猪IFN-α14的表达、纯化及抗病毒活性分析  

Expression, purification and antiviral activity analysis of recombinant porcine interferon-α14

作　　者：方剑玉[1] 白杰[1,2] 席燕燕 黄慧敏 徐引弟 王改利[1,2] 张青娴 徐彬[1,2] 李绍钰[1,2] 王克领 FANG Jianyu;BAI Jie;XI Yanyan;HUANG Huiming;XU Yindi;WANG Gaili;ZHANG Qingxian;XU Bin;LI Shaoyu;WANG Keling(Instituteof Animal Husbandryand Veterinary Science,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Zhengzhou 450002,China;Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou450002,China)

机构地区：[1]河南省农业科学院畜牧兽医研究所,河南郑州450002 [2]畜禽繁育与营养调控河南省重点实验室,河南郑州450002 [3]河南省农业科学院动物免疫学重点实验室,河南郑州450002

出　　处：《中国兽医学报》2022年第10期1994-2000,共7页Chinese Journal of Veterinary Science

基　　金：河南省农业科学院自主创新基金资助项目(2021ZC53)。

摘　　要：干扰素(interferon, IFN)是一类具有抗病毒和免疫调节功能的细胞因子,目前已经应用于多种人和动物病毒病的预防和治疗。不同猪干扰素亚型的抗病毒活性差异较大,为了解猪IFN-α14的抗病毒活性,将猪IFN-α14基因序列进行分析,去除信号肽、优化密码子,并采用基因合成的方法合成了猪IFN-α14亚型基因,将其克隆入pCSMH原核表达载体,优化诱导条件,利用Ni琼脂糖凝胶纯化柱进行亲和层析,获得高纯度的重组猪IFN-α14蛋白,Western blot检测证明重组蛋白具有较好的特异性。用不同浓度的重组猪IFN-α14处理Vero细胞后,接种1 000 TCID_(50)猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV),24 h收集细胞和上清,通过荧光定量检测细胞内病毒的RNA水平,同时测定细胞上清中病毒的TCID_(50)。猪IFN-α14蛋白被成功表达并纯化,证明了10μg/L的重组猪IFN-α14干扰素能够显著抑制PEDV的复制。与本实验室前期表达纯化的猪IFN-α2、8相比,猪IFN-α14抑制PEDV复制的活性显著提高,为新型抗病毒药物开发提供了重要的依据和数据支持。Interferons(IFNs) represent a family of functionally related cytokines and they can evoke antiviral and immunomodulatory cellular responses, and it has been applied to prevent and treat the human and animal viral diseases.According to their cell surface receptors and primary sequences, IFNs are classified into various subtypes, and IFN-α showed the strongest antiviral activity.Porcine IFN-α14 gene sequence was analyzed and signal peptide was removed, and then the IFN-α14 gene was further optimized and synthesized.The E.coli expression vector pCSMH porcine IFN-α14 was constructed and its expression was induced by temperature change.Affinity chromatography was used to obtain high purity porcine IFN-α14 protein.Western blot assay was used to examine the antibody specificity of expression recombinant porcine IFN-α14,and the recombinant porcine IFN-α14 could react with porcine interferon polyclonal antibody.The Vero cells were treated with different concentration recombinant porcine IFN-α14 for 24 h, then the cells were infected with PEDV(1 000TCID50).After 48h,the cells were collected to test the RNA level of PEDV in cells with fluorescence quantitative PCR,PEDV TCID50were also were examined.The result showed that 10μg/L recombinant porcine IFN-α14 could effectively inhibit the PEDV replication in Vero cells.And compared with the IFN-α2,8,the recombinant porcine IFN-α14 have showed higher anti-PEDV activity.This study provides the basis for the development of new antiviral drugs.

关 键 词：猪IFN-α14 抗病毒活性 大肠杆菌表达 猪流行性腹泻病毒 

分 类 号：S852.4[农业科学—基础兽医学]