野生型和突变型ABCB6红色荧光融合蛋白表达载体的构建

发布时间：2018-01-11 07:26

  本文关键词：野生型和突变型ABCB6红色荧光融合蛋白表达载体的构建　出处：《医学研究生学报》2016年09期 　论文类型：期刊论文

  更多相关文章： ABCB 融合蛋白 表达载体 细胞定位

【摘要】：目的 ABCB6基因突变与多种疾病相关,尤其是遗传性泛发型色素异常症。为进一步研究ABCB6在色素代谢中的作用,构建野生型和突变型ABCB6红色荧光蛋白融合蛋白表达载体p DsRed-wt-ABCB6和p DsRed-L356P-ABCB6,并观察ABCB6在人黑素细胞瘤细胞系A375中细胞定位情况。方法 以前期构建的p IRES2-Zs Green1-ABCB6表达载体为基础构建野生型和突变型ABCB6蛋白与红色荧光蛋白融合表达载体p DsRed-wt/L356P-ABCB6,将构建的质粒转染A375细胞,Western blot检测细胞中ABCB6的表达水平,激光共聚焦显微镜观察ABCB6在细胞中的定位。结果 p DsRed-wt/L356P-ABCB6质粒经菌落PCR、双酶切和测序鉴定正确,转染A375细胞后,Western blot检测细胞中ABCB6表达量显著增高,激光共聚焦显微镜观察到野生型和突变型ABCB6均定位于细胞质中。结论 p DsRed-wt/L356P-ABCB6融合蛋白表达载体成功构建,观察了ABCB6在细胞中的定位,为进一步研究ABCB6的功能及其在相关疾病中致病机制奠定了良好的基础。
[Abstract]:Objective to study the role of ABCB6 gene mutation in pigment metabolism, especially in hereditary generalized dystrophy. Wild-type and mutants of ABCB6 red fluorescent protein fusion protein expression vector p DsRed-wt-ABCB6 and p DsRed-L356P-ABCB6 were constructed. The localization of ABCB6 in human melanoma cell line A375 was observed. Construction of Fusion expression Vector of Wild and Mutant ABCB6 protein and Red fluorescent protein based on Green1-ABCB6 expression Vector. DsRed-wt/L356P-ABCB6. The expression of ABCB6 in A375 cells was detected by Western blot. The localization of ABCB6 in cells was observed by laser confocal microscope. Results the p DsRed-wt/L356P-ABCB6 plasmid was confirmed by colony PCR, double enzyme digestion and sequencing. After transfection of A375 cells, the expression of ABCB6 in A375 cells was detected by Western blot. Both wild-type and mutant ABCB6 were observed in cytoplasm by laser confocal microscopy. Conclusion the expression vector of p DsRed-wt/L356P-ABCB6 fusion protein was successfully constructed. The localization of ABCB6 in cells was observed, which laid a good foundation for further study on the function of ABCB6 and its pathogenesis in related diseases.
【作者单位】： 华中科技大学同济医学院附属同济医院皮肤科;华中科技大学同济医学院附属同济医院麻醉科;
【分类号】：R596
【正文快照】： 0引言ATP结合盒(ATP-binding cassette,ABC)转运蛋白是一类大型跨膜蛋白,具有多个结构域,可通过ATP依赖的途径促进多种生物化合物,如药物、胆酸、多肽、类固醇、离子和磷脂等物质的跨膜转运[1]。人类基因组可编码48种ABC转运蛋白,根据蛋白的功能和同源性可分为7个亚家族(ABCA，

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