稳定表达VP5蛋白BGC823细胞系的构建  

Construction of BGC823 Cell Line Stably Expressing VP5 Protein of Herpes Simplex Virus TypeⅡ

作　　者：肖雄 赵晓彤 李乐 周芹 汪洋 胡翰 刘滨磊 XIAO Xiong;ZHAO Xiaotong;LI Le;ZHOU Qin;WANG Yang;HU Han;LIU Binlei(School of Food and Biological Engin.,Hubei Univ.of Tech.,Wuhan 430068,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉430068

出　　处：《湖北工业大学学报》2024年第4期61-65,共5页Journal of Hubei University of Technology

摘　　要：溶瘤Ⅱ型单纯疱疹病毒oHSV2是对Ⅱ型单纯疱疹病毒HSV-2进行基因改造后得到的新型溶瘤病毒。UL19基因编码的VP5蛋白为oHSV2主要衣壳蛋白之一。在前期已经通过GST pull-down与质谱检测分析确定了oHSV2的VP5蛋白为人类白细胞抗原E(HLA-E)的相互作用蛋白的基础上,将HLA-E作为NK与CTL等免疫细胞表面CD94/NKG2A的强效抑制性配体,当它与CD94/NKG2A结合后,NK与CTL细胞的免疫功能会受到一定程度的抑制。前期研究已证实oHSV2会在体外上调部分肿瘤细胞系表面HLA-E表达,为探究oHSV2是否通过VP5蛋白上调肿瘤细胞系表面HLA-E的表达,进而影响HLA-E和CD94/NKG2A的结合,最终改变NK细胞的抗肿瘤能力,通过PiggyBac转座系统构建稳定表达VP5蛋白的人胃腺癌细胞BGC823-VP5细胞系。所构建的细胞系经流式检测单克隆细胞阳性率在99%以上;Western Blot检测到VP5蛋白在细胞内表达;实时无标记动态细胞分析技术(RTCA Real Time Cell AnaIysis)检测表明BGC823-VP5细胞系与亲本细胞BGC823细胞生长活性一致。稳定表达VP5蛋白的人胃癌细胞BGC823-VP5细胞系的构建,为进一步研究oHSV2上VP5蛋白在人胃癌BGC823细胞内影响HLA-E的表达,进而影响免疫细胞的抗肿瘤能力奠定了基础。Oncolytic herpes simplex virus typeⅡoHSV2 is a new type of oncolytic virus obtained by genetic modification on the basis of herpes simplex virus typeⅡHSV-2.VP5 protein encoded by UL19 gene is one of the main capsid proteins of oHSV2.Our team has determined that VP5 protein of oHSV2 is the interaction protein of human leukocyte antigen E(HLA-E)through GST pull down and mass spectrometry detection and analysis in the early stage.HLA-E is a potent inhibitory ligand of CD94/NKG2A on the surface of CTL cells and NK cells.When it binds with CD94/NKG2A,the immune function of CTL cells and NK cells will be inhibited.On the basis of previous studies that have confirmed that oHSV2 can up regulate the expression of HLA-E on the surface of some tumor cell lines in vitro,in order to explore whether oHSV2 can up regulate the expression of HLA-E on the surface of tumor cell lines through VP5 protein,thereby affecting the binding of HLA-E and CD94/NKG2A,and ultimately changing the anti-tumor ability of NK cells,a human gastric adenocarcinoma cell line BGC823-VP5 stably expressing VP5 was constructed through PiggyBac transposition system.The positive rate of monoclonal cells in the constructed cell line was more than 99%by flow cytometry;Western Blot detected VP5 protein expression in cells;RTCA Real Time Cell AnaIysis showed that the growth activity of BGC823-VP5 cell line was consistent with that of its parent cell BGC823.The construction of a human gastric cancer cell line BGC823-VP5 stably expressing VP5 lays a foundation for exploring the effect of VP5 protein on oHSV2 on the expression of HLA-E in tumor cells and thus on the anti-tumor activity of NK cells.

关 键 词：溶瘤Ⅱ型单纯疱疹病毒 VP5蛋白 BGC823细胞 

分 类 号：R735.2[医药卫生—肿瘤]