文章摘要
冯晓敏,卞颖华,徐 伟,刘新建,朱 进,管晓虹.狂犬病毒G蛋白基因的克隆?表达及生物学活性鉴定[J].南京医科大学学报,2011,(7):986~990
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
投稿时间:2011-01-24  
DOI:10.7655
中文关键词: 狂犬病毒  G蛋白基因  融合蛋白  可溶性表达
英文关键词: rabies virus  G protein gene  fusion protein  soluble expression
基金项目:国家“863”计划资助(2007AA02Z418)
作者单位
冯晓敏 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 
卞颖华 南京医科大学生物技术系,江苏 南京 210029 
徐 伟 南京医科大学第二附属医院消化内科,江苏 南京 210011 
刘新建 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 
朱 进 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 
管晓虹 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 
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中文摘要:
      目的:将狂犬病毒G蛋白(rabies virus G protein, 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?
英文摘要:
      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.
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