hAPG12-EGFP融合基因真核表达载体的构建及其在酵母中的表达

目的本研究旨在构建大肠杆菌-酿酒酵母真核表达载体pESC-URA-EGFP-hAPG12,并将构建好的载体转化到酵母。方法从pEGFP-C2中扩增出增强绿色荧光蛋白EGFP基因,定向亚克隆到真核表达载体pESC-URA;hAPG12插入到EGFP基因氨基末端;构建的载体转化到酵母。结果EGFP和hAPG12基因正确亚克隆到pESC-URA中,EGFP-hAPG12融合蛋白在酵母中表达。结论构建了具有报告基因及hAPG12的重组真核表达载体pESC-URA-EGFP-hAPG12。

Construction of hAPG12- EGFP fusion gene eukaryotic expression vector and its expression in yeast

Objective To investigate the construction, the hAPG12-EGFP fusion gene eukaryotic expression vector and its transformation into the yeast. Methods Enhanced green fluorescent protein(EGFP) gene was obtained from pEGFP-C2 via PCR. The PCR products were inserted into eukaryotic expression vector pESC-URA. Then the hAPG12 was subcloned into pESC-URA-EGFP to construct pESC-URA-EGFP-hAPG12. The plasmid constructed was transformed into the nutrient defective yeast YPH500 which was grown on minimal plates lacking uracil. The positive transformants were futher identified with fluorescence microscopy. Results a recombinant plasmid pESC-URA-EGFP-hAPG12 for eukaryotic expression was obtained after subcloning. By fluorescence microscopy, EGFP-hApg12 fusion protein was observed in cytosolic of yeast after being induced by galactose and reached fastigium at 24 hours. Conclusion A new E.Coli-S.cerevisiae expression vector with EGFP gene is constructed and EGFP- hAPG12 fusion protein is expressed. The fusion protein can be used to monitor the autophagy pathways in yeast in future study.