固始鸭白细胞介素-18全基因克隆与分子进化分析

根据GenBank发表的鸭IL-18cDNA基因序列设计、合成一对引物,应用RT-PCR技术,无需用非特异性免疫原如PHA等刺激脾淋巴细胞,直接提取脾淋巴细胞总RNA,扩增固始鸭IL-18基因,并克隆、测序。测序结果表明固始鸭IL-18基因全序列为610bp,包含1个完整阅读框,编码1条由200个氨基酸残基组成的多肽。序列分析发现,固始鸭IL-18基因与GenBank中两条鸭IL-18基因(AF336122和DQ490137)核苷酸同源性分别为98.8%、99.8%,与AF336122有5个碱基发生非同义变异,2个碱基为同义变异,氨基酸同源性为97.5%,与DQ490137有一个碱基发生非同义变异,氨基酸同源性为99.5%。固始鸭IL-18前体蛋白第30位谷氨酸处有一个IL-1β转换酶的caspase-1切割位点及在人和其他动物中已证实的IL-1标签序列,推测鸭IL-18成熟蛋白由170个氨基酸组成。固始鸭与人和其他动物的IL-18基因进化分析表明,IL-18基因存在着种的多样性,且亲缘关系越近,同源性越高。

Cloning and phylogenetic analysis of the interleukin-18 gene in gushi duck

A pair of primers was designed according to duck IL-18 gene sequences in GenBank.Duck IL-18 gene was amplified by RT-PCR from total RNA extracted from gushi duck splenocyte and the PCR product was cloned and sequenced.The result showed that the duck IL-18 gene is 603 bp in length encoding 200 amino acids.The gene is highly conserved and shared 98.8 %-99.8 % nucleotide homology and 97.5 %-99.5 % amino acid homology with other duck IL-18 genes(accession No.AF336122 and DQ490137).Thje gene contains a putative caspase-1 cleavage site of IL-1beta-converting enzyme(ICE) at aspartic acid 30 of the primary translation product and IL-1 signature-like sequences similar to those identified in human and others mammals IL-18,indicating that a mature DuIL-18 might comprise 170 amino acids.Phylogenetic tree analysis indicated that the IL-18 sequence variation was related to genus,the nearer relationship,the higher homology.