IL-13及其变异体真核表达载体的构建表达与亚细胞定位

Construction and expression of human interleukin-13 and its mutant and subcellular location

目的:体外构建野生型人白细胞介素-13(whIL-13)及变异型人白细胞介素-13(mhIL-13)与增强型绿色荧光蛋白(EGFP)融和蛋白真核表达载体,分析其在COS-7细胞中的表达和亚细胞定位。方法:以RT-PCR方法扩增whIL-13、mhIL-13全长编码基因,构建EGFP-whIL-13、EGFP-mhIL-13融和蛋白真核表达载体,转染C0S-7细胞,以激光扫描共聚焦显微镜观察融合蛋白的表达及其在细胞内的分布情况。结果:两种融和蛋白表达载体均构建正确,将其转染COS-7细胞后,在阳性克隆胞浆内均可见明亮的绿色荧光,胞核空虚。结论:成功构建EGFP-whIL-13、EGFP-mhIL-13融和蛋白表达载体,并在COS-7细胞中得到表达,表达的两种融和蛋白细胞内分布特征没有区别,均位于胞浆内。

Objective：To construct a eukaryotic expression vector for wild-type human Interleuldn-13（whIL-13）and its mutant （mhIL-13） gene fused with enhanced green fluorescent protein（EGFP） and to analyze the expression and subcellular location of the fusion protein in COS-7 cells. Methods：The whole whIL-13 and mhIL-13 coding gene was amplified by RT-PCR. And then the expression vector for EGFP-whIL-13, EGFP-mhIL-13 fusion protein were constructed. The recombinant fusion gene were transfected into COS- 7 cells. The expression and subcellular location of the fusion protein were detected by confocal microscopy. Results ：The construction of expression vector were all corrected. In the positive cells, green fluorescent was found to be localized in cytoplasm,while empty in nucleus. Conclusion：The expression vectors for EGFP-whIL-13, EGFP-mhIL-13 fusion protein were constructed successfully and expressed in COS-7 cells. Fusion proteins were distributed in cytoplasm and no significant difference was observed between them.