文章摘要
黑头软口鲦上皮瘤细胞IFN-1基因的表达及抗病毒活性研究
Expression and Antiviral Activity of IFN-1 Gene in Epithelioma papulosum cyprini cells
投稿时间:2019-06-26  修订日期:2019-12-01
DOI:
中文关键词: IFN-1  EPC  SVCV  原核表达  
英文关键词: IFN-1  EPC  SVCV  prokaryotic expression
基金项目:国家自然科学基金
作者单位E-mail
孙杰 华中农业大学 2047687285@qq.com 
郭雅娜 华中农业大学  
戴彩姣 华中农业大学  
陈孝煊 华中农业大学  
李莉娟 华中农业大学  
袁军法 华中农业大学 jfyuan@mail.hzau.edu.cn 
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中文摘要:
      为表达黑头软口鲦上皮瘤细胞(EPC)I型干扰素(IFN-1)及检测其抗病毒的活性,本实验通过RT-PCR从EPC中扩增IFN-1基因,构建重组表达质粒pET-32a-IFN-1,并转化到宿主菌Transetta,体外纯化后检测其抗病毒活性。结果显示,IFN-1编码区大小为552bp,编码184个氨基酸,与草鱼干扰素1(CiIFN1)亲缘关系最近。通过SDS-PAGE,重组表达质粒pET-32a-IFN-1在宿主菌中可明显表达约35KDa的融合蛋白条带,且部分呈可溶性表达,进而通过亲和纯化可溶性重组IFN-1(rIFN-1),免疫新西兰大白兔获得效价较高的抗IFN-1多克隆抗体,可用以检测细胞内源性的IFN-1。荧光定量PCR显示rIFN-1与EPC细胞孵育可以诱导抗病毒蛋白Mx1的表达,并抑制鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)引起的细胞病变(CPE)及SVCV的复制,表明rIFN-1具有抗病毒活性。
英文摘要:
      To clone type I interferon (IFN-1) from Epithelioma papulosum cyprini cells (EPC), the IFN-1 gene was amplified from EPC by RT-PCR, and the recombinant plasmid pET-32a-IFN-1 was constructed and transformed into the host strain Transetta for expression. Sequence analysis showed that the coding region of IFN-1 gene was 552 bp in length and encoded 184 amino acids, which was closely related to grass carp interferon 1(CiIFN1). Recombinant soluble IFN-1 (rIFN-1) was induced in Transetta and obtained by Ni-affinity purification. As well, anti-rIFN-1 polyclonal antibody was prepared by immunizing New Zealand white rabbits with rIFN-1 and could be used to detect endogenous IFN-1 in EPC cells. Quantitative PCR showed that the antiviral protein Mx1 was induced and Spring viremia of carp virus (SVCV) replication as well as cytopathic effect (CPE) was significantly inhibited when EPC was incubated with rIFN-1, which indicated that rIFN-1 harbored antiviral activity in EPC cells.
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