中文摘要：

目的 构建真核表达载体plRES2-EGFP／TRAIL和pinES2-EGFP／CD，为研究其联合表达对恶性胶质瘤的联合治疗作用提供基础。方法将pCMV／CD质粒和pcDNA3．1（＋）／TRAIL质粒行琼脂糖凝胶电泳检测，确定其完整性，并进行序列测定确定有无基因突变。pCMV／CD质粒用SacⅡ／XhoⅠ双酶切，pcDNA3．1（＋）TRAIL质粒用BamHⅠ／XhoⅠ双酶切，将目的基因定向克隆到真核细胞表达载体plRES2-EGFP中，转化E．coliDH5α感受态细胞，通过限制性内切酶双酶切、PCR及核酸序列分析等筛选、鉴定重组质粒。结果所构建的真核表达载体plRES2-EGFP／TRAIL经SacⅡ／XhoⅠ双酶切回收片段分别为1．0、5．2kb；plRES2-EGFP／CD经BamHⅠ／XhoⅠ双酶切回收片段分别为1．3、5．2kb。PCR及测序证实，plRES2-EGFP／TRAIL和plRES2-EGFP／CD质粒基因组中分别包含TRAIL和CD基因。结论成功构建了真核表达载体plRES2-EGFP／TRAIL和plRES2-EGFP／CD，为研究其联合表达对恶性胶质瘤的联合治疗作用奠定了基础。

英文摘要：

Objective To construct the eukaryotic expression vector encoding CD and TRAIL genes, pIRES2-EGFP/ TRAIL and pIRES2-EGFP/CD, and provide the research basis of the association between overexpression of CD and TRAIL genes in C6 glioma cells. Methods The received plasmids DNA were assessed by electrophoresis in 1% agarose gel and sequencing. CD and TRAIL genes were cloned directionally into eukaryotic expression vector, pIRES2-EGFP, through double enzyme-cutting by Sac Ⅱ/Xho Ⅰ and BamH Ⅰ/Xho Ⅰ . The recombinant plasmids were converted into E. coli DH5α competent cell, and then bolted and identified by double enzyme-cutting of restriction enzyme, PCR, and nucleic acid sequence analysis. Results The length of two pIRES2-EGFP/TRAIL fragmems after double-cutting by Sac Ⅱ/Xho Ⅰ were confident with theoretic length of 1.0 kb and 5.2 kb. The length of two pIRES2-EGFP/CD fragments after double-cutting by BamH I/Xho I were confident with theoretic length of 1.3 kb and 5.2 kb. pIRES2-EGFP/TRAIL and plRES2-EGFP/ CD were confirmed to be contained CD and TRAIL genes by PCR and nucleic acid sequence analysis. Conclusions Reconstructing pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD could successfully establish abasis of further research of the association between overexpression of CD and TRAIL genes in C6 glioma cells.