鲤鱼白细胞介素10(IL-10)基因cDNA克隆及序列分析

[目的]获取鲤鱼全长IL-10(interleukin 10)cDNA,并对其序列进行分析。[方法]利用DD-RTPCR(differential display RT-PCR)方法获得差异表达IL-10 cDNA片段,以地高辛标记做为探针,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行核酸杂交筛选,克隆鲤鱼IL-10全长cDNA,并对该序列进行序列分析和同源性比较。[结果]鲤鱼全长IL-10 cDNA共1 117 bp,包含55 bp的5'端非编码区,一个540 bp的编码179个氨基酸的开放阅读框及522 bp的3'端非编码区,其中,3'非编码区包含3个mRNA不稳定基序"ATTTA";该蛋白序列具有IL-10家族的典型序列特征;序列同源性比较表明,所获得的序列与GenBank上登录的鲤鱼IL-10基因同源性为89.1%。[结论]该试验为进一步研究IL-10在体内的表达方式、功能特点、调控机理及其在炎症反应和免疫应答中的作用机制奠定了基础。

Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

[Objective] This study aimed to obtain IL-10(interleukin 10) full-length cDNA of common carp(Cyprinus carpio L.) and conduct the sequence analysis.[Method] The differentially expressed cDNA fragment was obtained by DD-RTPCR(differential display RT-PCR) to get the full-length IL-10 cDNA.The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated with mitogen was screened by a probe labeled with DIG(digoxigenin).The IL-10 full-length cDNA was cloned from 0.8 ×104 recombinant phages,and the sequence analysis and homology comparison were carried out.[Result] Sequence analysis indicated that the IL-10 full-length cDNA of common carp was 1 117 bp long,containing a 55 bp 5′-UTR,a 522 bp 3′-UTR,and a 540 bp open reading frame(ORF) encoding 179 amino acids.In addition,there were three motifs for mRNA instability(ATTTA) in the 3’-untranslated region.The deduced protein sequence shared typical sequence features of the IL-10 family.Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-10 gene from GenBank.[Conclusion] This study laid foundation for further study of the expression manner,functional characteristic and regulation mechanism of IL-10 in vivo and the interaction mechanism in the inflammatory reaction and immune response.