宿主ARF4和ARF5基因敲除小鼠的构建及其在寨卡病毒感染中的作用  

Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice

作　　者：邓考 李明圆 张惠莹 邓永强 秦源[1] 秦成峰 DENG Kao;LI Mingyuan;ZHANG Huiying;DENG Yongqiang;QIN Yuan;QIN Chengfeng(College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Beijing 100071,China;Department of Chemical Pathology and Li Ka Shing Institute of Health Sciences,The Chinese University of Hong Kong,Hong Kong 999077,China)

机构地区：[1]福建农林大学生命科学学院,福建福州350002 [2]军事科学院军事医学研究院微生物流行病研究所、病原微生物生物安全全国重点实验室,北京100071 [3]香港中文大学化学病理系、李嘉诚健康科学研究所,中国香港999077

出　　处：《生物工程学报》2024年第12期4605-4615,共11页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2022YFC2303700);国家自然科学基金(82172271)。

摘　　要：本研究旨在构建宿主ADP-核糖基化因子4(ADP-ribosylation factor 4,ARF4)和ADP-核糖基化因子5(ADP-ribosylation factor 5,ARF5)基因敲除小鼠,明确ARF4和ARF5基因对寨卡病毒感染的作用。首先利用CRISPR-Cas9技术,获得ARF4和ARF5单基因敲除小鼠(ARF4KO和ARF5KO),并通过杂交繁育获得双基因敲除小鼠(ARF4KO/ARF5KO),通过PCR法鉴定小鼠基因型,定量RT-PCR法检测目标基因的敲除效果。之后,用寨卡病毒感染基因敲除小鼠,采集第2、4、6天小鼠血液和各组织样本,提取核酸后用RT-q PCR法检测病毒载量,用HE染色观察组织病理变化。结果显示,用ARF4和ARF5特异引物,分别在ARF4KO^(–/+)、ARF5KO^(–/–)以及ARF4KO^(–/+)/ARF5KO^(–/–)小鼠扩增到与目标基因大小一致的条带。RT-qPCR检测结果显示,ARF4KO^(–/+)小鼠各组织中ARF4mRNA水平显著降低,其敲除效率在37.8%-50.0%之间,ARF5表达水平无变化。ARF5KO^(–/–)小鼠组织中ARF5 mRNA完全敲除,ARF4无变化。ARF4KO^(–/+)/ARF5KO^(–/–)小鼠在肺、肾和睾丸中ARF4mRNA水平显著降低,ARF5完全敲除。最后,用寨卡病毒分别感染基因敲除小鼠和野生型小鼠。结果显示,与野生型小鼠相比,ARF4KO^(–/+)小鼠在各时间点血清中病毒载量均显著下降,但ARF5KO^(–/–)小鼠与ARF4KO^(–/+)/ARF5KO^(–/–)小鼠无明显变化。同时,与野生型小鼠相比,ARF4KO^(–/+)小鼠各组织病毒载量没有显著降低,但其大脑和睾丸的病理性改变减轻。本研究利用CRISPR-Cas9技术成功构建了ARF4和ARF5基因敲除小鼠,并证实ARF4是寨卡病毒感染必需的宿主蛋白,为后续深入研究ARF4和ARF5在寨卡病毒感染致病中的作用机制以及抗病毒药物研发奠定了基础。The effects of host factors ADP-ribosylation factor 4(ARF4)and ADP-ribosylation factor 5(ARF5)upon Zika virus(ZIKV)infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9.Firstly,ARF5 and ARF4 genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice.Fertilized eggs were transplanted to obtain ARF4 or ARF5 knockout(ARF4KO or ARF5KO)mice,and ARF4/5 double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice(ARF4KO/ARF5KO).Then,the mouse genotypes were identified by PCR to identify the positive knockout mice,and RT-qPCR was employed to examine the knockout efficiency.The mice were then infected with ZIKV and the blood and tissue samples were collected after 2,4,and 6 days.RT-qPCR was then employed to determine the virus load,and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue.The results showed that expected PCR bands were detected from ARF4KO^(–/+),ARF5KO^(–/–),and ARF4KO^(–/+)/ARF5KO^(–/–)mice,respectively.The results of mRNA transcription measurement indicated the significant knockdown of ARF4 by 37.8%–50.0%but not ARF5 in ARF4KO^(–/+)compared with the wild-type mice.Meanwhile,complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO^(–/–)mice.Additionally,completed knockout of ARF5 and down-regulated mRNA level of ARF4 in the lung,kidney,and testis were detected in ARF4KO^(–/+)/ARF5KO^(–/–)mice in comparison with the wild-type mice.The virus load in the serum decreased in ARF4KO^(–/+)mice,while it showed no significant change in ARF5KO^(–/–)or ARF4KO^(–/+)/ARF5KO^(–/–)mice compared with that in the wild type.Meanwhile,ARF4KO^(–/+)mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice.We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study.ARF4 rather than ARF5 is essential

关 键 词：CRISPR-Cas9技术 基因敲除小鼠 ADP-核糖基化因子4 ADP-核糖基化因子5 

分 类 号：R511[医药卫生—内科学]