CN106167511A - A kind of extracting method of Mycoplasma bovis biofilm total protein - Google Patents
A kind of extracting method of Mycoplasma bovis biofilm total protein Download PDFInfo
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- CN106167511A CN106167511A CN201610629037.XA CN201610629037A CN106167511A CN 106167511 A CN106167511 A CN 106167511A CN 201610629037 A CN201610629037 A CN 201610629037A CN 106167511 A CN106167511 A CN 106167511A
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- biofilm
- mycoplasma bovis
- supernatant
- total protein
- thalline
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
Abstract
A kind of method extracting Mycoplasma bovis biofilm total protein overcoming prior art not enough of disclosure of the invention.The extracting method of the Mycoplasma bovis biofilm total protein of the present invention be initially with mechanically collect in the Mycoplasma bovis biofilm thalline that Mycoplasma bovis biofilm thalline the most just obtains add lysate carry out cracking process, supernatant is isolated again after ice-bath ultrasonic processes in the Mycoplasma bovis after processing through cracking, last adds TCA/ acetone soln in the supernatant that previous step obtains, and abandons supernatant and obtain total protein after low-temperature precipitation.Instant invention overcomes the separation of polysaccharide component that prior art exists and thalline not exclusively, the residual such as polysaccharide and DNA, total protein be degradable and the problem such as loss；Add the step removing the impurity (removing lipid material by TCA/ acetone soln) such as lipid material in Sample Preparation Procedure, decreasing the interference of non-protein material during making the gross protein sample of extraction isoelectric focusing electrophoresis behind, beneficially total protein efficiently separates during isoelectrofocusing and polyacrylamide gel electrophoresis.
Description
Technical field
The present invention relates to a kind of protein extracting method, actually the present invention relates to a kind of total egg of Mycoplasma bovis biofilm
White extracting method.
Background technology
Currently, the research of life sciences comes into the genome times afterwards comprehensively, and protein science has become the genome times afterwards comprehensively and ground
Study carefully forward position and the study hotspot of organism.Two-dimensional electrophoresis is the most classical proteomic techniques.By two-dimensional electrophoresis, by protein
Sample (including microbial cells, cell, tissue etc.) separates with molecular size range according to isoelectric point, IP, then carries out separation albumen
Mass Spectrometric Identification, data are analyzed by application bioinformatics technique, obtain the qualitative and quantitative of multiple proteins contained by sample
Information.In bidirectional electrophoresis technique, protein extraction prepare most important, protein extract preparation quality quality,
Directly affect two-dimensional electrophoresis effect and follow-up Mass Spectrometric Identification.There is no a general method of total protein extraction at present.In mycoplasma
In the research of protein science, due to specimen types and the difference of growth conditions, the extracting method of albumen is different, and effect also difference is very
Greatly.High-quality mycoplasma protein extracting method, becomes the committed step in mycoplasma two-dimensional electrophoresis.Biofilm
(biofilm, BF) is that microorganism secretion goes out multiple polysaccharide protein complex by the microorganism parcel wherein microcolony formed
(Sauer K. The genomics and proteomics of biofilm formation. Genome Biol.
2003,4 (6): 219.).Mycoplasma under biofilm state has knot diverse with the mycoplasma under floating state
The features such as structure, growth and metabolism.It has now been found that, many mycoplasma species include that Mycoplasma bovis can form biofilm, different
Between bacterial strain formed biofilm ability exist larger difference (McAuliffe L, Ellis RJ, Miles K, Ayling RD,
Nicholas RA. Biofilm formation by mycoplasma species and its role in
environmental persistence and survival. Microbiology. 2006, 152(Pt 4):913-
922.).The mycoplasma thalline being under biofilm state wraps up due to polysaccharide protein complex, additionally, bacterium solution contains lipid
Deng metabolite, these factors can affect the effect of two-dimensional electrophoresis, and then affects follow-up Mass Spectrometric Identification.
Research currently, with respect to mycoplasma biofilm protein science is seldom reported.On the one hand it is owing to mycoplasma is formed
The more general antibacterial of biofilm content for lack a lot, cultivate with extract increasingly difficult；On the other hand due to mycoplasma without
There is very big difference in cell wall structure, biofilm phage structure and planktonic cells thalline, the GL-PP of parcel thalline is combined
Thing can affect the extraction effect of total protein, so that it is extremely difficult to obtain high-quality biofilm total protein.Biological in mycoplasma
During the extraction of tunicle total protein, not only need to affect the lipid etc. of thallus DNA, secretion the interference material of two-dimensional electrophoresis
Remove, in addition it is also necessary to the materials such as the GL-PP being wrapped in outside biofilm thalline are removed, additionally needs farthest to carry
The dissolubility of high sample, it is thus achieved that the maximum amount of total protein, reduces the loss of total protein.Also the most effectively for cattle in prior art
The extracting method of mycoplasma biofilm.
