CN108845145A - A method of detection histidine kinase activity - Google Patents
A method of detection histidine kinase activity Download PDFInfo
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- CN108845145A CN108845145A CN201810643829.1A CN201810643829A CN108845145A CN 108845145 A CN108845145 A CN 108845145A CN 201810643829 A CN201810643829 A CN 201810643829A CN 108845145 A CN108845145 A CN 108845145A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01—MEASURING; TESTING
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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Abstract
The present invention relates to a kind of methods for detecting histidine kinase activity, belong to field of biotechnology.Main technical schemes are as follows：Prepare phosphorylation histidine structure analog comlete antigen；Prepare phosphorylation histidine structure analog antibody；Prepare protein sample to be detected；Electrophoresis；Transferring film；Phosphorylation histidine structure analog antibody incubation, secondary antibody are incubated for：Pvdf membrane after closing is placed on equipped in 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids antibody-solutions box, shakes and is incubated for 2 hours under the conditions of 4 DEG C；Development.It is an object of the invention in place of overcome the deficiencies in the prior art, a kind of phosphorylation histidine structure analog antibody preparing convenience, low in cost, high specificity, efficient and sensible is provided, the antibody can be used to the detection of histidine kinase activity, it can also be used to the detection of histidine phosphorylated protein.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a method of detection histidine kinase activity.
Background technique
Protein phosphorylation is a kind of protein post-translational modification form being widely present in organism, this amino acid with
The modification mode that phosphate group is covalently attached plays important regulative to protein structure and function.For example, microorganism
Two component signal transduction system, the system by cross-film receptor histidine kinase (receptor histidine kinase,
RHK it) is formed with intracytoplasmic reaction regulatory protein (response regulator, RR).When RHK senses that external environment stimulates
Afterwards, autophosphorylation, the conservative asparagicacid residue being then transferred to phosphate group on RR occur for histidine residue.RR
The expression of downstream gene is regulated and controled after phosphorylation occurs.
Research shows that Two component signal transduction system not only has adjustment effect to the basic vital movement of bacterium, also participate in adjusting
The virulence of many pathogens and the generation of resistance mechanism are controlled, it is and pathogenic closely related, it is considered to be potential drug whip mark.By
Core protein of the body histidine kinase as Two component signal transduction system plays during microbial cell signal transduction
Vital effect.Therefore, easy histidine kinase activity analysis method quickly, highly sensitive is for medical diagnosis on disease, target
Screening etc. to drug is all of great significance.
Traditional protein kinase activity detection method is activity method, and this method utilizes radioactive element γ32The ATP of P label
Phosphorylation reaction is carried out as substrate, isolates and kinase activity is quantified by scinticounting after phosphorylated polypeptide product.However, should
Method needs complicated operating procedure, and radioactive waste has very big harm to environment and operator.It develops in recent years
A series of new analysis methods based on technologies such as fluorescence, electrochemistry, surface plasma resonance and mass spectrums, these methods pass through enrichment
And Selective Separation phosphated peptide section realizes the Activity determination to protein kinase in turn, especially in serine/threonine/junket ammonia
On the Activity determination of acid kinase, yield good result.But these methods are encountered when detecting histidine kinase activity
It is difficult.The associative key of histidine kinase catalytic phosphatase group and amino acid be N-P key, it is highly unstable in acid condition and
Abundance is very low.Have studies have shown that existing for 49 DEG C of 1mol/L HCl under the conditions of, τ-phosphorylation histidine half-life period is 18s,
τ-phosphorylation histidine half-life period is 25s.However, the method for common enrichment peptide fragment is to carry out under the conditions of lower pH, such as
Immobilized metal affinity chromatography, metal oxidation affinity chromatography etc., utilize Cu2+、Ti4+、Fe3+Equal metal ions or metal oxide exist
In acidic environment selectively in conjunction with electronegative phosphate group, effective enrichment to phosphated peptide section is realized.In addition, mass spectrum
The detection of phosphated peptide section is also required to carry out under acidic environment, often will cause histidine phosphated peptide section in detection process
Loss.This also means that the unstability due to natural phosphate histidine, many serine/threonine kinases/junket ammonia
The detection technique of acid kinase can not be applied on the activity research of histidine kinase, therefore be badly in need of developing novel, efficient detection
The analysis method of histidine kinase activity.
