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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 SGK3 kinase PX domain mutant vector and observe its expression and localization in human renal blastocysts 293T. Methods: The primers were designed according to the principle of overlap extension PCR. The SGK3 mutant with point mutation was synthesized and ligated into p EGFP vector. The mutant was transiently transfected into 293T cells. The fluorescence intensity was measured by fluorescence inverted microscopy Mutant expression in cells and functional realization. Results: Sequencing results showed that the SGK3 sequence with point mutation was synthesized. The inhomogeneous distribution of SGK3 mutant in wild-type SGK3 in 293T cells under fluorescence inverted microscope showed that the transfection was successful. The mutant vector was obtained in human renal blastocysts 293T Expression and positioning. CONCLUSION: SGK3 kinase can lose the function of localization by changing the amino acid at position 90 of PX domain sequence, so as to provide basis for studying the function of SGK3 in cells.