科微学术

生物工程学报

2025年4月5日 17:13 星期六
首页 > 过刊浏览>2024年第40卷第11期 >4138-4148. DOI:10.13345/j.cjb.240122
PDF HTML阅读 XML下载 导出引用 引用提醒
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
作者:
基金项目:

国家自然科学基金(32270969,81972308)


Construction of a stable 4T1 cell line expressing UL19 by the PiggyBac transposon system
Author:
  • 摘要
    • | |
  • 访问统计
    • |
  • 参考文献 [28]
    • |
  • 相似文献 [20]
    • | | |
  • 文章评论
    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.

    参考文献
    [1] KAUFMAN HL, KOHLHAPP FJ, ZLOZA A. Oncolytic viruses: a new class of immunotherapy drugs[J]. Nature Reviews Drug Discovery, 2015, 14: 642-662.
    [2] ZOLALY MA, MAHALLAWI W, KHAWAJI ZY, ALAHMADI MA. The clinical advances of oncolytic viruses in cancer immunotherapy[J]. Cureus, 2023, 15(6): e40742.
    [3] TANG TY, HUANG X, ZHANG G, LIANG TB. Oncolytic immunotherapy: multiple mechanisms of oncolytic peptides to confer anticancer immunity[J]. Journal for Immunotherapy of Cancer, 2022, 10(7): e005065.
    [4] ZHANG W, ZENG BB, HU X, ZOU LJ, LIANG J, SONG Y, LIU BL, LIU SM. Oncolytic herpes simplex virus type 2 can effectively inhibit colorectal cancer liver metastasis by modulating the immune status in the tumor microenvironment and inducing specific antitumor immunity[J]. Human Gene Therapy, 2021, 32(3/4): 203-215.
    [5] 商木岩, 郭帅, 张强, 朴浩哲. 中国乳腺癌筛查现状[J]. 实用癌症杂志, 2020, 35(11): 1911-1914. SHANG MY, GUO S, ZHANG Q, PIAO HZ. Current status of breast cancer screening in China[J]. Journal of Practical Cancer, 2020, 35(11): 1911-1914(in Chinese).
    [6] PASHAYAN N, ANTONIOU AC, IVANUS U, ESSERMAN LJ, EASTON DF, FRENCH D, SROCZYNSKI G, HALL P, CUZICK J, EVANS DG, SIMARD J, GARCIA-CLOSAS M, SCHMUTZLER R, WEGWARTH O, PHAROAH P, MOORTHIE S, de MONTGOLFIER S, BARON C, HERCEG Z, TURNBULL C, et al. Personalized early detection and prevention of breast cancer: ENVISION consensus statement[J]. Nature Reviews Clinical Oncology, 2020, 17: 687-705.
    [7] HOLMES CE, MUSS HB. Diagnosis and treatment of breast cancer in the elderly[J]. CA: a Cancer Journal for Clinicians, 2003, 53(4): 227-244.
    [8] 刘芳彤, 邢淑雁, 刘孝云, 叶冬雪, 杨佳, 张国英, 容蓉, 杨勇. 基于实时无标记细胞分析技术时间维度特征的体外抗肿瘤活性评价新策略[J]. 中国药理学通报, 2024, 40(3): 592-598. LIU FT, XING SY, LIU XY, YE DX, YANG J, ZHANG GY, RONG R, YANG Y. A new strategy for evaluating in vitro antitumor activity based on the time-dimensional characterization of real-time label-free cell analysis[J]. Chinese Journal of Pharmacology, 2024, 40(3): 592-598(in Chinese).
    [9] ANSBRO MR, JACOB CG, AMATO R, KEKRE M, AMARATUNGA C, SRENG S, SUON S, MIOTTO O, FAIRHURST RM, WELLEMS TE, KWIATKOWSKI DP. Development of copy number assays for detection and surveillance of piperaquine resistance associated plasmepsin 2/3 copy number variation in Plasmodium falciparum[J]. Malaria Journal, 2020, 19(1): 181.
    [10] FERREIRA ID, ROSÁRIO VE, CRAVO PVL. Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum[J]. Malaria Journal, 2006, 5: 1.
    [11] AITMAN TJ, DONG R, VYSE TJ, NORSWORTHY PJ, JOHNSON MD, SMITH J, MANGION J, ROBERTON-LOWE C, MARSHALL AJ, PETRETTO E, HODGES MD, BHANGAL G, PATEL SG, SHEEHAN-ROONEY K, DUDA M, COOK PR, EVANS DJ, DOMIN J, FLINT J, BOYLE JJ, et al. Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans[J]. Nature, 2006, 439: 851-855.
    [12] ANDTBACKA RHI, KAUFMAN HL, COLLICHIO F, AMATRUDA T, SENZER N, CHESNEY J, DELMAN KA, SPITLER LE, PUZANOV I, AGARWALA SS, MILHEM M, CRANMER L, CURTI B, LEWIS K, ROSS M, GUTHRIE T, LINETTE GP, DANIELS GA, HARRINGTON K, MIDDLETON MR, et al. Talimogene laherparepvec improves durable response rate in patients with advanced melanoma[J]. Journal of Clinical Oncology, 2015, 33(25): 2780-2788.
    [13] MA J, RAMACHANDRAN M, JIN C, QUIJANO-RUBIO C, MARTIKAINEN M, YU D, ESSAND M. Characterization of virus-mediated immunogenic cancer cell death and the consequences for oncolytic virus-based immunotherapy of cancer[J]. Cell Death & Disease, 2020, 11: 48.
