重组猪干扰素α的原核表达及抗病毒活性  

Prokaryotic expression and antiviral activity of recombinant porcine interferonα

作　　者：李秀丽[1,2] 韩颖 赵款[1,2,3] 张武超 梁飞 杨欢 雷白时 袁万哲 LI Xiuli;HAN Ying;ZHAO Kuan;ZHANG Wuchao;LIANG Fei;YANG Huan;LEIBaishi;YUAN Wanzhe(Hebei Agricultural University,Baoding,Hebei 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding,Hebei 071001,China;Shijiazhuang Animal Genetic Engineering Drug Technology Innovation Center,Shijiazhuang 050800,China;Hebei Birui Animal Pharmaceutical Co.,Ltd.,Zhengding,Hebei 050800,China;Shandong Junan Animal Disease Prevention and Control Center,Junan,Shandong 276600,China)

机构地区：[1]河北农业大学,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001 [3]石家庄市动物基因工程药物技术创新中心,河北石家庄050800 [4]河北伯瑞动物药业有限公司,河北正定050800 [5]山东省莒南县动物疫病预防控制中心,山东莒南276600

出　　处：《中国兽医学报》2023年第2期245-252,共8页Chinese Journal of Veterinary Science

基　　金：2019年河北省自然科学基金重点资助项目(C2019204219)。

摘　　要：为了探究重组猪干扰素(PoIFN-α)在体内外的抗病毒活性,利用pET-32a载体系统,构建了PoIFN-α的原核表达体系。在体外,MTT法在猪肾细胞(PK-15)和猪睾丸细胞(ST)上测定了其细胞毒性,并利用细胞病变抑制法测定其抗水泡性口炎病毒(VSV)、猪脑心肌炎病毒(EMCV)和猪伪狂犬病病毒(PRV)的活性,同时测定了重组蛋白作用PK-15细胞后干扰素刺激基因(ISGs)的转录水平;之后通过小鼠攻毒保护试验,测定了rPoIFN-α治疗后各组织器官的病毒载量,ELISA测定了小鼠血清中细胞因子TNF-α、IL-1β、IL-10和IL-6的动态变化,并对整个试验期间小鼠出现的临床症状进行打分。结果显示,成功获得了原核表达的特异性重组蛋白rPoIFN-α,在细胞上抗病毒比活性达到1.67×10^(6)IU,对细胞没有损害作用,且显著上调了Mx1、OAS等因子的转录水平;在小鼠体内能明显拮抗EMCV和PRV的增殖,减少促炎因子的产生,延缓临床症状的出现。结果表明,原核表达重组蛋白rPoIFN-α在体内外均具有抗病毒活性,为进一步推进蛋白药物在临床上的应用奠定了基础。In order to explore the antiviral activity of the recombinant porcine interferon(PoIFN-α)in vivo and in vitro,PoIFN-αwas constructed by using pET-32a vector system.In vitro,its cytotoxicity was measured on porcine kidney cells(PK-15)and porcine testis cells(ST)by MTT method,and its activities against vesicular stomatitis virus(VSV),porcine encephalomyocarditis virus(EMCV)and porcine pseudorabies virus(PRV)were measured by cytopathic inhibition method.At the same time,the transcription level of interferon stimulated genes(ISGs)was measured after the recombinant protein acted on PK-15 cells.Then rPoIFN-α was determined by mouse challenge protection test.The viral load of various tissues and organs were tested.The cytokine TNF-α,IL-1β,IL-10 and IL-6 in mouse serum were measured by ELISA.The dynamic changes and the clinical symptoms of mice during the whole experiment were scored.The results showed that the recombinant protein rPoIFN-α was successfully obtained by prokaryotic expression,its specific antiviral activity reached 1.67×10^(6) IU and it has no damaging effect on cells.It significantly upregulated the transcription levels of Mx1,OAS and other factors.It could significantly antagonize the proliferation of EMCV and PRV,reduce the production of proinflammatory factors and delay the occurrence of clinical symptoms in mice.The results showed that the recombinant protein rPoIFN-α expressed in prokaryotic cells has antiviral activity in vivo and in vitro,which lays a foundation for further promoting the clinical use of protein drugs.

关 键 词：重组猪干扰素α 大肠杆菌表达 干扰素刺激基因 细胞毒性 抗病毒活性 

分 类 号：S825.65[农业科学—畜牧学]