CN104502610A - Method for screening antibiotics binding proteins - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly discloses a method for screening antibiotics binding proteins. The method comprises the steps of stressing an organism by utilizing antibiotics to obtain a stress differential protein group of the organism, detecting the antibiotic content in a differential protein spot, and screening and identifying the antibiotics binding protein. The method is wide in application range, can be used for rapidly and accurately screening new antibiotics binding protein and has important significance on detection of the antibiotics and preventing drug-resistance bacteria.
Description
Technical field
The invention belongs to biological technical field, particularly, relate to a kind of protein-bonded method of qualification microbiotic.
Background technology
Microbiotic associated proteins may be used for antibiotic detection, simultaneously also for the research of microbiotic mechanism of action and Plasmid provides molecular target.The protein-bonded screening of current microbiotic mainly adopts classical isotope incorporation of markings method.This method is the organelle utilizing the microbiotic of cold labeling relevant to suppressed metabolic activity, and as ribosomes, cellular component, as cell wall extracts etc. is hatched, recycling radioautograph, the methods such as SDS-PAGE isolate microbiotic associated proteins.The advantage of this Research Thinking is " shooting the arrow at the target ", first studies the metabolic activity that microbiotic affects, then isolates the protein be combined with microbiotic in this metabolic activity, can reduce screening operation amount, save time and cost; Weak point is, after microbiotic enters into cell interior, also may affect the physiological activity of bacterium with other protein bound, if this protein is not within object organelle or cellular component, then be difficult to be found.Thus be difficult to find new microbiotic associated proteins, also directly affect the further investigation to microbiotic mechanism of action and Plasmid.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of screening microbiotic protein-bonded method.Described method is applied widely, can screen the microbiotic associated proteins made new advances quickly and accurately, has great importance to the control of antibiotic detection and drug-fast bacteria.
Above-mentioned purpose of the present invention is achieved by the following technical programs.
A kind of protein-bonded method of screening microbiotic, comprises the steps:
S1. use microbiotic stressed organisms, extract the whole cell protein of biosome and carry out electrophoretic separation analysis, obtain differential proteins group, comparison obtains differential protein spot;
S2. reclaim differential protein spot, carry out microbiotic detection respectively, according to content antibiotic in differential protein spot, screen and identify microbiotic associated proteins;
Wherein, microbiotic described in S2 detects and adopts indirect competitive, makes the microbiotic in differential protein spot and coupled antigen competition antibiotic antibody, using protein site corresponding during physiological saline process as negative control, and carries out light absorption value detection; Screen described light absorption value < 100%, and the protein site of significant difference p < 0.01 is microbiotic associated proteins;
Or microbiotic described in S2 detects and adopts direct measuring method, screening antibiotic content >=5 times negative control, and the protein site of significant difference p < 0.01 is microbiotic associated proteins; Corresponding protein site when described negative control is physiology saline treatment;
The minimum inhibition concentration of antibiotic concentration >=4 described in S1 times, the concentration of the described all biosomes of and antibiotic concentration≤kill.Described minimum inhibition concentration refers to hatch 24 hours in certain circumstances, and certain biosome can be suppressed to occur the lowest concentration of drug rised appreciably; When biosome is bacterium, minimum inhibition concentration is minimal inhibitory concentration (MIC), for quantitative measurement antibacterial activity in vitro.
The present invention finds that the microbiotic of high dose directly stress affect the protein-bonded abundance of biosome microbiotic, then based on the proteomic techniques of electrophoretic separation, protein electrophoresis collection of illustrative plates during employing physiological saline process biosome and differential proteins group are compared, found relevant difference albumen, the microbiotic detection method of binding specificity carries out screening and identification to microbiotic associated proteins then.The method of the invention is applicable to various microbiotic, and described biosome comprises microorganism, plant and animal cell, applied widely, can screen the microbiotic associated proteins made new advances quickly and accurately.
Preferably, electrophoretic separation described in S1 analyzes cell protein used is 50 μ g ~ 2 mg.
Preferably, antibiotic concentration described in S1 is the minimum inhibition concentration of 8 ~ 100 times.
