Method of extracting bovine mycoplasma outer membrane protein suitable for proteomics
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A proteomics, Mycoplasma bovis technology, applied in the extraction of outer membrane protein and the extraction of outer membrane protein of Mycoplasma bovis, can solve the problems of difficult transfer to second dimension electrophoresis, application limitations, etc. The effect of improving extraction efficiency and quality
Inactive Publication Date: 2015-04-01
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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For example, for the separation of extremely acidic, extremely alkaline, too large and too small, insoluble proteins, and poor repeatability and resolution of electrophoretic patterns, it is difficult to transfer hydrophobic proteins and macromolecular proteins to second-dimensional electrophoresis, trace regulatory proteins It is often covered by high content of structural proteins, and these defects are often related to protein extraction methods, which to some extent limit the application of 2-DE technology in proteomics
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example 1
[0030] Example 1: a method for extracting Mycoplasma bovis outer membrane protein suitable for proteomics, comprising the following steps:
[0031] (1) Cultivation and collection of bacteria, collect the bacteria grown on the mycoplasma medium, centrifuge at 2000 rpm for 10 min, remove the excess medium, wash with pre-cooled PBS (pH 7.0) buffer solution for 3 times; after cleaning , transfer the bacterial pellet to a 1.5 ml centrifuge tube (EP tube), the amount of bacteria loaded in each EP tube is 1 / 3 of the total volume of the tube, about 500 μl, wash off the excess PBS buffer with a pipette gun Liquid, seal the mouth of the tube with a parafilm, and freeze the bacteria at -80°C for future use.
[0032] (2) Take frozen cells at -80°C, grind them with liquid nitrogen, and accurately weigh a certain amount (0.1 g) of cell powder into a 1.5 ml centrifuge tube.
[0033] (3) Add an equal amount (0.1ml) of protein extract solution to the centrifuge tube. The formula of protein extr...
example 2
[0039] Example 2: The detection and verification method of Mycoplasma bovis outer membrane protein, the outer membrane protein obtained in Example 1 is detected, the specific method is as follows:
[0040] (1) The detection of protein concentration adopts the Bradford method, with bovine serum albumin as the standard, bovine serum protein (BSA) is prepared into standard solutions of different concentrations with sterile deionized water, and added to the Coomassie brilliant blue G-250 solution, With Coomassie Brilliant Blue G-250 staining solution as the control, mix and stand for 10 min. Measure the absorbance of the solution at 595 nm with a UV spectrophotometer, and draw a standard curve. Take the protein sample to be tested, and use the OD595 absorbance value at a wavelength of 595nm to calculate the protein content in the sample according to the standard curve.
[0041] (2) Sample hydration, use a pipette to absorb the sample, add the sample linearly from left to right al...
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Abstract
The invention relates to a method of extracting a bovine mycoplasma outer membrane protein suitable for proteomics. The method comprises the following steps: culturing and collecting a strain, grinding by liquid nitrogen, carrying out constant-temperature ultrasonic concussion, centrifugally collecting protein precipitate, rinsing with ethanol and acetone, then carrying out centrifugal drying by virtue of vacuum freezing to obtain protein powder, and carrying out cracking treatment; and uniformly mixing the cracked solution, carrying out centrifugation, and maintaining supernatant to obtain the bovine mycoplasma outer membrane protein. According to the method disclosed by the invention, the protein extraction steps and used reagents are optimized, so that the extraction efficiency and purity degree of the protein are greatly improved, and impurities interfering a first dimension and a second dimension in dimensional electrophoresis are effectively removed; compared with the traditional method, the method is simple and convenient, is capable of saving labor and time, is high in protein extraction efficiency and less in disruptors and can be used for obtaining a map with high resolution, good repeatability, uniform protein spot distribution and clear protein spots, and has important significance for research on contagious bovine pleuropneumonia mycoplasma omics and screening research of immune proteins.
Description
technical field [0001] The invention relates to a method for extracting outer membrane protein, in particular to a method for extracting Mycoplasma bovis outer membrane protein suitable for proteomics, and belongs to the field of biotechnology. Background technique [0002] Bovine infectious pleuropneumonia, also known as bovine pneumonia, is a highly contagious pneumonia of cattle caused by mycoplasma filamentous. The disease is characterized by exudative inflammation of pulmonary interstitial lymphatic vessels, knot tissue, and alveolar tissue, as well as serous fibrinous pleuropneumonia. Susceptible animals to this disease are mainly dairy cows, beef cattle, cattle, buffaloes, yaks, etc. Various cattle have different susceptibility to this disease, depending on their breed, lifestyle and individual resistance. The incidence rate is 60-70%, and the fatality rate is 30-50%. Other animals and humans are not susceptible. The source of infection is mainly sick cattle and b...
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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/30C07K1/30
CPCC07K14/30
Inventor 李海利魏成斌张震徐引弟朗利敏张青娴候自花蔺萍游一施巧婷杨素新崔忠华
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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