| 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。 |
| 关键词: 黑鲷 肿瘤坏死因子α(TNF α) 克隆 特征分析 |
| DOI： |
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| 基金项目: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。 |
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| MOLECULAR CLONING AND CHARACTERIZATION OF TUMOR NECROSIS FACTOR ALPHA (TNF α) IN BLACK SEABREAM ACANTHOPAGRUS SCHLEGELI |
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GAO Chun-Ping1, CAI Zhong-Hua2, SONG Lin-Sheng3, WU Long-Tao3, CHI Zhen-Ming1
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1.College of Life Sciences, Ocean University of China, Qingdao, 266003;2.Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055;3.Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071
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| Abstract: |
| 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 Nglycosylation site and signal peptide. InterPro software analysis indicated that the peptide included a TNF profile and a TNF family signature. The secondary structure analysis showed that the BS TNF was similar to mammalian TNF α; they were conserved in both sites in which should form a disulphide bridge and the biological activity trimeric proteins, respectively. Tertiary analysis revealed that the BS TNF was similar to mammalian TNF α which had β-sandwich structures containing two stacked β-pleated sheets each formed by five anti-parallel β strands that adopted a classical “jelly-roll” topology. All these structural properties are common in mammalian TNF α, but different to TNF β. The structural analysis suggested that the BS TNF should be TNF α, not TNF β.
RT-PCR revealed that the TNF transcript was constitutively expressed in black seabream, and the BS TNF transcript could be detected in most of the detected tissue both in control and stimulated fish, but the level of the expression was different. The BS TNF α was mainly expressed in head kidney, spleen and gill, whereas faint expression could be detected in blood, brain, heart, liver and kidney. This results harmonizes with the expressed TNF α transcript of seabream and channel catfish, but different from that of red seabream, flounder and rainbow trout. |
| Key words: Black seabream (Acanthopagrus schlegeli), Tumor necrosis factor alpha (TNF A), Cloning, Characterization |
