泥蚶血红蛋白异源四聚体酶解多肽抗菌活性及其机理研究

王娟娟; 包永波; 王素芳; 林志华

doi:10.3969/j.issn.0253-4193.2015.12.011

泥蚶血红蛋白异源四聚体酶解多肽抗菌活性及其机理研究

doi: 10.3969/j.issn.0253-4193.2015.12.011

1.
宁波大学海洋学院, 浙江宁波 315211;浙江海洋高效健康养殖协同创新中心, 浙江宁波 315211
2.
浙江万里学院浙江省水产种质资源高效利用技术研究重点实验室, 浙江宁波 315100
3.
宁波大学海洋学院, 浙江宁波 315211;浙江万里学院浙江省水产种质资源高效利用技术研究重点实验室, 浙江宁波 315100

基金项目: 国家高技术研究发展计划项目(2012AA10A410);浙江省自然科学重点基金项目(LZ12C190001);浙江省重中之重学科开放基金项目(KF2014002);宁波市自然科学基金(2015A610258)。

计量
- 文章访问数: 1115
- HTML全文浏览量: 7
- PDF下载量: 945
- 被引次数: 0
出版历程
- 收稿日期: 2015-07-20
- 修回日期: 2015-09-28

Antibacterial activity and mechanisms of heterotetramer hemoglobin derived polypeptide from Tegillarca granosa

1.
School of Marine Sciences, Ningbo University, Ningbo 315211, China;Zhejiang Collaborative Innovation Center of Ocean Efficient Healthy Culture, Ningbo 315211
2.
Zhejiang Key Laboratory of Aquatic Germplasm Resources, Zhejiang Wanli University, Ningbo 315100, China
3.
School of Marine Sciences, Ningbo University, Ningbo 315211, China;Zhejiang Key Laboratory of Aquatic Germplasm Resources, Zhejiang Wanli University, Ningbo 315100, China

摘要

摘要: 采用凝胶层析技术从泥蚶血细胞裂解物中纯化得到两种血红蛋白Tg-HbⅡ(异源四聚体)和Tg-HbⅠ(同源二聚体),回收率分别为48.15%和24.91%。胰蛋白酶酶解Tg-HbⅡ,酶解液经反相C-18色谱柱分离纯化得7种血红蛋白源多肽F1~F7。通过琼脂扩散法检测酶解多肽抑菌活性,结果发现7种多肽对金黄色葡萄球菌、大肠杆菌、恶臭假单胞菌、枯草芽孢杆菌和坚强芽孢杆菌均无抑菌作用,但多肽F2~F7对副溶血弧菌、溶藻弧菌和哈维氏弧菌具有明显抗菌活性,且多肽F2~F7对副溶血弧菌、溶藻弧菌和哈维氏弧菌的MIC值在0.012~0.200 mg/mL范围内。荧光显微镜观察酶解多肽杀菌效果发现多肽杀菌迅速,5 min内弧菌细胞几乎全部死亡。利用透射电子显微镜观察正常的和经抗菌肽处理后的溶藻弧菌和哈维氏弧菌细胞的超微结构,结果显示,对照组正常细胞内部结构完整,而经抗菌肽处理过的细胞其细胞壁完整,但因缺乏细胞内容物支持导致局部区域内陷,细胞质膜明显收缩,细胞内物质外流形成许多空泡从而导致细胞死亡。研究结果表明血红蛋白源抗菌肽对泥蚶致病菌弧菌有明显的抗菌活性,其通过破坏细胞内部结构而起到杀菌作用,这些发现为血红蛋白抗菌机制的研究奠定了基础。
- 泥蚶 /
- 酶解多肽 /
- 反相高效液相色谱 /
- 抗菌活性 /
- 超微结构 /
- 抗菌机理
Abstract: Tg-HbⅡ(heterogeneous tetramer) and Tg-HbⅠ(homodimer) were purified by gel chromatography with a recovery 48.15% and 24.91%. Seven major peptides(F1-F7) were purified by reverse phase C-18 column from trysin enzymatic hydrolysates of Tg-HbⅡ. The agar diffusion method was used to detect their antibacterial activity. Results showed that all peptide collections exhibited no antibacterial activity to Staphylococcus aureus,Pseudomonas putida,Eschetichia coli,Bacillus firmus and B. subtilis; but peptides F2-F7 showed antibacterial activity against Vibrio alginolyticus,V. harveyi and V. parahaemolyticus. MICs of peptide F2-F7 toward V. alginolyticus,V. parahaemolyticus and V. harveyi were 0.012-0.200 mg/mL Using fluorescence microscopy to observe the cell that treated with peptide,found that vibrio cells die rapidly in 5 min. Using Transmission electron microscopy to observe V. alginolyticus and V. harveryi after antibacterial peptides treatment,the results showed that the peptides changed permeability of cytoplasmic membrane,leading to leaking out of cytoplasmic contents. Finally,the affected Vibrio died due to leakage of the cell contents. Research results showed that the Tegillarca granosa hemoglobin source of antimicrobial peptides have obvious antibacterial activity against vibrio and antimicrobial peptides kill bacteria by damaging its internal structure,these findings laid a solid foundation to the research of antibacterial mechanism of hemoglobin.
- Tegillarca granosa /
- enzymolysis peptides /
- RP-HPLC /
- antibacterial activity /
- ultrastructure /
- antibacterial mechanism

HTML全文

参考文献(21)

顾莉娟,吴健伟,苏晓庆,等. 抗菌肽的研究进展[J]. 中国生化药物杂志,2006,27(6):383-386. Gu Lijuan,Wu Jianwei,Su Xiaoqing,et al. Progress on antimicrobial peptides[J]. Chinese Journal of Biochemical Pharmaceutics,2006,27(6):383-386.

