野生型和突变型 ABCB6红色荧光融合蛋白表达载体的构建

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pDsRed-wt-ABCB6和pDsRed-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pIRES2-ZsGreen1-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pDsRed-wt／L356P-ABCB6，将构建的质粒转染A375细胞，West-ern 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 pDsRed-wt／L356 P-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 pDsRed-wt／L356 P-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。%Objective The mutation of the ABCB6 gene is involved in a variety of diseases , including dyschromatosis univer-salis hereditaria (DUH).This study aimed to construct the expression vectors for the ABCB 6-DsRed fusion proteins, pDsRed-wt-AB-CB6 and pDsRed-L356P-ABCB6, detect its cellular localization in A375 cells, and thus facilitate future studies on the pathogenesis of ABCB6-related diseases . Methods The recombinant plasmids pDsRed-wt/L356 P-ABCB6 were constructed based on the previously constructed pIRES2-ZsGreen1-ABCB6 vector and then transfected into A 375 cells.At 48 hours after transfection , the expression of AB-CB6 was detected by Western blot and the cellular localization of ABCB 6 determined under the laser scanning confocal microscope . Results The expression vectors pDsRed-wt/L356P-ABCB6 were verified by colony PCR, enzyme digestion, and DNA sequencing. The expression of ABCB 6 was significantly increased in the A 375 cells after transfected with the recombinant plasmids .Confocal mi-croscopy showed the localization of both wild-type and mutant ABCB6 in the cytoplasm. Conclusion The expression vectors for wild-type and mutant ABCB6-DsRed fusion protein were successfully constructed and the localization of ABCB 6 in A375 cells was de-termined, which may serve as a basis for further studies of ABCB 6 and the pathogenesis of ABCB 6-related diseases .