人端粒酶催化亚单位(hTERT)特异性锤头状核酶真核表达载体的构建及鉴定  

Construction and identification of the eukaryotic expression vector containing genes of hammerhead ribozyme targeting hTERT mRNA

作　　者：樊丽[1] 许景伟 邹宇[1] 

机构地区：[1]齐齐哈尔医学院中心实验室,161006 [2]齐哈尔医学院第一附属医院普外一科,161006

出　　处：《齐齐哈尔医学院学报》2008年第21期2564-2566,共3页Journal of Qiqihar Medical University

摘　　要：目的设计并合成人TERT特异性的锤头状核酶(hammerhead ribozyme)基因并构建其真核表达载体。含有核酶靶基因hTERTcDNA保守序列的T载体pMD18-T-hTERT的构建。方法运用计算机软件人工设计并合成核酶基因(RZ),将核酶基因克隆到真核表达载体pcDNA 3.1(+)中,重组子由BamH I和EcoR I酶切,琼脂糖凝胶电泳鉴定及DNA测序。RT-PCR法扩增目的基因cDNA保守序列,与pMD-18进行T-A连接,重组子经菌液PCR及测序鉴定。结果RZ人工合成后与pcDNA 3.1(+)连接,酶切电泳及DNA测序证实合成的核酶基因序列正确并已被准确克隆入pcDNA 3.1(+)的BamH I和EcoR I位点之间,命名为pcDNA 3.1-RZ。220bp的hTERT基因cDNA保守序列准确克隆于pMD18-T,命名为pMD18-T-hTERT。结论成功合成hTERT特异性的锤头状核酶基因并构建了该基因的真核表达载体,以及核酶靶基因的pMD18-T-hTERT载体。Objective To construct and identify the eukaryotic expression vector with genes of hammerhead ribozyme targeting hTERT mRNA and construct pMD18-T-hTERT. Methods According to design of computer,two specific restriction site BamH I andEcoR I were added to both ends of the ribozyme gene,then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1.Results The positive recombinants were screened by tolerance of ampicillin,and plasmids were extracted from the positive recombinants and digested by BamH I and EcoRI,and then were analyzed by agarose gel electrophoresis and DNA sequencing and obtain objective gene consensus sequence 220bp by RT-PCR and construct pMD18-T-hTERT by T-A connect.Conclusions The eukaryotic expression vectors with genes of hammerhead ribozyme targeting hTERT mRNA and with genes of the substrate of the specific ribozyme were constructed successfully and respectively.

关 键 词：HTERT 锤头状核酶 真核表达载体pcDNA3.1(+) 

分 类 号：R735.8[医药卫生—肿瘤] R512.62[医药卫生—临床医学]