葡萄球菌肠毒素A真核表达质粒的构建及其在喉癌Hep-2细胞来源exosomes中的表达  

Construction of staphylococcal enterotoxin A eukaryotic expression plasmid and its expression in laryngocarcinoma Hep-2 cell-derived exosomes

作　　者：田涵斋 吉晓滨[1] 谢景华[1] 刘启才 

机构地区：[1]广州市第一人民医院耳鼻喉科,510180 [2]广州医科大学实验医学研究中心

出　　处：《中华生物医学工程杂志》2017年第3期183-188,共6页Chinese Journal of Biomedical Engineering

基　　金：广东省科技厅产业技术研究与开发资金计划项目（20128031800340）;广州市科技和信息化局社会发展应用基础研究专项（2013J4100024）

摘　　要：目的 构建葡萄球菌肠毒素A（SEA）与糖基化磷脂酰肌醇（GPI）的真核表达质粒pmiR-RB-REPORT-SEA-GPI并转染人喉癌细胞株Hep-2细胞,检测Hep-2细胞分泌的exosomes中SEA的表达.方法 利用NOT I酶切原核生物质粒pGSI-SEA-GPI粘性末端,琼脂糖凝胶电泳分离、提纯目的基因SEA-GPI,利用T4连接酶连接真核表达质粒pmiR-RB-REPORT与SEA-GPI构建真核表达质粒pmiR-RB-REPORT-SEA-GPI.脂质体法转染pmiR-RB-REPORT-SEA-GPI至Hep-2细胞,荧光定量实时PCR（qRT-PCR）检测Hep-2细胞SEA-GPI mRNA的表达.提取Hep-2细胞分泌的exosomes,免疫印迹测定SEA在exosomes中的表达.结果 琼脂糖凝胶电泳显示成功构建真核表达质粒pmiR-RB-REPORT-SEA-GPI.qRT-PCR检测显示,pmiR-RB-REPORT-SEA-GPI转染的Hep-2细胞SEA-GPI mRNA显著表达.免疫印迹显示Hep-2细胞分泌的exosomes中存在SEA蛋白表达.结论 SEA可能通过GPI结构锚定于exosomes膜上,为制备含有SEA-GPI锚定蛋白的SEA-eoxsomes瘤苗提供了依据.Objective To construct pmiR-RB-REPORT-SEA-GPI,the eukaryotic expression plasmid of staphylococcal enterotoxin A （SEA） and glycosylphosphatidylinositol （GPI）,transfect human laryngocarcinoma Hep-2 cells with this plasmid,and determine the expression of SEA in exosomes secreted by Hep-2 cells. Methods The cohesive end of prokaryotic plasmid pGSI-SEA-GPI was digested with NOT I. Separation and purification of the target gene SEA-GPI was completed by agarose gel electrophoresis. The eukaryotic expression plasmid pmiR-RB-REPORT-SEA-GPI was constructed by ligating the eukaryotic expression plasmid pmiR-RB-REPORT with SEA-GPI using T4 ligase. Hep-2 cells were transfected with pmiR-RB-REPORT-SEA-GPI by using lipofectamine method. The expression of SEA-GPI mRNA in Hep-2 cells was detected by fluorescent quantitative real-time PCR（qRT-PCR）. The exosomes secreted by Hep-2 cells were extracted and determined for expression of SEA by Western blotting. Results Agarose gel electrophoresis showed that the eukaryotic expression plasmid pmiR-RB-REPORT-SEA-GPI was successfully constructed. qRT-PCR showed that SEA-GPI mRNA was expressed in Hep-2 cells transfected with pmiR-RB-REPORT-SEA-GPI. Western blotting showed SEA protein expression in exosomes secreted by Hep-2 cells. Conclusion SEA may be anchored to exosomes membrane via GPI structure,which provides a basis for preparation of SEA-eoxsome tumor vaccines containing SEA-GPI anchoring protein.

关 键 词：葡萄球菌肠毒素A 糖基化磷脂酰肌醇 转染 EXOSOMES 荧光定量实时PCR 

分 类 号：R739.65[医药卫生—肿瘤]