狂犬病病毒糖蛋白膜外区表达及中和抗体间接ELISA检测方法的建立  

Development of Indirect ELISA for the Detection of Neutralizing Antibodies against Rabies Virus Based on Expression of Ectodomain of Glycoprotein

作　　者：高志强[1] 谷强[1] 张鹤晓[1] 赖平安[1] 柏亚铎[1] 蒲静[1] 张伟[1] 乔彩霞[1] 汪琳[1] 吴丹[1] 

机构地区：[1]北京出入境检验检疫局,北京101113

出　　处：《畜牧兽医学报》2009年第10期1514-1520,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：北京市科技计划:国家科技计划项目衔接-科技奥运专项(Z07001000560715)

摘　　要：采用RT-PCR方法克隆了狂犬病病毒CVS株G蛋白基因编码区,进而将完整编码区和膜外区编码基因分别克隆于pET-32a,将其中含膜外区编码基因的重组质粒pET-32a-PG转化BL21(DE3),经1.0 mmol.L-1IPTG诱导,外源基因获得高效表达。通过Western-blot以及ELISA试验证明表达产物具有良好的反应原性。以纯化后的表达产物作为抗原包被酶标板,用已知中和抗体效价的OIE参考血清对该方法进行了标准化,建立了检测狂犬病病毒中和抗体的间接ELISA方法。结果表明,抗原的最佳包被量为3.74μg,血清的最佳稀释度为1:100,待检血清阳性临界值为0.50。用此方法检测了418份血清样品,并与荧光抗体病毒中和试验(FAVN)方法进行了比较,符合率为98.61%。A DNA fragment, which encodes the glycoprotein of Rabies virus （RV） CVS strain, was amplified by RT-PCR. The ectodomain and complete glycoprotein genes were cloned into pET-32a respectively. Of two recombinant plasmids, only the ectodomain protein was expressed in very high level after the recombinant pET-32a-PG plasmid transformed into BL21（DE3） when induced with 1.0 mmol .L^-1 IPTG. The reactiongenicity of the expressed product was demonstrated by Western-blot and ELISA. By coating plates with purified recombinant protein as antigen, an indirect ELISA for the detection of neutralizing antibodies against RV was established, and was standardized further with titer-known OIE reference serum. The results obtained indicated that the amount of optimum coated antigen was 3.74 μg, and optimum serum dilution was 1： 100. The cut off value was determined as 0.50. By parallel detecting a total of 418 serum samples with developed ELISA and fluorescent antibody virus neutralization （FAVN）, the calculated coincidence rate was 98.61%.

关 键 词：狂犬病病毒 糖蛋白 膜外区表达 中和抗体 ELISA 

分 类 号：S852.659.5[农业科学—基础兽医学]