PAX3基因重组真核细胞表达质粒的构建、表达及意义

发布时间：2018-03-02 18:37

本文选题：Waardengurg综合征　切入点：PAX　出处：《听力学及言语疾病杂志》2014年01期 　论文类型：期刊论文

【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。
[Abstract]:Objective to construct the expression plasmid of PAX3 gene and its mutants to study the expression and localization of exogenous expression, for the study of Waardenburg syndrome (Waardenburg syndrome WS) to provide experimental basis for the pathogenesis. Methods by molecular cloning technique and double enzyme digestion of pECEPAX3 and pcDNA3.0-HA. The recombinant PAX3 gene eukaryotic expression plasmid pcDNA3.0-PAX3-HA, which was as a template we constructed PAX3 new gene mutation of H80D and H186fs expression plasmid pcDNA3.0-H80D-HA and pcDNA3.0-H186fs-HA DNA sequencing. PAX3, H80D and H186fs expression plasmids were transiently transfected with mouse embryonic fibroblast cells (NIH3T3 cells), were used to detect and observe the wild PAX3 protein and mutant H80D by Western blot and immunofluorescence, expression and distribution H186fs protein in NIH3T3 cells. The results of PAX3 and its mutant H80D and H186fs plasmid DNA sequencing identification Set the correct sequence, the correct expression of three in NIH3T3 cells, H80D and PAX3 distribution only in the nucleus, H186fs were distributed in cytoplasm and nucleus. Conclusion: This study successfully constructed PAX3 mutant gene and its eukaryotic expression plasmid, producing mutations that affect the subcellular localization of PAX3 protein in vitro, for further study in China the human PAX3 gene laid the foundation of molecular mechanism of mutagenesis induced by the pathogenesis of WS.

【作者单位】：新疆医科大学第一附属医院耳鼻咽喉科;中南大学医学遗传学国家重点实验室;中南大学湘雅医院耳鼻咽喉科;
【基金】：国家自然科学基金(81260160,81170923)资助
【分类号】：R764

【共引文献】