梅花鹿鹿角盘蛋白的分离纯化及其对大肠杆菌抑菌机制的初步研究

王懿璞; 周怡君; 胡薇

doi:10.16305/j.1007-1334.2021.2101042

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梅花鹿鹿角盘蛋白的分离纯化及其对大肠杆菌抑菌机制的初步研究

实验研究 | 更新时间：2022-08-03

- 梅花鹿鹿角盘蛋白的分离纯化及其对大肠杆菌抑菌机制的初步研究
- Isolation and purification of antler plate protein from Sika deer and preliminary study on its bacteriostatic mechanism against Escherichia coli
- 上海中医药杂志 2021年55卷第6期页码：72-78
- 作者机构：
  
  1.吉林农业大学生命科学学院（吉林　长春　130118）
- 作者简介：
  
  王懿璞，女，硕士研究生，主要从事生物化学与分子生物学方面的研究工作
  胡薇，教授，博士研究生导师；E-mail：huweilab@126.com
- 基金信息：
  
  吉林省发改委资助项目(2018C048-4)
- DOI：10.16305/j.1007-1334.2021.2101042
  中图分类号：
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王懿璞, 周怡君, 胡薇. 梅花鹿鹿角盘蛋白的分离纯化及其对大肠杆菌抑菌机制的初步研究[J]. 上海中医药杂志, 2021,55(6):72-78.

Yipu WANG, Yijun ZHOU, Wei HU. Isolation and purification of antler plate protein from Sika deer and preliminary study on its bacteriostatic mechanism against Escherichia coli[J]. Shanghai Journal of Traditional Chinese Medicine, 2021,55(6):72-78.
王懿璞, 周怡君, 胡薇. 梅花鹿鹿角盘蛋白的分离纯化及其对大肠杆菌抑菌机制的初步研究[J]. 上海中医药杂志, 2021,55(6):72-78. DOI： 10.16305/j.1007-1334.2021.2101042.

Yipu WANG, Yijun ZHOU, Wei HU. Isolation and purification of antler plate protein from Sika deer and preliminary study on its bacteriostatic mechanism against Escherichia coli[J]. Shanghai Journal of Traditional Chinese Medicine, 2021,55(6):72-78. DOI： 10.16305/j.1007-1334.2021.2101042.

摘要

目的,2,分离纯化梅花鹿鹿角盘蛋白并对其抑菌机制进行初步探究。,方法,2,以鹿角盘为原料，采用酸提醇沉提取鹿角盘总蛋白，SephacrylS-100HR和反向高效液相层析分离纯化鹿角盘抗菌蛋白，最终确定蛋白质分子质量。以大肠杆菌为研究对象，测定抗菌蛋白的最小抑菌浓度（MIC）；结合荧光显微镜、扫描电镜、流式细胞仪等方法从大肠杆菌细胞膜通透性、完整性和ROS活性氧的产生等方面探讨鹿角盘蛋白对大肠杆菌的抑制机制。,结果,2,SephadexG-25除盐后总蛋白含量为90.3％，经SephacrylS-100HR凝胶层析获得分子量<20.1 kDa的蛋白组分S2对大肠杆菌具有抑菌活性，将S2（<20.1 kDa）组分通过反相高效液相得到抗菌蛋白18.963 kDa。抗菌蛋白在浓度为5 mg/mL时对大肠杆菌具有最小抑菌能力；处理组在一定时间内电导率、β-半乳糖苷酶活性的显著差异表明细胞膜通透性受损；扫描电镜发现抗菌蛋白作用后菌体出现变形、塌陷、皱缩、破碎，荧光显微镜观察PI单染处理组荧光强度增强，说明抗菌蛋白破坏了大肠杆菌细胞膜、细胞壁的完整性；菌体经抗菌蛋白浓度为2倍最小抑菌浓度（2 MIC）处理2 h，流式细胞仪检测ROS表达量为69.3％，菌体氧化应激水平显著提高，从而对大肠杆菌起到一定的抑制作用。,结论,2,鹿角盘蛋白经过分离纯化后得到的抗菌蛋白可能通过改变大肠杆菌细胞膜通透性、细胞壁完整性和提高菌体氧化应激水平，从而起到抑菌的作用。

