【摘要】 为了解重组虹鳟Ⅰ型干扰素(interferon,IFN)IFNa的抗病毒效果,本研究根据NCBI中登录的虹鳟IFNa基因序列(AM489418)设计引物,PCR扩增IFNa基因,并构建重组质粒pET32a(+)-IFNa,在大肠杆菌中诱导表达。SDS-PAGE检测结果显示,获得的重组IFNa(rIFNa)蛋白以可溶性和包涵体两种形式表达,大小约为35 ku;重组蛋白纯化后经western blot检测,结果显示rIFNa蛋白大小符合预期;通过细胞病变抑制法检测了rIFNa蛋白的抗病毒活性,结果显示rIFNa蛋白的生物活性与其浓度呈正相关关系,抗病毒效价约为1×10~4U/mg。以上结果表明,原核表达的虹鳟rIFNa蛋白具备较好的抗病毒活性。本研究为后续研究rIFNa蛋白作为免疫增强剂抵抗病毒感染奠定了基础。更多还原

【Abstract】 In order to know the antiviral activity of the recombinant rainbow trout type Ⅰ interferon a(IFNa) in vitro, the rainbow trout IFNa gene was amplified by PCR and inserted into the prokaryotic expression vector pET-32 a(+). The obtained recombinant plasmid was expressed in Escherichia Coli. The primers designed based on the IFNa gene sequence(AM489418) published on NCBI website. The obtained recombinant IFNa protein(rIFNa) was expressed in forms of both soluble proteins and inclusion bodies with the size of 35 ku. The purified rIFNa was determinated by western blot. The results showed that the rIFNa size was accordance with the expected protein. The antiviral activity of rIFNa protein was examined using cytopathiceffect inhibition assay method. The results showed that the antiviral activity of rIFNa was positively correlated with its concentration. And its antiviral titer was 1×10~4 U/mg. This study showed that the rIFNa has good antiviral activity, which laid the foundation for the subsequent study of rIFNa as an immunopotentiator against viral infection.更多还原