中文摘要：

目的 构建携带胞嘧啶脱氨酶（CD）和血管内皮抑素（ES）基因的慢病毒载体。方法 根据GenBank提供大肠杆菌源CD和ES基因序列设计、合成2个基因并构建与荧光蛋白融合的真核表达载体pEGFP-CD和pDS-RED—ES。酶切和测序鉴定后，将载体瞬时转染HEK293细胞，运用实时PCR检测CD和ES基因表达情况。把载体中基因片段EGFP—CD和DS—RED．ES通过BP／LR重组分别构建慢病毒载体，并利用293FT细胞进行慢病毒的包装。将病毒原液浓缩，并测定病毒滴度。结果 酶切、测序及PCR鉴定证实构建出慢病毒载体pLenti6．3-CD-EGFP和pLenti6．3-ES—Monomer—DsRed。感染293FT细胞后荧光显微镜下观察到GFP和DsRed的表达。浓缩慢病毒滴度分别为2．2×10^8TU／ml和6．5×10^7TU／ml。结论 成功构建了CD和ES基因慢病毒载体。

英文摘要：

Objective To construct the lentiviral vectors with cytosine deaminase （CD） and endostatin （ES） genes. Methods The eukaryotic expression vectors pEGFP-CD and pDS- RED-ES encoding fluorescent proteins were constructed via designing and synthesizing the genes of CD and ES based on the sequence of E.coli provided by GenBank. After identification by digestion and sequencing, the vectors were immediately transfected into HEK293 cells followed by detection of CD and ES expression via real-time polymerase chain reaction. The lentiviral vectors were constructed using the gene segments EGFP-CD and DS- RED-ES via BP/LR recombination and were encapsulated by using 293FT cells. The viral stock solution was concentrated followed by detection of the viral titers. Results Construction of lentiviral vectors, pLenti6.3-CD-EGFP and pLenti6.3-ES-Monomer-DsRed, was verified by identification using enzyme digestion, gene sequencing and polymerase chain reaction. Expression of GFP and DsRed was noted via fluorescence microscope after transfection into 293FT cells. The concentrated titer of 2.2×10^8 TU/ml and 6.5× 10^7 TU/ml was revealed in respective lentivirus. Conclusion The lentiviral vectors of CD and ES are successfully constructed in this study.