表达外源基因SPAM1重组CAV-2溶瘤病毒的构建与拯救

doi:10.11843/j.issn.0366-6964.2024.03.034

摘要/Abstract

摘要： 旨在研究犬腺病毒-2型（canine adenovirus type 2，CAV-2）作为溶瘤病毒的潜力并进一步探索精子黏附因子（SPAM1）协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础，使用RED/ET同源重组系统构建携带SPAM1外源基因的中间载体，使用中间载体将CAV-2骨架载体E3区域缺失并替换SPAM1外源基因表达盒。再利用合成引物敲除kmccdB反向筛选表达盒构建P15A-CAV-2-mCMV-SPAM1-SV40 polyA感染性克隆质粒并在MDCK-E1A细胞系中拯救重组溶瘤病毒，对重组病毒体外溶瘤效应进行验证。根据测序与酶切验证结果证明，本研究成功构建并拯救出稳定表达外源基因SPAM1的重组溶瘤病毒1株；IFA试验证明，重组毒株能够高水平稳定表达外源基因且外源蛋白具有可弥散分布于细胞间的特点。缺失E3区域表达外源基因SPAM1的重组溶瘤病毒通过光镜观察和CCK8检测发现具有对犬癌细胞系A72强烈的杀伤效应。综上，本研究成功构建1株具有良好溶瘤效应的重组CAV-2，为后续应用于宠物肿瘤治疗奠定了基础。

关键词: 溶瘤病毒, 精子黏附因子, 病毒反向遗传操作, 犬腺病毒-2

Abstract: The aim of this study was to investigate the potential of canine adenovirus type 2 (CAV-2) as an oncolytic virus and to further explore the value of sperm adhesion factor (SPAM1) synergizing with CAV-2 to remodel the tumor microenvironment in tumor immunotherapy. Based on the P15-CAV-2 reverse genetic manipulation platform, the RED/ET homologous recombination system was used to construct an intermediate vector carrying the exogenous gene of SPAM1, and then the intermediate vector was used to deletion the E3 region of the CAV-2 backbone vector and replace the expression cassette of the exogenous gene of SPAM1. Then the synthetic primers was used to knockdown the kmccdB reverse screening expression cassette to construct the P15A-CAV-2-mCMV-SPAM1-SV40 polyA infectious clone plasmid and rescue the recombinant lysogenic viruses in the MDCK-E1A cell line, then the recombinant viruses' in vitro tumor lysis effect was validated. The results of sequencing and digestion showed that, a recombinant lyssavirus strain stably expressing the exogenous gene SPAM1 had been successfully constructed and rescued, and the IFA assay proved that the recombinant strain could stably express the exogenous gene at a high level, and the exogenous protein had the characteristics of diffusely distributed in the intercellular region. The recombinant lysovirus with the deletion of the E3 region expressing the exogenous gene SPAM1 was found to have a strong killing effect on the canine cancer cell line A72 by light microscopy and CCK8 assay. In conclusion, the current study successfully constructed a recombinant CAV-2 strain with a good tumor lysis effect, which lays the foundation for subsequent application in pet tumor therapy.

Key words: oncolytic virus, sperm adhesion factor, virus reverse genetic operation, canine adenovirus-2

中图分类号:

S852.655

高龙, 常心怡, 李程, 赵晓亚, 李汶洁, 范浩谦, 马静云. 表达外源基因SPAM1重组CAV-2溶瘤病毒的构建与拯救[J]. 畜牧兽医学报, 2024, 55(3): 1228-1237.

GAO Long, CHANG Xinyi, LI Cheng, ZHAO Xiaoya, LI Wenjie, FAN Haoqian, MA Jingyun. Construction and Rescue of Recombinant CAV-2 Oncolytic Virus Expressing Exogenous Gene SPAM1[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1228-1237.