Summary of the invention
To make up the deficiencies in the prior art, the present invention is using phosphorylation histidine structure analog as haptens, with animal
Immunization obtains phosphorylation histidine structure analog antibody, and based on it, obtains a kind of detection histidine kinase activity
Method.
Technical scheme is as follows：A method of detection histidine kinase activity includes the following steps：
(1) phosphorylation histidine structure analog comlete antigen is prepared
Weigh phosphorylation histidine structure analog, n-hydroxysuccinimide, 1- (3- dimethylamino-propyl) -3- ethyl
Carbodiimide hydrochloride is successively dissolved in n,N-Dimethylformamide, is stirred to react at 20-30 DEG C, and activated solution A is obtained；It weighs
Carrier protein is dissolved in sodium bicarbonate solution, and magnetic agitation obtains solution B；Under ice-water bath, activated solution A is slowly added dropwise
Into solution B, magnetic agitation obtains solution C；Solution C is fitted into bag filter, dialyse and is centrifuged to obtain comlete antigen；
The chemical name of the phosphorylation histidine structure analog is 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphine
The chemical formula of acid, the phosphorylation histidine structure analog is：
(2) phosphorylation histidine structure analog antibody is prepared
The Freund's complete adjuvant of the comlete antigen and equivalent that weigh step (1) preparation is fully emulsified for fundamental immunity, or
Fully emulsified for booster immunization with the incomplete Freund's adjuvant of equivalent, each immunization interval 20 days is immunized 3 times altogether；For the third time
Immune animal is put to death after 14 days immune and takes blood, obtains 100mL blood；First blood is placed under ice-water bath and stands 4 hours, then
6000rpm is centrifuged 10min, obtains antiserum, antiserum is obtained phosphorylation histidine structure analog through affinitive layer purification
Antibody；
(3) protein sample to be detected is prepared；
(4) electrophoresis；
(5) transferring film；
(6) phosphorylation histidine structure analog antibody incubation, secondary antibody are incubated for：Pvdf membrane after closing is placed on and is equipped with
In 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids antibody-solutions box, shakes and be incubated for 2 hours under the conditions of 4 DEG C；
(7) develop.
Beneficial effects of the present invention are as follows：It is an object of the invention to provide one kind in place of overcome the deficiencies in the prior art
The phosphorylation histidine structure analog antibody of convenient, low in cost, high specificity, efficient and sensible is prepared, the antibody is i.e. available
In the detection of histidine kinase activity, it can also be used to the detection of histidine phosphorylated protein.
Detailed description of the invention
Fig. 1 is testing result figure of the invention.
Specific embodiment
The present invention is described further combined with specific embodiments below, if without specified otherwise, the raw materials used in the present invention
And equipment is the ordinary skill in the art.
The preparation of 1 phosphorylation histidine structure analog comlete antigen of embodiment
Comlete antigen is prepared with bovine serum albumin (BSA) for carrier protein using carbodiimide reaction method.It weighs
21.9mg (0.1mmol) 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids, 11.5mg (0.1mmol) N- hydroxysuccinimidyl acyl
Imines (NHS), 19.1mg (0.1mmol) 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) are successively dissolved in
In 1.5mL n,N-Dimethylformamide (DMF), it is stirred to react 12h at 20 DEG C, obtains activated solution A.Weigh BSA 75mg
It is dissolved in the sodium bicarbonate solution of 7.5mL 0.1M (pH=9.6), magnetic agitation (300rpm) obtains solution B.In ice-water bath
Under, activated solution A is slowly dropped in solution B, magnetic agitation (400rpm) is reacted for 24 hours, and solution C is obtained.Solution C is packed into
It in bag filter, is dialysed with 1L 0.01M PBS (pH=7.2), every 8h is changed the liquid once, and is dialysed 3 days altogether.Dialyse product 6000rpm from
Heart 10min obtains comlete antigen B-BSA.
2 1- of embodiment (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids Antibody preparation (fundamental immunity)
The Freund's complete adjuvant for weighing above-mentioned comlete antigen B-BSA 2mg and equivalent is fully emulsified for fundamental immunity.Exempt from
Epidemic disease mode is to the intradermal multi-point injection in new zealand white rabbit back, each dosage 0.5mg/kg of every rabbit.
