bcl-2/EGFP融合基因真核表达质粒的构建

Construction of bcl-2/EGFP fused gene eukaryotic expressing plasmid

为深入研究bcl 2对细胞凋亡的调控机制和肿瘤发生发展的影响,应用基因重组手段,根据表达载体pEGFP C1上的多克隆位点和bcl 2基因序列设计了1对引物,对含有bcl 2基因的质粒pWB bcl进行了PCR扩增,得到了708bp的目的片断。将其克隆到pMD18 Tvector,筛选阳性克隆并测序,序列分析表明,其与原初序列同源性达99%。将bcl 2插入到载体启动子下游,与报告基因绿色荧光蛋白(EnhancerGreenFluorescentProtein,EGFP)融合,经限制性内切酶酶切和PCR鉴定,证明Bcl 2/EGFP真核表达质粒构建成功。

The pro-oncogene B cell lymphoma/leukemia-2 gene (bcl-2) plays an important role in the regulation of apoptosis.In order to study deeply the mechanism,the eukaryotic expressing vector of bcl-2/EGFP fused gene was constructed by the methods of gene recombinating.A pair of primer was designed according to multiple cloning sites of pEGFP-C1 vector and the gene sequence of bcl-2 cDNA.The pWB-bcl plasmid which contains bcl-2 gene was amplified by PCR.The product of PCR was 708 bp.bcl-2 was integrated into pMD18-T vector.The recombinant pMD-bcl plasmid was sequenced and analyzed,homology was 99.bcl-2 gene was inserted into the promoter downstream of pEGFP-C1 vector and fused with Enhancer Green Fluorescent Protein (EGFP) gene.The result affirmed that bcl-2/EGFP fused gene eukaryotic expressing vector was constructed successfully by the restricted endonuclease and PCR.