To construct the eukaryot ic expression system of E1 glycopr otein of rubella virus(RV)JR23 str ain and to study the biological an d immunological properties of the expressed product,the E1 gene was amplified by RT-PC...To construct the eukaryot ic expression system of E1 glycopr otein of rubella virus(RV)JR23 str ain and to study the biological an d immunological properties of the expressed product,the E1 gene was amplified by RT-PCR,sequenced and recombinated with the pBluescript ⅡSK+ vectorThe recombinated vecto r was co-transfected into BHK21 ce ll with vaccinia virus which carri es the gene of T7 RNA polymeraseT he expressed protein was characteri zed with hemadsorption,immunohisto chemistry and indirect immunofluor escence stainingThe results showe d that the recombinated vector,pBS K+-RV E1,was proved containing E1 gene by enzyme digestion and seque ncingThe expressed protein could be detected both in cytoplasm amd on membrane of the transfected cel ls by hemadsorption,immunohistoche mistry and indirect immunofluoresc enceThe E1 protein mainly focused in cytoplasm near the nucleusThes e data proved that the expression vector,pBSK+-RV E1,was successfull y constructed and the glycoprotein E1 of RV JR23 strain was effective ly expressed in BHK21 cell culture This study provides foundations f or studies on the relationships be tween structure and function of RV gene,between structure and functio n of RV proteins,and on the subuni t vaccine of rubella展开更多
目的真核细胞稳定表达水痘-带状疱疹病毒（VZV）糖蛋白E（g E）的胞外域基因,应用g E抗原建立VZV感染的血清学试验来评价VZV疫苗的免疫效果。方法构建含有VZV g E胞外域基因的真核表达质粒p CDNA3.1-g E,测序后脂质体转染COS-7细胞,经G...目的真核细胞稳定表达水痘-带状疱疹病毒（VZV）糖蛋白E（g E）的胞外域基因,应用g E抗原建立VZV感染的血清学试验来评价VZV疫苗的免疫效果。方法构建含有VZV g E胞外域基因的真核表达质粒p CDNA3.1-g E,测序后脂质体转染COS-7细胞,经G418筛选出稳定表达VZV g E的细胞株。采用RT-PCR法检测VZV g E的mRNA,Western blot和间接免疫荧光法检测g E的免疫反应性,表达产物经Ni2＋-NTA柱纯化后包被ELISA板,对127份0~10岁正常儿童血清中VZV-Ig G抗体水平进行检测。结果成功筛选出能够稳定表达VZV g E胞外域基因的COS-7细胞株,RT-PCR检测到g E的mRNA,经Western blot和间接免疫荧光鉴定,g E具有明显的免疫反应性,COS-7细胞株和其培养上清液中均有g E融合蛋白,表达量约为0.632 mg/ml,纯度约为90%。ELISA实验检测了127份0~10岁儿童血清中VZV-Ig G抗体,总阳性率为81.89%,特异度和灵敏度分别为93.75%和88.24%。结论本实验获得稳定高效表达VZV g E胞外域基因的COS-7细胞株,建立的ELISA血清学检测方法有助于VZV感染的流行病学研究和对易感人群VZV感染的诊断和预防。展开更多
文摘To construct the eukaryot ic expression system of E1 glycopr otein of rubella virus(RV)JR23 str ain and to study the biological an d immunological properties of the expressed product,the E1 gene was amplified by RT-PCR,sequenced and recombinated with the pBluescript ⅡSK+ vectorThe recombinated vecto r was co-transfected into BHK21 ce ll with vaccinia virus which carri es the gene of T7 RNA polymeraseT he expressed protein was characteri zed with hemadsorption,immunohisto chemistry and indirect immunofluor escence stainingThe results showe d that the recombinated vector,pBS K+-RV E1,was proved containing E1 gene by enzyme digestion and seque ncingThe expressed protein could be detected both in cytoplasm amd on membrane of the transfected cel ls by hemadsorption,immunohistoche mistry and indirect immunofluoresc enceThe E1 protein mainly focused in cytoplasm near the nucleusThes e data proved that the expression vector,pBSK+-RV E1,was successfull y constructed and the glycoprotein E1 of RV JR23 strain was effective ly expressed in BHK21 cell culture This study provides foundations f or studies on the relationships be tween structure and function of RV gene,between structure and functio n of RV proteins,and on the subuni t vaccine of rubella
文摘目的真核细胞稳定表达水痘-带状疱疹病毒（VZV）糖蛋白E（g E）的胞外域基因,应用g E抗原建立VZV感染的血清学试验来评价VZV疫苗的免疫效果。方法构建含有VZV g E胞外域基因的真核表达质粒p CDNA3.1-g E,测序后脂质体转染COS-7细胞,经G418筛选出稳定表达VZV g E的细胞株。采用RT-PCR法检测VZV g E的mRNA,Western blot和间接免疫荧光法检测g E的免疫反应性,表达产物经Ni2＋-NTA柱纯化后包被ELISA板,对127份0~10岁正常儿童血清中VZV-Ig G抗体水平进行检测。结果成功筛选出能够稳定表达VZV g E胞外域基因的COS-7细胞株,RT-PCR检测到g E的mRNA,经Western blot和间接免疫荧光鉴定,g E具有明显的免疫反应性,COS-7细胞株和其培养上清液中均有g E融合蛋白,表达量约为0.632 mg/ml,纯度约为90%。ELISA实验检测了127份0~10岁儿童血清中VZV-Ig G抗体,总阳性率为81.89%,特异度和灵敏度分别为93.75%和88.24%。结论本实验获得稳定高效表达VZV g E胞外域基因的COS-7细胞株,建立的ELISA血清学检测方法有助于VZV感染的流行病学研究和对易感人群VZV感染的诊断和预防。