3 1- of embodiment (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids Antibody preparation (booster immunization)
After fundamental immunity 20 days, the incomplete Freund's adjuvant for weighing above-mentioned comlete antigen B-BSA 2mg and equivalent is sufficiently newborn
Change and is used for booster immunization.Immunization ways are to carry out first time booster immunization to the intradermal multi-point injection in new zealand white rabbit back.Again
Every 20 days carry out second of booster immunization, each dosage 0.5mg/kg of every rabbit.It, will 14 days after second of booster immunization
Immune animal, which is put to death, takes blood.First blood is placed under ice-water bath and stands 4h, 6000rpm, centrifugation 10min, and measure antiserum effect
Value obtains 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids antibody, will make until its valence value reaches certain numerical value
The antibody got ready be stored in -20 DEG C it is spare.
The detection of 4 histidine kinase activity of embodiment
1, prepared by protein sample
60 μ g staphylococcus aureus arl Two component signal transduction system receptor protein ArlS (histidine kinase) are taken to dispense
In two 1.5mL centrifuge tubes, number C1 (phosphorylation) and C2 (dephosphorylation).5mM ATP, 5mM are sequentially added in C1
MgCl2, 10mM β-Me, 20mM HEPES, 150mM NaCl, react 30min at 37 DEG C.5mM MgCl is sequentially added in C22、
10mM β-Me, 20mM HEPES, 150mM NaCl react 30min under the conditions of 37 DEG C, 500mM azanol are then added, 37
60min is reacted under the conditions of DEG C.
60 μ g bovine serum albumin BSAs are taken to be divided in two 1.5mL centrifuge tubes, number B1 (phosphorylation) and B2 (remove phosphoric acid
Change).5mM ATP, 5mM MgCl are sequentially added in B12, 10mM β-Me, 20mM HEPES, 150mM NaCl, reacted at 37 DEG C
30min.5mM MgCl is sequentially added in B22, 10mM β-Me, 20mM HEPES, 150mM NaCl, reacted under the conditions of 37 DEG C
Then 30min adds 500mM azanol, react 60min under the conditions of 37 DEG C.
After reaction, C1, C2, B1 and B2 are heated 5 minutes under 100 DEG C of metal baths, make albuminous degeneration.
2, electrophoresis
2 pieces of SDS-PAGE running gel are prepared, number is J1, J2 respectively.Running gel J1 loading sequence is C1, B1；Running gel
J2 loading sequence is C2, B2.
3, transferring film
By taking running gel J1 as an example, impregnated 50 × 82mm pvdf membrane 30 seconds with 15mL anhydrous methanol, then with 15mL × TBS-T
Buffer impregnates pvdf membrane 15 minutes.Filter paper is impregnated 15 minutes with 20mL Transfer Buffer.Two are successively clamped with tweezers
It opens filter paper to overlay on transferring film instrument, a pvdf membrane is placed on filter paper, and a running gel is placed on pvdf membrane, and two filter paper cover
On running gel.Filter paper is smoothed with tweezers, removes bubble, opens transferring film instrument, and electric current 80mA is set, the time 40 minutes.
Running gel J2 transferring film method and step are consistent with running gel J1.Pvdf membrane after running gel J1, J2 transferring film is respectively
M1、M2。
4, phosphorylation histidine structure analog antibody incubation, secondary antibody are incubated for
By taking pvdf membrane M1 as an example, pvdf membrane is put into 20mL confining liquid after transferring film, closes half an hour.Then, will
Pvdf membrane after closing is placed on equipped with 5mL (antiserum B：Confining liquid=1:1000) 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4-
In the box of base phosphonic acids antibody-solutions, incubation 2 hours is slowly shaken under the conditions of 4 DEG C.Antibody is washed with 15mL × TBS-T buffer
Pvdf membrane after A incubation 5 minutes every time, washs 8 times altogether.Pvdf membrane after closing is placed on equipped with 5mL (secondary antibody：Confining liquid=
1:1000) in two corresponding anti-solution box, incubation 2 hours is slowly shaken under the conditions of 4 DEG C.Secondary antibody is washed with 15mL × TBS-T buffer
Pvdf membrane after incubation 5 minutes every time, washs 8 times altogether.
Pvdf membrane M2 transferring film method and step are consistent with pvdf membrane M1.
5, develop
By taking pvdf membrane M1 as an example, first 10 × 10cm preservative film is laid on testing stand, then the M1 after washing is placed on guarantor
On fresh film.1mL developer solution rinse pvdf membrane is pipetted M1 3 minutes with liquid-transfering gun, then is blotted extra developer solution with blotting paper, most
Pvdf membrane M1 is placed in Full-automatic chemiluminescence image analyzer afterwards and is developed.
