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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)25bp;3′-非编码区(3′-UTR)535bp,存在2个mRNA不稳定基序ATTTA;开放阅读框ORF长1632bp,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61.773ku。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。
In this study, based on the EST sequence of the tumor necrosis factor receptor-associated factor 6 (TRAF6) in the carp, diosgenin-labeled carp peripheral blood The white blood cell cDNA library was screened by nucleic acid hybridization, and two positive clones were obtained from the recombinant phage after two rounds of screening. Sequence analysis showed that the sequence contained 25bp 5’-UTR (5’-UTR) and 535bp in the 3’-UTR (3’-UTR). There were two ATTTA mRNA instability motifs. The open reading frame ORF is 1632bp long and encodes 543 amino acids. The predicted isoelectric point of protein was 5.88, the molecular mass was 61.773ku. Sequence homology comparison showed that the obtained sequence shared 99% identity with TRAF6a gene of Carp registered in GenBank. Protein sequence analysis revealed that it has typical sequence features of the TRAF family.