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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 the eukaryotic expression plasmid of signal regulatory protein-α (SIRP-α) gene and express it in A549 cells. Methods The total RNA was extracted from Hep G2 cells. The SIRP-α gene fragment was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 (+) - EGFP to construct eukaryotic expression vector pcDNA3.1 (+) - SIRP -α / EGFP. The recombinant plasmid was transfected into A549 cells and transfected into A549 cells 24 hours after transfection. The transfected cells were observed under a fluorescence microscope 48 hours after transfection, the transcription level of SIRP-α gene was detected by RT-PCR. SIRP-α protein expression was detected. Results The recombinant plasmid pcDNA3.1 (+) - SIRP-α / EGFP was identified by double enzyme digestion and sequencing. A549 cells transfected with pcDNA3.1 (+) - SIRP-α / EGFP were identified by fluorescence microscopy Visible green fluorescence; 243 bp target band can be seen by RT-PCR results, Western blot test showed the presence of SIRP-α. Conclusion The eukaryotic expression plasmid pcDNA3.1 (+) - SIRP-α / EGFP was successfully constructed and expressed in A549 cells, which laid the foundation for the further study on the relationship between SIRP-α and lung cancer.