【作者】 刘文生； 苏莲娇； 李美娟； 唐蕾； 李林； 潘庆；

【Author】 Liu Wensheng;Su Lianjiao;Li Meijuan;Tang Tei;Li Lin;Pan Qing;Department of Aquaculture,College of Animal Sciences,South China Agricultural University;

【机构】 华南农业大学动物科学学院水产养殖系；

【摘要】 根据已知鱼类的IGF-Ⅰ氨基酸序列,利用CODEHOP网站在线设计兼并性引物,用黄颡鱼(Pelteobagrus fulvidraco)肝胰脏总RNA反转录作为模板,克隆得到中间序列,通过SMART RACE技术获得了IGF-Ⅰc DNA全长,BLAST分析结果表明得到的序列为黄颡鱼IGF-Ⅰc DNA序列。其中开放阅读框为441bp,编码146个氨基酸残基。同源性分析发现,黄颡鱼IGF-Ⅰ的ORF核苷酸序列和氨基酸序列与斑点叉尾鮰(Ictalurus punctatus)同源性较高,分别为92.5%和93.8%。黄颡鱼IGF-Ⅰ蛋白质分子质量为16123.4Da,等电点为9.68。根据开放性阅读框,设计引物扩增得到成熟肽c DNA片段,并克隆至p ET-28a载体构建重组表达质粒PET-28a/IGF-Ⅰ,在37℃IPTG诱导4h的条件下成功在E.coli BL21(DE3)中融合表达。SDS-PAGE分析表明,融合蛋白大小为11.2k Da。重组蛋白主要以包涵体的形式存在,Western-blotting免疫印迹分析表明,融合蛋白可特异性地被6×His抗体识别。更多还原

【Abstract】 We cloned the partial c DNA of IGF-Ⅰin Pelteobagrus fulvidraco by using a pair of degenerate primers based on the amino acid sequence obtained from other species. The full length c DNA was then obtained by SMART RACE, which contained an ORF of 441 bp nucleotides that encoded a protein of 146 amino acid residues. The homology analysis showed that the ORF nucleotide and amino acid sequence shared 92.5% and 93.8% similarities with Ictalurus punctatus, respectively. The molecular weight of the IGF-Ⅰin P. fulvidraco was 16123.4Da and its p I was 9.68. The mature peptide fragment of the IGF-Ⅰ was amplified and cloned into the prokaryotic expression vector p ET-28 a to construct a recombinant plasmid. The recombinant plasmid was subsequently transformed into E. coli BL21(DE3), and it successfully expressed recombinant IGF-Ⅰmature peptide protein after 4 hours being induced with IPTG at 37℃. The fusion protein was expressed in the form of inclusion bodies. The SDS-PAGE analysis revealed that the IGF-I polypeptide with molecular weight of 11.2 KD. The Western-blotting indicated that the recombinant protein was specifically recognized by 6×His antibody after denaturation, purification and refolding.更多还原