Abstract

Objective,2,To isolate and purify antler plate protein of Sika deer and to explore its bacteriostatic mechanism.,Methods,2,Using antler plate as raw material, the total protein of antler plate was extracted by acid extraction and alcohol precipitation. The antibacterial protein of antler plate was isolated and purified by SephacrylS-100HR and reverse high performance liquid chromatography, and finally the molecular weight of the protein was determined. Taking ,Escherichia coli, as the research object, the minimal inhibitory concentration (MIC) of antimicrobial protein was determined. Combined with fluorescence microscope, scanning electron microscope and flow cytometry, the inhibitory mechanism of antler plate protein on ,Escherichia coli, was explored from the aspects of ,Escherichia coli, cell membrane permeability, integrity and the production of ROS reactive oxygen species.,Results,2,The total protein content after SephadexG-25 desalted was 90.3%. The protein component S2 with molecular weight ,<,20.1 kDa obtained by SephacrylS-100HR gel chromatography had antibacterial activity against ,Escherichia coli, and antibacterial protein 18.963 kDa was obtained from S2 (,<,20.1 kDa) component by reversed-phase high performance liquid chromatography. The minimal inhibitory concentration of antibacterial protein to ,Escherichia coli, was 5 mg/mL. The significant differences in electrical conductivity and β-galactosidase activity within a certain period of time in the treatment group indicated that the cell membrane permeability was impaired. Under scanning electron microscope the bacteria were deformed, collapsed, shrank and shattered after the action of antibacterial protein. The fluorescence intensity of PI single staining group was enhanced by fluorescence microscope, indicating that antibacterial protein destroyed the integrity of ,Escherichia coli, cell membrane and cell wall. When the bacteria were treated with 2 times the minimum inhibitory concentration (2 MIC) for 2 hours, the expression of ROS was 69.3% by flow cytometry, and the level of oxidative stress increased significantly, which played a certain inhibitory effect on ,Escherichia coli,.,Conclusion,2,Antimicrobial proteins obtained after the isolation and purification of antler plate proteins may achieve antibacterial effect by changing the permeability of cell membrane, the integrity of cell wall and improving the level of oxidative stress of ,Escherichia coli,.

关键词

鹿角盘蛋白分离纯化大肠杆菌抑菌机制

Keywords

antler plateproteinisolation and purificationEscherichia coliantimicrobial mechanism

references

LIU Y, LI R, XIAO X, et al. Antibiotic adjuvants: an alternative approach to overcome multi-drug resistant Gram-negative bacteria[J]. Crit Rev Microbiol, 2019, 45(3): 301-314.

ZASLOFF M. Antimicrobial peptides of multicellular organisms[J]. Nature, 2002, 415(6870): 389-395.

D’COSTA V M, MCGRANN K M, HUGHES D W, et al. Sampling the antibiotic resistome[J]. Science, 2006, 311(5759): 374-377.

HANCOCK R E, SAHL H G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies[J]. Nat Biotechnol, 2006, 24(12): 1551-1557.

田玉华. 梅花鹿角脱盘蛋白多肽的分离纯化及活性研究[D]. 长春：吉林农业大学，2011.

国家药典委员会. 中华人民共和国药典（2015年版）：一部[M]. 北京：中国医药科技出版社，2015:321.

张旭霞，田玉华，齐琳，等. 梅花鹿鹿角脱盘化学成分的研究[J]. 吉林畜牧兽医，2013,34(8):11-16.

宋波洋，王聪，姚昶. 鹿角在急性乳腺炎中的应用总结[J]. 中医药导报，2019,25(24):91-93.

张程程. 鹿角盘提取物抗炎作用的研究[D]. 大连：大连理工大学, 2011.

陈思，郜玉钢，臧埔，等. 鹿角脱盘5种提取部位对H22荷瘤小鼠免疫功能的影响[J]. 上海中医药杂志，2017,51(8):87-92.

吴菲菲. 鹿角盘提取物的体外抗骨质疏松作用[D]. 大连：大连理工大学，2013.