Therefore, in order to be better understood by the differential expression of Mycoplasma bovis biofilm and planktonic cells albumen, research biology
The Forming Mechanism of tunicle, resistance mechanism and immunologic mechanism, need a kind of total egg of mycoplasma biofilm simple, practical and efficient
White extracting method.
Summary of the invention
The present invention provide a kind of overcome prior art not enough, can simply, practicality and extract cattle efficiently and prop up former
The method of body biofilm total protein.
The extracting method of the Mycoplasma bovis biofilm total protein of the present invention is to prop up initially with mechanically collecting cattle
The Mycoplasma bovis biofilm thalline that substance biofilm thalline the most just obtains adds lysate and carries out cracking process, then
By through cracking process after Mycoplasma bovis in ice-bath ultrasonic process after isolate supernatant, last obtains in previous step
Supernatant in add TCA/ acetone soln, after low-temperature precipitation, abandon supernatant obtain total protein.
Currently preferred extracting method is:
A. mechanical means collects Mycoplasma bovis biofilm thalline is to remove supernatant from Mycoplasma bovis biofilm thalline, then
With 0.01M pH 7.2 PBS, then scrape the biofilm adhering to culture dish with cell scraper, centrifugal supernatant discarded
After again with 0.01M pH 7.2 PBS thalline；
B. method for cracking treatment be added lysate be the SDS of 4% by mass ratio, the Tris-HCL of 100 mM, 100
MM DTT forms；
C. the thalline vortex oscillation after cracking processes is resuspended, then carries out boiling water bath, uses ice after vortex oscillation Mechanical Crushing
Bath ultrasonication processes, and is then again separate out supernatant；
E. obtain supernatant enters 10%TCA/ acetone soln by the previous step of 10 times of volumes ,-20 DEG C of precipitation 12 h, 12,
000 g is centrifuged 10 min and abandons supernatant.Adding pre-cold acetone and clean precipitation 2 times, 12,000 g are centrifuged 10 min and abandon supernatant, room temperature
Air-dry precipitation.
The extracting method of the Mycoplasma bovis biofilm total protein of the present invention is to utilize cell scraper to scrape, and many times of PBS is clear
Wash and operate with high speed centrifugation, can effectively remove the impurity pollution to total protein such as medium component, bacterium solution top layer lipid；Utilize
Lysate cracking, low temperature ultrasonic crush, and in combination with Mechanical Crushing, overcome polysaccharide component that prior art exists and thalline
Separate not exclusively, the residual such as polysaccharide and DNA, total protein be degradable and the problem such as loss；Add in removing Sample Preparation Procedure
The step of the impurity such as lipid material (removing lipid material by TCA/ acetone soln), makes the gross protein sample of extraction behind
Isoelectric focusing electrophoresis during decrease the interference of non-protein material, beneficially total protein is at isoelectrofocusing and polypropylene
Acrylamide gel electrophoresis process efficiently separates.By follow-up two-dimensional electrophoresis Detection and Extraction effect, result shows, with prior art phase
Ratio, the inventive method can obtain greater protein point, protein site up to 1200, and prior art is 1000 ~ 1100 protein sites；
The image that the inventive method obtains is apparent, assembles more abundant, and hangover and disperse are less.
The extraction that method is mycoplasma biofilm total protein that the present invention provides provides good reference, can extensively answer
Extraction for mycoplasma biofilm total protein, it is simple to the research of mycoplasma biofilm protein science.
Accompanying drawing explanation
Fig. 1 adopts the two-dimensional electrophoresis figure of the Mycoplasma bovis biofilm total protein being obtained by the present invention.
Detailed description of the invention
Below in conjunction with the extracting method that embodiment is the Mycoplasma bovis biofilm total protein explaining orally the present invention in detail, implement
Involved by test apparatus it is:
First to isoelectrofocusing instrument Ettan IPGphor Isoelectric Focusing System, second to perpendicular plate electricity
Swimming instrument Hofer SE 600, power supply EPS-601 are GE Amersham product；Pure water device is Millpore Products；Figure
As scanner Typhoon FLA9000 is GE Products.IPG immobilized ph gradient strip (13cm, pH3-10), IPG Buffer(pH3-
10), CHAPS, dithiothreitol, DTT (DTT), iodoacetamide are purchased from GE healthcare company of the U.S.；SDS、Glycine、
Tris, Acrymide, BIS, APS, Urea, TEMED are BIO-RAD reagent；Protein quantification test kit is that Beijing thunder root is biological
Technology Co., Ltd.'s product；Dehydrated alcohol, acetone, trichloroacetic acid are domestic analytical pure；Other reagent are GE Products；
In two-dimensional electrophoresis, water used is ultra-pure water.
