诱导性鼠Smad7真核表达载体的构建与鉴定

Construction and identification of tetracycline-inducible rat Smad7 eukaryotic expression vector

目的构建四环素诱导性鼠Smad7真核表达载体。方法采用常规分子生物学方法,抽提SD大鼠肾脏总RNA,RT-PCR获得鼠Smad7cDNA,再定向克隆于四环素诱导性真核表达载体pBI-L多克隆位点NheI和HindIII之间,限制性内切酶酶切和测序鉴定。结果重组pBI-L-Smad7真核表达载体经限制性内切酶酶切分析和测序分析,与理论值相符。结论本实验成功构建鼠Smad7四环素诱导性真核表达载体pBI-L-Smad7,为今后临床开展组织器官纤维化Smad7基因治疗奠定实验基础。

Objective To construct a tetracycline-inducible eukaryotic expression vector of rat Smad7. Methods The total RNA was extracted from normal rat kidney with Trizol agent. Rat Smad7 cDNA fragment was cloned by RT-PCR, and was inserted into the restriction site between NheI and Hind III of the inducible eukaryotic expression vector pBI-L by tetracycline. pBI-L-Smad7 was constructed by digestion and ligation, and detected by restriction endonuclease digestion and sequencing. Results The recombinant eukaryotic expression vector pBI-L-Smad7 was constructed correctly as confirmed by restriction endonuclease digestion and sequencing. The fragment of pBI-L-Smad7 digested with restriction endonucleases and the sequence of inserted Smad7 cDNA were consistent with the results of theoretical analysis. Conclusion The tetracyclineinducible eukaryotic expression vector of rat Smad7, pBI-L-SmadT, is constructed successfully, which may facilitate further clinical study of Smad7 gene therapy for tissue and organ fibrosis.