信号调节蛋白-α基因真核表达质粒的构建及其在A549细胞中的表达

Construction of eukaryotic expression vector for signal regulatory protein-α gene and its expression in A549 cells

目的构建信号调节蛋白-α(signal regulatory protein-α,SIRP-α)基因真核表达质粒,并于A549细胞中进行表达。方法提取Hep G2细胞总RNA,RT-PCR扩增SIRP-α基因片段,克隆至真核表达载体pc DNA3.1(+)-EGFP中,构建真核表达质粒pc DNA3.1(+)-SIRP-α/EGFP。将重组表达质粒转染A549细胞,转染24 h后,荧光显微镜下观察转染情况;转染48 h后,RT-PCR检测SIRP-α基因的转录水平;转染72 h后,Western blot法检测SIRP-α蛋白的表达。结果经双酶切和测序鉴定真核表达质粒pc DNA3.1(+)-SIRP-α/EGFP构建正确;转染pc DNA3.1(+)-SIRP-α/EGFP的A549细胞于荧光显微镜下可见绿色荧光;RT-PCR检测结果可见243 bp目的条带,Western blot检测显示SIRP-α存在。结论成功构建了真核表达质粒pc DNA3.1(+)-SIRP-α/EGFP,并于A549细胞中有效表达,为进一步研究SIRP-α与肺癌的相关性奠定了基础。

Objective To construct a eukaryotic expression vector for signal regulatory protein-α（SIRP-α） gene and express in human lung cancer A549 cells. Methods Total RNA was extracted from Hep G2 cells, with which SIRP-αgene was amplified by RT-PCR and inserted into eukaryotic expression vector pc DNA3. 1（＋）-EGFP. A549 cells were transfected with the constructed recombinant plasmid pc DNA3. 1（＋）-SIRP-α∕EGFP in mediation of Lipofectamine 2000,observed by fluorescent microscopy 24 h later, and determined for transcription level of SIRP-α gene by RT-PCR 48 h later and for expression level of SIRP-α by Western blot 72 h later. Results Restriction analysis and sequencing proved that recombinant plasmid pc DNA3. 1（＋）-SIRP-α / EGFP was constructed correctly. Green fluorescence was observed in A549 cells 24 h after transfection with the plasmid. Target gene band at a length of 243 bp was proved by RT-PCR while the target protein band by Western blot. Conclusion Eukaryotic expression vector pc DNA3. 1（＋）-SIRP-α / EGFP was successfully constructed and expressed effectively in A549 cells, which laid a foundation of further study on the rela-tionship of SIRP-α to lung cancer.