Molecular identification, expression and function analysis on creatine kinase in Trachidermus fasciatus
-
摘要: 前期蛋白质质谱分析发现,松江鲈(Trachidermus fasciatus Heckel)经鳗弧菌(Vibrio anguillarum)刺激后,其皮肤黏液中的肌酸激酶(creatine kinase,CK)的表达明显上调。目前,肌酸激酶在鱼类中的功能研究尚未深入开展。本文利用RACE技术克隆获得了松江鲈CK基因的全长cDNA序列(命名为TfM-CK)。TfM-CK基因的cDNA全长为1 474 bp,开放阅读框为1 146 bp,编码381个氨基酸。序列比对分析显示,TfM-CK序列高度保守。实时荧光定量PCR显示,TfM-CK mRNA广泛表达于松江鲈各组织,在肌肉中的相对表达量最高,其次为血液。鳗弧菌刺激后,TfM-CK mRNA在肌肉、皮肤、脾脏和头肾中均上调表达。其中,脾脏中的表达量高达对照组的900多倍。原核重组表达纯化的TfM-CK (rTfM-CK)蛋白酶活测定实验结果显示,重组蛋白的酶活为22.0 U/mg。rTfM-CK对鳗弧菌、大肠杆菌(Escherichia coli)、巨大芽孢杆菌(Bacillus megaterium)和金黄色葡萄球菌(Staphylococcus aureus)有较强的凝集作用。由以上实验结果推测,TfM-CK可能通过细菌凝集作用参与到了松江鲈抵抗病原菌的先天免疫防御过程中。本研究为认识鱼类肌酸激酶的功能及其在病原菌防御过程中的分子免疫机制机制提供了参考。Abstract: In previous study, we found that creatine kinase (CK) was one of the proteins which were up-regulated in the skin mucus of roughskin sculpin Trachidermus fasciatus post Vibrio anguillarum injection. To date, the function of this gene has not been well studied in fish. In this study, the cDNA sequence of CK gene was cloned using 3' RACE and 5'RACE techniques from roughskin sculpin, named as TfM-CK. The full-length of TfM-CK cDNA was 1 474 bp, which contained an open reading frame (ORF) of 1 146 bp encoding a polypeptide of 381 amino acids. The sequence BLASTP in NCBI indicated that TfM-CK was highly conserved. Quantitative real-time PCR (qPCR) analysis showed that TfM-CK mRNA expressed in all the detected tissues, with the highest expression in the muscle. After Vibrio anguillarum infection, TfM-CK transcripts were up-regulated significantly in the muscle, spleen, skin and head kidney, with the highest increase of 900 fold in the spleen. The kinase activity of recombinant protein of TfM-CK (rTfM-CK) was relatively high with 22.0 U/mg protein. It displayed high agglutination activity to all 4 tested bacterial including 2 Gram-negative bacteria Escherichia coli, Vibrio Anguillarum and 2 Gram-positive bacteria Staphylococcus aureus and Bacillus megaterium. These data indicate that TfM-CK participate in the fish immune response against pathogen infection. The present results may provide a theoretical basis for further investigation into the functions of fish TfM-CK and its molecular mechanisms of regulating fish immune response to pathogens.
-
Key words:
- creatine kinase /
- Trachidermus Fasciatus /
- expression /
- innate immunity /
- bacterial agglutination
-
于诗群, 王世党. 松江鲈鱼的生物学特征及养殖技术[J]. 北京水产, 2008, 25(8):50-52. Yu Shiqun, Wang Shidang. Characterization and breeding technology of roughskin sculpin, Trachidermus Fasciatus[J]. Journal of Beijing Fisheries, 2008, 25(8):50-52. 陈学昭, 张雷, 于珊珊, 等. 松江鲈(Trachidermus fasciatus)泛素结合酶E2-D2基因的分子克隆及组织表达分析[J]. 海洋学报, 2016, 37(10):133-140. Chen Xuezhao, Zhang Lei, Yu Shanshan, et al. Molecular cloning and expression analysis of ubiquitin-conjugating enzyme E2-D2 in roughskin sculpin, Trachidermus Fasciatus[J]. Haiyang Xuebao, 2016, 37(10):133-140. Cai Yan, Lee J, Wang Wei, et al. Effect of Cd2+ on muscle type of creatine kinase:inhibition kinetics integrating computational simulations[J]. International Journal of Biological Macromolecules, 2016, 83:233-241. Ellington W R, Suzuki T. Early evolution of the creatine kinase gene family and the capacity for creatine biosynthesis and membrane transport[C]//Salomons G S, Wyss M. Creatine and Creatine Kinase in Health and Disease. Dordrecht:Springer, 2007, 46:17-26. Rao J K M, Bujacz G, Wlodawer A. Crystal structure of rabbit muscle creatine kinase[J]. FEBS Letters, 1998, 439(1/2):133-137. 范宁宁. 青岛文昌鱼肌酸激酶基因的克隆、表达分析和功能研究[D]. 