新城疫病毒HBNU/LSRC/F3株HN基因真核表达及其编码蛋白的抗肿瘤功能研究  

The eukaryotic expression of the hemagglutinin-neuraminidase gene of the HBNU/LSRC/F3 strain of the Newcastle disease virus and the antitumor function of the protein that gene codes for

作　　者：李冠华[1] 王静[1] 武彩霞[1] 刘华[1] 刘进军[1] 刘开扬[1] 

机构地区：[1]河北北方学院生命科学研究中心,河北张家口075000

出　　处：《中国病原生物学杂志》2016年第10期900-904,共5页Journal of Pathogen Biology

基　　金：河北省自然科学基金项目(No.H2014405044);河北北方学院创新人才培育项目(No.CXRC1318)

摘　　要：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 In order to explore the antitumor properties of the HN gene of the HBNU/LSRC/F3 strain of the Newcastle disease virus（NDV）,the HN gene was cloned and expressed in human esophageal cancer ECA109 cells.Methods The total RNA of NDV was extracted,primers were designed in accordance with the open reading frame（accession number：KC246549.1）of the HN gene and multiple cloning sites of the eukaryotic expression vector C-flag pcDNA3.The HN gene was amplified with reverse transcription PCR（RT-PCR）,ligated into the eukaryotic expression vector,and sequenced.The recombinant plasmids were transfected into ECA109 cells.HN protein and the gene that codes for it were detected with immunofluorescence and RT-PCR.The antitumor effect of the HN gene was measured with flow cytometry. Results The HN gene was ligated into the eukaryotic expression vector C-flag pcDNA3,and sequencing results indicated that the HN gene had the same sequence as that of the HBNU/LSRC/F3 strain registered in GenBank.The recombinant plasmids in the cell cytoplasm fluoresced yellow-green under a fluorescence microscope 36 hours after transfection.The mRNA of HN was amplified with the RNA of cancer cells using RT-PCR 48 hours after transfection,and the blank plasmid group displayed evidence of transfected cells.According to flow cytometry,the rate of apoptosis in the recombinant plasmid group was 8.2±1.56%,so the rate increased significantly（P〈0.05）compared to the rate of4.6±0.83%in the blank plasmid control group. Conclusion The expression of HN protein was detected in ECA109 cells,and the protein displayed antitumor properties when expressed eukaryotically.

关 键 词：HN蛋白 真核表达 抗肿瘤 

分 类 号：R373.9[医药卫生—病原生物学]