【作者】 赵刚；

【导师】 陈铁桥； 李刚；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2010， 硕士

【摘要】 本试验旨在建立一种检测狂犬病抗体的方法。通过传代培养BHK21细胞扩增狂犬病毒LEP-Flury株病毒,并根据GenBank发表的狂犬病病毒(Rabies Virus, RV) LEP-Flury株的基因序列设计一对引物,通过RT-PCR扩增得到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,阳性重组表达质粒转化原核表达宿主菌BL21(DE3),用IPTG诱导表达P蛋白。SDS-PAGE和Western-blot分析确定蛋白表达量和特异性。使用镍琼脂糖凝胶FF对P蛋白进行纯化,纯化的蛋白作为诊断抗原,通过对反应条件的优化,初步建立间接ELISA方法用于检测狂犬病抗体。结果扩增得到了RV P基因,构建了克隆质粒pGM-T-P和原核表达质粒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蛋白建立了用于检测RV抗体的间接ELISA方法。更多还原

【Abstract】 In order to established a kind of method to detect the antibody of rabies virus.Amplied rabies virus of LEP-Flury strain by BHK21.The complete length of P gene from rabies virus was amplified by RT-PCR using a pair of specific 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-digested by NotI and EcoRI, the product was transferred into prokaryotic expression vetor pET-32a (+) to abstain the prokaryotically expressed plasmid pET-32a-P. The target gene was then expressed in the E.coli BL21(DE3) cell by inducted with IPTG. 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, indirect ELISA for the detection of rabies virus antibodies in canine serum was applied after management of the optional woking condition.更多还原