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.
Construction of BGC823 Cell Line Stably Expressing VP5 Protein of Herpes Simplex Virus Type Ⅱ
Oncolytic herpes simplex virus type Ⅱ oHSV2 is a new type of oncolytic virus obtained by genet-ic modification on the basis of herpes simplex virus type Ⅱ HSV-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 in-teraction 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 sur-face 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 regu-late 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 Re-al Time Cell Analysis 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.
oncolytic herpes simplex virus type ⅡVP5 proteinBGC823 cells
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