狂犬病毒G蛋白基因的克隆、表达及生物学活性鉴定  

Cloning and expression of G protein gene in rabies virus and biological activity analysis

作　　者：冯晓敏[1] 卞颖华[2] 徐伟[3] 刘新建[1] 朱进[1] 管晓虹[1] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室病理学系,江苏南京210029 [2]南京医科大学卫生部抗体技术重点实验室生物技术系,江苏南京210029 [3]南京医科大学第二附属医院消化内科,江苏南京210011

出　　处：《南京医科大学学报（自然科学版）》2011年第7期986-990,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家"863"计划资助(2007AA02Z418)

摘　　要：目的:将狂犬病毒G蛋白(rabies virus Gprotein,RVG)基因片段克隆到原核表达载体中,表达并纯化GST融合G蛋白,为研制狂犬病新型ELISA抗体检测试剂盒提供诊断抗原。方法:根据GenBank发表的狂犬病病毒CVS-11株G蛋白结构基因序列设计引物,利用RT-PCR扩增出RVG基因片段,并定向克隆于pGEX-6P-1载体中,构建原核表达载体pGEX-RVG。阳性重组质粒转化宿主菌BL21(DE3),IPTG诱导表达,通过对表达条件的优化,确定可溶性表达的最佳条件。利用谷胱甘肽转移酶(GST)亲和层析法获得纯化的目的蛋白,Western blot对表达的蛋白进行鉴定。结果:构建了原核表达载体pGEX-RVG,经IPTG诱导后可表达分子量约36 000融合蛋白,经纯化获得高纯度的目的蛋白。Western blot检测表明重组的融合蛋白有较好的生物学活性。结论:成功表达并纯化狂犬病毒G蛋白,为进一步研制狂犬病新型ELISA抗体检测试剂盒提供抗原。Objective：To clone the rabies virus G protein（RVG） gene fragments into prokaryotic expressing vector,and to express and purify RVG,which could be applied as diagnostic antigen for indirect ELlSA assay to detect rabies virus antibodies.Methods：The G gene of rabies virus was amplified by RT-PCR with a pair of specific primers designed according to the relevant sequence of GenBank.The PCR product was cloned into prokaryotic expression vector pGEX-6P-1 to construct the expression plasmid pGEX-RVG.The positive recombinant plasmid was transformed into host bacteria E.coli BL21（DE3） and induced with IPTG.In order to determine the optimal induction condition for soluble expression,optimization condition was studied.The glutathione S-transferase（GST） affinity chromatography was used to purify the recombinant protein,and the biological activity of RVG was analyzed by Western blot.Results：The prokaryotic expression vector pGEX-RVG were successfully constructed.After inducted by IPTG,the fusion protein about 36 000 was observed,which was the same as expected.The recombinant fusion protein has a fine biological activity which was confirmed by Western blot.Conclusion：The rabies virus G protein was successfully expressed and purified,which could be provided diagnostic antigen for indirect ELlSA assay for the detection of rabies virus antibodies.

关 键 词：狂犬病毒 G蛋白基因 融合蛋白 可溶性表达 

分 类 号：Q786[生物学—分子生物学]