黑鲷(Acanthopagrus schlegeli)肿瘤坏死因子α cDNA的克隆及特征分析  

MOLECULAR CLONING AND CHARACTERIZATION OF TUMOR NECROSIS FACTOR ALPHA (TNF α) IN BLACK SEABREAM ACANTHOPAGRUS SCHLEGELI

作　　者：高春萍[1] 蔡中华[2] 宋林生[3] 吴龙涛[3] 池振明[1] 

机构地区：[1]中国海洋大学生命学院 [2]清华大学深圳研究生院,深圳518055 [3]中国科学院海洋研究所

出　　处：《海洋与湖沼》2005年第4期326-334,共9页Oceanologia Et Limnologia Sinica

基　　金：863国家高技术研究发展计划资助;2001AA628118号;国家自然科学基金项目资助;40476075号;中国科学院海洋研究所实验海洋生物学重点实验室资助。

摘　　要：采用同源克隆的方法,从黑鲷中获得肿瘤坏死因子(TNFα)全长cDNA序列。该序列含1288个核苷酸,可编码253个氨基酸的TNF前体蛋白。它是一种跨膜蛋白,无糖基化位点和信号肽结构。黑鲷TNFα与其它鱼类的TNFα的相似性很高,占据进化上独立的分支;与哺乳类TNFα和TNFβ源于共同的祖先。基因结构分析显示,该基因含有TNF家族profile和TNF家族的标签序列;在参与TNFα基因二硫键形成的两个半胱氨酸和TNFα三聚体形成的位点高度保守;空间结构模拟显示,它与哺乳类TNFα的空间结构相似,都是由两个β折叠片组成,每个折叠片包含5个反向平行的β链。表达研究结果表明,黑鲷TNFα在检测的各个组织中均为组成型表达,表现为在刺激与非刺激鱼体中,都可以检测到黑鲷TNFα的表达,但是其表达水平在不同组织中有很大差异。黑鲷TNFα在头肾、脾脏和鳃的表达量较高,而在心脏、肝脏、血液、肾脏和大脑中表达量较低。A partial homologue sequence of tumor necrosis factor alpha (TNF α) had been identified in black seabream by homology strategy using a pair of degenerated primers according to the conserved regions of other animal TNF α genes. Subsequently the sequence was elongated by RACE PCR to get full cDNA sequence with adapter primer and gene specific primer. The cDNA of black seabream TNF α (BS TNF) had 1288bp nucleotides including a short 5′ UTR of 38bp, 3′ UTR of 491bp and an open reading frame (ORF) of 759bp that could encode propeptide of 253 amino acids with putative MW of 28kDa and putative PI of 4.94. In 3′ UTR, there were several mRNA instability motifs of ATTTA and three endotoxin responsive motifs of TTATTTAT or ATATTTAT which should be involved in TNF α gene transcript regulation and endotoxin response. The BS TNF amino acids sequence shared highest identity with seabream TNF α of 94.8%, followed by red seabream TNF α of 78.8% and then with other piscine TNF α. The BS TNF had about 25%—26% identity with mammalian TNF α, and 20%—23% identity with mammalian TNF β homolog. Using CLUST W software, all the piscine TNF α and parts of mammalian TNF α and TNF β amino acids were clustered, and a phylogenic tree was constructed by Neighbor-Joint (NJ) algorithm. Phylogenic analysis showed that all of the piscine TNF α located in an independent clade which was different from both mammalian TNF α and TNF β. The black seabream, seabream and red seabream that belong to the same family, were clustered together firstly, then the Japanese flounder was clustered as the first sister group. While the trout and carp were some far from it, they were located as a near branch. These results are in common with the traditional taxonomy. The deduced peptide belongs to the membrane protein which has a transmembrane domain but lacks clearly N-glycosylation site and signal peptide. InterPro software analysis indicated that the peptide included a TNF profile and a TNF family signature. The secondary structure analysis

关 键 词：黑鲷 肿瘤坏死因子α(TNFα) 克隆 特征分析 

分 类 号：Q789[生物学—分子生物学]