野生型和突变型ABCB6红色荧光融合蛋白表达载体的构建

Construction of wild-type and mutant ABCB6-DsRed fusion protein expression vectors

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。

Objective The mutation of the ABCB6 gene is involved in a variety of diseases, including dyschromatosis universalis hereditaria （DUH）. This study aimed to construct the expression vectors for the ABCB6-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/L356P-ABCB6 were constructed based on the previously constructed plRES2-ZsGreenl-ABCB6 vector and then transfected into A375 cells. At 48 hours after transfection, the expression of AB- CB6 was detected by Western blot and the cellular localization of ABCB6 determined under the laser scanning eonfocal microscope. Results The expression vectors pDsRed-wt/L356P-ABCB6 were verified by colony PCR, enzyme digestion, and DNA sequencing. The expression of ABCB6 was significantly increased in the A375 cells after transfeeted 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 ABCB6 in A375 ceils was de- termined, which may serve as a basis for further studies of ABCB6 and the pathogenesis of ABCB6-related diseases.