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目的构建CD47靶向shRNA慢病毒干扰载体,并观察其对Hep-2细胞CD47基因的抑制效应。方法设计CD47靶点特异性的寡核苷酸序列,利用酶切反应合成含特异性系列线性化的pGCSIL-GFP载体,包装293T细胞产生慢病毒并行滴度测定,再转染人喉癌Hep-2细胞,通过实时荧光定量PCR及Western Blotting检测重组慢病毒对CD47靶基因在mRNA和蛋白水平上的沉默抑制情况。结果阳性克隆PCR及测序证实针对CD47基因shRNA慢病毒干扰载体构建成功,RT-PCR及Western Blotting检测载体在mRNA和蛋白水平上可抑制喉癌Hep-2细胞的CD47表达。结论成功构建针对CD47基因的慢病毒干扰载体,为进一步行喉癌CD47基因功能研究提供了较好的实验工具。
Objective To construct a lentivirus vector targeting CD47 targeting shRNA and observe its inhibitory effect on CD47 gene in Hep-2 cells. Methods CD47 target-specific oligonucleotide sequences were designed. Specific series of linearized pGCSIL-GFP vectors were synthesized by restriction endonuclease digestion. 293T cells were packaged for simultaneous lentivirus titration and then transfected into human laryngeal carcinoma Hep- 2 cells were detected by real-time fluorescent quantitative PCR and Western Blotting recombinant lentivirus on CD47 target gene silencing at the mRNA and protein levels. Results Positive cloned PCR and sequencing confirmed that shRNA targeting lentiviral vector CD47 was constructed successfully. RT-PCR and Western Blotting could inhibit the expression of CD47 on Hep-2 cells. Conclusion The successful construction of lentivirus vector targeting CD47 gene provides a good experimental tool for the further study of CD47 gene function in laryngeal cancer.