摘要
猪α干扰素(PoIFN-α)可用于治疗和预防病毒感染,但天然猪α干扰素抗病毒活性低,寻找高生物活性的突变体序列对猪传染性疾病的防治措施研究具有重要意义。运用生物信息学方法分析和比对PoIFN-α的各亚型氨基酸序列,对天然PoIFN-α序列的八个位点进行定点突变构建PoIFN-α8s突变体并进行原核表达纯化;获得重组蛋白后在PK-15细胞上针对水疱性口炎病毒(VSV)进行活性测定,同时在小鼠体内验证PoIFN-α8s突变体对猪伪狂犬病病毒(PRV)的抑制能力。结果发现,重组PoIFN-α8s测得抗病毒活性为1.13×10^(7)IU·mg^(-1),对比PoIFN-α产品,PoIFN-α8s可降低PK-15细胞上VSV的病毒滴度和基因拷贝数达10倍以上,具有更高的体外抑制VSV增殖能力;动物试验表明,PoIFN-α8s可降低感染PRV小鼠体内各器官的病毒载量。综上,本研究成功构建高效的PoIFN-α8s突变体,并验证了其良好的体内外抗病毒活性。
Porcineαinterferon(PoIFN-α)can be used to treat and prevent viral infection.However,the natural pigαinterferon has low antiviral activity,so it is of great significance to find the mutant sequence of high biological activity for the prevention and control measures of swine infectious diseases.After using bioinformatics methods to analyze and compare the amino acid sequences of various subtypes of PoIFN-α,PoIFN-α8s mutants were constructed by site-directed mutagenesis of eight sites in the native PoIFN-αsequence and purified by prokaruclear expression.After obtaining the recombinant protein,activity assays were conducted against the vesicular stomatitis virus(VSV)in PK-15 cells and validated the inhibitory effect of the PoIFN-α8s mutant on the pseudorabies virus(PRV)in mice.The results showed that,antiviral activity of recombinant PoIFN-α8s was 1.13×10^(7)IU·mg^(-1),which reduced the viral load and gene copy number of VSV on PK-15 cells by more than 10 times compared to the PoIFN-αproduct.Animal experiments indicated that PoIFN-α8s can reduce the viral load in various organs of mice infected with PRV.Thus,this study successfully constructed an efficient PoIFN-α8s mutant and verified its good antiviral activity both in vivo and in vitro.
作者
吴琼
李凌丹
袁辉
宾晨
邓可
李伟
叶十一
李国攀
沈青春
熊涛
WU Qiong;LI Lingdan;YUAN Hui;BIN Chen;DENG Ke;LI Wei;YE Shiyi;LI Guopan;SHEN Qingchun;XIONG Tao(College of Life Sciences,Yangtze University,Jingzhou 434025,China;Institute of Animal Husbandry and Veterinary Medicine,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)
出处
《畜牧兽医学报》
北大核心
2025年第5期2413-2423,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
兽医生物技术国家重点实验室开放课题资助青年基金(SKLVBF202002)。
关键词
猪Α干扰素
原核表达
突变
生物信息学分析
抗病毒活性
porcineαinterferon
prokaryotic expression
mutations
bioinformatics analysis
antiviral activity
