大海马转化生长因子β1(TGF-β1)基因的克隆鉴定及功能研究  

Molecular cloning and primary functional analysis of seahorse(Hip-pocampus kuda) transforming growth factor-β1(TGF-β1) gene

作　　者：平海林[1] 吴金英[1] 徐胜威[1] 胡开顺[1] 段志刚[1] 

机构地区：[1]中山大学生命科学学院水生经济动物研究所暨广东省水生经济动物良种繁育重点实验室,广东广州510275

出　　处：《中国水产科学》2011年第5期1021-1031,共11页Journal of Fishery Sciences of China

基　　金：国家海洋863计划项目(2002AA624010);广州市科技计划项目(2003Z3-C7321)

摘　　要：转化生长因子β1(Transforming growth factor-β1,TGF-β1)是一种多效应细胞因子,在免疫系统中,成熟的TGF-β1多肽是一种强有力的细胞分化调节剂和免疫调节剂。本研究通过基因克隆得到了大海马(Hippocampus kuda)TGF-β1 cDNA全长序列(GenBank登录号为HM564035),全长为1 941 bp,开放阅读框为1 161 bp,编码1种含386个氨基酸的多肽。同源性分析表明,大海马TGF-β1与斜带石斑鱼TGF-β1的亲缘关系最近。Real-time PCR结果显示,TGF-β1基因在健康大海马肾中表达量最高,其次是肝、大脑、小脑、皮肤、中脑、卵巢,在鳃和肠道中表达很少,在心脏和肌肉中检测不到表达。大海马TGF-β1重组蛋白在TNT快速偶联的转录/翻译系统中高效表达,用BS3交联后形成了大量的可溶性二聚体蛋白,Western blot检测结果显示该重组蛋白具有良好的免疫学活性。利用得到的大海马rTGF-β1刺激HeLa细胞,结果显示刺激细胞中TGF-β1下游Smad信号通路中的Smad3、Smad4基因表达量上升,Smad7基因表达量下降,表明所得到的大海马TGF-β1重组蛋白具有特定的生物活性,能够诱导人细胞中Smad信号通路的活化,延续了TGF-β1蛋白功能的保守性。Transforming growth factor-β1（TGF-β1） is a pleiotropic cytokine.In the immune system,the mature TGF-β1 peptide is a potent differentiation modulator and immunomodulator,with the ability to down-regulate the expression of many cytokines.In this study,we have cloned the full-length cDNA of TGF-β1 from seahorse（Hippocampus kuda） using homology cloning approach combined with RACE method.The full length cDNA is 1 941 bp,which contains an ORF of 1 161 bp,encoding a 386 amino acid protein.Homology analysis showed that the seahorse TGF-β1 had the closest relationship with the TGF-β1 of the orange-spotted grouper（Epinephelus coioides）.Real-time PCR results showed that the highest expression of seahorse TGF-β1 mRNA was found in the kidney,followed by the liver,cerebrum,cerebellum,skin,midbrain,ovary,and the expression level were low in the gill and intestine,while no expression was detected in the heart and muscle.Recombinant seahorse TGF-β1（rTGF-β1） was expressed in the TNT？ Quick Coupled Transcription/Translation Systems,then cross-linked using bis（sulfosuccinimidyl） subetate（BS3） and formed a large number of soluble dimeric rTGF-β1.Western-blot test results showed that the recombinant protein has a good immunological activity.The acquired rTGF-β1 protein was used to stimulate HeLa cells.Expression level of Smad signaling pathway downstream genes showed Smad3,Smad4 were markedly expressed,Smad7 was weakly expressed.The results indicate that the function of TGF-β1 protein of H.kuda is conserved as other TGF-β1 and that TGF-β1 of H.kuda has a specific biological activity which can induce the activation of Smad signaling pathway in human cells.

关 键 词：TGF-Β1 大海马 克隆 组织分布 重组蛋白 信号通路 

分 类 号：Q78[生物学—分子生物学] S917[农业科学—水产科学]