Construction of Smad eukaryotic expression plasmid and its expression in EL4 cells

WANG Jingfan; JI Ran; LI Xingyu; CHEN Jinling

doi:10.3969/j.issn.1002-266X.2018.21.003

Abstract：Objective To construct eukaryotic expression plasmids of pcDNA3. 1-Smad2,pcDNA3. 1-Smad3,and pcDNA3. 1-Smad4 and to investigate whether Smad can be stably expressed in the mouse T lymphoma cells (EL4). Meth-ods The target CDS fragment of transcription factors Smad2,Smad3,and Smad4 was obtained by PCR using EL4 cells cDNA as the template;then pMD19-T-Smad2,pMD19-T-Smad3,and pMD19-T-Smad4 were constructed,respectively;the pMD19-T-Smad2 and pcDNA3. 1 vector were cut by restriction endonucleases Hind Ⅲ and EcoR Ⅰ,while the pMD19-T-Smad3,pMD19-T-Smad4 and pcDNA3. 1 vector were cut by restriction endonucleases Xho Ⅰ and EcoR Ⅰ,and then we ligated the purified enzyme-digested products by using the T4 DNA ligase;subsequently,the connection productions were used to transform DH5α competent cells,and the positive clones were picked up;finally,the recombinant plasmids were i-dentified through PCR,double-restrict-enzyme digestion,and DNA sequencing. The expression plasmids of pcDNA3. 1-Smad2,pcDNA3. 1-Smad3,and pcDNA3. 1-Smad4 were transfected into EL4 cells,respectively. The expression of target proteins was detected by Western blotting. Results The recombinant plasmids of pcDNA3. 1-Smad2,pcDNA3. 1-Smad3, and pcDNA3. 1-Smad4 were constructed successfully,which were confirmed by PCR and double-restrict-enzyme. DNA se-quencing results were analyzed by DNAMAN,and we further confirmed that there was no mutation base in the insertion fragment of pcDNA3. 1-Smad2,pcDNA3. 1-Smad3,and pcDNA3. 1-Smad4. Transfection of the recombinant plasmids of pcDNA3. 1-Smad2,pcDNA3. 1-Smad3,and pcDNA3. 1-Smad4 into EL4 cells could remarkably up-regulate the expression of Smad2,Smad3,and Smad4,respectively. Conclusions The eukaryotic expression plasmids of pcDNA3. 1-Smad2,pcDNA3. 1-Smad3,and pcDNA3. 1-Smad4 are constructed successfully. Transfection of the recombinant plasmids into EL4 cells induces the high expression of target proteins.