虹鳟Ⅰ型干扰素e7在胖头鳜肌肉细胞中的表达及其抗传染性造血器官坏死病毒活性检测

Expression of Rainbow trout type Ⅰ interferon e7 in Fathead minnow cell line and its anti-virus activity assay

为表达虹鳟Ⅰ型干扰素e7(IFNe7),并检测其抗传染性造血器官坏死病毒(IHNV)的活性,本实验采用RT-PCR从虹鳟肾脏组织中扩增虹鳟IFNe7基因,克隆至p MD19-T载体,测序分析后构建重组真核表达载体p EGFP-IFNe7,并转染至胖头鳜肌肉细胞(FHM)检测其抗病毒活性。结果显示,虹鳟IFNe7基因序列为561 bp,编码186个氨基酸,与大西洋鲑Ⅰ型干扰素(IFN-I)亲缘关系最近。通过荧光观察、RT-PCR及western blot方法鉴定显示,真核表达载体p EGFP-IFNe7能在FHM细胞中表达49 ku的重组蛋白;采用细胞病变抑制法表明IFNe7具有抗IHNV的活性。本研究为进一步研究虹鳟病毒性疾病奠定基础。

In order to express type I interferon e7 （IFNe7） of rainbow trout and identify its anti-infection hematopietic necrosis virus （IHNV） activity, the IFNe7 gene was amplified from kidney tissue of rainbow trout by RT-PCR and cloned into pMD19-T vector for sequencing and analysis. The IFNe7 gene was further subcloned into pEGFP-N1 expression vector to construct recombinant plasmid of pEGFP-N1-IFNe7, which was transfected into Fathead minnow cell line （FHM）. The results showed that IFNe7 was 561bp encoding 186 amino acid, showing similar relationship with type I interferon of Atlantic salmon. The expressed recombinant protein was about 49ku, which could be expressed correctly in FHM cells transfected with pEGFP-N1-IFNe7 verified by observation under fluorescence microscope and western blot. FHM cells with pEGFP-N1-IFNe7 had anti-IHNV activity proved by cytopathic inhibition assay. This study provided a basis for the further research for viral disease in rainbow trout.