[1]范嘉琦,张慧,李乐,等.稳转oHSV2主要衣壳蛋白VP5人宫颈癌细胞系的构建[J].湖北工业大学学报,2024,39(4):50-56.
　FAN Jiaqi,ZHANG Hui,LI Le,et al.Construction of A Human Cervical Cancer Cell Line Containingthe Main Capsid Protein VP5 of Stable OncolyticHerpes Simplex Virus Type Ⅱ[J].,2024,39(4):50-56.
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稳转oHSV2主要衣壳蛋白VP5人宫颈癌细胞系的构建()
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《湖北工业大学学报》[ISSN:1003-4684/CN:42-1752/Z]

卷:
39
期数:
2024年第4期
页码:
50-56
栏目:
出版日期:
2024-08-28

文章信息/Info

Title:
Construction of A Human Cervical Cancer Cell Line Containingthe Main Capsid Protein VP5 of Stable OncolyticHerpes Simplex Virus Type Ⅱ
文章编号:
1003-4684(2024)04-0050-07
作者:
范嘉琦;  张慧;  李乐;  周芹;  汪洋;  胡翰;  刘滨磊
(湖北工业大学生物工程与食品学院，湖北 武汉 430068)
Author(s):
FAN Jiaqi ;  ZHANG Hui;  LI Le;  ZHOU Qin;  WANG Yang; HU Han;  LIU Binlei
(School of Biological Engineering and Food Science, Hubei Univ. of Tech., Wuhan 430068, China)
关键词:
溶瘤Ⅱ型单纯疱疹病毒;  主要衣壳蛋白;  稳转细胞系
Keywords:
oncolytic herpes simplex virus type Ⅱ;  Main capsid protein;  Stable cell line
分类号:
R153
文献标志码:
A
摘要:
为建立稳定表达溶瘤Ⅱ型单纯疱疹病毒主要衣壳蛋白（VP5）的人子宫颈癌HeLaVP5细胞系，探究VP5蛋白是否调控自然杀伤细胞（NK）的细胞毒性。运用piggyBac转座系统，通过lipofectamine 3000TM 脂质体转染法，将 pPBDPUL19和pSPBT质粒转入HeLa细胞，利用嘌呤霉素(Puro)筛选稳定细胞株，用有限稀释法得到单克隆细胞，利用western blot鉴定HeLaVP5细胞系中VP5蛋白的表达，以及利用流式细胞术检测HeLaVP5中GFP的表达，最后实时监测法比较改造前后细胞系的生长活性变化。通过嘌呤霉素抗性筛选和有限稀释挑取单克隆，获得3株HeLaVP5单克隆细胞；western blot鉴定发现3株HeLaVP5细胞系VP5蛋白均有表达；流式检测单克隆细胞系GFP阳性率均达到99%；活细胞实时监测法检测发现HeLaVP5细胞系与亲本细胞HeLa细胞具有一致的生长活性。HeLaVP5稳转细胞系构建成功，作为评估VP5蛋白是否调控自然杀伤细胞（NK）细胞毒性的靶细胞，为揭示oHSV2发挥抗肿瘤作用机制提供理论基础。
Abstract:
This paper aims to establish a human cervical cancer HeLa VP5 cell line stably expressing the main capsid protein (VP5) of oncolytic herpes simplex virus type Ⅱ, and to lay a foundation for exploring whether VP5 protein regulates the cytotoxicity of natural killer cells (NK). To this end, pPBDPUL19 and pSPBT plasmids were transferred into HeLa cells by using piggyBac transposition system and lipofectamine 3000 TM liposome transfection method; Purinomycin (Puro) was used to screen stable cell lines; Monoclonal cells were obtained by finite dilution method; Western blot was used to identify the expression of VP5 protein in HeLa VP5 cell line; The expression of GFP in HeLa VP5 was detected by flow cytometry; Live cell real time monitoring method was used to compare the growth activity changes of cell lines before and after transformation. The results show that three HeLa VP5 monoclone cells were obtained by purinomycin resistance screening and limited dilution selection; Western blot showed that VP5 protein was expressed in three HeLa VP5 cell lines; The positive rate of GFP was 99% by flow cytometry; The live cell real time monitoring method showed that the HeLa VP5 cell line had the same growth activity as the parent cell HeLa cells. The HeLa VP5 stably transfected cell line has been successfully constructed as a target cell to evaluate whether VP5 protein regulates the cytotoxicity of natural killer cells (NK), providing a theoretical basis for revealing the mechanism of oHSV2’s antitumor effect.

