene were connected to the pMD18-T cloning vector and expression vector pGEX-6p-1.Recombinant expression plasmids were induced by IPTG and successful expression of the four recombinant proteins.The results of SDS-PAGE electrophoresis showed that the four target fusion proteins mainly existed in the form of inclusion bodies,with its size of 40 300,39 000,38 700 and 37 800 approximately.All of four recombinant proteins were purified by using GSTrap HP chromatography and identified using the methods of Western blot and indirect ELISA.The gB3 and gB4 recombinant proteins had better antigenic reactivity and specificity.

关 键 词：牛传染性鼻气管炎病毒 GB基因 截短表达 抗原性分析 

分 类 号：S852.653[农业科学—基础兽医学]