大鼠血栓调节蛋白基因的克隆及在原核细胞中的表达

目的研究大鼠血栓调节蛋白基因的扩增、克隆及在原核细胞中的表达。方法以大鼠基因组DNA为模板,进行PCR扩增,用pMD18-Tsimple vector进行T克隆并测序。经EcoRⅠ和SalⅠ双酶切,将目的基因克隆到pET-28a(+)表达载体质粒中,转化E.coliBL21(DE3)表达重组蛋白,通过SDS-PAGE分析表达产物。结果含有大鼠血栓调节蛋白基因的PCR产物约为1850bp。重组pMD18-Tsimple vector质粒和重组pET-28a(+)质粒经EcoRⅠ和SalⅠ双酶切后,有相应大小的目的片段,测序结果正确。经IPTG诱导,重组pET-28a(+)质粒在E.coliBL21(DE3)中有相对分子质量约为72000的融合蛋白表达。结论成功克隆了大鼠血栓调节蛋白基因,并成功表达了该基因编码的蛋白。

Cloning of rat thrombomodulin gene and its prokaryotic expression

Purpose To study the PCR amplification,cloning and protein expression of rats thrombomodulin(TM)gene.MethodsWith the genome of rats as template,TM gene was amplified by means of PCR and its T-A was cloned by pMD18-T simple vector and sequenced.After EcoRⅠ and SalⅠ digestion,the target gene fragment was linked to pET-28a(+)vector to construct the recombination plasmid.After identification with restriction digestion and DNA sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3)for expression of the recombinant protein.The target protein was identified by SDS-PAGE.ResultsThe length of the PCR product of TM gene was about 1 850 bp.The TM gene was successfully inserted into pMD18-T simple vector with correct sequence and the cloning of the TM gene into the pET-28a(+)protein expression plasmid was achieved.Expression of the protein of pET-28a(+)recombination plasmid and TM gene with a new band about 72 kd in weight was detected after IPTG induction.ConclusionThe TM gene has been successfully cloned and expressed in E.coli BL21(DE3).