Preferably, microbiotic described in S2 detects and adopts indirect competitive, screen described light absorption value 0 ~ 90%, and the protein site of significant difference p < 0.01 is microbiotic associated proteins.
Preferably, microbiotic described in S2 detects and adopts direct measuring method, and screening antibiotic content is the protein site of 5 ~ 100 times of negative controls is microbiotic associated proteins; Corresponding protein site when described negative control is physiology saline treatment.
The method of the invention can be suitable for all types of microbiotic, because when utilizing different types of microbiotic to carry out processing to biosome (comprising microorganism, plant and animal cell), all can obtain and process protein accordingly, carry out proteome analysis, find and identify that microbiotic stress differential protein.Adopt microbiotic detection method again, detecting wherein whether containing processing microbiotic used, identifying corresponding microbiotic associated proteins thus.Therefore, this principle can adapt to all types of microbiotic.
Preferably, microbiotic described in S1 is new fluoroquinolones, aminoglycoside antibiotics, beta-lactam antibiotic, macrolide antibiotics, amphenicols microbiotic, TCs, lincosamides, polypeptide antibiotics, antifungal antibiotic microbiotic, how Phosphorus microbiotic.
More preferably, microbiotic described in S1 is penicillin, new fluoroquinolones.
Biosome in the method for the invention is antibiotic effective object, because all types of biosome all can produce stress reaction to antibiotic effect, therefore comprise microorganism, all biosomes of plant and animal cell are all applicable to method of the present invention.
Preferably, biosome described in S1 is bacterium, and described minimum inhibition concentration refers to minimal inhibitory concentration.Bacterium is easy to cultivate and extract albumen, is that object is convenient to experimental study with bacterium.
Preferably, bacterium described in S1 is Escherichia coli and Wdwardsiella tarda.
Preferably, electrophoretic separation analysis described in S1 is Two-dimensional Electrophoresis Analysis.
Compared with prior art, beneficial effect of the present invention is: the present invention is applied widely, can screen the microbiotic associated proteins made new advances quickly and accurately, has great importance to the control of antibiotic detection and drug-fast bacteria.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 ampicillin stress the result of IEF/SDS-PAGE bidimensional electrophoretic separation of Edwardsiella tarda; A is control group, and B is microbiotic stress group.
Fig. 2 is that embodiment 1 ampicillin stress the IEF/SDS-PAGE profile variation protein site of Edwardsiella tarda.
Fig. 3 is embodiment 1 ampicillin content and percentage absorbance standard curve.
Fig. 4 is the percentage absorbance of embodiment 1 protein site ETAE_0175, ETAE_3367 and control group.
Fig. 5 is that embodiment 2 Balofloxacin stress the result of IEF/SDS-PAGE bidimensional electrophoretic separation of Escherichia coli K12 BW25113; A is control group, and B is microbiotic stress group.
Fig. 6 is that embodiment 2 Balofloxacin stress the IEF/SDS-PAGE profile variation protein site of Escherichia coli K12 BW25113.
Fig. 7 is embodiment 2 Balofloxacin content and Standardization curve for fluorescence intensity.
Fig. 8 is the Balofloxacin concentration of embodiment 2 protein site AceF.
Embodiment
Below in conjunction with Figure of description and specific embodiment, the present invention is described in further details, but embodiment does not limit in any form the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Present embodiment respectively for ampicillin stress Edwardsiella tarda, Balofloxacin stress Escherichia coli, but conduct is not to the present invention's antibiotic restriction used for described ampicillin, Balofloxacin, and described Edwardsiella tarda, Escherichia coli be not also as the restriction to used organism of the present invention.The inventive method is applicable to all kinds of microbiotic stress different kind organism body.
embodiment 1the protein-bonded screening of Edwardsiella tarda ampicillin and qualification
S1. IEF/SDS-PAGE analyze ampicillin stress Edwardsiella tarda differential proteomics:
The ampicillin being 20 times of minimal inhibitory concentrations by concentration stress Edwardsiella tarda EIB202, extract the whole cell protein of Edwardsiella tarda, and get 500 μ g and carry out IEF/SDS-PAGE Two-dimensional Electrophoresis Analysis, obtain ampicillin differential proteins group, experimental result is shown in Fig. 1.Protein electrophoresis collection of illustrative plates during employing physiological saline process Edwardsiella tarda and differential proteins group are compared, after analysis is compared to collection of illustrative plates, the protein site that finds differences altogether adds up to 59, comparing result is shown in Fig. 2, these differential proteins and ampicillin stress Edwardsiella tarda EIB202 relevant.
