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A method for extracting the outer membrane protein of Mycoplasma hyopneumoniae suitable for two-dimensional electrophoresis

A technology of mycoplasma hyopneumoniae and its extraction method, which is applied to the preparation method of peptides, chemical instruments and methods, and decapeptides. Effect of denaturation and improvement of protein yield

Active Publication Date: 2017-01-04
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Claims
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Problems solved by technology

However, with the widespread use of a large number of antimicrobials, especially broad-spectrum antibacterials, in the pig industry, the problem of bacterial resistance to antimicrobials has become increasingly prominent. Metabolic process to change the virulence of bacteria or the adaptability of bacteria to the environment, etc.
[0005] In the 20th century, humans systematically studied the human genome and sequenced the entire genome. Most of the genetic codes have been deciphered, and the functions of some genes have not been clearly studied. The pathogenic mechanisms of some major diseases have not yet been fully resolved.
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.

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  • A method for extracting the outer membrane protein of Mycoplasma hyopneumoniae suitable for two-dimensional electrophoresis
  • A method for extracting the outer membrane protein of Mycoplasma hyopneumoniae suitable for two-dimensional electrophoresis

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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 ...

example 2

[0048] 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:

[0049] (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.

[0050] (2) Sample hydration, use a pipette to draw the sample, add the sample linearly along the...

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Abstract

The invention relates to a method for extracting the outer membrane protein of Mycoplasma hyopneumoniae suitable for two-dimensional electrophoresis, comprising the steps of cell culture and collection, liquid nitrogen grinding, protein extraction and cracking, thawing, ultrasonic vibration and ammonium acetate ethanol solution treatment, and then The protein precipitate was collected, rinsed with acetone, and vacuum-frozen; then the sample was lysed, and finally the supernatant was obtained after centrifugation. The invention can greatly improve the efficiency and purity of protein extraction, and has the characteristics of simple operation, labor-saving and time-saving, high extraction efficiency, and less interfering substances, and is more suitable for materials with difficult protein extraction, low extraction yield, and many interfering impurities; At the same time, the obtained protein is easy to be dissolved for the second time, with few impurities and interferences, and fully meets the requirements of the first and second dimensions of two-dimensional electrophoresis. It can obtain a map with high resolution, good repeatability, uniform distribution of protein spots, and clear protein spots. It is of great significance to study the omics of Mycoplasma hyopneumoniae, the screening of immune proteins and the screening of vaccine candidate proteins.

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 Patents(China)
IPC IPC(8): C07K14/30C07K1/30
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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