Construction of wild-type and mutant ABCB6-DsRed fusion protein expression vectors
10.16571/j.cnki.1008-8199.2016.09.003
- VernacularTitle:野生型和突变型 ABCB6红色荧光融合蛋白表达载体的构建
- Author:
Hongwen LI
;
Luzhu CHEN
;
Yunhua DENG
;
Caie ZHANG
- Publication Type:Journal Article
- Keywords:
ABCB6;
Fusion protein;
Expression vector;
Cellular localization
- From:
Journal of Medical Postgraduates
2016;29(9):906-909
- CountryChina
- Language:Chinese
-
Abstract:
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 .
