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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 the eukaryotic expression vector of staphylococcal enterotoxin A (SEA) gene and establish its stable expression of laryngeal carcinoma cell line. Methods: According to the sequence of SEA gene of Staphylococcus aureus strain ATCC13565, the coding sequence was synthesized and subcloned into eukaryotic expression vector pIRES2-EGFP to construct recombinant plasmid pSEA-IRES-EGFP. The recombinant plasmid was transfected into laryngeal carcinoma Hep-2 cells were screened by G418 to obtain monoclonal antibodies. The expression of SEA in laryngeal carcinoma cells was identified by RT-PCR and ELISA. Results: The recombinant SEA gene was subcloned into pires-EGFP vector and confirmed by sequencing. The sequence of the SEA sequence was identical with that of the standard strain Staphylococcus aureus ATCC13565 in GenBank. After transfected with Hep-2 cells , Screened for 2 weeks to obtain resistant monoclonal, picked monoclonal, RT-PCR amplification of specific gene fragments. After ELISA analysis, it was found that the cell culture supernatant SEA protein content level pg level. Conclusion: The recombinant eukaryotic expression vector of SEA gene of superantigen was constructed successfully. After transfection into Hep-2 laryngeal squamous cell carcinoma, the SEA gene can express and persistently secrete SEA protein.