重组猫IFN-α的质粒构建、真核表达及体外抗病毒活性分析  

Plasmid Construction and Eukaryotic Expression of FeIFN-αand Its Antiviral Activity in vitro

作　　者：李沛恒 邹万成 陈竞 张国庆[2] 崔春梅 蒋嵘 金宁一 李昌 LI Peiheng;ZOU Wancheng;CHEN Jing;ZHANG Guoqing;CUI Chunmei;JIANG Rong;JIN Ningyi;LI Chang(Jilin Agricultural University,College of animal science and technology,Changchun 130122,China;Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses,Chinese Academy of Medical Sciences,Changchun Institute of veterinary medicine,CAAS,Changchun 130122,China)

机构地区：[1]吉林农业大学动物医学院,长春130122 [2]中国农业科学院长春兽医研究所,中国医学科学院人兽共患病毒病防控关键技术研究创新单元,长春130122

出　　处：《中国动物传染病学报》2024年第6期17-24,共8页Chinese Journal of Animal Infectious Diseases

基　　金：中国医学科学院与健康科技创新工程项目(2020-I2M-5-001)。

摘　　要：本文为探究猫IFN-α(FeIFN-α)在体外MDCK、F81细胞中的抗病毒效果,为后续体内实验提供参考。根据猫IFN-α(GenBank登录号:NP_001027000.1)序列并添加6×His标签,合成后通过pUC57载体进行克隆,然后亚克隆至pcDNA3.1载体,获得含有目的基因的真核表达质粒,转染293T细胞,通过Western blot检测FeIFN-α的表达情况。通过重组绿色荧光蛋白水疱性口炎病毒(VSV-GFP)感染细胞实验,利用结晶紫染色法进行FeIFN-α抗病毒活性初步分析并确定最佳稀释倍数,通过荧光观察法、流式细胞术检测其抗病毒活性,最后通过相对荧光定量qPCR技术检测猫IFN-α对干扰素刺激基因(ISG15、MX1、OAS1)的调控作用。结果表明,成功表达FeIFN-α并具有生物学活性,且可以明显抑制VSV-GFP病毒侵染F81、MDCK细胞,qPCR结果分析表明猫IFN-α在F81、MDCK细胞中均可有效诱导ISG15、MX1、OAS1基因上调。本研究结果为后续犬猫体内抗病毒实验提供参考依据。This article was to explore the in vitro antiviral effect of feline IFN-α(FeIFN-α)in MDCK and F81 cells and provide data for subsequent in vivo experiments.The FeIFN-αsequence deposited in GenBank(NP_001027000.1)with addition of the 6×His tag cloned to PUC57 vector and sub-cloned to pcDNA3.1 to get the eukaryotic expression plasmid for transfection into 293T cells.The FeIFN-αexpression was detected by Western blotting.Preliminary experiments were performed to determine the optimal dilution ratio by infecting cells with vesicular stomatitis virus-green fluorescent protein(VSV-GFP)and examining the antiviral activity of FeIFN-αby crystal violet staining.The antiviral activity was detected by fluorescence observation and flow cytometry.Finally,interferon stimulation to genes(ISG15,MX1,OAS1)controlled by FeIFN-αwas detected by relative fluorescence quantitative qPCR.The results showed successful expression of the FeIFN-αwith biological activity and significant inhibition of VSV-GFP infection in F81 and MDCK cells.The qPCR results also showed that FeIFN-αeffectively induced the increase of ISG15,MX1 and OAS1 genes in F81 and MDCK cells.This research provided a reference for subsequent antiviral experiments in dogs and cats.

关 键 词：猫IFN-α 构建与表达 抗病毒活性 干扰素刺激基因 

分 类 号：S859.7[农业科学—临床兽医学]