    [14] WALKER JD, SEHGAL I, KOUSOULAS KG. Oncolytic herpes simplex virus 1 encoding 15-prostaglandin dehydrogenase mitigates immune suppression and reduces ectopic primary and metastatic breast cancer in mice[J]. Journal of Virology, 2011, 85(14): 7363-7371.
    [15] WATANABE I, KASUYA H, NOMURA N, SHIKANO T, SHIROTA T, KANAZUMI N, TAKEDA S, NOMOTO S, SUGIMOTO H, NAKAO A. Effects of tumor selective replication-competent herpes viruses in combination with gemcitabine on pancreatic cancer[J]. Cancer Chemotherapy and Pharmacology, 2008, 61(5): 875-882.
    [16] ELDE NC, CHILD SJ, GEBALLE AP, MALIK HS. Protein kinase R reveals an evolutionary model for defeating viral mimicry[J]. Nature, 2009, 457: 485-489.
    [17] TOMAZIN R, HILL AB, JUGOVIC P, YORK I, van ENDERT P, PLOEGH HL, ANDREWS DW, JOHNSON DC. Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP[J]. The EMBO Journal, 1996, 15(13): 3256-3266.
    [18] GALOCHA B, HILL A, BARNETT BC, DOLAN A, RAIMONDI A, COOK RF, BRUNNER J, McGEOCH DJ, PLOEGH HL. The active site of ICP47, a herpes simplex virus-encoded inhibitor of the major histocompatibility complex (MHC)-encoded peptide transporter associated with antigen processing (TAP), maps to the NH2-terminal 35 residues[J]. The Journal of Experimental Medicine, 1997, 185(9): 1565-1572.
    [19] AHN K, MEYER TH, UEBEL S, SEMPÉ P, DJABALLAH H, YANG Y, PETERSON PA, FRÜH K, TAMPÉ R. Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47[J]. The EMBO Journal, 1996, 15(13): 3247-3255.
    [20] PIETRA G, ROMAGNANI C, MORETTA L, MINGARI MC. HLA-E and HLA-E-bound peptides: recognition by subsets of NK and T cells[J]. Current Pharmaceutical Design, 2009, 15(28): 3336-3344.
    [21] ANDRÉ P, DENIS C, SOULAS C, BOURBON- CAILLET C, LOPEZ J, ARNOUX T, BLÉRY M, BONNAFOUS C, GAUTHIER L, MOREL A, ROSSI B, REMARK R, BRESO V, BONNET E, HABIF G, GUIA S, LALANNE AI, HOFFMANN C, LANTZ O, FAYETTE J, et al. Anti-NKG2A MAb is a checkpoint inhibitor that promotes anti-tumor immunity by unleashing both T and NK cells[J]. Cell, 2018, 175(7): 1731-1743.e13.
    [22] LIU XW, SONG JN, ZHANG H, LIU XY, ZUO FL, ZHAO YN, ZHAO YJ, YIN XM, GUO XY, WU X, ZHANG H, XU J, HU JP, JING J, MA XL, SHI HB. Immune checkpoint HLA-E: CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance[J]. Cancer Cell, 2023, 41(2): 272-287.e9.
    [23] MYERS JA, MILLER JS. Exploring the NK cell platform for cancer immunotherapy[J]. Nature Reviews Clinical Oncology, 2021, 18: 85-100.
    [24] IWASZKO M, BOGUNIA-KUBIK K. Clinical significance of the HLA-E and CD94/NKG2 interaction[J]. Archivum Immunologiae et Therapiae Experimentalis, 2011, 59(5): 353.
    [25] PRAŠNIKAR E, PERDIH A, BORIŠEK J. Nonameric peptide orchestrates signal transduction in the activating HLA-E/NKG2C/CD94 immune complex as revealed by all-atom simulations[J]. International Journal of Molecular Sciences, 2021, 22(13): 6670.
    [26] WANG Y, JIN J, LI YY, ZHOU Q, YAO RY, WU Z, HU H, FANG ZZ, DONG S, CAI Q, HU S, LIU BL. NK cell tumor therapy modulated by UV-inactivated oncolytic herpes simplex virus type 2 and checkpoint inhibitors[J]. Translational Research, 2022, 240: 64-86.
    [27] YUAN S, WANG JL, ZHU DJ, WANG N, GAO Q, CHEN WY, TANG H, WANG JZ, ZHANG XZ, LIU HR, RAO ZH, WANG XX. Cryo-EM structure of a herpesvirus capsid at 3.1Å[J]. Science, 2018, 360(6384): eaao7283.
    [28] 姚若一, 范嘉琦, 肖雄, 周芹, 汪洋, 胡翰, 刘滨磊. HLA-E与oHSV2 VP5蛋白相互作用研究[J]. 湖北工业大学学报, 2023, 38(5): 82-87. YAO RY, FAN JQ, XIAO X, ZHOU Q, WANG Y, HU H, LIU BL. Study on the interaction between HLAE and oHSV2 VP5 protein[J]. Journal of Hubei University of Technology, 2023, 38(5): 82-87(in Chinese).
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

赵晓彤,王欣雅,刘滨磊,胡翰,汪洋. 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[J]. 生物工程学报, 2024, 40(11): 4138-4148

复制
分享
文章指标
  • 点击次数:146
  • 下载次数: 269
  • HTML阅读次数: 212
  • 引用次数: 0
历史
  • 收稿日期:2024-02-19
  • 在线发布日期: 2024-11-07
  • 出版日期: 2024-11-25
文章二维码
您是第5997206位访问者
生物工程学报 ® 2025 版权所有

通信地址：中国科学院微生物研究所    邮编：100101

电话：010-64807509   E-mail：cjb@im.ac.cn

技术支持：北京勤云科技发展有限公司