表达超抗原葡萄球菌肠毒素A的喉癌细胞的建立  

Construction of the superantigen SEA transfected laryngocarcinoma cells

作　　者：吉晓滨[1] 吕景礼[2] 刘启才[2] 谢景华[1] 

机构地区：[1]广州市第一人民医院耳鼻咽喉科,广州510180 [2]广州医学院实验医学研究中心

出　　处：《临床耳鼻咽喉头颈外科杂志》2013年第7期376-378,381,共4页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：广东省科技厅产业技术研究与开发资金计划项目(No:2012B031800340);广州市科技局社会发展应用基础研究专项(No:2009JI-C501-2)

摘　　要：目的:构建葡萄球菌肠毒素A(SEA)基因真核表达载体并建立其稳定表达的喉癌细胞系。方法:根据标准葡萄球菌菌株ATCC13565中SEA基因序列,人工合成其编码区序列,然后亚克隆至真核表达载体pIRES2-EGFP,构建pSEA-IRES-EGFP重组质粒,再将重组质粒转染至喉癌Hep-2细胞,G418筛选获得抗性单克隆,用RT-PCR和ELISA法鉴定SEA在喉癌细胞中的表达。结果:合成的SEA基因亚克隆至真核表达载体pires-EGFP后,测序证实克隆的SEA序列与GenBank中标准葡萄球菌菌株ATCC13565的编码区序列完全一致;重组质粒转染喉癌Hep-2细胞后,经筛选2周获得抗性单克隆,挑选单克隆,RT-PCR扩增获得特异的基因片段。经ELISA分析,发现细胞培养上清液中SEA蛋白的含量达pg级水平。结论:成功构建了超抗原SEA基因的重组真核表达载体,转染至喉癌Hep-2细胞后,SEA基因能够在细胞表达并持续分泌SEA蛋白。Objective：To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A（SEA）gene,and to identify its expression in laryngeal squamous carcinoma cells.Method：SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus,referencing standard strains producing SEA.Coding sequence of SEA was artificially synthetized.Than,SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP.The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells.Resistant clones were screened by G418.The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method.Result：The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP.Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank.After recombinant plasmid transfected to laryngocarcinoma cells,the resistant clones was obtained after screening for two weeks.The clones were selected.The specific gene fragment was obtained by RT-PCR amplification.ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level.Conclusion：The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed,and is capable of effective expression and continued secretion of SEA protein in laryngocarcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.

关 键 词：超抗原 葡萄球菌肠毒素A 真核载体 基因克隆 喉肿瘤 

分 类 号：R378.11[医药卫生—病原生物学]