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《Journal of Clinical Otorhinolaryngology Head and Neck Surgery》 2013-07
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Construction of the superantigen SEA transfected laryngocarcinoma cells

JI Xiaobin1 LV Jingli2 LIU Qicai2 XIE Jinghua1(1Department of Otorhinolaryngology,Guangzhou First People′s Hospital,Guangzhou,510180,China;2Experimental Medical Research Center,Guangzhou Medical College)  
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.
【Fund】: 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)
【CateGory Index】: R739.65
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