参考文献

[1] SUNG H, FERLAY J, SIEGEL R L, et al.Global Cancer Statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin, 2021, 71(3):209-249.
[2] HWANG C C, IGASE M, SAKURAI M, et al.Oncolytic reovirus therapy:pilot study in dogs with spontaneously occurring tumours[J].Vet Comp Oncol, 2018, 16(2):229-238.
[3] MALIK D, MAHENDIRATTA S, KAUR H, et al.Futuristic approach to cancer treatment[J].Gene, 2021, 805:145906.
[4] 王乐.肿瘤免疫疗法及临床应用的研究进展[J].河北医药, 2022, 44(16):2520-2525.
WANG L.Research progress of tumor immunotherapy and its clinical application[J].Hebei Medical Journal, 2022, 44(16):2520-2525.(in Chinese)
[5] 肖月, 陈玮琳.溶瘤病毒抗神经胶质瘤治疗研究中的问题与对策[J].中国肿瘤生物治疗杂志, 2023, 30(4):286-295.
XIAO Y, CHEN W L.Problems and countermeasures in the research of oncolytic virus anti-glioma treatment[J].Chinese Journal of Cancer Biotherapy, 2023, 30(4):286-295.(in Chinese)
[6] HUANG Z J, GUO H E, LIN L, et al.Application of oncolytic virus in tumor therapy[J].J Med Virol, 2023, 95(4):e28729.
[7] BRU T, SALINAS S, KREMER E J.An update on canine adenovirus type 2 and its vectors[J].Viruses, 2010, 2(9):2134-2153.
[8] 张慧莹, 王田田, 王运航, 等.犬腺病毒2型载体及其应用的研究进展[J].黑龙江畜牧兽医, 2022(16):24-28.
ZHANG H Y, WANG T T, WANG Y H, et al.Research progress of Canine adenovirus type 2 vector and its application[J].Heilongjiang Animal Science and Veterinary Medicine, 2022(16):24-28.(in Chinese)
[9] KREMER E J.CAR chasing:canine adenovirus vectors-all bite and no bark?[J].J Gene Med, 2004, 6 Suppl 1:S139-S151.
[10] DEL RIO D, BEUCHER B, LAVIGNE M, et al.CAV-2 vector development and gene transfer in the central and peripheral nervous systems[J].Front Mol Neurosci, 2019, 12:71.
[11] TIAINEN S, TUMELIUS R, RILLA K, et al.High numbers of macrophages, especially M2-like (CD163-positive), correlate with hyaluronan accumulation and poor outcome in breast cancer[J].Histopathology, 2015, 66(6):873-883.
[12] PATHRIA P, LOUIS T L, VARNER J A.Targeting tumor-associated macrophages in cancer[J].Trends Immunol, 2019, 40(4):310-327.
[13] KOBAYASHI T, CHANMEE T, ITANO N.Hyaluronan:metabolism and function[J].Biomolecules, 2020, 10(11):1525.
[14] MARTIN-DELEON P A.Epididymal SPAM1 and its impact on sperm function[J].Mol Cell Endocrinol, 2006, 250(1-2):114-121.
[15] LIU Y Q, XU D L, LIU Y, et al.Remotely boosting hyaluronidase activity to normalize the hypoxic immunosuppressive tumor microenvironment for photothermal immunotherapy[J].Biomaterials, 2022, 284:121516.
[16] 杜峰, 任国胜.透明质酸酶PH-20和肿瘤[J].重庆医学, 2005, 34(12):1794-1796.
DU F, REN G S.Hyaluronidase PH-20 and tumors[J].Chongqing Medical Journal, 2005, 34(12):1794-1796.(in Chinese)
[17] SHI T, SONG X R, WANG Y, et al.Combining oncolytic viruses with cancer immunotherapy:establishing a new generation of cancer treatment[J].Front Immunol, 2020, 11:683.
[18] FUKUHARA H, INO Y, TODO T.Oncolytic virus therapy:a new era of cancer treatment at dawn[J].Cancer Sci, 2016, 107(10):1373-1379.
[19] MA W Q, HE H B, WANG H M.Oncolytic herpes simplex virus and immunotherapy[J].BMC Immunol, 2018, 19(1):40.
[20] 王立朋, 孙芳, 程晋霞, 等.表达PD1抗体的重组溶瘤流感病毒株的构建及效果评价[J].免疫学杂志, 2021, 37(5):446-453.
WANG L P, SUN F, CHENG J X, et al.Generation and oncolytic efficacy evaluation of recombinant influenza virus carrying PD1 antibody[J].Immunological Journal, 2021, 37(5):446-453.(in Chinese)
