青鳉干扰素的克隆与表达分析

The Cloning and Expression Analysis of Interferon from Oryzias latipes

干扰素(interferons,IFNs)是一类能够诱导细胞进入抗病毒状态的分泌蛋白,是脊椎动物抵抗病毒入侵的第一道屏障。本实验以青鳉(Oryzias latipes)为实验材料,通过polyⅠ:C诱导处理,克隆得到一个Ⅰ型干扰素c DNA序列,命名为Ol IFNβ。Ol IFNβ基因含有4个外显子和3个内含子,具有典型的Ⅰ型干扰素的结构特点,如含有2个半胱氨酸残基和m RNA非稳定序列。氨基酸序列比对分析显示,Ol IFNβ与三刺鱼IFN1同源性最高,达54%。系统进化树分析表明,Ol IFNβ与三刺鱼干扰素的亲缘关系最近,且和其它鱼类Ⅰ型干扰素聚成一个分支。荧光定量PCR分析表明,Ol IFNβ表达具有时间和组织特异性。PolyⅠ:C诱导处理后,随着处理时间的延长Ol IFNβ表达量逐渐上调,6 h后达到最高,随后逐渐下调。其中脾脏变化最为显著,鳃和肠次之。本研究显示,Ol IFNβ在青鳉先天免疫应答过程中起重要作用,为将来进一步探索Ol IFNβ的功能提供了帮助。

Interferons （IFNs） are a class of secreted proteins that induce vertebrate cells into antiviral state, which is the first defence of vertebrate to resist virus invasion. Oryzias latipes were used as experimential materials in this study, and the cDNA sequence of the type Ⅰ IFN, which was termed OllFNfl, was cloned after polyinosinic： polycytidylic acid （poly Ⅰ： C） treatment. The OlIFNβ gene contained 4 exons and 3 introns, and possessed the typical structural features of type Ⅰ IFNs such as containing two cysteine residues and mRNA unstable sequences Amino acid alignment analysis indicated that the OlIFNβ had the highest homology with the IFN1 of Gasterosteus aculeatus and it was up to 54%. Phylogenetic tree analysis showed that the OlIFNβ had the closest relationship with Gasterosteus aculeatus IFN and gathered into a branch with the type Ⅰ IFNs of other fishes. Real-time PCR analysis revealed that the OlIFNβ expression displayed time-specific and organs-specific. After stimulated by poly Ⅰ ： C, the expression of the OlIFNβ increased gradually with time, and reached the peak at 6 h post-treatment and declined gradually afterward. Among all tissues, spleen showed the most significant changes, followed by gill and intestine. The study indicated that the OlIFNβ played a key role in innate immune response in Oryzias latipes, which shed light on the the function analysis of OlIFNβ in the future.