| p21-EGFP 融合基因表达载体与卵母细胞的表达定位 |
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| 引用本文： | 武迪迪,张杰,具英花,宗志宏,李雪松,于秉治. p21-EGFP 融合基因表达载体与卵母细胞的表达定位[J]. 贵阳医学院学报, 2007, 32(1): 5-8 |
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| 作者姓名： | 武迪迪 张杰 具英花 宗志宏 李雪松 于秉治 |
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| 作者单位： | 中国医科大学基础医学院,生物化学与分子生物学教研室,辽宁,沈阳,110001 |
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| 基金项目： | 国家重点基础研究发展计划(973计划) , 国家自然科学基金 |
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| 摘 要： | 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Western Blot 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.
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| 关 键 词： | 基因,p21 卵母细胞 荧光抚体技术 小鼠 绿色荧光蛋白 |
| 文章编号： | 1000-2707(2007)01-0005-04 |
| 修稿时间： | 2006-11-20 |
Construction of p21/EGFP Fusion Gene Expression Vector and Its Expression in Mouse Oocyte |
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| WU Didi,ZHANG Jie,JU Yinghua,ZONG Zhihong,LI Xuesong,YU Bingzhi. Construction of p21/EGFP Fusion Gene Expression Vector and Its Expression in Mouse Oocyte[J]. Journal of Guiyang Medical College, 2007, 32(1): 5-8 |
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| Authors: | WU Didi ZHANG Jie JU Yinghua ZONG Zhihong LI Xuesong YU Bingzhi |
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| Affiliation: | Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, China Medical University, Shenyang 110001, Liaoning, China |
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| Abstract: | 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. |
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| Keywords: | genes,p21 oocytes fluorescent antibody technique mice green fluorescent protein |
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