基于天然poIFN-α17突变的高活性猪干扰素α17m的制备及其体外抗病毒活性  

Preparation and antiviral activity study of high-activity porcine IFN-α17m protein

作　　者：方剑玉[1] 游一[1] 席燕燕 张青娴[1] 毛展展 徐彬[1,2] 郎丽敏 陈红英[4] 李绍钰[1,2] FANG Jianyu;YOU Yi;XI Yanyan;ZHANG Qingxian;MAO Zhanzhan;XU Bin;LANG Limin;CHEN Hongying;LI Shaoyu(Institute of Animal Husbandry and Veterinary Science,Henan Academ y of Agricultural Sciences,Zhengzhou 450002,China;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Zhengzhou 450002,China;Bodn Synbio Technology Inc.,Richamond,British Columbia 1501-1120,Canada;College of Animal Medicine,Henan Agricultural University,Zhengzhou 450046,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]河南省农业科学院畜禽繁育与营养调控河南省重点实验室,河南郑州450002 [3]Bodn Synbio生物技术公司,加拿大不列颠哥伦比亚列治文1501-1120 [4]河南农业大学动物医学院,河南郑州450046

出　　处：《中国兽医学报》2024年第5期921-927,共7页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关基金资助项目(222102110054);河南省农业科学院自主创新基金资助项目(2023ZC059)。

摘　　要：为获得高抗病毒活性的猪α亚型干扰素(porcine interferon α,poIFN-α),将天然的poIFN-α17进行突变和合成,并将突变后的猪poIFN-α17基因命名为poIFN-α17m,合成poIFN-α17和poIFN-α17m基因后将其克隆入pVB220大肠杆菌表达载体,将该载体转化DH5α感受态细胞,进行温度诱导表达后,收集菌体进行SDS-PAGE。将表达的蛋白采用Ni琼脂糖凝胶纯化后,采用Western blot检测重组poIFN-α17和poIFN-α17m的反应原性,在PK-15细胞上检测其对VSV和PRV的抗病毒活性,检测重组poIFN-α17和poIFN-α17m蛋白对下游干扰素刺激基因(interferon stimulating genes, ISGs)Mx1、OAS1、ISG15的诱导激活作用。结果显示,成功构建了pVB220-IFN-α17和pVB220-IFN-α17m表达载体,实现了poIFN-α17和poIFN-α17m在大肠杆菌中的表达。该蛋白具有良好的反应原性,且重组poIFN-α17m在PK-15细胞上对水泡性口炎病毒(vesicular stomatitis virus, VSV)和猪伪狂犬病病毒(pseudorabies virus, PRV)的抗病毒活性明显高于天然的poIFN-α17,重组poIFN-α17m能有效激活ISGs的表达。The aim of this study is to obtain the high antiviral activity of porcine interferonα(poIFN-α).Porcine IFN-α17(poIFN-α17)gene was mutated and synthesized,and mutated poIFN-α17 was named as poIFN-α17m,then the gene was further digested and cloned into the pVB220 plasmid to construct E.coli expression vectors pVB220-IFN-α17 and pVB220-IFN-α17m.The expression vectors were transformed into E.coli DH5αand recombinant poIFN-α17 and poIFN-α17m were expressed with temperature change.The recombinant protein was purified using affinity chromatography and identified by Western blot.The antiviral activity of recombinant poIFN-α17 and poIFN-α17m was tested in PK-15 cells against PRV and VSV.The expression of interferon stimulating genes(ISGs)induced by recombinant poIFN-α17 and poIFN-α17m was further detected and compared.The results demonstrated that the pVB220-IFN-α17 and pVB220-IFN-α17m expression vector were successfully constructed,and inclusion expression of poIFN-α17 and poIFN-α17m protein in E.coli DH5αwas obtained after the induction with temperature change,and the recombinant poIFN-α17and poIFN-α17mcould react with porcine interferon polyclonal antibody.Furthermore,the result demonstrated that the antiviral activity of poIFN-α17magainst PRV and VSV was superior to poIFN-α17.Moreover,recombinant poIFN-α17m could effectively induce the expression of ISGs.

关 键 词：猪干扰素α17m 大肠杆菌表达 干扰素刺激基因 抗病毒 

分 类 号：S858.28[农业科学—临床兽医学]