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Mycoplasmal pneumonia of swine mycoplasma outer membrane protein extraction method applicable to 2-dimensional electrophoresis

A technology of Mycoplasma hyopneumoniae and extraction method, which is applied to the preparation method of peptides, chemical instruments and methods, depsipeptides, etc., can solve the difficulty in transferring hydrophobic proteins and macromolecular proteins into second-dimension electrophoresis, electrophoresis pattern repeatability and resolution problems such as poor efficiency and concealment, to achieve the effect of good grinding effect, improved efficiency and purity, and improved efficiency

Active Publication Date: 2014-12-17
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Application Information

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Problems solved by technology

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 other problems. These defects are often related to protein extraction methods. These defects limit the application of 2-DE technology in proteomics to a certain extent.

Method used

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  • Mycoplasmal pneumonia of swine mycoplasma outer membrane protein extraction method applicable to 2-dimensional electrophoresis
  • Mycoplasmal pneumonia of swine mycoplasma outer membrane protein extraction method applicable to 2-dimensional electrophoresis

Examples

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example 1

[0038] Example 1: A method for extracting Mycoplasma hyopneumoniae outer membrane protein suitable for two-dimensional electrophoresis, comprising the following steps:

[0039] (1) Cultivation and collection of bacteria. Resuscitate the freeze-dried Mycoplasma hyopneumoniae on the mycoplasma medium, pick a single colony and inoculate it in the mycoplasma medium, and culture it with shaking at 37°C; collect it on the mycoplasma medium The growing bacteria were centrifuged at 2000 rpm for 10 min to remove excess medium and washed three times with pre-cooled PBS (pH 7.4) buffer solution; The amount of bacteria loaded in the EP tube is 1 / 3 of the total volume of the EP tube (about 500 μl), remove the excess PBS buffer with a pipette gun, seal the tube opening with a parafilm, and store at -80°C for later use;

[0040] (2) Take frozen bacteria at -80°C, grind with liquid nitrogen, and accurately weigh 0.1 g of bacteria into a 1.5 ml centrifuge tube;

[0041] (3) Add an equal amo...

example 2

[0049] Example 2: The detection and verification method of Mycoplasma hyopneumoniae outer membrane protein, the outer membrane protein that example 1 obtains is detected, and specific method is as follows:

[0050] (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. The absorbance at 595 nm was measured with a UV spectrophotometer, and a standard curve was drawn. Take the protein sample to be tested, measure the absorbance value of OD595 at a wavelength of 595 nm, and calculate the protein content in the sample according to the standard curve.

[0051] (2) Sample hydration, use a pipette to draw the sample, add the sample linearly along the hy...

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Abstract

The invention relates to a mycoplasmal pneumonia of swine mycoplasma outer membrane protein extraction method applicable to 2-dimensional electrophoresis. The method comprises the steps of mycoplasma breeding and collection, liquid nitrogen grinding, protein extraction and splitting, defreezing, ultrasonic concussion, ammonium acetate ethanol solution treatment and the like and further comprises collecting protein precipitate, rinsing the protein precipitate through acetone, and performing vacuum freezing, sample splitting and then centrifugal treatment to obtain liquid supernatant. The mycoplasmal pneumonia of swine mycoplasma outer membrane protein extraction method applicable to the 2-dimensional electrophoresis can greatly improve the efficiency and the degree of purity of protein extraction, has the advantages of being simple in operation, labor-saving, time-saving, high in extracting efficiency, low in interference and the like and is particularly applicable to materials with protein extracting difficulty, low extracting efficiency and high impurity interference; meanwhile, obtained protein is easy to secondary-dissolve and few in impurities and interferents and fully meets the requirements of bidirectional electrophoresis on a first direction and a second direction; maps with high definition, good repeatability, uniform protein spot distribution and clear protein spots can be obtained and are significant to mycoplasmal pneumonia of swine proteome research and immunity protein and vaccine candidate protein screening research.

Description

technical field [0001] The invention relates to a method for extracting protein, in particular to a method for extracting outer membrane protein of Mycoplasma hyopneumoniae suitable for two-dimensional electrophoresis, and belongs to the field of biotechnology. Background technique [0002] Mycoplasma pneumonia of swine (MPS), also known as porcine endemic pneumonia, commonly known as swine asthma, is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae, the main clinical symptoms are cough and wheezing, the main pathological The change is characterized by meaty or shrimp-like consolidation in the apical lobe, heart lobe, middle lobe, and anterior edge of the diaphragmatic lobe. The disease is widely distributed all over the world. The diseased pigs have long-term poor growth and development, low feed conversion rate, and high mortality. If secondary infection can cause serious death, the resulting economic loss is very large, and it will seriously har...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/30C07K1/30
CPCC07K14/30
Inventor 李海利王克领郎利敏徐引弟朱文豪施巧婷游一张清娴张立宪郑万录许峰
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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