Concrete operation step is as follows:
(1) cultivation of Mycoplasma bovis biofilm: will cultivate the Mycoplasma bovis planktonic cells of 20 h, inoculates 30 with 1:10 ratio
ml
MTB culture medium (21 g/L PPLO broth, 1 g/L glucose, 2 g/L sodium pyruvate, 100
mL/L 25% yeast extract, 20% horse serum, 0.4% phenol red 5 mL/L, 100 units of
Penicillin, and 0.01% acetic acid thallium, pH 7.4), it is placed on the training of a diameter of 150 mm
Support in ware, 30 ml/ wares, 37 DEG C of constant temperature culture 72 h.
(2) collection to Mycoplasma bovis biofilm: remove the culture fluid in culture dish, clear with 0.01M pH 7.2 PBS
Wash, each 40 ml, after cleaning 3 times, scrape the biofilm adhering to culture dish with cell scraper, with 0.01M pH 7.2
PBS resuspended about 5 × 108Individual cell, 4 DEG C 12,000 g is centrifuged 30 min, abandons supernatant, with 0.01M pH 7.2 PBS
Thalline 3 times.
(3) thalline cleaned adds 1 ml lysate (4% SDS, 100 mM Tris-HCL, 100 mM DTT), whirlpool
Rotation is vibrated resuspended thalline, boiling water bath 5 min, Mechanical Crushing 3 times (MP sample preparation instrument, 6 m/s, 60 s/time), ice-bath ultrasonic
Broken 10 times (power 80W, ultrasonic 10s, intermittently 15s), boiling water bath 5 min, 4 DEG C 12,000 g takes supernatant after being centrifuged 30 min
To new centrifuge tube.
(4) adding 10%TCA/ acetone soln with 10 times of albumen supernatant volume in new centrifuge tube, every 100 μ L of supernatant add
1 ml 10%TCA/ acetone soln ,-20 DEG C of precipitation 12 h, 4 DEG C 12,000 g abandons supernatant after being centrifuged 10 min.The most effective 1 ml
Pre-cold acetone cleans albumen 2 times, and 4 DEG C 12,000 g is centrifuged 10 min, and room temperature air-dries 5 min, collect be dried total protein,
Obtain described total protein.
(5) be dried albumen in add 800 μ L dissolve buffer (8M urea, 4% CHAPS, 40 mM Tris, 65
MM DTT, cocktail) 30 min, vibrate once every 10 min, 4 DEG C 12,000 g is centrifuged 30 min, takes supernatant.With
Total protein in supernatant is carried out quantitatively by protein quantification test kit, about obtains 800 μ g mycoplasma total proteins.
The biofilm total protein extracted in previous embodiment is carried out two-dimensional electrophoresis test, and step is as follows with result:
By embodiment extract total protein 200 μ g and Chong Pao rise liquid (8 M Urea, 2% CHAPS, 18 mM DTT, 0.5%
IPG Buffer, Bromophenol blue trace) it is sufficiently mixed, heavily bubble rises 12 hours, and cumulative volume is 250 μ L.Add
Holding glue groove, take out adhesive tape, use 13 cm, pH3-10 IPG immobilized ph gradient strip, glue surface is put into from one end down and is held glue groove, makes heavily to steep to rise
Immersion moistens whole adhesive tape, covers with mineral oil, every adhesive tape about 3 ml.First is carried out to isoelectrofocusing, deposition condition after loading
For: 30V 12h, 500V 1h, 1000V 1h, 8000V 8h, 500V 4h.The balance of adhesive tape is carried out: by adhesive tape after isoelectrofocusing
After taking-up, first put into (50 mM Tris-HCl pH 8.8,6 M urea, 2% SDS, 30% in level pad I
Glycerol, 1% DTT), it is placed on shaking table after balancing 15 minutes, then proceeds to (50 mM Tris-in level pad II
HCl pH 8.8,6 M urea, 2% SDS, 30% glycerol, 4% iodoacetamide), it is placed on shaking table balance
15 minutes.After two steps balances, carrying out second to SDS-PAGE, concentration is 12.5% gel, fills to from glass plate top with gel
Living with water seal after holding at 0.5 cm, polymerization about needs 1 hour.Suck water after polymerization, with electrophoresis wash buffer 2 times, blot.Then
Seal with the agarose of 0.5%；Finally carry out SDS-PAGE electrophoresis, first by 15 mA/ gel electrophoresis 30 min, afterwards by 30 mA/ glue
To bromophenol blue under glue along away from 0.5 cm.After electrophoresis terminates, silver nitrate dye liquor dyes, and uses scanner Typhoon after decolouring
Glue figure after electrophoresis is scanned by TMFLA 9500, is analyzed image with Image-master software.
