| Antler plate is the bone part left after the antler of sika deer or red deer was sawed off.It will fall off naturally when the antler grows out in the next spring.Because of its shape,it is called antler plate or deer tray.As early as in the Han Dynasty,one of the four major works of Chinese medicine,there is a record of antler plate in the Shennong Materia Medica Classic.Compared with other rare Chinese medicinal materials,antler plate is economical and easy to obtain.However,as a natural medicine raw material,antler plate has the advantages of mild action,non-toxicity and no side effects,but it has not received enough attention and more in-depth and extensive research.Therefore,the research on antler plate is conducive to the development of antler plate industry.Proteins,one of the main functional components of the antler plate,have also attracted extensive attention because they are the material basis of life and an important component of body cells and tissues.At present,the research on antler plate protein is mainly focused on the exploration of biological activity,but there are few reports on the antibacterial aspects of it,and the antibacterial mechanism is not clear Therefore,this project takes the protein as the entry point to search for the bioactive protein in the antler plate and conduct a preliminary study on its antimicrobial mechanism of action,aiming to provide data support for the development and utilization of the valuable animal medicine.The total protein of sika deer antler plate was obtained by cold extraction with aqueous solution and desalination with Sephadex G-25 gel,the antibacterial protein of the antler plate was isolated by Sephacryl S-100 HR and reverse high-performance liquid chromatography(RP-HPLC).The total protein and antimicrobial protein of deer antler plate were identified and analyzed by the following methods: SDS-PAGE electrophoresis,matrix assisted laser desorption ionization time of flight mass spectrometry,reverse high performance liquid phase,etc.Escherichia coli was selected as the experimental strain according to the work basis of the early experiment of antibacterial activity in the laboratory,Broth microdilution method was used to determine the bacteriostatic effect of antler plate protein,and the minimum bacteriostatic concentrations of total protein and antibacterial protein were determined.On this basis,the paper diffusion method is used to more intuitively observe the bacteriostatic degree,Combined with the fluorescence microscope,scanning electron microscope,flow cytometry and other methods to explore the inhibition mechanism of antler plate protein on Escherichia coli from the aspects of Escherichia coli integrity and Oxidative stress of bacteria produces toxic reactive oxygen species.The results showed that the total protein content of the antler plate after desalination was90.3%.The protein component S2 with a molecular weight of lt<20.1 k Da was obtained by Sephacryl S-100 HR gel chromatography.The antimicrobial protein of the S2 was obtained by reversed-phase HPLC,Its molecular weight was 18.963 k Da and its purity was 81.92%.At the concentration of 5 mg/m L,the antimicrobial protein had the minimum inhibitory ability against Escherichia coli.The significant differences in conductivity and β-galactosidase activity in the treatment group indicated that the membrane permeability was impaired.Scanning electron microscopy showed that the bacteria were deformed,collapsed,shrunk and broken after the antibacterial proteins were treated.Fluorescence microscopy showed that the fluorescence intensity of PI single staining group was enhanced,which indicated that the antibacterial proteins destroyed the integrity of the E.coli cell membrane.After 2MIC treatment,the ROS expression level detected by flow cytometry was 69.3%,which significantly increased the oxidative stress level of the bacteria and thus had a certain inhibitory effect on Escherichia coli. |
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