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晚期糖基化终末产物受体不同突变体真核表达载体的构建和表达

Construction of different mutants of HA-tagged human RAGE gene and their eukaryotic expression
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摘要 目的构建带HA标签的晚期糖基化终末产物受体(RAGE)不同突变体的真核细胞表达载体。方法采用定点突变技术将亚克隆在载体pcDNA3-HA上的野生型RAGE构建成不同突变体pcDNA3-HA-RAGE(S391A)、pcDNA3-HA-RAGE(S399A)、pcDNA3-HA-RAGE(S400A)、pcDNA3-HA-RAGE(T401A),经测序鉴定正确后转染HEK293细胞,利用Westernblotting检测各突变体的表达。结果RAGE不同突变体经测序鉴定正确无误后,在HEK293细胞中得到高量表达。结论成功构建了RAGE不同突变体的真核表达载体,并在真核细胞中进行了表达,为进一步研究RAGE在细胞信号转导中的作用奠定了基础。 Objective To construct eukaryotic expression vectors for HA-tagged receptor for advanced glycation end products （RAGE） mutants. Methods Site-directed mutagenesis was applied to wild-type RAGE gene cloned in the pcDNA3 vector with HA tag to obtain the mutants pcDNA3-HA-RAGE（S391A）, pcDNA3-HA-RAGE（S399A）, pcDNA3-HA-RAGE（S400A）, and pcDNA3-HA-RAGE（T401A）. After identification by sequencing, the mutants were transfected into HEK293 cells, and the expression of these mutants were detected by Western blotting using anti-HA antibody. Results The HA-tagged RAGE mutants constructed were verified successfully by sequencing, and highly expressed in HEK293 cells. Conclusion The success in constructing HA-tagged RAGE mutants, which are highly expressed in eukaryotic cells, may facilitate the functional study of RAGE in cell signal transduction.
作者 程蔚蔚 李煜生 龚小卫 赵琳琳 王继刚 邓鹏 姜勇
机构地区 南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室
出处 《南方医科大学学报》 CAS CSCD 北大核心 2008年第10期1779-1781,1785,共4页 Journal of Southern Medical University
基金 教育部创新团队项目(IRT0731) 国家自然科学基金委-广东省人民政府自然科学联名基金重点项目(U0632004) 广东省科技计划项目(A1090202) 广东省自然科学基金重点项目(13058)
关键词 晚期糖基化终末产物受体 定点突变 载体构建 基因表达 receptor, advanced glycation end products site-directed mutagenesis vector construction gene expression
分类号 Q78 [生物学—分子生物学]
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参考文献13

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南方医科大学学报
2008年 第10期

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