Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand
Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand
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DOI：
10.1016/j.dci.2006.08.006
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发表时间：
2007-01-01
影响因子：
2.9
通讯作者：
Aoki, Takashi
中科院分区：
文献类型：
--
作者：
Kurobe, Tomofumi;Hirono, Ikuo;Aoki, Takashi
我们从牙鲆 (Paralichthys olivaceus) 中分离并测序了 Fas 配体 cDNA 及其基因。 JF-Fas配体cDNA由1016bp组成，编码230个氨基酸残基。推导的JF-Fas配体氨基酸序列与人Fas配体、肿瘤坏死因子-a和淋巴毒素-a的同一性分别为26.1％、24.5％和23.0％。在 N 末端区域发现了一个对于蛋白质定位很重要的富含脯氨酸的结构域 (PRD)，并且形成二硫键的两个半胱氨酸残基是保守的。 JF-Fas配体基因长度为1.8 kb，由四个外显子和三个内含子组成。 JF-Fas配体第二内含子的长度比人和猪Fas配体基因中的长度短。然而，外显子和内含子的组织与哺乳动物的相似。对12个组织进行RT-PCR，在肾脏、胸腺、鳃、胃和脾中检测到JF-Fas配体mRNA的表达。在大肠杆菌蛋白表达系统中制备的重组JF-Fas配体对日本牙鲆细胞系HINAE表现出细胞毒活性，并导致基因组DNA断裂。通过MTT法测定细胞毒活性。这些结果表明鱼类拥有 Fas 配体系统。 (c) 2006 年，爱思唯尔有限公司出版。
We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-a and Lymphotoxin-a were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and intorons is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system. (c) 2006 Published by Elsevier Ltd.