Construction of human Bad gene eukaryotic expressing vector and Bad gene expression in human basal cell carcinima cell line
- VernacularTitle:促凋亡分子Bad真核表达载体构建及其在人基底细胞癌细胞中的表达
- Author:
Bin HU
;
Xinghua FENG
;
Fang LIU
- Publication Type:Journal Article
- Keywords:
Human Bad gene;
Clone;
Eukaryotic expression vector;
Basal cell carcinoma
- From:
Journal of Practical Stomatology
2001;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct eukaryotic expressing vector of the full length coding sequence of Bad gene and to express the gene in the basal cell carcinima A431 cells.Methods:Bad gene was amplified from Hela cell line by RT-PCR and the fragment of the cDNA was cloned into eukaryotic expressing vector pcDNA3.1-myc by ligating the fragment into XhoI and EcoRI site.The recombinant plasmid pcDNA3.1-myc-Bad was identified by DNA sequencing and restriction enzyme analysis.The gene transfection mediated by lipofectin was used to introduce the eukaryotic expressing vector of pcDNA3.1-myc-Bad into human basal cell carcinima A431 cells. After selection with G418, resistant colonies were obtained.Trasfection efficiency was identified by Western blot and SABC-FITC assay.Cell proliferation was examined by cell counting and colonogenic assay after transfection.Results:A 500 bp DNA fragment was amplified with RT-PCR.Sequence and restriction enzyme analysis showed that the recombinant plasmid pcDNA3.1-myc-Bad was constructed successfully.In human basal cell carcinima cell line A431 Bad gene was expressed.The cell proliferation was inhibited by 62.6% and colonogenesis by 39.9% by the transfection of the gene.Conclusion: Human Bad gene was successfully cloned.Transfection of basal cell carcinoma cells with the gene may inhibit the cell proliferation and colonogenesis.
