GST融合蛋白表达系统在实验教学中的应用

Application of Glutathione-S-transferase Fusion Protein Expression System in Experimental Teaching

以GST-AQP7(Aquaporin 7)融合蛋白的原核表达为例,进行了生物学开放性实验教学的实践与探讨。利用PCR技术快速扩增小鼠AQP7-C末端亲水性肽段区基因片段,构建重组表达载体pGEX-4T-1∕AQP7,转化表达菌株E. coli BL21,诱导表达GST-AQP7融合蛋白,并对目的蛋白亲和层析纯化。PCR鉴定和DNA测序结果表明,pGEX-4T-1∕AQP7重组表达载体构建正确,SDS-PAGE蛋白电泳结果证实GST-AQP7高效表达并成功纯化。经过上述实验过程,学生系统掌握了从基因操作到蛋白表达一系列基本的分子生物学实验技能,很好地将课本所学理论知识应用于实验研究,提高了学生的综合科研素质,激发了学生的实验热情。

Taking prokaryotic expression of GST-AQP7 fusion protein as an example, the practice and discussion of open experimental teaching were carried out with the combination of scientific research task. The carboxyl terminus gene fragment of AQP7 hydrophilic peptide was amplified and inserted into expression vector pGEX-4T-1. The recombinant expression vector was transformed into Escherichia coli BL21 cells. The GST-AQP7 fusion protein was induced to be expressed and further purified. PCR amplification and DNA sequence analysis results showed that the recombinant vector pGEX-4T-1∕AQP7 was constructed with those expected. The result of SDS-PAGE assay indicated that the fusion protein of GST-AQP7 was successfully expressed and purified. On the basis of experiments, students mastered the molecular biology experiment skills about gene manipulation and protein expression. The theoretical knowledge was applied to experimental research, the student’s comprehensive quality was improved and their enthusiasm for research was cultivated.