【摘要】 目的采用原核表达系统获得HCMV pp65目的蛋白,免疫家兔制备该蛋白特异性抗血清。方法构建表达GST-HCMV pp65融合蛋白的质粒,在大肠杆菌中诱导表达,经GST亲和层析纯化后的pp65蛋白免疫家兔,获得兔多克隆抗血清。采用ELISA、SDS-PAGE电泳及Western blot检测纯化后的蛋白,并以间接免疫荧光染色法检测鉴定该蛋白的抗原性。结果成功构建PGEX-5X-pp65重组质粒,转化E.coli BL21(DE3)感受态细胞,获得可溶性的融合蛋白;GST亲和层析纯化后的蛋白免疫家兔获得特异性抗血清。结论在原核细胞中成功获得了融合蛋白pp65,免疫家兔后获得具有高度特异性抗血清。为进一步揭示该蛋白的功能及作用机制奠定了重要基础。更多还原

【Abstract】 Objective Prokaryotic expression system to obtain the purpose of HCMV pp65 protein,preparation of the target protein-specific immune rabbit antiserum.Methods Construction of GST-HCMV pp65 expression plasmid of fusion protein in E.coli-induced expressed,GST affinity chromatography purified,then immuned rabbits with purified pp65 protein to obtain polyclonal rabbit antiserum and use affinity chromatography;we used ELISA,SDSPAGE electrophoresis,Western blot and immunofluorescence staining identified.Results Prokaryotic expression constructed at HCMV pp65 functional domain,PGEX-5X-pp65 had been successfully generated,which expressed this recombinant protein in the main form of solubility fusion protein.After the affinity chromatograph of GST,using this recombinant protein to immunize rabbit,we got specific polyclonal antiserum after the affinity chromatograph.Conclusion We have obtained the solubility fusion protein of HCMV pp65,and after immunizing rabbit obtaining specific polyclonal antiserum,which can be used to detect the interest protein HCMV pp65.更多还原