摘要
目的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 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。
Objective To isolate and purify antler plate protein of Sika deer and to explore its bacteriostatic mechanism. Methods 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-100 HR 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 The total protein content after SephadexG-25 desalted was 90.3%. The protein component S2 with molecular weight <20.1 kDa obtained by SephacrylS-100 HR 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
作者
王懿璞
周怡君
胡薇
WANG Yipu;ZHOU Yijun;HU Wei(College of Life Sciences,Jilin Agricultural University,Changchun,Jilin 130118,China)
出处
《上海中医药杂志》
2021年第6期72-78,共7页
Shanghai Journal of Traditional Chinese Medicine
基金
吉林省发改委资助项目(2018C048-4)。
关键词
鹿角盘
蛋白
分离纯化
大肠杆菌
抑菌机制
antler plate
protein
isolation and purification
Escherichia coli
antimicrobial mechanism
