真鲷肿瘤坏死因子α(TNFα)cDNA的克隆与表达  

Cloning and Expression of Tumor Necrosis Factor (TNFα) cDNA from Red Seabream Pagrus major

作　　者：蔡中华[1] 宋林生[1] 高春萍[2] 吴龙涛[1] 邱丽华[1] 相建海[1] 

机构地区：[1]中国科学院海洋研究所实验海洋生物重点实验室 [2]中国海洋大学生命科学院,青岛266003

出　　处：《生物化学与生物物理学报》2003年第12期1111-1116,共6页

基　　金：国家重点基础研究发展规划项目(973计划) (No .G19990 12 0 0 8);国家高技术研究发展计划项目(863计划 )(No .2001AA628180 );王宽城博士后奖励基金 (No .2001100 2110140)资助~~

摘　　要：采用同源克隆和末端快速扩增 (RACE)的方法 ,从真鲷中克隆到 12 6 4bp的TNFα全cDNA编码序列 (Gen Bank登录号为AY314 0 10 )。该序列包括 6 6 6bp可读框 (ORF) ,85bp的 5′末端非编码区 (UTR)以及 5 14bp的 3′末端非编码区。序列的 3′UTR含 5个mRNA不稳定模体 (motif)和 3个内毒素应答序列 ,但是未发现基因的PolyA加尾信号。由该序列推导的多肽含有明确的跨膜域 ,TNF家族的标签序列 (signature) ,TNF2家族分布 (profile)文件 ,粘多糖结合位点和细胞粘附位点。进化分析显示 ,真鲷TNF与哺乳类TNFα和TNFβ具有较高的相似性 ,源于共同的祖先。但结构及表达分析表明 ,它是TNFα而不是TNFβ。表达研究显示 ,真鲷TNFα存在组成型和诱导型两种表达形式 ,表现在刺激与非刺激的真鲷中 ,TNFα均可在部分组织中表达 ,但是在受刺激鱼体中基因表达的组织分布显著增多。A fragment of TNFα cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3′ and 5′ RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5′ UTR of 85 bp, a 3′ UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3′ UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFα family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFα and TNFβ by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFα were located independently in a different branch compared with mammalian TNFα and TNFβ. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFα, not TNFβ. RT-PCR was used to study TNFα transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFα transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen. Results showed that TNFα mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

关 键 词：真鲷 肿瘤坏死因子Α TNFΑ CDNA 克隆 表达 

分 类 号：S943[农业科学—水产养殖]