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

目的:克隆Bcl-2相关促凋亡基因Bad的全长编码序列,构建Bad基因的真核表达载体并在人基底细胞癌细胞系(A431)中表达,为研究该基因在人基底细胞癌治疗中的作用奠定基础。方法:根据已发表的Bad基因的核苷酸序列设计并合成引物,用Hela细胞抽提的RNA进行逆转录聚合酶链反应(RT-PCR),扩增产物用XhoI和EcoRI双酶切后定向克隆到真核细胞表达载体pcDNA3.1-myc中,用限制性内切酶酶切重组质粒pcDNA3.1-myc-Bad并对其DNA测定序列。用脂质体法将pcDNA3.1-myc-Bad导入人基底细胞癌细胞系A431中,G418选择培养,Western blot及SABC-FITC分析鉴定其表达。瞬时转染A431细胞,通过细胞计数和集落形成实验观察其对A431细胞生长的影响。结果:RT-PCR扩增出长500bp的特异性片段,经克隆至pcDNA3.1-myc后酶切鉴定证实,并测序表明序列与GenBank报道完全一致。pcDNA3.1-myc-Bad在A431细胞中有表达并可影响细胞生长,与对照组有明显差异。结论:成功克隆了Bad的编码序列,构建了其真核细胞表达载体pcDNA3.1-myc-Bad,并在瞬时转染肿瘤细胞后观察到细胞生长受抑制。

Construction of human Bad gene eukaryotic expressing vector and Bad gene expression in human basal cell carcinima cell line

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.