PAX3基因重组真核细胞表达质粒的构建、表达及意义

Generation and Expression of Recombinant Eukaryotic Expression Plasmids of PAX3 Gene and Its Significance

张华;李家大;罗浑金;陈红胜;梅凌云;贺楚峰;冯永;

1:新疆医科大学第一附属医院耳鼻咽喉科

2:中南大学医学遗传学国家重点实验室

3:中南大学湘雅医院耳鼻咽喉科

摘要
目的通过构建PAX3基因及其突变体表达质粒初步研究其外源性表达和定位表达,为研究Waardenburg综合征(Waardenburg syndrome,WS)发病机制提供实验基础。方法通过分子克隆技术双酶切pECEPAX3和pcDNA3.0-HA后连接构建PAX3基因重组真核细胞表达质粒pcDNA3.0-PAX3-HA,以其为模板分别构建PAX3基因新发突变H80D和H186fs表达质粒pcDNA3.0-H80D-HA和pcDNA3.0-H186fs-HA,DNA测序鉴定。将PAX3、H80D和H186fs表达质粒分别瞬时转染小鼠胚胎成纤维细胞(NIH3T3细胞),采用Western blot和细胞免疫荧光法分别检测和观察野生PAX3蛋白和突变H80D、H186fs蛋白在NIH3T3细胞中的表达和分布。结果 PAX3及其突变体H80D和H186fs表达质粒经DNA测序鉴定序列正确,三者在NIH3T3细胞中正确表达,H80D与PAX3仅在细胞核中分布,H186fs在细胞质与细胞核中均有分布。结论本研究成功构建了PAX3基因及其突变体真核细胞表达质粒,突变对PAX3蛋白的亚细胞定位产生影响,为在体外实验进一步研究国人PAX3基因突变致WS发病的分子机制奠定了实验基础。
关键词
Waardengurg综合征;PAX3;基因突变;体外实验
基金项目(Foundation):
国家自然科学基金(81260160,81170923)资助
作者
张华;李家大;罗浑金;陈红胜;梅凌云;贺楚峰;冯永;
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