鲤鱼白细胞介素-1β全长cDNA的克隆·鉴定及其差异表达分析

Cloning,Identification and Differential Expression Analysis of Full-length cDNA of Carp Interleukin-1β

[目的]进行鲤鱼白细胞介素-1β(IL-1β)全长cDNA的克隆、鉴定及其差异表达分析。[方法]利用DD-RTPCR方法获得差异表达cDNA片段,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行筛选,克隆了鲤鱼IL-1β的全长cDNA,并进行了序列分析和差异表达分析。[结果]获得的阳性克隆含有1个大小为831 bp编码276个氨基酸的完整开放阅读框。聚类分析表明,鲤鱼IL-1β氨基酸序列与日本鲤鱼紧密聚为一支,氨基酸序列的同源性达95%,之后聚类顺序依次为鲫鱼、斑马鱼、猪、牛、马、人和小鼠。差异表达分析表明,经有丝分裂原刺激后前期(4 h)白细胞中IL-1β的表达量显著增大,但随着时间推移(12,24 h)并非一直较同期大,表达量总体趋势成峰形。[结论]为进一步研究IL-1β在体内的表达方式、功能特点和调控机理以及在炎症反应、应急反应和免疫应答的作用机制奠定了基础。

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of full-length cDNA of carp interleukin-1β（IL-1β）.[Method] By using DD-RTPCR method,the differential expression cDNA fragment was gained.cDNA library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the whole length of carp interleukin-1β cDNA was cloned.Moreover,the sequence analysis and differential expression analysis were carried out.[Result] The positive clone which had a whole ORF encoded 276 amino acids in 831 bp was obtained.The cluster analysis showed that the amino acid sequence of carp interleukin-1β and Japanese carp were a group,and the sequence homology of amino acid reached 95%.The clustering order was carassius,zebrafish,pig,cow,horse,human and mouse in turn.The differential expression analysis showed that the expressed amount of IL-1β in the leucocytes obviously increased in the prior period（4 h） after the mitogen stimulated.But as the time went by（12 and 24 h）,it wasn＇t bigger than that in the same period.The total trend of expressed amount presented the peak type.[Conclusion] The research laid the foundation for further studying the expression manner,function characteristic and regulation mechanism of IL-1β in vivo and the action mechanisms in the inflammatory reaction,emergency reaction and immune response.