FHIT基因真核细胞表达载体的构建与鉴定

Construction and identification of eukaryotic cell expression vector of human FHIT gene

目的 构建人FHIT基因真核细胞表达载体。方法 采用RT PCR方法 ,从人新鲜甲状腺组织的总cDNA中扩增出 45 6bp的人FHITcDNA片段 ,然后用KpnⅠ和BstXⅠ双酶切后定向克隆到真核细胞表达载体pcDNA3中 ,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒 ;用免疫细胞化学法检测FHIT基因的表达情况。结果 人FHIT基因cD NA已经正确克隆到真核细胞表达载体pcDNA3中 ;体外转染MM96L细胞后 ,可见转染细胞胞浆中有较高量的Fhit蛋白表达。

Objective To construct eukaryotic cell expression vector of human frangible histone triad (FHIT) gene. Methods A 456 bp cDNA fragment was amplified from the total RNA of normal human thyroid tissue by RT PCR method and cloned into plasmid pcDNA3. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes Kpn Ⅰ and Bst XⅠ and sequenced by Sanger dideoxy mediated chain termination. The expression of FHIT gene was detected by immunocytochemical methods. Results The results showed that the cDNA fragment included 456 bp entire coding region. The recombinant eukaryotic cell expression vector of pcDNA3 FHIT was constructed, and the sequence of the insert was identical to the published sequence. MM96L cells transfected with the pcDNA3 FHIT plasmid expressed high level of Fhit protein in cytoplasm. Conclusion The recombinant plasmid pcDNA3 FHIT can provide a strong molecular tool for the studies of neoplasm pathogenesis.