重组犬IFN-α的原核表达及抗病毒活性研究  

Prokaryotic expression and antiviral activity of recombinant canine IFN-α

作　　者：李乐琴 曹众达 李海珠 杜吉革 朱真[2] 陈小云[2] 罗玉峰[2] 胡仕凤[1] 印春生[2] LI Leqin;CAO Zhongda;LI Haizhu;DU Jige;ZHU Zhen;CHEN Xiaoyun;LUO Yufeng;HU Shifeng;YIN Chunsheng(College of Animal Medicine,Hunan Agricultural University,Changsha 410128,China;China Institute of Veterinary Drug Control,Beijing 102629,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]中国兽医药品监察所,北京102629

出　　处：《中国兽医科学》2024年第5期632-640,共9页Chinese Veterinary Science

基　　金：国家重点研发计划“动物疫病综合防控关键技术研发与应用”重点专项“兽用生物制品标准物质研制”项目(2022YFD1800600)。

摘　　要：本试验旨在建立犬α干扰素(CaIFN-α)原核表达及纯化方法,并对其抗病毒活性进行研究。通过构建pET-28a/CaIFN-α表达载体,高效表达和纯化rCaIFN-α;采用CCK-8法在犬肾细胞(MDCK)上测定rCaIFN-α的细胞毒性,在MDCK/VSV系统中测定其抗病毒活性效价;RT-qPCR检测rCaIFN-α作用MDCK细胞后下游ISGs的表达水平;进一步利用TCID 50和间接免疫荧光(IFA)测定rCaIFN-α抗犬副流感病毒(CPIV)和犬瘟热病毒(CDV)的感染能力。电泳和Western-blot结果显示,成功制备原核表达的特异性rCaIFN-α;CCK-8结果显示重组蛋白无明显细胞毒性,细胞病变抑制法测定抗VSV病毒活性效价为1.39×107 U/mL;RT-qPCR显示rCaIFN-α可显著上调ISG15、Mxl等ISGs的转录水平;IFA等结果显示,rCaIFN-α对CPIV和CDV具有良好的抗病毒作用。结果表明,本试验制备的rCaIFN-α具有优良的抗病毒感染活性,为进一步研发新型宠物抗病毒制剂奠定了基础。The aim of this experiment was to establish a method for the prokaryotic expression and purification of canine interferon-α(CaIFN-α)and to research its antiviral activity.rCaIFN-αwas efficiently expressed and purified by constructing pET-28a/CaIFN-αexpression vector.The cytotoxicity of rCaIFN-αwas determined on canine kidney cells(MDCK)by CCK-8 assay,and its antiviral activity potency was determined in the MDCK/VSV system.RT-qPCR was used to detect the expression level of downstream ISGs after rCaIFN-αacted on the MDCK cells.TCID50 and indirect immunofluorescence(IFA)were further utilized to determine the ability of rCaIFN-αto combat infection of canine parainfluenza virus(CPIV)and canine distemper virus(CDV).The results of electrophoresis and Western-blot showed that specific rCaIFN-αwas successfully expressed.CCK-8 results showed that the recombinant protein was not obviously cytotoxic,and the potency of anti-VSV viral activity determined by cytopathic inhibition assay was 1.39×10^(7)U/mL.rCaIFN-αwas significantly up-regulated in the transcript levels of ISG15,Mxl,and other ISGs as determined by RT-qPCR.IFA and other results showed that rCaIFN-αhad excellent antiviral effects on CPIV and CDV.In conclusion that of the rCaIFN-αprepared in this experiment have excellent antiviral activity,which lays the foundation for further advancement of novel pet antiviral agents.

关 键 词：犬IFN-α 原核表达 干扰素刺激基因(ISGs) 抗病毒活性 

分 类 号：S852.659.5[农业科学—基础兽医学]