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目的在毕赤酵母(Pichia pastoris)表达系统中表达结核分枝杆菌ESAT-6基因。方法以重组质粒pET23a-ESAT-6为模板,亚克隆目的片段ESAT-6至酵母菌分泌表达载体pPICZaA,重组质粒经线性化后电转化转染至毕赤酵母菌GS115菌株,经抗生素Zeocin筛选得到高拷贝转化子。经甲醇诱导表达,取细胞裂解上清进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法分析。结果成功构建了pPICZaA-ESAT-6毕赤酵母表达重组质粒。经甲醇诱导,在酵母细胞内表达分子质量单位为18kDa的蛋白。蛋白质印迹(Western blot)显示,18kDa蛋白被活动性肺结核病人血清抗体识别。结论在毕赤酵母菌中成功表达了带有信号肽ESAT-6基因,为进一步研究新型单位疫苗打下坚实的基础。
Objective To express Mycobacterium tuberculosis ESAT-6 gene in Pichia pastoris expression system. Methods The recombinant plasmid pET23a-ESAT-6 was subcloned into Escherichia coli to express the plasmid pPICZaA. After linearization, the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The recombinant plasmid was screened by Zeocin High-copy transformants. After induced by methanol, the cell lysate supernatant was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results The recombinant plasmid pPICZaA-ESAT-6 was successfully constructed. Methanol-induced expression of a molecular mass unit of 18 kDa in yeast cells. Western blot showed that 18 kDa protein was recognized by serum antibody of active pulmonary tuberculosis patients. Conclusion The ESAT-6 gene with signal peptide was successfully expressed in Pichia pastoris, laying a solid foundation for further research on new unit vaccine.