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目的 克隆金黄色葡萄球菌肠毒素A(SEA)基因、构建其真核表达系统及目的重组蛋白的表达情况。方法 采用高保真PCR从金黄色葡萄球菌ATCC135 6 5菌株中扩增全长SEA ,目的扩增片段T -A克隆后测序。经核酸内切酶酶切重组质粒 pUCm -T -SEA获得的 pUCm -T -SEA基因片段与真核表达载体 pPIC9K连接。采用电融合法将重组真核载体pPIC9K -SEA转化入P pastorisGS115株 ,构建成真核表达系统 pPIC9K -SEA -P pastorisGS115。采用含G4 18抑制剂的YPD琼脂平板和PCR筛选并鉴定 pPIC9K -SEA -P pastorisGS115。 0 5 % (V∶V)甲醇诱导下 ,采用SDS -PAGE检查BMMY液体培养基上清中重组SEA(rSEA)的表达情况。结果 可从S aureusATCC135 6 5株DNA中扩增获得SEA目的片段。与报道的SEA基因序列 (GenBankNo :AP0 0 4 82 8andL2 2 5 6 6 )比较 ,所克隆的SEA基因核苷酸及氨基酸序列的相似性分别高达 98 84 %～ 10 0 %和 10 0 %。在甲醇的诱导下 ,pPIC9K -SEA -P pastorisGS115能表达在SDS -PAGE图谱位于预期位置的rSEA。结论 我们成功地构建了能表达SEA的真核表达系统 ,为进一步分析SEA分子中毒性相关活性位点、定位突变获得减毒或无毒的突变体奠定了基础。
Objective To clone the Staphylococcus aureus enterotoxin A (SEA) gene and construct its eukaryotic expression system and target recombinant protein expression. Methods High-fidelity PCR was used to amplify full-length SEA from Staphylococcus aureus ATCC13565. The objective of this study was to amplify the fragment of T-A cloned and sequenced. The pUCm-T-SEA gene fragment obtained by restriction endonuclease digestion of the recombinant plasmid pUCm-T-SEA was ligated with the eukaryotic expression vector pPIC9K. The recombinant eukaryotic vector pPIC9K-SEA was transformed into P pastoris GS115 by electrofusion method and constructed into eukaryotic expression system pPIC9K-SEA-P pastorisGS115. The pPIC9K-SEA-P pastoris GS115 was screened and identified using YPD agar plates containing G418 inhibitors and PCR. The expression of recombinant SEA (rSEA) in the supernatant of BMMY liquid medium was examined by SDS-PAGE under the induction of 0 5% (V: V) methanol. Results The target fragment of SEA was obtained by amplification of 5 strains of S. aureus ATCC13565. Compared with the reported sequence of SEA gene (GenBankNo: AP0 0 4 82 8 and L 2 2 5 6 6), the similarity of nucleotide and amino acid sequence of the cloned SEA gene was 98 84% -100% and 100% respectively. Under the induction of methanol, pPIC9K-SEA-P pastorisGS115 can express rSEA at the expected position in the SDS-PAGE map. Conclusions We have successfully constructed a eukaryotic expression system capable of expressing SEA, which lays the foundation for further analysis of active sites of toxicity-related SEA molecules and localization mutations to obtain attenuated or non-toxic mutants.