鸡白细胞介素5基因的克隆与原核表达

Cloning and prokaryotic expression of chicken interleukin-5 gene

根据GenBank中登录的鸡白细胞介素5(IL-5)基因序列,设计并合成1对特异性引物,首次从ConA激活的鸡脾淋巴细胞中扩增出了鸡IL-5基因cDNA。序列分析表明,该基因全长381bp,编码126个氨基酸,蛋白质分子质量约14.63ku,包括22个氨基酸长度的信号肽。与其他哺乳动物IL-5的氨基酸同源性低于25%,显示较强的种属特异性。但该蛋白的三维结构与人IL-5蛋白结构相似,主要由4个α螺旋构成。进而构建了此基因的原核表达载体pET-28a-IL-5,并在大肠杆菌Rosetta(DE3)中通过IPTG的诱导成功实现了表达,为进一步研究禽IL-5的结构和生物学功能奠定了良好的基础。

According to a forecast sequence of chicken interleukin-5（ChIL-5） gene published in GenBank,a pair of primers was designed and used for cloning ChIL-5 gene by RT-PCR from chicken spleen lymphocytes stimulated by ConA for the first time.The complete nucleotide sequence of the cloned gene contained a 381bp open reading frame encoding 126 amino acids,including the sequence encoding 22-amino-acid signal peptide.The protein encoded by the gene was about 14.63ku in molecular weight.The amino acid sequence deduced from the gene had less than 25% similarity to that of other mammals.However,the predicted tertiary structure of ChIL-5 protein was similar to that of the human,and the structure of the protein mainly consisted of four α-helix.This gene was cloned into pET-28a（＋） vector to construct recombinant expression plasmid pET-28a-IL-5,then the recombinant plasmid was transformed into Escherichia coli Rosetta（DE3） and expressed under induction of IPTG.The prokaryotic expression of ChIL-5 gene provided foundation for further studies of the structure and biological function of avian IL-5.