摘要
【目的】对狂犬病毒Rmg基因进行克隆、表达、纯化及复性,以期获得表达量高、反应原性好的Rmg蛋白,为研制快速、直观、简便检测狂犬病毒抗体的胶体金试纸条奠定基础。【方法】用常规PCR从狂犬病毒基因组中克隆出狂犬病毒G蛋白的主要抗原表位基因Rmg,并将其插入原核表达载体pET-Trx中,构建原核表达质粒pET-Trx-Rmg,将pET-Trx-Rmg转化BL21(DE3)plysS,在IPTG诱导下进行表达。【结果】SDS-PAGE分析结果表明,Rmg基因在BL21(DE3)plysS中获得高效表达,表达量占菌体总蛋白的30.5%左右,并主要以不溶的包涵体形式存在,分子量约为51.7kDa。将表达的蛋白以亲和层析法进行纯化并通过谷胱甘肽还原法进行复性,纯化后蛋白纯度达99.1%。经Western blotting检测,发现表达的Rmg蛋白能够与狂犬病毒高免血清发生特异反应,但与犬细小病毒(CPV)、犬瘟热病毒(CDV)阳性血清不发生反应。【结论】Rmg蛋白具有良好的抗原性,可用于研制检测狂犬病毒抗体的胶体金试纸条。
[Objective]The present study was aimed to clone,purify,express the main antigen epitope gene Rmg and to obtain highly expressed and reactive Rmg protein in order to develop collaurum test paper for quick,direct and convenient detection of rabies virus.[Method]The main antigen epitope Rmg gene was cloned from rabies virus genome using RT-PCR and inserted into the prokaryotic expression vector pET-Trx to construct recombinant plasmid pET-Trx-Rmg and its transformation into competent cells BL21（DE3） plysS.The target protein was expressed with IPTG induction and identified on SDS-PAGE.[Result]The Rmg gene showed high expression of protein in BL21（DE3） plysS accounting for about 30.5% of bacterial total protein and presented as inclusion bodies with 51.7 kD of weight.The purity of expressed protein reached 99.1% followed by chromatography purification and glutathione renaturation.There were no specific reactions of expressed Rmg protein with positive serum of CPV or CDV,while it showed specific reaction with RV in Western blotting.[Conclusion]It was suggested that Rmg protein has good antigenicity and can be used for developing collaurum test paper for detection of rabies virus.
出处
《南方农业学报》
CAS
CSCD
2011年第10期1276-1279,共4页
Journal of Southern Agriculture
基金
广西基本科研业务费专项项目(桂科专项11-4)
关键词
狂犬病病毒
Rmg基因
原核表达
纯化
rabies virus
Rmg gene
prokaryotic expression
purification