The Mycoplasma bovis biofilm total protein using the present invention to extract is clear at two-dimensional electrophoresis glue figure epigraph, and background is done
Only, hangover and diffusing phenomenon are less, the protein site obtained up to 1200, and protein site is circular or oval, and protein site focuses on
Fully, can be used for protein science research.And prior art obtains protein site and is only 1000 ~ 1100.The inventive method obtains
Compared with prior art, image is apparent for image, and protein site is assembled more abundant, and hangover and disperse are less.
Claims (2)
1. an extracting method for Mycoplasma bovis biofilm total protein, mechanically collects Mycoplasma bovis biofilm bacterium
Body, it is characterised in that extracting method is further comprising the steps of:
(1) cracking process is carried out by the upper Mycoplasma bovis biofilm thalline mechanically obtained adds lysate；
(2) by through cracking process after Mycoplasma bovis in ice-bath ultrasonic process after isolate supernatant；
(3) add TCA/ acetone soln in the supernatant that previous step obtains, after low-temperature precipitation, abandon supernatant obtain total egg
In vain.
The extracting method of Mycoplasma bovis tunicle total protein the most according to claim 1, it is characterised in that described:
A. mechanical means collects Mycoplasma bovis biofilm thalline is to remove supernatant from Mycoplasma bovis biofilm thalline, then
With 0.01M pH 7.2 PBS, then scrape the biofilm adhering to culture dish with cell scraper, centrifugal supernatant discarded
After again with 0.01M pH 7.2 PBS thalline；
B. method for cracking treatment be added lysate be the SDS of 4% by mass ratio, the Tris-HCL of 100 mM, 100
MM DTT forms；
C. the thalline vortex oscillation after cracking processes is resuspended, then carries out boiling water bath, uses ice after vortex oscillation Mechanical Crushing
Bath ultrasonication processes, and is then again separate out supernatant；
D. obtain supernatant enters 10%TCA/ acetone soln by the previous step of 10 times of volumes ,-20 DEG C of precipitation 12 h, 12,
000 g is centrifuged 10 min and abandons supernatant, adds pre-cold acetone and cleans precipitation 2 times, and 12,000 g are centrifuged 10 min and abandon supernatant, room temperature
Air-dry precipitation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690319A (en) * | 2011-12-08 | 2012-09-26 | 河南科技大学 | Extraction method of porcine streptococcus total protein in biofilm state |
| CN103319566A (en) * | 2013-06-26 | 2013-09-25 | 昆明理工大学 | Method for extracting fungus total protein |
| CN104478999A (en) * | 2014-11-28 | 2015-04-01 | 河南省农业科学院畜牧兽医研究所 | Method of extracting bovine mycoplasma outer membrane protein suitable for proteomics |
| CN104697831A (en) * | 2015-02-16 | 2015-06-10 | 同济大学 | Extraction method of biological membrane proteins and preparation method of electrophoresis sample |
-
2016
- 2016-08-04 CN CN201610629037.XA patent/CN106167511A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690319A (en) * | 2011-12-08 | 2012-09-26 | 河南科技大学 | Extraction method of porcine streptococcus total protein in biofilm state |
| CN103319566A (en) * | 2013-06-26 | 2013-09-25 | 昆明理工大学 | Method for extracting fungus total protein |
| CN104478999A (en) * | 2014-11-28 | 2015-04-01 | 河南省农业科学院畜牧兽医研究所 | Method of extracting bovine mycoplasma outer membrane protein suitable for proteomics |
| CN104697831A (en) * | 2015-02-16 | 2015-06-10 | 同济大学 | Extraction method of biological membrane proteins and preparation method of electrophoresis sample |
Non-Patent Citations (2)
| Title |
|---|
| LAURA MCAULIFFE: "Biofilm formation by mycoplasma species and its role in environmental persistence and survival", 《MICROBIOLOGY》 * |
| 陈维: "牛支原体蛋白质组学双向电泳技术的建立及初步分析", 《东北农业大学学报》 * |
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