p21-EGFP融合基因表达载体与卵母细胞的表达定位

Construction of p21/EGFP Fusion Gene Expression Vector and Its Expression in Mouse Oocyte

目的:通过构建绿色荧光蛋白p21-EGFP融合基因表达载体,观察其在小鼠卵母细胞中的表达及定位,为研究细胞周期蛋白激酶抑制因子p21对小鼠卵母细胞细胞周期的影响提供实验基础。方法:以含有p21的全长序列的质粒为模板PCR扩增p21cDNA,应用基因重组技术与EGFP基因融合,构建以绿色荧光蛋白EGFP为报告基因的重组表达质粒pEGFP-p21。利用显微注射方法注射到小鼠卵母细胞中进行表达,用荧光显微镜直接观察pEGFP-p21融合蛋白在细胞中的分布和定位,Western Blot方法检测其蛋白的表达。经酶切证实插入片断的正确性。结果:荧光显微镜观察结果显示在空载体pEGFP-C3转染的小鼠卵母细胞中,绿色荧光呈弥散分布,而经注射重组质粒pEGFP-p21的小鼠卵母细胞中,绿色荧光主要集中在细胞核内。同时用WesternBlot证实了pEGFP-p21融合基因的表达。结论:成功的构建了pEGFP-p21融合表达质粒。同时证明小鼠卵母细胞能高效表达pEGFP-p21融合基因,且主要定位于细胞核内。

To construct p21-EGFP expression vector, and to observe its expression in mouse oocyte. Methods： p21 ORF was amplified from p21 WAF1 by PCR and inserted into plasmid pEGFP-C3. The recombinant expression plasmid pEGFP-p21 was transfected into mouse oocyte by microinjecting method. Western blotting was used to analyze the expression product of pEGFP-p21 fusion protein and the expression location was observed under fluorescent microscope. Results： pEGFP-p21 was constructed successfully. Fusion protein expressed in mouse oocyte and localized in nuclear. Conclusion： Fusion protein expresseds in nuclear.