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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FHIT cDNA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EcoR Ⅰ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。
Objective To clone human fragile histidine triad (FHIT) gene and construct its eukaryotic expression plasmid pRcCMV-FHIT. Methods Total RNA of human peripheral blood was extracted and the full-length open reading frame of human FHIT cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was ligated with the intermediate vector pGEM-T and transformed into E. coli DH5α. Then single colony colony PCR, restriction enzyme digestion and sequencing. The fragment of interest was cut with EcoR I. Inserted into the corresponding sites of the eukaryotic expression vector pRc / CMV2, and constructed the expression plasmid pRc / CMV2-FHIT. The recombinant plasmid was transformed into Escherichia coli DH5α, and then colony PCR was carried out to identify the recombinant plasmid. Results The sequencing results showed that FHIT cDNA obtained from normal human peripheral blood mononuclear cells contained 781 bases, which was completely consistent with the sequence of Genbank (NM_002012.1). Identification of pRc / CMV2-FHIT by restriction enzyme was consistent with the expected results. Conclusion The recombinant eukaryotic expression plasmid pRcCMV-FHIT was successfully constructed, which laid the foundation for the further study on the effect of FHIT gene transfection on the apoptosis of colon cancer cell line SW480.