赤点石斑鱼<em>HSP60</em>基因克隆及弧菌应激前后的组织表达特性分析

曲朦; 施晓峰; 张之文; 丁少雄

赤点石斑鱼HSP60基因克隆及弧菌应激前后的组织表达特性分析

厦门大学海洋与环境学院, 福建厦门 361005

基金项目: 福建省自然科学基金(2060203);福建省新世纪人才资助计划。

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出版历程
- 收稿日期: 2010-08-23

Cloning of HSP60 gene from Epinephelus akaara and its express characterization before and after vibrionic stressed

College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China

摘要

摘要: HSP60(heat shock protein 60,热休克蛋白60)作为高度保守的热休克蛋白家族的一员,不仅可以在应激状态下帮助其他蛋白正确折叠并恢复天然构象,还可作为危险信号作用于天然免疫系统。基于HSP60的EST序列设计特异引物,采用RACE技术成功克隆获得赤点石斑鱼HSP60 cDNA全序列及基因组全序列。其cDNA序列全长2 351 bp,包括75 bp的5'UTR,539 bp的3'UTR和1 737 bp的开放阅读框,共编码578个氨基酸。核苷酸及氨基酸序列比对结果显示该基因与其他脊椎动物的相似性很高。基于氨基酸序列构建的系统进化树与传统物种树基本吻合,说明HSP60可能是1个潜在的可用于物种系统进化研究的良好标记。所获得的赤点石斑鱼HSP60基因组全序列含有8个内含子和9个外显子,也与已知其他硬骨鱼类基本一致。这种在序列特征、蛋白空间结构及基因组结构上的高度保守性意味着HSP60在动物生命活动中可能发挥着至关重要的作用。通过实时PCR检测结果发现HSP60在赤点石斑鱼各组织中均有表达,而攻毒后鱼体肝的表达量显著增高(P<0.05),鳃、脾、胃和脑等重要组织器官的表达量也有不同程度的提高,该结果证实了HSP60基因在鱼类非特异性免疫应答中发挥了作用。
- 赤点石斑鱼 /
- HSP60 /
- 克隆 /
- 组织表达特性
Abstract: As a major member of heat shock protein families, HSP60 can not only be served as a molecular chaperone to promote protein disaggregation and proper refolding, but also be regarded as a threatened signal under stress to elicit a potent proinflammatory response in cells of the innate immune system. Based on the EST sequences from cDNA library, the full length cDNA of HSP60 from E.akaara was successfully obtained by RACE technique. The full-length of HSP60 is 2351bp, including 75bp of 5'UTR, 539bp of 3'UTR, and a 1737bp open reading frame encoding a protein of 578 amino acids. The alignment result shows that the nuclear acid sequence and amino acids sequence of HSP60 from E. akaara share high identity with other vertebrate homologs. The phylogenetic tree based on the HSP60 amino acid sequences is congruent with their evolutionary relationships, which indicates that HSP60 may be an appropriate marker for the phylogenetic research among interspecies. The E. akaara HSP60 gene contains 9 introns and 10 exons, the same as other teleosteans. The conserved sequence, gene structure and 3D protein structure show that HSP60 may play an important role in life activities. Real-time PCR was also conducted to detect the express characterization of the E. akaara HSP60 gene. The result shows that HSP60 can be detected in various tissues, and after Vibrio harveyi-challenging, the expression of HSP60 in the liver increases dramatically, most of other tissues are also detected increasing to a great extent, which supports that HSP60 maybe play a role in fish innate immune response.
- Epinephelus akaara /
- heat shock protein60 /
- cloning /
- express characterization

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参考文献(28)

龚兴国,于红.热休克蛋白60的研究进展[J].中国病理生理杂志,2004,20 (11):2151-2154.

邬堂春,贺涵贞,张国高.热应激蛋白研究的进展与前景[J].中华预防医学杂志,2000,34(1):6-7.

HABICLT C, BURKART V.Heat shock protein 60:regulatory role on innate immune cells[J].Cellular and Molecular Life Sciences,2007,64:742-751.

