人FHIT基因的克隆及其真核表达质粒的构建

目的克隆人脆性组氨酸三联体（FHIT）基因，构建其真核表达质粒pRcCMV-FHIT。方法提取人外周血总RNA，用逆转录-聚合酶链反应（RT—PCR）技术扩增人FHITcDNA全长开放阅读框序列，PCR产物与中间载体pGEM-T连接后转化到大肠杆菌DH5α。然后对单菌落进行菌落PCR、限制性内切酶酶切鉴定和测序。用EcoRI将目的片段切下，插入真核表达载体pRc／CMV2的相应位点，构建表达质粒pRc／CMV2-FHIT，将该重组质粒转化到大肠杆菌DH5α后进行菌落PCR、酶切鉴定。结果测序结果表明从正常人外周血单个核细胞中所获得的FHITcDNA含有781个碱基，与Genbank（NM_002012．1）序列完全一致。pRc／CMV2-FHIT经酶切鉴定与预期结果一致。结论成功构建重组真核表达质粒pRcCMV-FHIT，为进一步研究脂质体转染FHIT基因对结肠癌细胞株SW480凋亡作用的影响奠定基础。

Cloning of human FHIT gene and construction of eukaryotic expression plasmid

Objective To clone the gene of Fragile histidine triad（FHIT） and to construct its eukaryotic expression plasmid pRcCMV-FHIT. Methods Total RNA was extracted from peripheral blood mononuclear cells （PBMCs）. RT-PCR was used to amplify FHIT eDNA open reading frame （ORF） sequence. The PCR products were inserted into pGEM-T, and the resulted plasmid was transformed into bacillus coli DH5α. The positive clone was selected and identified by colony PCR, digestion of internal restriction enzyme, and sequencing. The FHIT gene fragment obtained from pGEM- T-FHIT was cloned into eukaryotic expression plasmid pRe/CMV2. The positive clone was selected and identified by colony PCR and digestion of internal restriction enzyme. Results The PCR amplification yielded a single band of about 781 base pairs, sequence analysis of the PCR products revealed that it is consistent with that of the references published （GenBank NM_002012.1）. Conclusions The human FHIT gene was cloned, and its eukaryotic expression plasmid pRcCMV-FHIT was constructed. It will be beneficion for the further studies on effects of liposome-mediated transfeetion of FHIT gene to colon Cancer Cell Line SW480.