运用重叠延伸PCR技术构建SGK3激酶PX结构域突变体

Construction of Mutant PX Domain of SGK3 by Splice Overlap Extension PCR

目的:构建SGK3激酶PX结构域突变体载体,观察其在人肾胚细胞293T中的表达与定位。方法:利用重叠延伸PCR原则设计引物,合成具有点突变的SGK3突变体,将其连接到p EGFP载体上,测序验证;将所获突变载体瞬时转染人肾胚细胞293T,利用荧光倒置显微镜检验突变体在细胞中的表达及功能实现。结果:测序结果表明合成了具有点突变的SGK3序列;荧光倒置显微镜下SGK3突变体相较野生型SGK3在293T细胞内的不均匀分布表明转染成功,突变体载体在人肾胚细胞293T中获得表达且有所定位。结论:通过改变PX结构域序列上第90位氨基酸可以使SGK3激酶失去定位的功能,从而为研究SGK3在细胞中的功能提供基础。

Objective： To construct the mutant vector of PX domain of SGK3, and to observe its expression and location in 293T cell Methods： mutant premiers were designed according to the splice overlap extension PCR method, SGK3 mutant sequence was then constructed, and linked to the pEGFP vector. The mutant vector was transient transfected into 293T cell, the expression and function realization of the SGK3 protein was verified by GFP fluorescence labeling system. Results： The SGK3 vector contends site directed mutant of PX domain was suc- cessful constructed verified by gene sequencing, GFP fluorescence result showed that mutant vector was successful transfected and expressed, protein of expression of mutant vector was allocated compared to the wild type of SGK3 vector. Conclusion： The location ability can be deprived by changing the 90th amino acid on the sequence of PX domain of SGK3, providing a fundamental for the study of function of SGK3 in cell.