摘要
目的:构建并鉴定大鼠Smad7/pcDNA3.1(+)真核表达质粒,为进一步研究Smad7的抗肝纤维化作用提供实验基础。方法:TRIzol法抽提大鼠肝组织总RNA,琼脂糖凝胶电泳检测其完整性,紫外分光核酸蛋白分析仪测定其浓度与纯度,两步法获取大鼠Smad7cDNA片段,CaCl2法制备感受态细胞,应用EcoRI及XhoI在pcDNA3.1(+)多克隆位点处进行双酶切,切胶回收载体及目的片段酶切产物,将回收的线性化pcDNA3.1(+)与Smad7cDNA连接,构建Smad7/pcDNA3.1(+)重组质粒,转化DH5α大肠杆菌,测序鉴定双酶切证实的阳性克隆。结果:电泳检测RNA的完整性良好,28S约为18S的2倍,A260/A280=1.986,纯度较好,阳性克隆酶切后电泳检测在DNAMarker1.3kb处见Smad7目的片段,5.4kb处见线性化pcDNA3.1(+),与预期结果相符,质粒重组成功,并测序证实。结论:大鼠Smad7真核表达质粒构建成功,为进一步单独或联合其他质粒从TGF-β/SMAD信号传导的不同环节干预肝纤维化提供了实验基础。
Objective：To construct rat Smad 7/pcDNA3.1（＋） eukaryotic expressing plasmid and provide the experimental foundation for further studying the role of Smad 7 in anti-hepatic fibrosis.Methods：Total RNA was obtained from rat liver tissues by TRIzol.The integrality,concentration and purity of samples were detected by ultraviolet spectrophotometry and a-garose electrophoresis.The fragment of Smad 7 cDNA was obtained by RT-PCR,and then amplified by PCR.The competent cells were induced by CaCl2.Multiple cloning sites of pcDNA3.1（＋）,the fragment cloning sites,and the fragment of Smad 7,were digested by EcoRI and XhoI,and then,purified and retrieved by agarose electrophoresis separation.The Smad 7 /pcD-NA3.1（＋） was constructed by joining with the purified linear pcDNA3.1（＋） and the purified fragment of Smad 7.This recombi-nant plasmid was transfected into DH5α by heat shock and identified by restriction endonuclease digestion and sequence analysis.Results：Agarose electrophoresis showed that the strips of 28 S and 18 S were integral.Furthermore,the luminosity of the 28 S strip was twice as high as 18 S.The ratio of A260/A280 was 1.986,so the total RNA was considered to be in good integrity and purity.The two fragments digested from Smad 7 /pcDNA3.1（＋） by EcoRI and XhoI represented 1.3 kb and 5.4 kb after agarose electrophoresis,indicating the successful construction of Smad 7 /pcDNA3.1（＋）.DNA sequence analysis showed the same re-sult.Conclusion：Smad 7/pcDNA3.1（＋） eukaryotic expressing plasmid was constructed successfully.It provides the experi-mental foundation for further studying that transfection of this plasmid alone or combined with other plasmids to block the TGF-β /SMAD signaling in different steps of hepatic fibrosis.
出处
《天津医药》
CAS
北大核心
2008年第11期891-893,共3页
Tianjin Medical Journal
基金
兵团博士资金资助项目(项目编号:05JC08)
