鲤鱼肿瘤坏死因子受体相关因子6全长cDNA克隆及序列分析

本试验以鲤鱼外周血白细胞肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)EST序列为基础,经地高辛标记后作为探针,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,从重组噬菌体中经过两轮筛选获得阳性克隆。序列分析结果显示,该序列包含有5'-非编码区(5'-UTR) 25 bp;3'-非编码区(3'-UTR) 535 bp,存在2个mRNA不稳定基序ATTTA;开放阅读框ORF长1632 bp,编码543个氨基酸。预测蛋白质等电点为5.88,分子质量大小为61.773 ku。序列同源性比对结果表明,所获得的序列与GenBank上登录的鲤鱼TRAF6a基因同源性达99%。蛋白质序列分析结果发现,其具有TRAF家族的典型序列特征。

Clonging and Sequence Analysis of Tumor Necrosis Factor Receptor-associated Factor 6 Full-length cDNA from Common Carp

The common carp tumor necrosis factor receptor-associated factor 6 (TRAF6) EST sequence was picked out from the cDNA library of peripheral blood leukocytes that was constructed. The cDNA library were separated from carp and stimulated with mitogen was screened by a probe labeled with DIG. The full-length TRAF6 cDNA was cloned from recombinant phages. Sequence analysis indicated that it encompasses with a 25 bp 5'-UTR and a 535 bp 3'-UTR,the open reading frame (ORF) of which was 1632 bp putatively coding 543 amino acids,and there were two motifs for mRNA instability ATTTA in the 3'-untranslated region. The predicted theoretical isoelectric point and molecular weight were 5.88 and 61.773 ku,respectively. Its nucleotide homology with carp TRAF6 from GenBank was up to 99%.The protein sequence analysis showed that it shared typical sequence features of the TRAF family.