S2. the protein-bonded screening of Edwardsiella tarda ampicillin: reclaim above-mentioned 59 differential protein spots, carry out ampicillin detection respectively, according to ampicillin content in differential protein spot, filter out the Edwardsiella tarda ampicillin associated proteins of candidate.
Ampicillin content standard Drawing of Curve: use the ampicillin content of ampicillin quick detection kit to differential protein spot to detect.Kit principle is the method utilizing indirect competitive ELISA, coupled antigen is coated with in plate hole, after the ampicillin in sample liquid differential protein spot and ampicillin antibody join plate hole, ampicillin in sample will compete antibody with coupled antigen, and the light absorption value of sample becomes negative correlation with the content of ampicillin contained by it.Absorbance detection is carried out to the ampicillin standard items of 0.1,0.3,0.9,2.7,8.1 bbp, according to absorbance drawing standard curve, the results are shown in Figure 3.Along with the rising of ampicillin concentration, the percentage absorbance of sample also declines thereupon.This typical curve can be used for the qualitative analysis of ampicillin content.
The preparation of sample: add after enzymolysis liquid carries out enzymolysis to differential protein spot, add sample diluting liquid in kit, microbiotic was discharged from micelle in ultrasonic 2 minutes minutes.To sample liquid carry out centrifugal after, Aspirate supernatant be used for antibiotic content detect.
The ampicillin assay of differential protein spot: utilize the method for above-mentioned indirect competitive ELISA to measure 59 discrepancys, find that No. 1 discrepancy percentage light absorption value be 67.1%, No. 19 discrepancys is 78.7%, the results are shown in Figure 4, all be starkly lower than control value (100%), be defined as the ampicillin associated proteins of candidate thus.According to Mass Spectrometric Identification result, can determine that No. 1 discrepancy is protein ETAE_0175, ETAE_0175(RpoB) be the β subunit of RNA polymerase, the binding site containing ribonucleoside triphosphote, produces relevant to Initial R NA chain; No. 19 protein sites are ETAE_3367, ETAE_3367 is glutathione disulfide bond reduction enzyme (glutathione reductase), and its effect is by reducing substances important in body---glutathione, is reduction-state from oxidized state.
embodiment 2the protein-bonded screening of Escherichia coli Balofloxacin and qualification
S1. IEF/SDS-PAGE analyze Balofloxacin stress Escherichia coli differential proteomics:
Be that 50 times of minimal inhibitory concentration Balofloxacins stress Escherichia coli K12 BW25113 by concentration, extract the whole cell protein of Escherichia coli K12 BW25113, and get 1 mg and carry out IEF/SDS-PAGE Two-dimensional Electrophoresis Analysis, obtain its differential proteins group, experimental result is shown in Fig. 5.Protein electrophoresis collection of illustrative plates during employing physiological saline process Escherichia coli and differential proteins group are compared, after analysis is compared to collection of illustrative plates, the protein site that finds differences altogether adds up to 24, comparing result is shown in Fig. 6, the generation of these differential proteins all to Balofloxacin stress Escherichia coli K12 BW25113 relevant.
S2. the screening of Escherichia coli Balofloxacin conjugated protein: reclaim above-mentioned 24 differential protein spots, in the differential proteomics that these Balofloxacins stress produce after bacterial classification, some protein directly can be combined with Balofloxacin, these Balofloxacins be combined on protein can adopt Balofloxacin detection kit to detect its content, and further by the Balofloxacin comparision contents of differential protein spot, filter out Balofloxacin associated proteins.
Balofloxacin content standard Drawing of Curve: Balofloxacin belongs to carbostyril antibiotic, due to the special chemical structure that it has, can inspire fluorescence under given conditions, fluorospectrophotometer thus can be used to carry out quantitative test.First set up the typical curve between Balofloxacin concentration and fluorescence intensity, Balofloxacin solution adopts 1,2,4,8,16 and 32ng/3mL, and the fluorescence intensity according to measuring draws out typical curve, the results are shown in Figure 7.
