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改良重叠区扩增法构建Rac1相关质粒

, PP. 177-182

Keywords: Rac1,重叠区扩增法,基因定点突变,融合蛋白

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Abstract:

采用改良重叠区扩增法实现Rac1基因定点突变,构建人Rac1基因相关真核表达质粒,观察EGFP-Rac1融合蛋白在人正常肝细胞LO2中的表达。利用RT-PCR的方法得到Rac1基因,使用改良重叠区扩增法实现Rac1基因的定点突变,获得Rac1突变基因。将Rac1基因及Rac1突变基因通过限制性酶切技术及DNA连接技术插入真核表达载体pEGFP-C1中,获得重组质粒pEGFP-C1-Rac1,pEGFP-C1-Rac1V12和pEGFP-C1-Rac1N17。将上述质粒瞬时转染入LO2细胞中,采用荧光技术及Westernblotting检测目的基因的表达。结果显示,限制性酶切及基因测序证实质粒构建正确,转染LO2细胞后,融合蛋白EGFP-Rac1在细胞中高效表达。成功构建真核表达质粒,在LO2细胞中,该质粒能成功表达融合蛋白EGFP-Rac1。

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