Smad真核表达质粒构建及其在小鼠T淋巴瘤细胞中的表达

摘要

目的 构建并鉴定pcDNA3.1-Smad2、pcDNA3.1-Smad3及pcDNA3.1-Smad4真核表达质粒,探讨Smad在小鼠T淋巴瘤细胞(EL4)中能否稳定表达.方法 以EL4细胞cDNA为模板,PCR法获取转录因子Smad2、Smad3、Smad4 mRNA CDS区即目的基因片段;构建含有目的基因的T载体pMD19-T-Smad2、pMD19-T-Smad3、pMD19-T-Smad4;用HindⅢ和EcoRⅠ双酶切pcDNA3.1载体、pMD19-T-Smad2,用XhoⅠ和EcoRⅠ双酶切pcDNA3.1载体、pMD19-T-Smad3及pMD19-T-Smad4,T4 DNA连接酶连接纯化后的酶切产物;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;通过PCR、双酶切及DNA测序等方法鉴定重组质粒.将构建好的pcDNA3.1-Smad2、pcDNA3.1-Smad3、pcDNA 3.1-Smad4电转入EL4细胞,72 h后用Western blotting法检测EL4细胞中目的蛋白的表达.结果 PCR和双酶切结果均提示重组质粒pcDNA3.1-Smad2、pcDNA3.1-Smad3、pcDNA 3.1-Smad4构建成功;测序结果确定插入片段无突变,序列完全正确.重组质粒pcDNA3.1-Smad2、pcDNA3.1-Smad3、pcDNA3.1-Smad4转入EL4细胞后上调目的蛋白Smad2、Smad3、Smad4蛋白的表达.结论 成功构建pcD-NA3.1-Smad2、pcDNA3.1-Smad3和pcDNA3.1-Smad4真核表达质粒;将Smad真核表达质粒成功转染入EL4细胞,诱导细胞内目的蛋白高表达.%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.