The preparation of sample: add after enzymolysis liquid carries out enzymolysis to differential protein spot, add sample diluting liquid (0.2mol/L KH
2pO
4), microbiotic was discharged from micelle in ultrasonic 2 minutes.To sample liquid carry out centrifugal after, Aspirate supernatant be used for antibiotic content detect.
The Balofloxacin assay of differential protein spot: utilize above-mentioned same procedure to measure the fluorescence intensity of all differences protein site, the formula drawn by typical curve, counts out the Balofloxacin content of each differential protein spot., found that with the impact of cancellation protein by deducting protein site fluorescent value in control group, the Balofloxacin content of No. 8 discrepancys is 12.130ng/3mL, sees Fig. 8.Determine that No. 8 protein sites are Balofloxacin associated proteins thus.Determine that No. 8 differential protein spots are AceF albumen through Mass Spectrometric Identification; it is the E2 subunit of pyruvic dehydrogenase multienzyme complex; i.e. dihydro sulphur decoyl CAT, its effect is that catalysis hydroxyethyl-TPP produces acetyl dihydrolipoamide with lipoamide, then produces acetyl coenzyme A molecule by decomposing.
Claims (6)
1. screen the protein-bonded method of microbiotic, it is characterized in that, comprise the steps:
S1. use microbiotic stressed organisms, extract the whole cell protein of biosome and carry out electrophoretic separation analysis, obtain differential proteins group, comparison obtains differential protein spot;
S2. reclaim differential protein spot, carry out microbiotic detection respectively, according to content antibiotic in differential protein spot, screen and identify microbiotic associated proteins;
Wherein, microbiotic described in S2 detects and adopts indirect competitive, makes the microbiotic in differential protein spot and coupled antigen competition antibiotic antibody, using protein site corresponding during physiological saline process as negative control, and carries out light absorption value detection; Screen described light absorption value < 100%, and the protein site of significant difference p < 0.01 is microbiotic associated proteins;
Or microbiotic described in S2 detects and adopts direct measuring method, screening antibiotic content >=5 times negative control, and the protein site of significant difference p < 0.01 is microbiotic associated proteins; Corresponding protein site when described negative control is physiology saline treatment;
The minimum inhibition concentration of antibiotic concentration >=4 described in S1 times, the concentration of the described all biosomes of and antibiotic concentration≤kill.
2. method according to claim 1, is characterized in that, it is 50 μ g ~ 2 mg that electrophoretic separation described in S1 analyzes cell protein used.
3. method according to claim 1, is characterized in that, antibiotic concentration described in S1 is the minimum inhibition concentration of 8 ~ 100 times.
4. method according to claim 1, is characterized in that, microbiotic described in S2 detects and adopts indirect competitive, screen described light absorption value 0 ~ 90%, and the protein site of significant difference p < 0.01 is microbiotic associated proteins.
5. method according to claim 1, is characterized in that, microbiotic described in S2 detects and adopts direct measuring method, and screening antibiotic content is the protein site of 5 ~ 100 times of negative controls is microbiotic associated proteins; Corresponding protein site when described negative control is physiology saline treatment.
6. method according to claim 1, is characterized in that, biosome described in S1 is bacterium.
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Patent Citations (5)
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| CN1425075A (en) * | 2000-02-22 | 2003-06-18 | 牛津生物医学（英国）有限公司 | Differential expression screening method |
| CN1793933A (en) * | 2004-12-27 | 2006-06-28 | 山东大学 | Method of screening action target of cardiac glycoside medicine using protein composition technology |
| CN1952168A (en) * | 2006-10-30 | 2007-04-25 | 复旦大学 | Method for screening mycobacterium tuberculosis multidrug-resistance protein |
| CN101613675A (en) * | 2009-01-07 | 2009-12-30 | 中国人民解放军第二军医大学 | Bortezomib drug resistant myeloma cell strain and construction process thereof and application |
| US20110091991A1 (en) * | 2009-10-21 | 2011-04-21 | Atomic Energy Council-Institute Of Nuclear Energy Research | Protein Biomarkers for Alzheimer's Disease Detection |
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