基于PiggyBac转座系统构建稳定表达UL19基因的小鼠乳腺癌细胞系4T1

Construction of a stable 4T1 cell line expressing UL19 by the PiggyBac transposon system

摘要:

为研究溶瘤Ⅱ型单纯疱疹病毒(oncolytic herpes simplex virus type 2, oHSV2)主要衣壳蛋白VP5蛋白(由UL19基因编码)调控免疫细胞抗肿瘤的机制，本研究通过PiggyBac转座系统构建了稳定表达VP5蛋白、近红外荧光蛋白(near-infrared fluorescent protein, iRFP)和绿色荧光蛋白(green fluorescent protein, GFP)的小鼠乳腺癌细胞4T1-iRFP-VP5-GFP细胞系。通过流式细胞术及免疫印迹法筛选GFP及VP5高表达的单克隆细胞系，检测所构建细胞系中UL19基因表达稳定性。经SYBR Green I实时荧光定量PCR、免疫印迹法检测，连续传代至15代的4T1-iRFP-VP5-GFP细胞中的UL19基因拷贝数及VP5蛋白表达量均显著高于瞬转UL19基因的4T1细胞，证明UL19基因稳定插入至4T1细胞基因组中。实时无标记动态细胞分析技术(real time cellanalysis, RTCA)监测4T1-iRFP-VP5-GFP细胞显示具有与其亲代细胞4T1相似的生长活性。进一步研究证实，NK92细胞对4T1-iRFP-VP5-GFP细胞的杀伤能力显著高于4T1细胞。本研究为阐述VP5蛋白在4T1细胞中通过HLA-E分子调控包括T细胞、NK细胞在内的免疫细胞发挥抗肿瘤功能奠定了基础。

Abstract:

To investigate the mechanism of the major capsid protein VP5 (encoded by the UL19 gene) of oncolytic herpes simplex virus type II (oHSV2) in regulating the antitumor function of immune cells, we constructed a mouse breast cancer cell line 4T1-iRFP-VP5-GFP stably expressing VP5 protein, near-infrared fluorescent protein (iRFP), and green fluorescent protein (GFP) by using the PiggyBac transposon system. Flow cytometry and Western blotting were employed to screen the monoclonal cell lines expressing both GFP and VP5 and examine the expression stability of UL19 in the constructed cell line. The results of SYBR Green I real-time PCR and Western blotting showed that the copies of UL19 and the expression level of VP5 protein in the 15th passage of 4T1-iRFP-VP5-GFP cells were significantly higher than those in the 4T1 cells transiently transfected with UL19, demonstrating the stable insertion of UL19 into the 4T1 cell genome. The real-time cell analysis (RTCA) was employed to monitor the proliferation of 4T1-iRFP-VP5-GFP cells, which showed similar proliferation activity to their parental 4T1 cells. Further studies confirmed that NK92 cells exhibited stronger cytotoxicity against 4T1-iRFP-VP5-GFP cells than against 4T1 cells. This study layed a foundation for elucidating the role of VP5 protein in regulating immune cells, including T cells and NK cells, via HLA-E in 4T1 cells to exert the anti-tumor function.