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

作　　者：赵刚[1,2] 李刚[2] 陈铁桥[1] 范晓娟[2] 

机构地区：[1]湖南农业大学,长沙410000 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国人兽共患病学报》2010年第2期163-167,共5页Chinese Journal of Zoonoses

基　　金：国家高技术研究发展计划"863"计划(2008AA10Z411);中央级公益性科研院所基本业务费专项(0032007008);北京市科委基金项目(Z07010501780701);国家农业行业公益项目(2008-3);联合资助

摘　　要：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.

关 键 词：狂犬病病毒LEP—Flury株 P基因 P蛋白 原核表达 间接ELISA 

分 类 号：R373.9[医药卫生—病原生物学]