帮助 关于我们

返回检索结果

石斑鱼β-防御素的酵母表达及其产物抗菌活性分析
THE YEAST EXPRESSION OF GROUPER β-DEFENSIN AND ANTIBACTERIAL ACTIVITY OF THE RECOMBINANT PROTEIN

查看参考文献31篇

文摘 防御素是一类阳离子抗菌肽。研究从石斑鱼垂体SMARTcDNA文库中扩增出129 bp石斑鱼β-防御素成熟肽序列,将其克隆到毕赤酵母表达载体pPCIZαA中,构建了石斑鱼β-防御素的真核表达载体,电击转化毕赤酵母GS115。Western Blot分析表明石斑鱼β-防御素在酵母菌中获得了表达。体外抗菌实验表明纯化的重组蛋白具有抑制大肠杆菌以及嗜水气单胞菌的作用,但是对革兰氏阳性菌,如金黄色葡萄球菌和藤黄微球菌的生长没有抑制作用。实验结果表明酵母表达的石斑鱼β-防御素能够特异地抑制革兰氏阴性菌的生长。
其他语种文摘 Defensin, a small cationic peptide, is an antimicrobial peptide, exhibit broad-spectrum antibacterial activity. They spread widely in plants, invertebrate and vertebrate animals, which can rapidly kill bacteria, fungi and viruses etc. Bacteria and viruses limited the production of grouper (Epinephelus coioides). In this study, we cloned grouper β-defensin open reading frame, to express in the Pichia pastoris, and detect the bioactive of recombinant protein against bacteria. First, grouper β-defensin cDNA was amplified by polymerase chain reaction (PCR) from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper. The 129 bp DNA fragment encoding mature grouper β-defensin peptide was subcloned into vector PMD18-T, then inserted into the yeast expression vector pPCIZαA and transfected into Pichia pastoris GS115 expression host by electroporation. The genome of Pichia pastoris clone was extracted as template for screened the positive clone by using PCR. After grouper β-defensin was secreted by GS 115 by small-scale culture, the recombinant grouper β-defensin was induced by methanol for large scale. The supernatant was collected in 24h, 48h and 72h post induction for the recombinant protein detection. Subsequently, the grouper β-defensin peptide was detected in the supernatant of transfected yeast by Western Blot analysis. The purified recombinant grouper β-defensin crude extract from transfected P. pastoris showed antimicrobial activities against Escherichia coli and Aeromonas hydrophila. However, recombinant protein did not inhibit the growth of Gram-positive bacteria, such as Staphylococcus aureus and Micrococcus luteus. Bioactivity analysis indicated that the antibacterial activity of the recombinant grouper β-defensin expressed in P. pastoris specific to Gram-negative bacteria. Grouper β-defensin have antibacterial activity-specific to Gram-negative bacteria, this specificity is in correspondence with the environment of grouper.
来源 水生生物学报 ,2011,35(5):739-744 【核心库】
关键词 石斑鱼β-防御素 ; 酵母表达 ; 抗菌活性
地址

中国科学院水生生物研究所, 淡水生态与生物技术国家重点实验室, 武汉, 430072

语种 中文
文献类型 研究性论文
ISSN 1000-3207
学科 分子生物学
基金 国家自然科学基金项目 ;  国家973计划 ;  中国科学院水生生物研究所淡水生态与生物技术国家重点实验室基金
文献收藏号 CSCD:4378886

参考文献 共 31 共2页

1.  Lee K K. Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish. Marine Biotechnologyl (NY),2002,4:267-277 被引 6    
2.  Lu D Q. Interleukin-1β gene in orange-spotted grouper, Epinephelus coioides: Molecular cloning, expression, biological activities and signal transduction. Molecular Immunology,2008,45:857-867 被引 6    
3.  Yin Z X. Cloning, expression and antimicrobial activity of an antimicrobial peptide, epinecidin-1, from the orange-spotted grouper, Epinephelus coioides. Aquaculture,2006,253:204-211 被引 8    
4.  Chi S C. Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae. Virus Research,1999,63:107-114 被引 2    
5.  Selsted M E. Mammalian defensins in the antimicrobial immune response. Nature Immunology,2005,6:551-557 被引 47    
6.  Zou J. Discovery of multiple beta-defensin like homologues in teleost fish. Molecular Immunology,2007,44:638-647 被引 17    
7.  Falco A. Expression and antiviral activity of a β-defensin-like peptide identified in the rainbow trout (Oncorhynchus mykiss) EST sequences. Molecular Immunology,2008,45:757-765 被引 4    
8.  Zhao J G. Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-κB and Sp1 of a medaka β-defensin. Developmental and Comparative Immunology,2009,33:624-637 被引 3    
9.  Nam B H. Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus. Fish & Shellfish Immunology,2010,28:267-274 被引 3    
10.  Guo C. Recombinant expression and characterization of an epididymis-specific antimicrobial peptide BIN1b/SPAG11E. Journal of Biotechnology,2009,139:33-37 被引 3    
11.  Diao H. Intein-mediated expression is an effective approach in the study of beta-defensins. Biochemical and Biophysical Research Communications,2007,357:840-846 被引 9    
12.  Sreekrishna K. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene,1997,190:55-62 被引 111    
13.  Cereghino J L. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews,2000,24:45-66 被引 311    
14.  Li Z. Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris. Protein Expression and Purification,2009,67:175-181 被引 5    
15.  Koliaraki V. Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris. Biochimie,2008,90:726-735 被引 5    
16.  Huang W. Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper. Molecular and Cellular Endocrinology,2009,307:224-236 被引 11    
17.  Peng J X. Evolutionary conservation of Dazl genomic organization and its continuous and dynamic distribution throughout germline development in gynogenetic gibel carp. Journal of Experimental Zoology Part B,2009,312:855-871 被引 19    
18.  Wu N. Histone H2A has a novel variant in fish oocytes. Biology of Reproduction,2009,81:275-283 被引 10    
19.  李辰昱. 赤点石斑鱼促甲状腺激素TSHβ启动子的克隆及其在斑马鱼胚胎原始性腺和垂体中定位表达. 水生生物学报,2010,34(4):822-827 被引 4    
20.  刘沙. 银鲫dmrt2b基因的分子特征及功能分析. 水生生物学报,2011,35(3):379-383 被引 3    
引证文献 8

1 史雨红 虹鳟LECT2的酵母表达、纯化及生物活性分析 动物学研究,2013,34(1):33-38
被引 0 次

2 王鹤 褶纹冠蚌防御素基因特征与表达 中国水产科学,2013,20(6):1188-1196
被引 1

显示所有8篇文献

论文科学数据集
PlumX Metrics
相关文献

 作者相关
 关键词相关
 参考文献相关

iAuthor 链接
周莉 0000-0002-3511-3449
版权所有 ©2008 中国科学院文献情报中心 制作维护:中国科学院文献情报中心
地址:北京中关村北四环西路33号 邮政编码:100190 联系电话:(010)82627496 E-mail:cscd@mail.las.ac.cn 京ICP备05002861号-4 | 京公网安备11010802043238号