BASU N, TODGHAM A E, ACKERMAN P A, et al.Heat shock protein genes and their functional significance in fish[J].Gene,2002,295:173-183.

MAUREEN E Claytom, ROLAND S, KARL F.Different expression patterns of heat shock proteins HSP 60 and HSP 70 in zebra mussels (Dreissena polymorpha) exposed to copper and tributyltin[J].Aquatic Toxicology,2000,47:213-226.

BARRY P, CHAO Z.The heat shock proteins: their roles as multi-component machines for protein folding[J].Fungal Biology Reviews,2008,22:110-119.

曹智,马骏,袁文俊,等.热休克蛋白60与细胞凋亡[J].生理科学进展,2008,39(3):267-270.

袁志强,彭毅志.热休克蛋白--细胞的内源性保护蛋白[J].生命的化学,2001,21(6):445-446.

KOJI O,VOLKER B,STEFANIE F, et al.Heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex[J].The Journal of Immunology, 2010,9:558-561.

汪宇辉.HSP60在免疫反应中的作用及信号传导通路[J].四川生理科学杂志,2002,24(2):49-51.

杨明金.热休克蛋白60和免疫反应[J].国外医学免疫分册,2004,27(5):249-252.

ROBERT P A Wallin, ANDREAS Lundqvist, SOLVEIG H Moré,et all.Heat-shock proteins as activators of the innate immune system[J].Trends in Immunology,2002,23(3):130-135.

MAKINO S G. WHITEHEAD G G, LIEN Ching-Ling, et all.Heat-shock protein 60 is required for blastema formation and maintenance during regeneration[J].PNAS,2005,102(41):14599-14604.

MARTN Monserrat, CELIA Herna’ndez, GUILLERMO Bodega, et all.Heat-shock proteins expression in fish central nervous system and its possible relation with water acidosis resistance[J].Neuroscience Research,1998,31:97-106.

RANDALL H.Groupers of the world[M].Rome: FAO Species Catalogue, 1993:106-107.

李明德.鱼类分类学[M].北京:海洋出版社, 1998:174-175.

徐晓津,徐斌,王军,等.大黄鱼感染哈维氏弧菌后血液生化指标的变化及组织病理学观察[J].水产学报.2010,34(4):618-625.

郑仲承.寡核苷酸的优化设计[J].生命的化学,2001,21(3):254-256.

任亮,朱宝芹,张轶博,等.利用软件Primer Premier 5.0进行PCR引物设计的研究[J].锦州医学院学报,2004, 25(6):43-46.

萨姆布鲁克 J,拉塞尔 D W.分子克隆实验指南[M],第3版.北京:科学出版社,2002.

ALTSCHUL S F, MADDEN T L, SCHFFER A A, et al. Gapped BLAST and PSI-BLAST:a new generation of protein database search programs[J].Nucl Acids Res,1997,25(17):3389-3402.

SAITOU N, NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4:406-425.

FENTON W A, KASHI Y, FURTAK K,et al. Residues in chaperonin GroEL required for polypeptide binding and release[J].Nature,1994,371:614-619.

XU Zhao-hui, HORWICH A L, SIGLER P B.The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex[J].Nature,1997,388(21):741-750.

LI Jiang, LIU Zhe-yun, TAN Chuang, et al. Dynamics and mechanism of repair of UV-induced (6-4) photoproduct by photolyase[J]. Nature,2007,450(7167): 203-218.

MANSI Srivastava, EMINA Begovic, JARROD Chapman. The Trichoplax genome and the nature of placozoans[J]. Nature,2008,454:955-960.

OHASHI K, BURKART V, FLOHE S.,et al. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex[J]. Immunol. 2000,164:558-561.

BULUT Y, FAURE E, THOMAS L, et al.Chlamydial heat shock protein 60 activates macrophages and endothelial cells through toll-like receptor 4 and MD2 in a MyD88-dependent pathway[J].Immunol, 2002,168(3):1435-1440.

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