反义Ki-67和Bcl-2真核细胞双表达载体的构建及鉴定

Construct and Identify the Eukaryotic Co-expression Vector of Human Ki-67 and Bcl-2 Genes

目的构建反义Ki-67和Bcl-2双表达载体用于肿瘤基因治疗的研究。方法从SGC-7901细胞总RNA中逆转录扩增Ki-67和Bcl-2 cDNA,T-A克隆到pMD18-T Simple载体。Ki-67经BamHⅠ和ClaⅠ双酶切,Bcl-2经EcoRⅠ和XhoⅠ双酶切后,分别反向插入pVITRO2的多克隆位点1(mcs1)和2(mcs2),构建单表达质粒pVITRO2-AsKi-67和pVITRO2-AsBcl-2,再将Bcl-2EcoRⅠ和XhoⅠ双酶切,反向插入pVITRO2-AsKi-67的mcs2,构建pVITRO2-AsKi-67-AsBcl-2双表达质粒。结果限制性内切酶和测序表明单表达质粒pVITRO2-AsKi-67和pVITRO2-AsBcl-2以及双表达质粒pVITRO2-AsKi-67-AsBcl-2构建成功。结论本研究成功构建了pVITRO2-AsKi-67和pVITRO2-AsBcl-2单表达质粒,及pVITRO2-AsKi-67-AsBcl-2双表达质粒。

Aim To construct the recombinant co-expression vector carrying antisense RNA to Ki-67 and Bcl-2 genes and which will be used to resist tumers. Methods RT-PCR was used to amplify the ORF of Ki-67 and Bcl-2 cDNA from total RNA of Gastric carcinoma cell line SGC-7901. The Ki-67 cDNA fragment and the Bcl-2 cDNA fragment were inserted into pMD18-T simple vector respectively. The Ki-67 was digested with BamH I and Cla I and the Bcl-2 was digested with EcoR I and Xho I. Then the Ki-67 cDNA fragment and the Bcl-2 cDNA fragment were inverted insertion into the multiple cloning sites 1 and 2 （mcs1 and mcs2） of the eukaryotic co-expression vector pVITRO2 respectively. To construct the single expression plasmid pVITRO2-AsKi-67 and pVITRO2-AsBcl-2. Then the Bcl-2 cDNA fragment was inverted insertion into the mcs2 of the vector pVITRO2-AsKi-67.To construct the co-expression vector pVITRO2-AsKi-67-AsBcl-2.Results The restriction endonucleases digestion and sequencing suggested that the eukaryotic co-expression vector pVITRO2-AsKi-67-AsBcl-2 and the single gene expression plasmids pVITRO2-AsKi-67 and pVITRO2-AsBcl-2. Conclusion The co-expression vector pVITRO2-AsKi-67-AsBcl-2 and the single gene expression plasmids pVITRO2-AsKi-67 and pVITRO2-AsBcl-2 were constructed successfully.