pCGN-HAM-N1-wcAPC质粒的构建及其功能

Construction of recombinant plasmid pCGN-HAM-N1-wcAPC and this plasmid inhibition of activation of T-cell factor/lymphocyte enhancer factor promoter

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.

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.