人抑癌基因FHIT真核表达质粒的构建与鉴定

Construction and Identification of Eukaryotic Cell Expression Vector of Human Tumor Suppressor Gene FHIT

[目的]构建人抑癌基因FHIT真核细胞表达载体。[方法]采用PCR方法,从人胎脑组织的总cDNA中扩增出444bp的人FHITcDNA片段,然后定向克隆到真核细胞表达载体pcDNA3.1/myc-His(-)B中,用限制性内切酶EcoRⅠ和BamHⅠ双酶切后酶切分析和DNA序列分析鉴定重组质粒;用该表达质粒转染COS-7细胞,Westernblot法检测FHIT蛋白的表达情况。[结果]人FHIT基因cDNA以正确方向插入到真核细胞表达载体pcDNA3.1/myc-His(-)B中;转染COS-7细胞后,可见转染细胞有Fhit蛋白的表达。[结论]本实验成功地构建了人抑癌基因FHIT的真核表达质粒pcDNA3.1/myc-His(-)B-FHIT,为研究FHIT基因在肿瘤的发生中的作用提供了有效的工具。

[Purpose] To construct eukaryotic cell expression vector of human fragile histidine triad（FHIT） gene. [Methods] A 444bp cDNA fragment was amplified from the human fetal brain by PCR method and cloned into plasmid pcDNA3.1/myc-His（-）B. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes EcoR I and BamH I and by DNA sequencing analysis, peDNA3.1/mye-His（-）B-FHIT was transfeeted into COS-7 cell line, the expression of FHIT protein was detected by Western blot method. [ Results ] The results showed that the human FHIT cDNA fragment was cloned correctly into plasmid pcDNA3.1/myc-His （-）B. COS-7 cells which were transfected with the pcDNA3.1/myc-His （-）B-FHIT plasmid expressed FHIT protein. [Conclusions] The eukaryotic cell expression vector of human FHIF gene pcDNA3.1/ myc-His （-）B-FHIT were constructed successfully which can provide an effective tool for the research of FHIT gene in carcinogenesis.