含人自噬基因hAPG12穿梭载体构建及其在酵母自噬中的作用

Construction of shuttle vector consisted of human autophagic gene hAPG12 and its expression in yeast

目的该研究旨在构建大肠杆菌-酿酒酵母穿梭载体pESC-URA-EGFP-hAPG12,将构建好的载体转化到酵母菌内,以此研究hAPG12在酵母自噬过程中的作用。方法从pEGFP-C2-hAPG12中扩增出EGFP-hAPG12融合基因,鉴定正确后定向亚克隆到大肠杆菌-酿酒酵母穿梭载体pESC-URA中,构建载体pESC-URA-EGFP-hAPG12并将其转化到酵母,荧光显微镜下观察,用自噬诱导剂诱导酵母自噬以观察hAPG12在酵母内的表达定位。结果证实EGFP-hAPG12基因已完全正确亚克隆到pESC-URA中,荧光显微镜观察结果证明EGFP-hAPG12融合蛋白在酵母中成功表达,以诱导培养24h的荧光最强,EGFP-hAPG12定位于酵母的自噬体中。结论成功构建了具有报告基因的重组真核表达载体pESC-URA-EGFP-hAPG12;EGFP-hAPG12融合蛋白在酵母中得到表达,hAPG12在酵母自噬体形成过程中起作用。

[Objective] To investigate the function of the hAPG12 in the process of autophagy in yeast, the hAPG12-EGFP fusion gene shuttle vector was constructed and The plasmid constructed above was transformed into the yeast. [Methods] EGFP- hAPG12 Fusion protein gene was obtained from pEGFP-C2- hAPG12 via PCR, The PCR products were inserted into E. coli-E, cerevisiae shuttle vector pESC-URA. The plasmid constructed above was transformed into the nutrient defective yeast, YPH500, and grow on minimal plates lacking uracil. The positive transformants were futher identified with fluorescence microscopy. The cells expressing EGFP-hApgl2p were further treated with rapamycin to induce autophagy to investigate the function of the hAPG12 in the process of autophagy in yeast. [Resets] A recombinant plasmid pESC-URA-EGFP-hAPG12 for eukaryotic expression was obtained after subcloning. By fluorescence microscopy, EGFP-hApgl2 fusion protein was observed in cytosolic of yeast after being induced by galactose and reached fastigium at 24 hours. EGFP-hApgl2p were found in autophagic bodies and cytoplasm. [Conclusion] A new E.Coli-S. cerevisiae shuttle vector with EGFP gene is constructed,and fluorescence microscopic analyses indicate that EGFP- hAPG12 fusion protein is expressed. The hAPG12 is related to the formation of autophagic bodies in the process of autophagy.