中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
6期
1072-1075,封3
,共5页
杨欢%陈志强%叶章群%王博涵%艾丽娅%姚炜敏
楊歡%陳誌彊%葉章群%王博涵%艾麗婭%姚煒敏
양환%진지강%협장군%왕박함%애려아%요위민
产甲酸草酸杆菌%慢病毒载体%高草酸尿症
產甲痠草痠桿菌%慢病毒載體%高草痠尿癥
산갑산초산간균%만병독재체%고초산뇨증
Oxalobacter formigenes%Lentiviral vector%Hyperoxalurias
目的 构建国人肠道来源产甲酸草酸杆菌(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-T Simple载体连接获得FRC和OXC的克隆载体,并转化大肠杆菌DH5α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pMD18T simple-FRC和pMD18T simple-OXC,用BamH Ⅰ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/v5 DEST-IRES-DsRED,转化DH5α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HEK293T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1.3kb和1.7kb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FRC cDNA和OXC cDNA,转染HEK293T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1.15×108 TU/ml和9.75×107 TU/ml.结论 成功构建表达OxCF中草酸分解关键基因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.
目的 構建國人腸道來源產甲痠草痠桿菌(OxCF)甲酰輔酶A轉移酶(FCoAT)基因(FRC)和草酰輔酶A脫羧酶(OCoAD)基因(OXC)的重組慢病毒pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED,為高草痠尿癥的基因治療奠定基礎.方法 採用Taq高保真DNA聚閤酶從OxCF基因組中擴增FRC基因和OXC基因片段,用DNA凝膠迴收試劑盒切膠迴收,用DNA連接試劑盒將迴收產物分彆與pMD18-T Simple載體連接穫得FRC和OXC的剋隆載體,併轉化大腸桿菌DH5α感受態細胞,挑取暘性剋隆併測序,篩選攜帶完整目的基因質粒pMD18T simple-FRC和pMD18T simple-OXC,用BamH Ⅰ酶切穫得目的基因,連接FRC和攜帶綠色熒光蛋白的pLenti6.3/v5DEST-IRES-EGFP,連接OXC與錶達紅色熒光蛋白的pLenti6.3/v5 DEST-IRES-DsRED,轉化DH5α後挑取暘性剋隆併測序.構建的慢病毒過錶達載體pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED轉染HEK293T細胞,包裝併測滴度.結果 聚閤酶鏈反應(PCR)從OxCF基因組中擴增分穫得1.3kb和1.7kb的DNA片段,與Genbank中FRC( U82167)間堿基序列匹配率為95.88％,存在53箇堿基的變異；與Genbank中OXC( M77128)間堿基序列匹配率為93.61％,存在109箇堿基的變異；測序證實pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED分彆含有大小正確的正嚮FRC cDNA和OXC cDNA,轉染HEK293T細胞實驗24h後,在熒光顯微鏡下相應可見大量綠色熒光和紅色熒光,兩種慢病毒滴度分彆為1.15×108 TU/ml和9.75×107 TU/ml.結論 成功構建錶達OxCF中草痠分解關鍵基因FRC和OXC的重組慢病毒載體,併通過轉染293細胞製備瞭高滴度慢病毒.
목적 구건국인장도래원산갑산초산간균(OxCF)갑선보매A전이매(FCoAT)기인(FRC)화초선보매A탈최매(OCoAD)기인(OXC)적중조만병독pLenti6.3-FRC-IRES-EGFP화pLenti6.3-OXC-IRES-DsRED,위고초산뇨증적기인치료전정기출.방법 채용Taq고보진DNA취합매종OxCF기인조중확증FRC기인화OXC기인편단,용DNA응효회수시제합절효회수,용DNA련접시제합장회수산물분별여pMD18-T Simple재체련접획득FRC화OXC적극륭재체,병전화대장간균DH5α감수태세포,도취양성극륭병측서,사선휴대완정목적기인질립pMD18T simple-FRC화pMD18T simple-OXC,용BamH Ⅰ매절획득목적기인,련접FRC화휴대록색형광단백적pLenti6.3/v5DEST-IRES-EGFP,련접OXC여표체홍색형광단백적pLenti6.3/v5 DEST-IRES-DsRED,전화DH5α후도취양성극륭병측서.구건적만병독과표체재체pLenti6.3-FRC-IRES-EGFP화pLenti6.3-OXC-IRES-DsRED전염HEK293T세포,포장병측적도.결과 취합매련반응(PCR)종OxCF기인조중확증분획득1.3kb화1.7kb적DNA편단,여Genbank중FRC( U82167)간감기서렬필배솔위95.88％,존재53개감기적변이；여Genbank중OXC( M77128)간감기서렬필배솔위93.61％,존재109개감기적변이；측서증실pLenti6.3-FRC-IRES-EGFP화pLenti6.3-OXC-IRES-DsRED분별함유대소정학적정향FRC cDNA화OXC cDNA,전염HEK293T세포실험24h후,재형광현미경하상응가견대량록색형광화홍색형광,량충만병독적도분별위1.15×108 TU/ml화9.75×107 TU/ml.결론 성공구건표체OxCF중초산분해관건기인FRC화OXC적중조만병독재체,병통과전염293세포제비료고적도만병독.
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 cDNA 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 DH5α.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 ligated 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 × 108 TU/ml and 9.75 × 107 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.