青岛:中国海洋大学, 2008. Fan Ningning. Cloning, expression and functional analysis of creatine kinase gene in amphioxus Branchiostoma belcheri tsingtauense[D]. Qingdao:Ocean University of China, 2008. Wyss M, Braissant O, Pischel I, et al. Creatine and creatine kinase in health and disease-a bright future ahead?[C]//Salomons G S, Wyss M. Creatine and Creatine Kinase in Health and Disease. Dordrecht:Springer, 2007. Ozawa E, Hagiwara Y, Yoshida M. Creatine kinase, cell membrane and Duchenne muscular dystrophy[J]. Molecular and Cellular Biochemistry, 1999, 190(1/2):143-151. Lopes R D, Lokhnygina Y, Hasselblad V, et al. Methods of creatine kinase-MB analysis to predict mortality in patients with myocardial infarction treated with reperfusion therapy[J]. Trials, 2013, 14:123. 高媛媛. 文昌鱼应激反应的蛋白质组学及creatine kinase的免疫功能研究[D]. 厦门:厦门大学, 2007. Gao Yuanyuan. Proteomics approach for stress response and immune function investigation for creatine kinase in amphioxus[D]. Xiamen:Xiamen University, 2007. Gao Yuanyuan, Zhang Danfeng, Li Hui, et al. Proteomic approach for caudal trauma-induced acute phase proteins reveals that creatine kinase is a key acute phase protein in amphioxus humoral fluid[J]. Journal of Proteome Research, 2007, 6(11):4321-4329. An Yan, Fan Ningning, Zhang Shicui. Creatine kinase is a bacteriostatic factor with a lectin-like activity[J]. Molecular Immunology, 2009, 46(13):2666-2670. 安艳. 肌酸激酶是具有凝集素活性的抑菌因子[D]. 青岛:中国海洋大学, 2009. An Yan. Creatine kinase is a bacteriostatic factor with a lectin-like activity[D]. Qingdao:Ocean University of China, 2009. Sun H W, Hui C F, Wu J L. Cloning, characterization, and expression in Escherichia coli of three creatine kinase muscle isoenzyme cDNAs from carp (Cyprinus carpio) striated muscle[J]. Journal of Biological Chemistry, 1998, 273(50):33774-33780. 王家庆, 李代宗, 郭冉, 等. 虹鳟脑型肌酸激酶基因cDNA全长的克隆与序列分析[J]. 上海海洋大学学报, 2009, 18(4):385-390. Wang Jiaqing, Li Daizong, Guo Ran, et al. The cloning and analysis of CKB full length cDNA derived from Oncorhynchus mykiss[J]. Journal of Shanghai Ocean University, 2009, 18(4):385-390. Silva C M B E, Tavares N N, Giovanni-De-Simone S, et al. Purification and partial characterization of creatine kinase from electric organ of Electrophorus electricus (L.)[J]. International Journal of Biochemistry and Cell Biology, 2000, 32(4):427-433. Harder G, Mcgowan R. Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish, Danio rerio[J]. Biochemistry and Cell Biology, 2001, 79(6):779-782. 张敏, 赵金良, 邓燕飞. 鳜肌酸激酶M-CK cDNA的克隆与组织表达分析[J]. 动物学研究, 2010, 31(1):77-83. Zhang Min, Zhao Jinliang, Deng Yanfei. Cloning and Tissue Expression Analysis of Creatine Kinase (M-CK) cDNA from the Mandarin Fish, Siniperca chuatsi[J]. Zoological Research, 2010, 31(1):77-83. Liu Zhanjiang, Kim S, Kucuktas H, et al. Multiple isoforms and an unusual cathodic isoform of creatine kinase from channel catfish (Ictalurus punctatus)[J]. Gene, 2001, 275(2):207-215. Winnard P Jr, Cashon R E, Sidell B D, et al. Isolation, characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 2003, 134(4):651-667. Sun H W, Liu Chengwen, Hui C F, et al. The carp muscle-specific sub-isoenzymes of creatine kinase form distinct dimers at different temperatures[J]. Journal of Biological Chemistry, 2003, 368(3):799-808. Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 2001, 25(4):402-408. 李莉, 李春梅. 鱼类非特异性免疫研究进展[J]. 河南农业科学, 2012, 41(2):26-32. Li Li, Li Chunmei. Research progress on non-specific immune of fish[J]. Journal of Henan Agricultural Science, 2012, 41(2):26-32.
点击查看大图
计量
- 文章访问数: 339
- HTML全文浏览量: 11
- PDF下载量: 242
- 被引次数: 0