中国水产科学2011，Vol.18Issue(5)：1021-1031,11.DOI:10.3724/SP.J.1118.2011.01021
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 (Hippocampus kuda) transforming growth factor-β1 (TGF-β1) gene
摘要
Abstract
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-pi had the closest relationship with the TGF-pi 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-pi (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-P 1. 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-pi protein of H, kuda is conserved as other TGF-β1 and that TGF-pi 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Key words
TGF-β1/ Hippocampus kuda/ cloning/ tissue distribution/ recombinant protein/ signaling pathway分类
生物科学引用本文复制引用
平海林,吴金英,徐胜威,胡开顺,段志刚..大海马转化生长因子β1(TGF-β1)基因的克隆鉴定及功能研究[J].中国水产科学,2011,18(5):1021-1031,11.基金项目
国家海洋863计划项目(2002AA624010) （2002AA624010）
广东省自然科学基金项目(05003313). （05003313）
