表达外源基因SPAM1重组CAV-2溶瘤病毒的构建与拯救  

Construction and Rescue of Recombinant CAV-2 Oncolytic Virus Expressing Exogenous Gene SPAM1

作　　者：高龙 常心怡 李程 赵晓亚[1] 李汶洁 范浩谦 马静云[1] GAO Long;CHANG Xinyi;LI Cheng;ZHAO Xiaoya;LI Wenjie;FAN Haoqian;MA Jingyun(School of Animal Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院,广州510642

出　　处：《畜牧兽医学报》2024年第3期1228-1237,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广州市科技计划项目(2023B03J1310);广东省基金(2022A1515012473)。

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。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.

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 

分 类 号：S852.655[农业科学—基础兽医学]