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试验以鲤鱼外周血白细胞肿瘤坏死因子α1(tumor necrosis factor alpha 1,TNFα1)EST序列为基础,经地高辛标记作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,从0.9×104个重组噬菌体中,经过2轮筛选获得3个阳性克隆。序列分析结果显示,该序列包含128 bp的5′非编码区(5-′UTR),423 bp的3′非编码区(3-′UTR),开放阅读框ORF长768 bp,共编码255个氨基酸,在其3′非编码区存在几个ATTTA不稳定基序。预测蛋白等电点为8.20,分子质量大小为28.1 ku。序列同源性比较结果表明,所获序列与GenBank上登录的鲤鱼TNFα基因的同源性达94%。蛋白质的序列和结构分析结果发现,其具有TNF家族的典型序列特征、1个跨膜区结构和1个假定的产生成熟肽的裂解位点。
Based on the EST sequences of tumor necrosis factor alpha 1 (TNFα1) from carp, the cDNA library of mitogens stimulated carp peripheral blood leucocytes was screened by digoxigenin × 104 recombinant phage, after 2 rounds of screening to obtain three positive clones. Sequence analysis showed that the sequence contained 5 ’UTR (128 bp) and 3’ UTR (423 bp). The ORF of the open reading frame (ORF) was 768 bp in length, encoding a total of 255 Amino acids, there are several ATTTA labile motifs in its 3 ’non-coding region. The predicted isoelectric point was 8.20 and the molecular mass was 28.1 ku. Sequence homology comparison showed that the obtained sequence shared 94% identity with the TNFα gene of Carp registered in GenBank. Sequence and structural analysis of the protein revealed that it has the typical sequence characteristics of the TNF family, a membrane-spanning structure and a putative cleavage site for the mature peptide.