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日本鳗鲡脾脏抗菌肽抗菌活性检测方法的比较

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  • 1. 集美大学水产学院, 厦门 361021;
    2. 集美大学鳗鲡现代产业技术教育部工程研究中心, 厦门 361021
梁 英(1979- ),女,福建莆田人,讲师,博士,主要从事水产动物病害防治及抗菌肽资源开发研究。E-mail:liangyingnanping@jmu.edu.cn

收稿日期: 2014-05-13

  网络出版日期: 2014-11-17

基金资助

福建省中青年教师教育科研项目(科技B类)(FB2013003);集美大学李尚大学科建设基金(ZC2013004);鳗鲡现代产业技术教育部工程研究中心开放基金(ZK2013002);集美大学科研基金(ZQ2012010)

Comparative Study on Test Methods for Antibacterial Activities of Antibacterial Peptides from Spleen of Japanese Eel (Anguilla japonica)

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  • 1. Fisheries College, Jimei University, Xiamen 361021, China;
    2. Engineering Research Centre of Eel Modern Technical Industry, Ministry of Education, Jimei University, Xiamen 361021, China

Received date: 2014-05-13

  Online published: 2014-11-17

摘要

本试验旨在建立一种在日本鳗鲡脾脏抗菌肽分离纯化过程中简便、灵敏的检测其抗菌活性的方法。试验以日本鳗鲡脾脏分子质量小于10 ku的蛋白质为抗菌肽样品,选择琼脂板孔穴扩散法和微孔液体培养法2种传统方法及微量液体培养法为检测方法,比较这3种方法在抗菌肽对3种鳗鲡常见致病菌株(迟钝爱德华菌、气单胞菌、嗜水气单胞菌)抗菌活性检测中的灵敏度和优缺点。结果表明:与琼脂板孔穴扩散法及微孔液体培养法相比,微量液体培养法简便、易于观察结果,具有最高的灵敏度且样品用量最少,适合在鳗鲡脾脏抗菌肽分离纯化过程中跟踪检测抗菌活性,尤其适合分离纯化后期获得微量单一样品抗菌活性的检测。由此得出,微量液体培养法适用于检测日本鳗鲡脾脏抗菌肽的抗菌活性。

本文引用格式

梁英, 黄文树, 关瑞章 . 日本鳗鲡脾脏抗菌肽抗菌活性检测方法的比较[J]. 动物营养学报, 2014 , 26(11) : 3461 -3468 . DOI: 10.3969/j.issn.1006-267x.2014.11.036

Abstract

The study was to establish a simple and sensitive test method for antibacterial activity analysis in the separation and purification of antibacterial peptides from the spleen of Japanese eel (Anguilla japonica). Experiment by using spleen protein of molecular mass<10 ku from Japanese eel as antibacterial peptide samples, to compare the sensitivity, advantages and disadvantages of three test methods. In these three test methods, two conventional methods were agar plate fovea diffusion method and microporous liquid culture method and another was trace liquid culture method, for testing antibacterial activities of antibacterial peptides against three common pathogenic bacterial strains of eel, including Edwardsiella tarda, Aeromonas sp. and Aeromonas hydrophila. The results showed that compared with the agar plate fovea diffusion method and microporous liquid culture method, the trace liquid culture method had the advantages of simple operation and easy to observe result. Furthermore, this method with the best sensitivity and using the least sample quantity could be suitable for testing antibacterial activities in the separation and purification of antibacterial peptides from the spleen of eel, especially for identifying antibacterial activity of the a few and single fraction obtained in late stage of separation and purification. This study suggests that the trace liquid culture method is suitable for testing antibacterial activities of antibacterial peptides from the spleen of Japanese eel.

参考文献

[1] LAUTH X,SHIKE H,BUMS J C,et al.Discovery and characterization of two isoforms of moronecidin,a novel antimicrobial peptides from hybrid striped bass[J].The Journal of Biological Chemistry,2002,277(7):5030-5039.  

[2] 梁英.抗菌肽的来源及应用[J].动物营养学报,2014,26(1):7-16.

[3] 黎观红,洪智敏,贾永杰,等.抗菌肽的抗菌作用及其机制[J].动物营养学报,2011,23(4):546-555.

