白纹伊蚊中肠组织和C6/36细胞与登革病毒相互作用蛋白的筛选

摘要

目的 筛选登革Ⅱ型病毒与白纹伊蚊中肠组织和C6/36细胞中的连接分子.方法 富集纯化登革Ⅱ型病毒,用12%SDS-PAGE分析提取的C6/36细胞膜蛋白和白纹伊蚊中肠组织总蛋白,将分离胶上的蛋白转于硝酸纤维素膜(PVDF)上,应用病毒覆盖蛋白结合试验(VOPBA)方法筛选白纹伊蚊中肠组织和C6/36细胞中表达连接登革Ⅱ型病毒的分子.结果 白纹伊蚊中肠组织总蛋白经12% SDS-PAGE分离后转膜,分别与纯化的病毒液和阴性对照液孵育,用抗登革病毒单克隆抗体检测,结果显示病毒孵育组在分子量30 kDa的位置有特异性的条带出现,而阴性对照组无此条带;用同样的方法筛选C6/36细胞上表达蛋白,病毒孵育组在分子量30 kDa和40 kDa的位置出现特异条带,而阴性对照组无.结论 VOPBA结果显示分子量为30 kDa的分子在中肠组织与C6/36细胞中都能表达,具有连接登革Ⅱ型病毒的作用,对其氨基酸序列的鉴定和功能分析工作正在进行中.%To screen the molecules binding dengue Ⅱ virus expressed in Aedes albopiclus midgut tissue and C6/36 cells, C6/36 cells were infected with dengue Ⅱ virus, and the virus were collected and purified. The total proteins of Aedes albopiclus midgut tissue and membrane proteins of C6/36 cells were extracted and analyzed using12% SDS-polyacrylamide gel (PAGE). After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and virus overlay protein binding assay (VOPBA) was carried out using an anti-dengue virus monoclonal antibody; one specific band of 30 kDa occurred after VOPBA of the proteins from Aedes albopiclus midgut tissue incubated with the virus, while the negative control group did not show this specific band. Two specific bands of 30 kDa and 40 kDa occurred after VOPBA of the proteins from the cells incubated with the virus, while the negative control group did not show these specific bands; the 30 kDa molecular binding with dengue type 2 was successfully expressed in both Aedes albopiclus midgut tissue and C6/36 cell, and its identification of amino acid sequence and biological functions is in progress.