[21] MONDAL M, GUO J G, HE P, et al.Recent advances of oncolytic virus in cancer therapy[J].Hum Vaccin Immunother, 2020, 16(10):2389-2402.
[22] GUEDAN S, ROJAS J J, GROS A, et al.Hyaluronidase expression by an oncolytic adenovirus enhances its intratumoral spread and suppresses tumor growth[J].Mol Ther, 2010, 18(7):1275-1283.
[23] HONG Y, NAM G H, KOH E, et al.Exosome as a vehicle for delivery of membrane protein therapeutics, PH20, for enhanced tumor penetration and antitumor efficacy[J].Adv Funct Mater, 2018, 28(5):1703074.
[24] GONG H, CHAO Y, XIANG J, et al.Hyaluronidase to enhance nanoparticle-based photodynamic tumor therapy[J].Nano Lett, 2016, 16(4):2512-2521.
[25] HONG Y, KIM Y K, KIM G B, et al.Degradation of tumour stromal hyaluronan by small extracellular vesicle-PH20 stimulates CD103⁺ dendritic cells and in combination with PD-L1 blockade boosts anti-tumour immunity[J].J Extracell Vesicles, 2019, 8(1):1670893.
[26] CLOQUELL A, MATEO I, GAMBERA S, et al.Systemic cellular viroimmunotherapy for canine high-grade gliomas[J].J Immunother Cancer, 2022, 10(12):e005669.
[27] MARTÍN-CARRASCO C, DELGADO-BONET P, TOMEO-MARTÍN B D, et al.Safety and efficacy of an oncolytic adenovirus as an immunotherapy for canine cancer patients[J].Vet Sci, 2022, 9(7):327.
[28] HEMMINKI A, KANERVA A, KREMER E J, et al.A canine conditionally replicating adenovirus for evaluating oncolytic virotherapy in a syngeneic animal model[J].Mol Ther, 2003, 7(2):163-173.
[29] KREMER E J, BOUTIN S, CHILLON M, et al.Canine adenovirus vectors:an alternative for adenovirus-mediated gene transfer[J].J.Virol, 2000, 74(1):505-512.
[30] 王海龙.DNA直接克隆的优化和ccdB反向筛选在Red/ET重组工程中的应用[D].长沙:湖南师范大学, 2013.
WANG H L.Optimization of direct DNA cloning an application of ccdB counter-selection in red/ET recombineering[D].Changsha:Hunan Normal University, 2013.(in Chinese)
[31] 杨志辉, 裴捷, 郭靖, 等.重组麻疹病毒沪191株反向遗传系统的建立[J].中国病原生物学杂志, 2023, 18(6):637-642, 649.
YANG Z H, PEI J, GUO J, et al.Establishment of reverse genetic system to construct recombinant MV-S191[J].Journal of Pathogen Biology, 2023, 18(6):637-642, 649.(in Chinese)
[32] WANG H L, LI Z, JIA R N, et al.ExoCET:exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes[J].Nucleic Acids Res, 2018, 46(5):e28.
[33] 井汇源, 李华玮, 王金合, 等.表达PRRSV GP2a-GP4的重组LCMV的构建与鉴定[J].动物医学进展, 2023, 44(3):32-37.
JING H Y, LI H W, WANG J H, et al.Establishment and identification of a recombinant LCMV expressing PRRSV GP2a-GP4[J].Progress in Veterinary Medicine, 2023, 44(3):32-37.(in Chinese)
[34] 周井祥, 薛江东, 刘晔, 等.表达猪繁殖与呼吸综合征病毒GP5蛋白基因重组犬2型腺病毒的构建及鉴定[J].中国预防兽医学报, 2010, 32(12):920-923.
ZHOU J X, XUE J D, LIU Y, et al.Construction of recombinant canine adenovirus expressing GP5 protein of porcine reproductive and respiratary syndrome virus[J].Chinese Journal of Preventive Veterinary Medicine, 2010, 32(12):920-923.(in Chinese)
[35] 李秀博, 崔尚金, 王春仁, 等.犬腺病毒作为载体在疾病治疗和预防中的作用研究进展[J].现代畜牧兽医, 2017(9):54-58.
LIU X B, CUI S J, WANG C R, et al.Research progress on canine adenovirus as vector in the treatment and prevention of disease[J].Modern Journal of Animal Husbandry and Veterinary Medicine, 2017(9):54-58.(in Chinese)