人NIT1基因原核表达载体的构建和鉴定  

Construction and identification of procaryotic expression vector of human NIT1 gene

作　　者：吴汉林[1] 王允[1] 周清华[1] 朱文[1] 王艳萍[1] 车国卫[1] 陈晓禾[1] 孙芝琳[1] 

机构地区：[1]四川大学华西医院四川省肺癌分子重点实验室,肿瘤中心,成都610041

出　　处：《中国肺癌杂志》2005年第2期81-84,共4页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金(No.30100181);四川大学青年科技基金资助~~

摘　　要：背景与目的　目前已知FHIT基因为抑癌基因,其结构和功能的异常与肺癌的发生、发展有密切关系,而且可能与NIT1基因存在功能上的相关性。但是,FHIT基因与NIT1基因调控肺癌发生发展的分子机制尚未明了。本研究旨在构建人NIT1基因原核表达载体,为进一步研究人肺癌细胞株中NIT1 基因和FHIT基因的关系打下基础。方法　应用RT PCR法获得NIT1基因cDNA,定向克隆到融合表达载体pET 32a中,作双酶切和测序鉴定。结果　①克隆的cDNA片断包含了人NIT1基因翻译区的全部序列,翻译区序列与GenBank登录的人NIT1 cDNA序列完全一致。②对构建的原核表达载体做双酶切,获得了预期的目的条带。结论　通过RT PCR和定向克隆的方法,成功构建了人NIT1 基因的原核表达载体,为研究人NIT1蛋白的表达和进一步研究人肺癌细胞中NIT1基因和抑癌基因FHIT的关系奠定了基础。Background and objective The human FHIT gene at chromosome 3p14.2 is a tumor suppressor gene, and its abnormality in structure and function is related to carcinogenesis and progression of lung cancer. It is postulated that FHIT and NIT1 likewise collaborate in biochemical or cellular pathway in mammalian cells, but their molecular mechanisms in lung cancer cell are still unknown. The aim of this study is to construct procaryotic expression vector of human NIT1, providing a foundation to explore the expression of human NIT1 protein and to study the interaction of NIT1 and FHIT genes in lung cancer cell lines. Methods The NIT1 cDNA was acquired by RT-PCR. EcoRⅠ/NotⅠ digested PCR product was directly cloned into procaryotic expression vector pET-32a. Results The sequence of NIT1 cDNA clone exactly corresponded with the sequence of NIT1 cDNA in GenBank. The expectant fragments of DNA were obtained after recombinant procaryotic expression vector was digested by EcoRⅠ and NotⅠ. Conclusion The procaryotic expression vector of human NIT1 is successfully constructed by RT-PCR and direct clone. It provides an important basis to detect the expression of NIT1 protein and to further explore the relationship between NIT1 and FHIT genes in oncogenesis and development of human lung cancer.

关 键 词：原核表达载体 人NIT1基因 FHIT基因 肺肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]