The major envelope glycoprotein E of dengue (DEN) virus plays a central role in the biology of flaviviruses. It is capable of inducing a protective immune response in vivo and responsible for the viral binding to the ...The major envelope glycoprotein E of dengue (DEN) virus plays a central role in the biology of flaviviruses. It is capable of inducing a protective immune response in vivo and responsible for the viral binding to the cellular receptor. The crystal structures of glycoprotein E ectodomains have already been determined. However, it is still un-clear where the well-defined B-cell epitopes for glycoprotein E which induce the neutralizing an-tibodies locates. Thus, in order to characterize the role of glycoprotein E in the pathogenesis of dengue virus infection, we first used network servers (http://bio.dfci. harvard.edu/Tools/ &http://www. imtech. res. in) to predict and analyze the well defined B-cell and T-cell epitopes of the glycoprotein of the DEN-1 HAWAII strain. Then based on the highly conserved envelop glyco-protein amino acids, the hydrophilicity, antigenic-ity, accessibility and flexibility of envelop glyco-protein E were further predicted by using Biotic softwares (DNASTAR) and network servers (http://bio. dfci.harvard.edu/Tools/), the secondary structure was putatively obtained. In our study, the sequence at 281-295 amino acid (aa) for den-gue virus type 1 HAWAII strain and the sequence at 345-359, 383-397 for dengue virus type 2 NGC strain were predicted as the more prevalent epi-topes by using multiple parameters and different analysis softwares, respectively. Two epitopes of DEN-2 and one of DEN-1 locate on the domain Ш and domainⅡ of the protein E, respectively. Sub-sequently, further studies will be carried out to examine the antigenicity and protection of the synthetic peptides with higher scores in the av-erage antigen index (AI) and better hydrophilic properties determined by our data.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The major envelope glycoprotein E of dengue (DEN) virus plays a central role in the biology of flaviviruses. It is capable of inducing a protective immune response in vivo and responsible for the viral binding to the cellular receptor. The crystal structures of glycoprotein E ectodomains have already been determined. However, it is still un-clear where the well-defined B-cell epitopes for glycoprotein E which induce the neutralizing an-tibodies locates. Thus, in order to characterize the role of glycoprotein E in the pathogenesis of dengue virus infection, we first used network servers (http://bio.dfci. harvard.edu/Tools/ &http://www. imtech. res. in) to predict and analyze the well defined B-cell and T-cell epitopes of the glycoprotein of the DEN-1 HAWAII strain. Then based on the highly conserved envelop glyco-protein amino acids, the hydrophilicity, antigenic-ity, accessibility and flexibility of envelop glyco-protein E were further predicted by using Biotic softwares (DNASTAR) and network servers (http://bio. dfci.harvard.edu/Tools/), the secondary structure was putatively obtained. In our study, the sequence at 281-295 amino acid (aa) for den-gue virus type 1 HAWAII strain and the sequence at 345-359, 383-397 for dengue virus type 2 NGC strain were predicted as the more prevalent epi-topes by using multiple parameters and different analysis softwares, respectively. Two epitopes of DEN-2 and one of DEN-1 locate on the domain Ш and domainⅡ of the protein E, respectively. Sub-sequently, further studies will be carried out to examine the antigenicity and protection of the synthetic peptides with higher scores in the av-erage antigen index (AI) and better hydrophilic properties determined by our data.
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。
