pcDNA3.1-Smac真核表达载体的构建及表达

Construction and expression of eukaryotic expression plasmid pcDNA3.1-smac

目的:构建人的smac基因真核表达载体pcDNA3.1-Smac,并在肺腺癌A549细胞中表达。方法:采用逆转录聚合酶链式反应(RT-PCR)技术从人睾丸组织中扩增到smac基因,构建重组真核表达载体pcDNA3.1-Smac。酶切及测序鉴定重组质粒正确后,脂质体介导转染至肺腺癌A549细胞中。采用RT-PCR、Westernblot法检测外源基因smac的表达,MTT法检测细胞生长抑制率。结果:PCR扩增片段与预期片段大小相符,插入片段测序结果与GenBank公布的一致,表明人smac基因克隆及真核表达载体pcDNA3.1-Smac构建成功。在mRNA水平和蛋白水平,转染后的细胞中外源基因smac表达均明显增加。转染smac质粒72h后,细胞的生长抑制率较转染空质粒组显著增加。结论:成功构建重组真核表达载体pcDNA3.1-Smac,并在肺癌A549细胞中进行了表达;验证了转染后对肺癌细胞生长有抑制作用。

AIM： To construct the eukaryotic expression vector of human gene Smac pcDNA3. 1-Smac and express it in the lung adenocarcinoma A549 ceils, METHODS： The Smac was amplified from human testis tissue by reverse transcriptase polymerase chain reaction （RT-PCR）. Then recombined eukaryotic expression vector pcDNA3, l-Smac was constructed. After the reconbinant plasmid was proved to be constructed correctly by endonucleases digesting and DNA sequencing, we trasfected it into lung adenocarcinama cells A549 through liposome inducing. The expression of Smac in transfectant A549 was detected by RT-PCR and Western blot. And the cell growth inhibition ratio after trasfection was detected by MTT. RESULTS： The amplified fragment by PCR was coincident with the anticipated result, and its sequence was in concordance with that published on GenBank. Therefore, the gene Smac was cloned successfully, and the recombinant plasmid pcDNA3. 1-Smac was also constructed successfully. Both on the mRNA level and the protein level, the expression of Smac gene was increased obviously in the transfected A549 detected by RTPCR and Western blot respectively. The cell growth inhibition ratio in the group transfected pcDNA3. l-Smac was significantly higher compared with the pcDNA3. l group after 72 hours. CONCLUSION： The recombinant eukaryotic expression vector pcDNA3. 1-Smac was constructed , and it could be obviously expressed in lung adenocarcinoma cells A549. It is also proven that Smac has the function of growth inhibition.