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
To construct an expression vector containing the E1 glycoprotein gene of rubella virus for the study on the effect of mutation of the E1 gene glycoprotein and the analysis of phylogenetic differences of sequences, the...To construct an expression vector containing the E1 glycoprotein gene of rubella virus for the study on the effect of mutation of the E1 gene glycoprotein and the analysis of phylogenetic differences of sequences, the gene encoding the E1 envelope glycoprotein was amplified from rubella virus, Jinan strain JR23, by RT-PCR and ligated into PMD-18T vector. The clones that carried the E1 gene were identified after amp r selection and analysis of restriction enzyme digestion. After sequencing this gene was analyzed by Danstar and Winstar programs, and the map of phylogenetic tree was drawn. The clone of E1 glycoprotein was thus constructed. It was found that the sequence differences between JR23 strain and the TCRB strain from Japan and those between JR23 strain and Thomas strain of England were rather small with difference values of 0.9% and 1.2% respectively. Yet those between JR23 strain and BRD2 strain from Beijing and those between JR23 strain and XG379 strain from Hong Kong were comparatively larger with difference values of 7.6% and 7.3% respectively. The sequence of JR23 strain with other strains was less than 3% except the NC strain (3.7%). It concludes that the construction of E1 glycoprotein gene offers an approach to study the relationship between structures and functions of E1 gene and its gene products. In the phylogenetic tree, it shows that there are significant differences in the sequences of rubella virus isolated in China, and this might be helpful to develop an effective subunit vaccine.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
基金This study was supported by grants from the Natural Science Foundationof Shandong Province (No.Q99C10) and Key University Teachers of Educa tion Ministry, China
文摘To construct an expression vector containing the E1 glycoprotein gene of rubella virus for the study on the effect of mutation of the E1 gene glycoprotein and the analysis of phylogenetic differences of sequences, the gene encoding the E1 envelope glycoprotein was amplified from rubella virus, Jinan strain JR23, by RT-PCR and ligated into PMD-18T vector. The clones that carried the E1 gene were identified after amp r selection and analysis of restriction enzyme digestion. After sequencing this gene was analyzed by Danstar and Winstar programs, and the map of phylogenetic tree was drawn. The clone of E1 glycoprotein was thus constructed. It was found that the sequence differences between JR23 strain and the TCRB strain from Japan and those between JR23 strain and Thomas strain of England were rather small with difference values of 0.9% and 1.2% respectively. Yet those between JR23 strain and BRD2 strain from Beijing and those between JR23 strain and XG379 strain from Hong Kong were comparatively larger with difference values of 7.6% and 7.3% respectively. The sequence of JR23 strain with other strains was less than 3% except the NC strain (3.7%). It concludes that the construction of E1 glycoprotein gene offers an approach to study the relationship between structures and functions of E1 gene and its gene products. In the phylogenetic tree, it shows that there are significant differences in the sequences of rubella virus isolated in China, and this might be helpful to develop an effective subunit vaccine.
