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目的:探索红色糖多孢菌红霉素eryG基因在大肠杆菌中的最佳表达条件。方法:以红色糖多孢菌总DNA为模板,PCR扩增出eryG基因序列,克隆到原核表达载体pET-22b(+)上,并使其在大肠杆菌BL21(DE3)中表达。从诱导剂IPTG的终浓度和诱导时间两个因素来优化eryG基因的表达条件。结果:eryG基因可以在大肠杆菌中表达,表达产物EryG经SDS-PAGE分析相对分子质量约为33×103。表达条件经优化后,重组蛋白EryG表达量占菌体总蛋白的55%以上。结论:红色糖多孢菌红霉素eryG基因可以在大肠杆菌中高效地表达,为进一步研究EryG的相关酶学性质、分析其对红霉素发酵产物的影响奠定了基础。
Objective: To explore the optimum conditions for erythromycin redG gene expression in Escherichia coli. Methods: The eryG gene was amplified by PCR using the total DNA of Saccharomyces cerevisiae as a template and cloned into prokaryotic expression vector pET-22b (+) and expressed in E. coli BL21 (DE3). The conditions of eryG gene expression were optimized from the final concentration of IPTG and the induction time. Results: The eryG gene was expressed in E. coli. The expression product of EryG was analyzed by SDS-PAGE and the relative molecular mass was about 33 × 103. After the expression conditions were optimized, the expression of recombinant protein EryG accounted for more than 55% of the total bacterial protein. Conclusion: erythromycin redG gene can be expressed efficiently in Escherichia coli, which lays the foundation for the further study on the related enzyme properties of EryG and its effect on erythromycin fermentation product.