利用杆状病毒表达系统表达猪IFN-L3及其抗PEDV活性的研究  

Expression of porcine IFN-L3 using baculovirus expression system and its inhibitant activity on PEDV

作　　者：郭珊珊 陈伟业[1] 薛美[1] 尹灵丹 罗毅 冯力[1] 刘平黄[1] GUO Shan-shan;CHEN Wei-ye;XUE Mei;YIN Ling-dan;LUO Yi;FENG Li;LIU Ping-huang(State Key Laboratory of Veterinary Biotec hnology/Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences ,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

出　　处：《中国兽医科学》2019年第10期1269-1276,共8页Chinese Veterinary Science

基　　金：哈尔滨市科技创新人才研究专项资金(2017RAXYJ050)

摘　　要：利用Bac-to-Bac杆状病毒表达系统在昆虫细胞上表达重组猪干扰素L3(rPoIFN-L3)进行抗猪流行性腹泻病毒(PEDV)的研究。首先通过间接免疫荧光和Western-blot验证rPoIFN-L3表达,并用Protein A亲和层析柱进行蛋白纯化;采用表达绿色荧光蛋白的水疱性口炎病毒(VSV*GFP)检测rPoIFN-L3的抗病毒活性,含rPoIFN-L3的细胞上清的抗病毒活性为1×10^3AU/mL,纯化的rPoIFN-L3抗病毒活性为5×10^5AU/mg。进一步对纯化的rPoIFN-L3蛋白在IPEC-J2细胞上进行抗PEDV活性探究,结果显示,rPoIFN-L3可呈剂量依赖性抑制PEDV复制,并且上调相关干扰素-刺激基因(ISGs)的表达。由此证明,本研究表达的rPoIFN-L3具有抗病毒活性,并可有效抑制PEDV在IPEC-J2上的复制,对预防和治疗PEDV感染有重要意义。In this paper,the recombinant porcine interferon L3(rPoIFN-L3) was expressed in insect cells by using the Bac-to-Bac baculovirus expression system for anti-porcine epidemic diarrhea virus(PEDV) investigation.First,the expression of rPoIFN-L3 was verified by indirect immunofluorescence and Western-blot,and then we purified rPoIFN-L3 by protein A affinity chromatography column. The antiviral activity of rPoIFN-L3 was detected by vesicular stomatitis virus expressing green fluorescent protein(VSV*GFP).The antiviral activity of the supernatant containing rPoIFN-L3 was 1×10^3 AU/m L,and the purified rPoIFN-L3 antiviral activity was 5×10^5 AU/mg. Furthermore,we verified that r Po IFN-L3 had a strong antiviral activity by inhibiting the replication of PEDV and up-regulated the expression of interferon-stimulated genes(ISGs) in a dose-dependent manner in IPEC-J2 cells.These results showed that rPoIFN-L3 expressed in this study has antiviral activity and can effectively inhibit the replication of PEDV in IPEC-J2.It is important to the prevention and treatment of PEDV infection.

关 键 词：猪干扰素L3 Bac-to-Bac杆状病毒表达系统 昆虫细胞 猪流行性腹泻病毒 

分 类 号：S852.42[农业科学—基础兽医学]