HLA-C 和HLA-G基因真核细胞双表达载体的克隆.docVIP

反义Ki-67 和Bcl-2真核细胞双表达载体的构建及鉴定 卢天龙，陈建国，韩跃武，张庆华 (兰州大学基础医学院医学生物化学与分子生物学研究所，甘肃 兰州 730000) Construct and identif of the eukaryotic co-expression vector of human Ki-67 and Bcl-2 genes LU Tian-long, CHEN Jian-guo, HAN Yue-wu, ZHANG Qing-hua Department of Biochemistry and Molecular Biology, Lanzhou Medical College, Lanzhou University, Lanzhou 730000, China Abstract: 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-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. Key words: Ki-67；Bcl-2；Bicistronic vector pVITRO2；antisense RNA 摘 要：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BamH Ⅰ和Cla Ⅰ双酶切，Bcl-2经EcoR Ⅰ和Xho Ⅰ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-2 EcoR Ⅰ和Xho Ⅰ双酶切，反向插入pVITRO2-AsKi-67的mcs2，构建pVITRO2-AsKi-67-