转录因子Smad2高表达载体的构建和鉴定

Construction and Identification of the overexpression vector of transcription factor Smad 2

目的构建并鉴定大鼠转录因子Smad2的高表达载体(简称pLJM1-Smad2)。方法首先以E3大鼠肝脏cDNA为模板、PCR法获取转录因子Smad2 mRNA CDS区(即目的基因片段);然后用Age I、EcoR I双酶切pLJM1-MGFP载体和目的基因片段,用T4DNA连接酶连接纯化后的酶切产物;之后再将连接产物转化DH5α大肠杆菌感受态细胞并挑选阳性克隆,扩大培养并提取重组质粒;最后用PCR、Age I和EcoR I双酶切以及DNA测序鉴定重组质粒。结果 PCR、Age I和EcoR I双酶切结果均提示pLJM1-Smad2重组载体中插入了目的片段Smad2;将含有目的片段的阳性重组体克隆送上海生工进行DNA测序,并将测序结果与NCBI数据库的进行比对,确定插入片段无突变、序列完全正确,证实pLJM1-Smad2重组载体中成功插入了目的基因片段Smad2。结论成功构建了转录因子Smad2的高表达载体pLJM1-Smad2。

Objective To construct and identify the overexpression vector of rat transcription factor Smad2 using eukaryotic expression vector pLJM1.Methods The target CDS fragment of transcription factor Smad2,which is the downstream molecule of TGF-βsignal pathway,was obtained by PCR using E3 rat liver cDNA as template.The target fragment and pLJM1 vector were cut by restriction endonucleases Age I and EcoR I,and then ligated the purified enzyme-digested products using the T4 DNA ligase.Subsequently,the connection productions were used to transform DH5αcompetent cells,and the positive clones were picked up from the agarose plate with ampicillin.Finally,the recombinant plasmids were identified through PCR、double-restrict-enzyme digestion of Age I and EcoR I and DNA sequencing.Results The results of PCR and double-restrict-enzyme digestion of Age I and EcoR I both confirmed that the target fragment have been successfully inserted into the recombinant vector pLJM1-Smad2.The BLAST analysis of DNA sequencing results further confirmed that there was no mutation base in the insertion fragment of pLJM1-Smad2.Conclusion The overexpression of rat transcription factor Smad2,pLJM1-Smad2,was constructed successfully.