抗凋亡基因bcl-2 cDNA表达型质粒的构建及序列分析

The construction and sequencing of the expression plasmid of the apoptosis-resistent gene bcl-2

目的 :克隆抗凋亡基因bcl-2,并构建其表达型质粒 ;进行序列分析 ,为基因转染作准备。方法 :应用PCR定向克隆技术 ,克隆SD大鼠鼠脑bcl-2全编码cDNA序列 ;应用双脱氧末端终止法进行重组质粒中bcl-2的cDNA序列分析 ;利用基因重组技术 ,将bcl-2全编码cDNA序列 ,导入pCMV -Tag -1表达型质粒。结果 :成功地克隆SD大鼠bcl-2全编码cDNA序列 ,并将其成功地导入pCMV -Tag -1表达型质粒 ;经序列分析 ,发现bcl-2全编码序列与GeneBank中的相应序列相比 ,有3个碱基出现差别 ,分别为171位a→g,233位a→c ,530位 g→a,均为同义突变。结论

Objective:Cloning the full length cDNA of bcl-2gene,which believed to be has the effect of resisting the apoptosis,and constructing the expression plasmid;sequencing the bcl-2gene,to prepare for gene transfection.Methods:Cloning the full length cDNA of bcl-2gene of SD rat brain tissue by PCR target cloning method;Sequencing the full length cDNA of bcl-2gene by dideoxy-nucleotide chain termination method;Putting the the full length cDNA of bcl-2gene into expression plasmid pCMV-Tag-1by gene recombination method.Results:Full length cDNA of bcl-2gene was cloned and constructed into the expresˉsion plasmid of pCMV-Tag1successfully.The cDNA sequence showed that only three bases were different from that in Gene Bank but the difference was believed to be synonymous muˉtation.Conclusion:This work provided the base for the processing study that was on the transfection of the anti-apoptosis bcl-2gene to the islet cell.