Boman H G. Peptide antibiotics and their role in innate immunity[J]. Annu Rev Immunol,1995,13:61-92.

蒋菁莉,任发政,蔡华伟. 牛乳酪蛋白降血压肽的超滤分离[J]. 食品科学,2006,27(7):124-128. Jiang Jingli,Ren Fazheng,Cai Huawei. Separation of antihypertensive peptides derived from Yak Milk casein with ultra-filtration technology[J]. Food Science,2006,27(7):124-128.

Adje E Y,Balti R,Kouach M,et al. Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin[J]. International Journal of Biological Macromolecules,2011,49(2):143-153.

赵玲,曹荣,刘淇,等. 南极磷虾酶解多肽的抑菌活性[J]. 渔业科学进展,2011,32(4):112-116. Zhao Ling,Cao Rong,Liu Qi,et al. Antimicrobial activity of polypeptides from Antarctic krill[J]. Progress in Fishery Sciences,2011,32(4):112-116.

王军,庞广昌. 抗菌肽抗菌机理的研究现状及趋势[J]. 食品科学,2005,26(8):526-529. Wang Jun,Pang Guangchang. Review on the current status and development trend of mechanism of antimicrobial peptides[J]. Food Science,2005,26(8):526-529.

汪青. 泥蚶血红蛋白克隆、表达与免疫相关性研究[D]. 上海:上海海洋大学,2012. Wang Qing. Molecular cloning,expression and immune-related research on the hemoglobin from blood clam Tegillarca granosa[D]. Shanghai:Shanghai Ocean University,2012.

王素芳,包永波,施淼江,等. 泥蚶血红蛋白的制备及其抗菌活性研究[J]. 海洋学报,2014,36(12):67-73. Wang Sufang,Bao Yongbo,Shi Miaojiang,et al. Purification and antibacterial activity of hemoglobin from Tegillarca granosa[J]. Haiyang Xuebao,2014,36(12):67-73.

Xu Bin,Zhao Jun,Jing Zhao,et al. Role of hemoglobin from blood clam Scapharca kagoshimensis beyond oxygen transport[J]. Fish & Shellfish Immunology,2015,44(1):248-256.

荆昭. 毛蚶(Scapharca kagoshimensis)血红蛋白的类酚氧化酶性质及其抗菌活性研究[D]. 青岛:中国海洋大学,2011. Jing Zhao. Study on phenoloxidase-like properties and antibacterial activity of hemoglobin from blood clam Scapharca kagoshimensis[D]. Qingdao:Ocean University of China,2011.

Bao Yongbo,Wang Qing,Lin Zhihua. Hemoglobin of the bloody clam Tegillarca granosa(Tg-HbI) is involved in the immune response against bacterial infection[J]. Fish & Shellfish Immunology,2011,31(4):517-523.

朱晓囡,苏志国. 反相液相色谱在蛋白质及多肽分离分析中的应用[J]. 分析化学,2004,32(2):248-254. Zhu Xiaonan,Su Zhiguo. Application of reversed-phase liquid chromatography in separation and analysis of proteins and peptides[J]. Chinese Journal of Analytical Chemistry,2004,32(2):248-254.

杨化新,张培培,徐康森. 重组人生长激素胰肽图谱分析[J]. 药物分析杂志,1994,14(3):10-13. Yang Huaxin,Zhang Peipei,Xu Kangsen. HPLC analysis of tryptic mapping of recombinant human growth hormone[J]. Chinese Journal of Pharmaceutical Analysis,1994,14(3):10-12.

Boman H G, Steiner H,Hultmark D,et al. Sequence and specificity of two antibacterial proteins involved in insect immunity[J]. Nature,1981,292(5820):246-248.

肖建光,刘文生. 水产动物抗菌肽的研究进展[J]. 水利渔业,2005,25(1):4-5. Xiao Jianguang,Liu Wensheng. Advances in immune peptide studies on aquaculture animals[J]. Reservoir Fisheries,2005,25(1):4-5.

Daoud R,Dubois V,Bors-Dodita L,et al. New antibacterial peptide derived from bovine hemoglobin[J]. Peptides,2005,26(5):713-719.

Lee S Y,Lee B L,Söderhäll K. Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus[J]. Biological Chemistry,2003,278(10):7927-7933.

陈彤彤,宋晓妍,解树涛,等. 植物源抗菌肽的研究进展[J]. 西北植物学报,2006,26(2):420-426. Chen Tongtong,Song Xiaoyan,Xie Shutao,et al. Research advances about anti-microbial peptides of plant origins[J]. Acta Botanica Boreali-Occidentalia Sinica,2006,26(2):420-426.

Viljanen P,Koski P,Vaara M. Effect of small cationic leukocyte peptides(defensins) on the permeability barrier of the outer membrane[J]. Infect Immunity,1988,56(9):2324-2329.

Uyterhoeven E T,Butler C H,Ko D,et al. Investigating the nucleic acid interactions and antimicrobial mechanism of buforin II[J]. Febs Letters,2008,582(12):1715-1718.

翟培,韩晋辉,候丽霞,等. 家蝇抗菌肽的抑菌动力学研究及其机理初探[J]. 中国生物工程杂志,2006,26(11):33-39. Zhai Pei,Han Jinhui,Hou Lixia,et al. Investigate antibiosis dynamics and antibacterial mechanisms of antibacterial peptides from Musca domestica Larvae[J]. China Biotechnology,2006,26(11):33-39.

施引文献

资源附件(0)

访问统计