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目的在人食管癌ECA109细胞中表达新城疫病毒HBNU/LSRC/F3株HN基因,观察其编码蛋白的抗肿瘤特性。方法提取新城疫病毒总RNA,以根据HN基因开放阅读框(登录号为KC246549.1)和真核表达载体C-flag pcDNA3的多克隆位点设计的带酶切位点引物通过逆转录PCR(RT-PCR)扩增HN基因,然后连接到真核表达载体上,测序正确的重组质粒转染ECA109细胞,运用免疫荧光及RT-PCR检验HN基因的表达,通过流式细胞术检测其表达产物的抗肿瘤效果。结果 HN基因正确连接到真核表达载体C-flag pcDNA3上,测序显示HN基因序列与GenBank上已发表的HBNU/LSRC/F3株的HN序列一致。重组质粒转染ECA109细胞36h后,其胞质内可见黄绿色荧光,转染48h后扩增到HN基因,而空质粒转染细胞扩增阴性。经流式细胞术检测,重组质粒转化肿瘤细胞凋亡率为(8.2±1.56)%,空质粒对照组为(4.6±0.83)%,差异有统计学意义(P<0.05)。结论 HN基因重组质粒转化ECA109细胞表达HN蛋白,该蛋白具有一定的抗肿瘤活性。
Objective To express HN gene of Newcastle disease virus (HBNU / LSRC / F3) in human esophageal carcinoma ECA109 cells and observe the antitumor properties of the encoded protein. Methods The total RNA of Newcastle disease virus (NDV) was extracted. Based on the HN gene open reading frame (accession number KC246549.1) and the multi-cloning site of the eukaryotic expression vector C-flag pcDNA3, HN gene was amplified by RT-PCR and then ligated into eukaryotic expression vector. The correct recombinant plasmid was transfected into ECA109 cells. The expression of HN gene was detected by immunofluorescence and RT-PCR. The expression of HN gene was detected by flow cytometry Anti-tumor effect. Results The HN gene was correctly ligated to the eukaryotic expression vector C-flag pcDNA3. The sequence of HN gene was consistent with the published HN sequence of HBNU / LSRC / F3 strain in GenBank. The recombinant plasmid transfected ECA109 cells 36h, the cytoplasm can be seen yellow-green fluorescence, 48h after transfection to HN gene amplification, empty plasmid transfected cells were negative. The results of flow cytometry showed that the apoptotic rates of the recombinant plasmids transformed into tumor cells were (8.2 ± 1.56)% and those in the empty plasmid control group were (4.6 ± 0.83)%, respectively (P <0.05). Conclusion The HN gene recombinant plasmid transforms ECN109 cells to express HN protein, which has some antitumor activity.