CAJ | 학술논문

目的构建带有HAM标签的真核表达重组质粒pCGN-HAM-N1-wcAPC及探讨其功能.方法制备高效率感受态；用聚合酶链反应(PCR)扩增,将腺瘤息肉病(APC)基因截短的片段APC1克隆于T载体；同时将pCMV-neo-bam-APC酶切,得到APC基因的另一个8300 bp截短片段kAPC亚克隆于pCGN-HAM-N1真核表达载体,得重组质粒pCGN-HAM-N1-kAPC；再运用双酶Sal Ⅰ和Kpn Ⅰ同时酶切TA克隆、pCGN-HAM-N1-kAPC,将APC1片段插入载体pCGN-HAM-N1-kAPC,获含野生型全长APC基因重组质粒pCGN-HAM-N1-wcAPC.用酶切,测序鉴定.脂质体法分别将含全长,截短的重组质粒,空载体与WNT信号系统的β-连环蛋白(β-catenin)、topflash和荧光素酶共同转染293T细胞,利用双荧光报告测试系统,检测转染后荧光素酶活性.结果重组质粒经酶切其大小与预期一致,经测序比对证实与GeneBank中给出的序列一致.转染pCGN-HAM-N1-wcAPC组的荧光素酶活性明显低于转染空载体组pCGN-HAM-N1(P＜0.05).结论含野生型全长9000 bp大片段APC基因分段成功插入pCGN-HAM-N1载体,带有HAM标签的真核表达重组质粒pCGN-HAM-N1-wcAPC构建成功,pCGN-HAM-N1-wcAPC抑制T细胞因子/淋巴增强因子(Tcf/Lef)启动子的活性.
목적 구건대유HAM표첨적진핵표체중조질립pCGN-HAM-N1-wcAPC급탐토기공능.방법 제비고효솔감수태；용취합매련반응(PCR)확증,장선류식육병(APC)기인절단적편단APC1극륭우T재체；동시장pCMV-neo-bam-APC매절,득도APC기인적령일개8300 bp절단편단kAPC아극륭우pCGN-HAM-N1진핵표체재체,득중조질립pCGN-HAM-N1-kAPC；재운용쌍매Sal Ⅰ화Kpn Ⅰ동시매절TA극륭、pCGN-HAM-N1-kAPC,장APC1편단삽입재체pCGN-HAM-N1-kAPC,획함야생형전장APC기인중조질립pCGN-HAM-N1-wcAPC.용매절,측서감정.지질체법분별장함전장,절단적중조질립,공재체여WNT신호계통적β-련배단백(β-catenin)、topflash화형광소매공동전염293T세포,이용쌍형광보고측시계통,검측전염후형광소매활성.결과 중조질립경매절기대소여예기일치,경측서비대증실여GeneBank중급출적서렬일치.전염pCGN-HAM-N1-wcAPC조적형광소매활성명현저우전염공재체조pCGN-HAM-N1(P＜0.05).결론 함야생형전장9000 bp대편단APC기인분단성공삽입pCGN-HAM-N1재체,대유HAM표첨적진핵표체중조질립pCGN-HAM-N1-wcAPC구건성공,pCGN-HAM-N1-wcAPC억제T세포인자/림파증강인자(Tcf/Lef)계동자적활성.
Objective To construct an eukaryotic expression recombinant plasmid named pCGN-HAM-N1-wcAPC and transfected it into cell line to simply analysis it function.Methods Insert a big one fragment 9000 bp Adenomatous polyposis coli (APC) gene into pCGN-HAM-N1,which is a eukaryotic expression vector with a HA epitope tag by Cloning polymerase chain reaction (PCR) products into pMD-18T and cutting one fragment insert into another plasmid.Then the recombinant vector was identified by incision enzyme and DNA sequence.Transfected 293T cell with the indicated amounts of vectors,including β-catenin,topflash,and Renilla luciferase,by Lipofectamine 2000.Analyzed luciferase activity and checked the function of pCGN-HAM-N1-wcAPC.Results 9000 bp APC gene insets into 5.2 kb vector pCGN-HAM-N1,DNA sequence was identical with APC cDNA in NCBI.The transfected pCGN-HAM-N1-wcAPC group luciferase activity was significantly lower than empty vector group.Conclusion The eukaryotic expression recombinant plasmid and transfection of it were successfully constructed,which will be used further study.pCGN-HAM-N1-wcAPC inhibits of Activation of T-cell factor/lymphocyte enhancer factor (Tcf/Lef) Promoter.