鸡白介素17A的克隆表达及活性测定

Cloning and functional characterization of chicken interleukin-17A

从经人工感染柔嫩艾美尔球虫孢子化卵囊(1×104个/只鸡)的盲肠上皮间淋巴细胞(IELs)提取总RNA,用RT-PCR方法成功扩增了鸡白介素17A(IL-17A)基因,测序结果显示,开放阅读框为453bp,编码150个氨基酸,与GenBank上鸡IL-17A基因(AM773756)的核苷酸和氨基酸序列完全一致(100%),与报道的哺乳动物IL-17A的氨基酸相似性达37%～46%。构建的pGEX-6p1-chIL-17A原核表达载体经1mmol/L IPTG诱导后表达出43kDa左右的融合蛋白,与预期大小一致,且Western-blot鉴定表达产物为目的蛋白。功能试验表明,表达的重组鸡IL-17A能够刺激鸡胚成纤维细胞产生IL-6。这些结果表明,鸡IL-17A在结构和功能方面与哺乳动物的IL-17A都有一定的相似性,可能在鸡的免疫应答过程中起到重要的作用。

Chicken IL-17A was cloned from IELs in the cecums of Eimeria tenella infected AA broilers.The full-length chIL-17A cDNA consisted of a 453 nucleotide sequence encoding an ORF of 150 AA.The sequence of nucleotide and amino acid is completely consistent with the reported chIL-17A in GenBank（AM773756）.ChIL-17 shared 37%~46% amino acid sequence identity to the previously described mammalian homologs.The constructed pGEX-6p1-chIL-17A expression vector expressed a 43 kDa fusion protein as expected which was detected by western blot assay.Treatment of CEFs with chIL-17A induced the expression of IL-6.Collectively,these results suggest that chIL-17A has structural and functional similarities to mammalian IL-17A and may play an important role in chicken immunity.