参考文献/References:

［1］ZHAO Q, ZHANG W, NING Z F，et al. A novel oncolytic herpes simplex virus type 2 has potent anti -tumor activity. J PloS one [J ], 2014, 9(03):e93103. [2 ]WANG Y, JIN J, LI Y, et al. NK cell tumor therapy modulated by UV -inactivated oncolytic herpes simplex virus type 2 and checkpoint inhibitors [J ]. Translational Research, 2022, 240: 64 -86. [3 ]HARDCASTLE J, KUROZUMI K, DMITRIEVA N, et al. Enhanced Antitumor Efficacy of Vasculostatin (Vstat120) Expressing Oncolytic HSV -1 [J ]. Molecular Therapy, 2010, 18(02): 285 -294. [4 ]赵倩. 新Ⅱ型单纯疱疹溶瘤病毒的构建和功能研究 [D ]. 北京：北京协和医学院, 2014. [5 ]SELIGER B, JASINSKI -BERGNER S, QUANDT D, et al. HLA -E expression and its clinical relevance in human renal cell carcinoma [J ]. Oncotarget. 2016，7(41):67360 -67372. [6 ]DAI H -S, GRIFFIN N, BOLYARD C, et al. The Fc domain of immunoglobulin is sufficient to bridge NK cells with virally infected cells [J ]. Immunity, 2017, 47(01): 159 -170. [7 ]ULBRECHT M, MARTINOZZI S, GRZESCHIK M, et al. Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA -E and prevents NK cell -mediated lysis [J ]. The Journal of Immunology, 2000, 164(10): 5019. [8 ]NATTERMANN J, NISCHALKE H D, HOFMEISTER V, et al. The HLA -A2 restricted T cell epitope HCV Core 35 -44 Stabilizes HLA -E expression and inhibits cytolysis mediated by natural killer cells [J ]. The American Journal of Pathology, 2005, 166(02): 443 -453. [9 ]YUAN S, WANG J, ZHU D, et al. Cryo -EM structure of a herpesvirus capsid at 3.1  [J ]. Science, 2018, 360(6384): eaao7283. [10 ] CHEN Y -T, FURUSHIMA K, HOU P -S, et al. Piggybac transposon -mediated, reversible gene transfer in human embryonic stem cells [J ]. Stem Cells and Development, 2009, 19(06): 763 -771. [11 ] Sandoval -Villegas N, Nurieva W, Amberger M, et al. Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering [J ]. International Journal of Molecular Sciences, 2021，22(10):5084. [12 ] RUSSELL S J, PENG K -W. Viruses as anticancer drugs [J ]. Trends in Pharmacological Sciences, 2007, 28(07): 326 -333. [13 ] TOGNARELLI E I, PALOMINO T F, CORRALES N, et al. Herpes simplex virus evasion of early host antiviral responses [J ]. Frontiers in Cellular and Infection Microbiology, 2019，30（09）:127. [14 ] DENG M, GUI X, KIM J, et al. LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration [J ]. Nature, 2018, 562(7728): 605 -609. [15 ] DUAN Q, ZHANG H, ZHENG J, et al. Turning Cold into Hot: Firing up the Tumor Microenvironment [J ]. Trends in Cancer, 2020, 6(07): 605 -618. [16 ] LIU Z, RAVINDRANATHAN R, KALINSKI P, et al. Rational combination of oncolytic vaccinia virus and PD -L1 blockade works synergistically to enhance therapeutic efficacy [J ]. Nature Communications, 2017, 8(01): 14754. [17 ] RIBAS A, DUMMER R, PUZANOV I, et al. Oncolytic virotherapy promotes intratumoral t cell infiltration and improves Anti -PD -1 Immunotherapy [J ]. Cell, 2017, 170(06): 1109 -1119. [18 ] MARTIN N T, BELL J C. Oncolytic Virus Combination Therapy: Killing One Bird with Two Stones [J ]. Molecular Therapy, 2018, 26(06): 1414 -1422. [19 ] SHI G, YANG Q, ZHANG Y, et al. Modulating the tumor microenvironment via oncolytic viruses and CSF -1R inhibition synergistically enhances Anti -PD -1 immunotherapy [J ]. Molecular Therapy, 2019, 27(01): 244 -260. [20 ] KONTERMANN R E, UNGERECHTS G, NETTELBECK D M. Viro -antibody therapy: engineering oncolytic viruses for genetic delivery of diverse antibody -based biotherapeutics [J ]. mAbs, 2021, 13(01): 1982447. [21 ] HONG B, SAHU U, MULLARKEY M P, et al. Replication and spread of oncolytic herpes simplex virus in solid tumors [J ]. Viruses, 2022, 14(1)：118. [22 ] PERERA MOLLIGODA ARACHCHIGE A S. Human NK cells: From development to effector functions [J ]. Innate Immunity, 2021, 27(03): 212 -229. [23 ] MBIRIBINDI B, PENA J K, ARVEDSON M P, et al. Epstein–Barr virus peptides derived from latent cycle proteins alter NKG2A+NK cell effector function [J ]. Scientific Reports, 2020, 10(01): 19973. [24 ] HAMMER Q, DUNST J, CHRIST W, et al. SARS -CoV -2 Nsp13 encodes for an HLA -E -stabilizing peptide that abrogates inhibition of NKG2A -expressing NK cells [J ]. Cell Reports, 2022, 38(10): 110503.

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备注/Memo

备注/Memo:
收稿日期］ 20221027［第一作者］ 范嘉琦（1997-）， 女， 山西晋中人， 湖北工业大学硕士研究生， 研究方向为生物与医药。［通信作者］ 刘滨磊（1962-）， 男， 湖北武汉人， 湖北工业大学教授， 研究方向为生物制药。
更新日期/Last Update: 2024-08-23