产甲酸草酸杆菌甲酰辅酶A转移酶基因和草酰辅酶A脱羧酶基因慢病毒表达载体的构建及鉴定  

Construction and identification of recombinant lentiviral expressing vectors formyl-CoA transferase gene and Oxalyl-CoA decarboxylase gene of oxalobacter formigenes from Chinese feces

作　　者：杨欢[1] 陈志强[1] 叶章群[1] 王博涵[1] 艾丽娅[1] 姚炜敏[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030

出　　处：《中华实验外科杂志》2012年第6期1072-1075,F0003,共5页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家自然科学基金资助项目（30872564）

摘　　要：目的构建尉人肠道米源产甲酸草酸杆菌（OxCF）甲酰辅酶A转移酶（FCoAT）基因（FRC）和草酰辅酶A脱羧酶（OCoAD）基因（OXC）的重组慢病毒pLenti6．3-FRC—IRES—EGFP和pLenti6．3-OXC—IRES—DsRED，为高草酸尿症的基因治疗奠定基础。方法采用Taq高保真DNA聚合酶从OxCF基因组中扩增FRC基因和OXC基因片段，用DNA凝胶回收试剂盒切胶回收，用DNA连接试剂盒将回收产物分别与pMD18-TSimple载体连接获得FRC和OXC的克隆载体，并转化大肠杆菌DH5a感受态细胞，挑取阳性克隆并测序，筛选携带完整目的基因质粒pMD18Tsimple—FRC和pMD18Tsimple—OXC，用BamHI酶切获得目的基因，连接FRC和携带绿色荧光蛋白的pLenti6．3／v5DEST—IRES—EGFP，连接OXC与表达红色荧光蛋白的pLenti6．3／v5DEST—IRES—DsRED，转化DH5a后挑取阳性克隆并测序。构建的慢病毒过表达载体pLenti6．3-FRC—IRES—EGFP和pLenti6．3-OXC—IRES—DsRED转染ttEK293T细胞，包装并测滴度。结果聚合酶链反应（PCR）从OxCF基因组中扩增分获得1．3kh和1．7kh的DNA片段，与Genbank中FRC（U82167）间碱基序列匹配率为95．88％，存在53个碱基的变异；与Genbank中OXC（M77128）间碱基序列匹配率为93．61％，存在109个碱基的变异；测序证实pLenti6．3-FRC—IRES—EGFP和pLenti6．3-OXC—IRES—DsRED分别含有大小正确的正向FRCcDNA和OXCcDNA，转染ttEK293T细胞实验24h后，在荧光显微镜下相应可见大量绿色荧光和红色荧光，两种慢病毒滴度分别为1．15×10^8TU／ml和9．75×10^7TU／ml。结论成功构建表达OxCF中草酸分解关键基闵FRC和OXC的重组慢病毒载体，并通过转染293细胞制备了高滴度慢病毒。Objective To clone the formyl-CoA transferase gene （FRC）, and Oxalyl-CoA decarboxylase gene （OXC）, in oxalobacter formigenes from Chinese feces （OxCF）, and construct the recombinant lentiviral expressing： vectors pLenti6. 3-FRC-IRES-EGFP and pLenti6. 3-OXC-IRES-DsRED, providing a basis for further study on gene therapy for hyperoxalurias. Methods Total RNA was isolated from OxCF, and the FRC and OXC eDNA was amplified by polymerase chain reaction （PCR） and then ligated with pMD18T simple vectors after retrieve and purification. The ligation products were transformed into competent DH5a. The positive recombinant clones were selected and identified by a complementation, restriction endonuclease digestion.＂ The cloning vector and the lentiviral vectors pLenti6. 3/v5 DEST-IRES-EGFP and pLenti6.3/v5 DEST-IRES-DsRED first digested with BamHI were hgated and transformed respectivly. The enzyme and PCR analyses were performed to confirm the recombinant vector, and then DNA sequence were analysis. The titer of constucted lentivirus vectors were tested. Results Two fragments of 1287 bp and 1707 bp were obtained by PCR respectivly. The enzyme and PCR analyses revealed that the correct FRC and OXC cDNA was cloned. The sequence of pLenti6. 3-FRC-IRES-EGFP and pLenti6. 3-OXC-IRES- DsRED was identical to that of cloned cDNA respectivly. HEK293T cells had green and red fluorescence 24 h after transfection. The titers of two types of virus were 1.15 × 10^8 TU/ml and 9. 75 × 10^7 TU/ml respectively. Conclusion FRC and OXC are cloned correctly and the recombinant lentiviral vectors pLenti6.3-FRC-IRES-EGFP and pLenti6. 3-OXC-IRES-DsRED are constructed successfully.

关 键 词：产甲酸草酸杆菌 慢病毒载体 高草酸尿症 

分 类 号：R589[医药卫生—内分泌]