反义Ki-67 和Bcl-2真核细胞双表达载体的构建及鉴定

im 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-Bcl-2.Results The restriction endonucleases digestion and sequencing suggested that the eukaryotic co-expression vector pVITRO2-AsKi-67-Bcl-2 and the single gene expression plasmids pVITRO2-AsKi-67 and pVITRO2-AsBcl-2 Conclusion The co-expression vector pVITRO2-AsKi-67-Bcl-2 and the single gene expression plasmids pVITRO2-AsKi-67 and pVITRO2-AsBcl-2 were constructed successfully.