IFN-α对山羊副流感病毒3型的抗病毒活性  

The Antiviral Activity of Goat Interferon Alpha to Caprine Parainfluenza Virus 3

作　　者：孙敏 郝飞[1] 张纹纹[1] 李文良[1,3,4] 杨蕾蕾 毛立[1,2] 程子龙[1] 刘茂军 SUN Min;HAO Fei;ZHANG Wenwen;LI Wenliang;YANG Leilei;MAO Li;CHENG Zilong;LIU Maojun(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture,Nanjing 210014,China;School of Life Sciences,Jiangsu University,Zhenjiang 212013,China;School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室,南京210014 [2]江苏大学生命科学学院,镇江212013 [3]江苏大学食品与生物工程学院,镇江212013 [4]南京农业大学动物医学院,南京210095

出　　处：《畜牧兽医学报》2023年第2期736-743,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省自然科学基金(BK20190272);国家自然科学基金(31902269)。

摘　　要：旨在探讨原核表达纯化的山羊α干扰素(IFN-α)对山羊副流感病毒3型(CPIV3)的抗病毒活性。通过分析山羊IFN-α的序列特点,比对不同种属IFN-α的同源性,进而构建山羊IFN-α成熟蛋白编码基因(去除信号肽基因序列)的原核表达载体pET-30a-gIFN-α,将其转化至感受态细胞Rosetta(DE3),IPTG诱导后镍柱及亲和纯化获得山羊IFN-α。利用RT-qPCR测定山羊IFN-α作用于牛肾细胞(Madin-Darby bovine kidney cell,MDBK cell)后6种干扰素刺激基因(interferon-stimulated genes,ISGs)的相对表达水平;同时,利用TCID50及Western blot测定其对CPIV3的抗病毒活性。结果表明,原核表达的山羊IFN-α蛋白含量为0.20 mg·mL^(-1),Western blot结果表明表达产物相对分子质量约为20 ku,与预期结果相符。RT-qPCR结果显示,山羊IFN-α孵育MDBK细胞后,可显著刺激RSAD2、STAT1及ISG15等6种ISGs基因的转录上调。与对照组细胞相比,山羊IFN-α显著下调MDBK细胞中CPIV3的病毒滴度,Western blot结果显示,山羊IFN-α孵育显著下调CPIV3非结构蛋白C的表达水平。山羊IFN-α对CPIV3在MDBK细胞中的增殖具有显著抑制作用。本研究扩展了I型干扰素的研究范围,为CPIV3等反刍动物疫病的防控提供新的思路。The aim of this study was to investigate the antiviral activity of goat interferon α(IFN-α) to caprine parainfluenza virus 3(CPIV3). The sequence characteristics of goat IFN-α was analyzed, and the homology of IFN-α from different species was compared. Then the recombinant expression plasmid pET-30a-gIFN-α by inserting goat IFN-α gene(removing the signal peptide gene sequence) into vector pET-30a was constructed and transformed into Rosetta(DE3), after induced by IPTG, the goat IFN-α was expressed and purified. Then the relative expression level of interferon-stimulated genes(ISGs) was measured, and the anti-CPIV3 activity was measured after exogenous goat IFN-α treatment on Madin-Darby bovine kidney cell(MDBK cell) by TCID50and Western blot. The results showed that, the protein concentration of goat IFN-α was 0.20 mg·mL^(-1), and Western blot analysis showed that the relative molecular weight was about 20 ku, which was consistent with expectations. The RT-qPCR result showed that the incubation of goat IFN-α on MDBK cells induced the up-regulation of the six ISGs including RSAD2, STAT1 and ISG15. Meanwhile, the goat IFN-α inhibited the CPIV3 replication, and down-regulated the expression level of the viral nonstructural protein C. All above results indicated that goat IFN-α could significantly inhibit the proliferation of CPIV3 on MDBK cells. This study extended the research type I interferon, and promoted the prevention and control of ruminant diseases.

关 键 词：山羊IFN-α 原核表达 ISGs 山羊副流感病毒3型 抗病毒活性 

分 类 号：S858.27[农业科学—临床兽医学]