狂犬病病毒P基因的原核表达及间接ELISA方法的应用

Prokaryotic expression of P gene from rabies virus and application of the indirect ELISA assay in the detection of its gene products

目的以狂犬病病毒P蛋白作为检测抗原,用间接ELISA方法检测狂犬病病毒抗体。方法根据GenBank发表的狂犬病病毒(Rabies Virus,RV)LEP-Flury株的基因序列设计引物,通过RT-PCR扩增出P基因的全长序列,克隆于pGM-T载体中,获得重组质粒pGM-T-P,将重组质粒用限制性内切酶NotI和EcoRI进行双酶切,酶切产物定向克隆于原核表达载体pET-32a(+)中,构建原核重组表达质粒pET-32a-P,阳性重组质粒转化原核表达宿主菌BL21(DE3),用IPTG诱导表达。SDS-PAGE和Western-blot分析确定蛋白表达量和特异性。用纯化的蛋白作为诊断抗原,通过对反应条件的优化,初步将间接ELISA方法应用于狂犬病病毒抗体的检测中。结果扩增RV P基因,构建了克隆质粒pGM-T-P、原核表达质粒pET-32a-P,高效表达了主要以可溶性形式存在的P蛋白,并能与RV阳性血清发生特异性反应。用表达的狂犬病病毒重组P蛋白建立了用于RV抗体检测的间接ELISA方法。结论成功表达了磷蛋白,用其作为固化抗原以间接ELISA方法检测RV抗体。

The complete length of P gene from rabies virus was amplified by RT-PCR using a pair of specifie primers designed according to the relevant sequences from GenBank. The PCR product was cloned into cloning expression vestor pGM-T to obtain the cloning expressed plasmid pGM-T-P. After double-digestion by NotI and EcoRI, the product was transferred into prokaryotic expression vetor pET-32a（＋ ）to obtain the prokaryotically expressed plasmid pET B2a-P. The target gene was then expressed in the E. coli BL21（DE3） cell with IPTG induction. The highest expression of target protein was analysed by SDS- PAGE, and the good immunoreactivity to rabies virus antibodies was proved by Western-blot analysis. By using purified protein, the indirect ELISA assay for the detection of rabies virus antibodies in canine serum was applied after management of the optional working condition.