[4] 岳昌武,莫宁萍,刘坤祥,等.抗菌肽的结构特点·作用机理及其应用前景[J].安徽农业科学,2008,36(5):1736-1739.

[5] 金莉莉,丁忠福,王秋雨.中国林蛙皮抗菌肽提取条件的优化研究[J].食品科学,2008,29(10):223-227.

[6] 赖仞,叶文娟,冉永禄,等.大蹼铃蟾皮肤分泌液中抗菌活性肽的分离纯化及其性质[J].动物学研究,1998,19(4):257-262.

[7] SIMMACO M,MIGNOGNA G,BARRA D.Antimicrobial peptides from amphibian skin:what do they tell us?[J]Biopolymers:Peptide Science,1998,47(6):435-450.

[8] SONNER A,KNOOP F C,PATEL M,et al.Antimicrobial properties of the frog skin peptide,ranatuerin-1 and its [Lys28] substituted analog[J].Peptides,2004,25(1):29-36.  

[9] 邓小娟,曹阳,钟仰进,等.SDS-PAGE凝胶原位检测抗菌蛋白的活性——生物自显影技术[J].西南农业大学学报:自然科学版,2005,27(1):136-137.

[10] 马瑜,孙燕,李治.两栖类皮肤抗菌肽的分离纯化与抗菌活性检测技术[J].药物生物技术,2010,17(1):83-86.

[11] 邬晓勇,何钢,颜军,等.抗菌肽抑菌活性测定方法的研究[J].生物学通报,2011,46(4):4-7.

[12] 齐珂珂,康相涛,李国喜,等.鸡白细胞中抗菌肽的分离纯化及活性鉴定方法的建立[J].西北农林科技大学学报:自然科学版,2008,36(9):27-39.

[13] 李秀兰,戴祝英,张双全.抗菌肽琼脂糖孔穴扩散法与比浊法测活比较及其相关性[J].南京师大学报:自然科学版,1998,21(2):81-83.

[14] ZHANG J J,YAN Q P,JI R X,et al.Isolation and characterization of a hepcidin peptide from the head kidney of large yellow croaker,Pseudosciaena crocea[J].Fish & Shellfish Immunology,2009,26(6):864-870.  

[15] FERNANDES J M O,MOLLE G,KEMP G D,et al.Isolation and characterization of oncorhyncin Ⅱ,a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout,Oncorhynchus mykiss[J].Developmental and Comparative Immunology,2004,28(2):127-138.  

[16] BIRKEMO G A,LVDERS T,ANDERSEN ,et al.Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)[J].Biochimica et Biophysica Acta,2003,1646(1/2):207-215.

[17] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1/2):248-254.

[18] ANDERSON R S,BEAVEN A E.Antibacterial activities of oyster (Crassostrea virginica) and mussel (Mytilus edulis and Geukensia demissa) plasma[J].Aquatic Living Resources,2001,14(6):343-349.  

[19] 陆婕,汪俊汉,钟雅,等.弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究[J].微生物学报,2006,46(3):406-411.

[20] 夏明明,徐上,李伟,等.高敏感薄层平板抗菌肽活性检测[J].农业生物技术学报,1995,3(2):19-24.

[21] 吴琦,杨美薷,钟德钰,等.琼脂糖弥散抗菌实验在检测腮腺唾液抗菌活性中的应用[J].华西口腔医学杂志,1995,13(3):201-206.

[22] 邬晓勇,何钢,颜军,等.蛙皮抗菌肽活性检测方法的筛选及其分离纯化[J].西南农业学报,2011,24(4):1564-1567.

[23] ISSACSON T,SOTO A,IWAMURO S,et al.Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica[J].Peptides,2002,23(3):419-425.  

[24] LIANG Y,GUAN R Z,HUANG W S,et al.Isolation and identification of a novel inducible antibacterial peptide from the skin mucus of Japanese eel,Anguilla japonica[J].The Protein Journal,2011,30(6):413-421.  

[25] HUANG W S,WANG K J,YANG M,et al.Purification and part characterization of a novel antibacterial protein scygonadin,isolated from the seminal plasma of mud crab,Scylla serrata (Forskl,1775)[J].Journal of Experimental Marine Biology and Ecology,2006,339(1):37-42.  

[26] 沈萍,陈向东.微生物学[M].北京:高等教育出版社,2006:130-135.
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