武迪迪,张杰,具英花,宗志宏,李雪松,于秉治
WU Didi,ZHANG Jie,JU Yinghua,ZONG Zhihong,LI Xuesong,YU Bingzhi(Department of Biochemistry and Molecular Biology

• 1:中国医科大学基础医学院生物化学与分子生物学教研室
• 2:中国医科大学基础医学院生物化学与分子生物学教研室
• 3:中国医科大学基础医学院生物化学与分子生物学教研室
• 4:中国医科大学基础医学院生物化学与分子生物学教研室

摘要(Abstract)：

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。
Objective: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.

关键词(KeyWords)： 基因,p21;卵母细胞;荧光抚体技术;小鼠;绿色荧光蛋白
genes,p21;oocytes;fluorescent antibody technique;mice;green fluorescent protein

Abstract:

Keywords:

基金项目(Foundation): 973规划项目(G1999055900-2);; 国家自然科学基金重点资助项目(39730460)

作者(Author): 武迪迪,张杰,具英花,宗志宏,李雪松,于秉治
WU Didi,ZHANG Jie,JU Yinghua,ZONG Zhihong,LI Xuesong,YU Bingzhi(Department of Biochemistry and Molecular Biology

DOI: 10.19367/j.cnki.1000-2707.2007.01.003

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