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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 high expression vector of rat transcription factor Smad2 (pLJM1-Smad2). Methods The CD3 region of Smad2 mRNA was obtained by PCR using the cDNA of E3 rat liver as a template. The gene fragment of pLJM1-MGFP was digested by Age I and EcoR I and ligated with T4 DNA ligase Ligation of the purified product after digestion; then the ligation product was transformed into DH5α E. coli competent cells and positive clones were selected to expand the culture and extract the recombinant plasmid; Finally, PCR, Age I and EcoR I double digestion and DNA sequencing identified the recombinant plasmid . Results The results of PCR, Age I and EcoR I double digestion showed that the target fragment Smad2 was inserted into pLJM1-Smad2 recombinant vector, and the positive recombinant clone containing the target fragment was sent to Shanghai Biosystems for DNA sequencing. The sequencing results were compared with NCBI database The sequence of the inserted fragment was confirmed to be correct and the gene fragment Smad2 was successfully inserted into the pLJM1-Smad2 recombinant vector. Conclusion The high expression vector pLJM1-Smad2 of transcription factor Smad2 was successfully constructed.