牛放. 花鹿鹿角脱盘蛋白质成分药理活性研究[D]. 长春：吉林农业大学, 2011.

史小青，刘金哲，姚艳飞，等. 梅花鹿鹿花盘对小鼠抗疲劳作用的研究[J]. 吉林农业大学学报，2011,33(4):408-410.

牛放，赵雨，杨菲，等. 梅花鹿鹿角脱盘提取物抗疲劳作用研究[J]. 食品科技，2011,36(11):218-220.

徐格格. 鹿角提取物通过NF-κB通路抑制三阴型乳腺癌侵袭和迁移的机制研究[D]. 长春：吉林大学，2020.

唐智佳，胡薇. 梅花鹿鹿角盘小肽的提取及体外抑菌活性研究[J]. 黑龙江畜牧兽医，2018(7):17-19.

李月，陈丹阳，王懿璞，等. 梅花鹿鹿角盘胶原肽制备及其活性作用研究[J]. 上海中医药杂志，2020,54(7):80-85.

黄凤杰，吉静娴，钱璟，等. 鹿角脱盘多肽的分离纯化及其降糖活性的研究[J]. 药物生物技术，2010,17(2):151-156.

苏凤艳，李慧萍，王艳梅，等. 鹿花盘蛋白质的提取与生物活性测定[J]. 动物科学与动物医学，2001,18(2):18-20.

杨言慧. 鹿茸干细胞和鹿角盘水提取物生物活性的研究[D]. 长春：吉林大学，2020.

关晴，赵海平，王东旭，等. 鹿角盘水提物对大鼠乳腺增生的作用效果及其机制研究[J]. 中国畜牧兽医，2019,46(1):296-304.

王海珍. 鹿角粉联合止痛消炎软膏治疗产褥性急性乳腺炎临床研究[J]. 新中医，2019,51(2):173-175.

高青，高红. 鹿角霜治疗乳头皲裂38例分析[J]. 山西医药杂志，2004,33(11):989.

VIEIRA T M, DOS SANTOS I A, SILVA T S, et al. Antimicrobial activity of monoketone curcuminoids against cariogenic bacteria[J]. Chem Biodivers, 2018, 15(8): e1800216.

杨斯琴，敖日格乐，王纯洁，等. 3种蒙药复方对致病性Escherichia coli O1的抑菌机理及对小鼠的保护率[J]. 微生物学通报，2016,43(8):1766-1773.

DIAO W R, ZHANG L L, FENG S S, et al. Chemical composition, antibacterial activity, and mechanism of action of the essential oil from Amomum kravanh[J]. J Food Prot, 2014, 77(10): 1740-1746.

NINGANAGOUDA S, RATHOD V, SINGH D, et al. Growth kinetics and mechanistic action of reactive oxygen species released by silver nanoparticles from Aspergillus niger on Escherichia coli[J/OL]. Biomed Res Int, 2014[2021-01-02]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083831/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083831/.

KANG S, KONG F, SHI X, et al. Antibacterial activity and mechanism of lactobionic acid against Pseudomonas fluorescens and Methicillin-resistant Staphylococcus aureus and its application on whole milk[J/OL]. Food Control, 2020[2020-12-12]. https://www.sciencedirect.com/science/article/abs/pii/S0956713519304657https://www.sciencedirect.com/science/article/abs/pii/S0956713519304657.

DIAO W R, ZHANG L L, FENG S S, et al. Chemical composition, antibacterial activity, and mechanism of action of the essential oil from Amomum kravanh[J]. J Food Prot, 2014, 77(10): 1740-1746.

LIU G, REN G, ZHAO L, et al. Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes[J/OL]. Food Control, 2017[2020-12-12].https://www.sciencedirect.com/science/article/abs/pii/S096713516305357https://www.sciencedirect.com/science/article/abs/pii/S096713516305357.

BELENKY P, YE J D, PORTER C B, et al. Bactericidal antibiotics induce toxic metabolic perturbations that lead to cellular damage[J]. Cell Rep, 2015, 13(5): 968-980.

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