As a result as shown in Figure 1, after ArlS kinases autophosphorylation, the present invention prepared by antibody can specificity to it
It is identified.And BSA does not have kinase activity, can not autophosphorylation, therefore antibody to its without identification.When ArlS albumen
After dephosphorylation, antibody can not also be identified it.The above results show that antibody prepared by the present invention being capable of successful detection group
The activity of histidine kinase.
Above-described embodiment is only intended to citing and explanation of the invention, and is not intended to limit the invention to described
In scope of embodiments.Furthermore it will be appreciated by persons skilled in the art that the present invention is not limited to the above embodiment, according to this hair
Bright introduction can also make more kinds of variants and modifications, these variants and modifications all fall within present invention model claimed
In enclosing.
Claims (1)
1. a kind of method for detecting histidine kinase activity, which is characterized in that include the following steps：
(1) phosphorylation histidine structure analog comlete antigen is prepared
Weigh phosphorylation histidine structure analog, n-hydroxysuccinimide, 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Inferior amine salt hydrochlorate is successively dissolved in n,N-Dimethylformamide, is stirred to react at 20-30 DEG C, and activated solution A is obtained；Weigh carrier
Albumen is dissolved in sodium bicarbonate solution, and magnetic agitation obtains solution B；Under ice-water bath, activated solution A is slowly dropped to molten
In liquid B, magnetic agitation obtains solution C；Solution C is fitted into bag filter, dialyse and is centrifuged to obtain comlete antigen；
The chemical name of the phosphorylation histidine structure analog is 1- (2- aminobutyl) -1- hydrogen-pyrazoles -4- base phosphonic acids,
The chemical formula of the phosphorylation histidine structure analog is：
(2) phosphorylation histidine structure analog antibody is prepared
The Freund's complete adjuvant of the comlete antigen and equivalent that weigh step (1) preparation is fully emulsified for fundamental immunity, or with etc.
The incomplete Freund's adjuvant of amount is fully emulsified to be used for booster immunization, and each immunization interval 20 days is immunized 3 times altogether；Third time is immune
Immune animal is put to death after 14 days and takes blood, obtains 100mL blood；First blood is placed under ice-water bath and stands 4 hours, then
6000rpm is centrifuged 10min, obtains antiserum, antiserum is obtained phosphorylation histidine structure analog through affinitive layer purification
Antibody；
(3) protein sample to be detected is prepared；
(4) electrophoresis；
(5) transferring film；
(6) phosphorylation histidine structure analog antibody incubation, secondary antibody are incubated for：Pvdf membrane after closing is placed on equipped with 1- (2-
Aminobutyl) in -1- hydrogen-pyrazoles -4- base phosphonic acids antibody-solutions box, shakes and be incubated for 2 hours under the conditions of 4 DEG C；
(7) develop.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130245038A1 (en) * | 2012-03-13 | 2013-09-19 | Abbvie Inc. | Method For Selecting Or Identifying A Subject For V1B Antagonist Therapy |
| EP2778176A1 (en) * | 2013-03-12 | 2014-09-17 | Sanofi | Compositions and methods for analyzing histidine phosphorylation |
| WO2015033120A1 (en) * | 2013-09-04 | 2015-03-12 | University Of Sheffield | Phosphohistidine and phosphotyrosine analogues |
| WO2015051079A2 (en) * | 2013-10-02 | 2015-04-09 | The Trustees Of Princeton University | Phosphohistidine mimetics and antibodies to same |
| WO2016018562A1 (en) * | 2014-07-31 | 2016-02-04 | Salk Institute For Biological Studies | Generation and use of polyclonal and monoclonal antibodies specific for 3-phosphohistidine |
| CN106008361A (en) * | 2015-07-01 | 2016-10-12 | 北京维德维康生物技术有限公司 | Albendazole artificial antigen and preparation method and application thereof |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130245038A1 (en) * | 2012-03-13 | 2013-09-19 | Abbvie Inc. | Method For Selecting Or Identifying A Subject For V1B Antagonist Therapy |
| EP2778176A1 (en) * | 2013-03-12 | 2014-09-17 | Sanofi | Compositions and methods for analyzing histidine phosphorylation |
| WO2015033120A1 (en) * | 2013-09-04 | 2015-03-12 | University Of Sheffield | Phosphohistidine and phosphotyrosine analogues |
| WO2015051079A2 (en) * | 2013-10-02 | 2015-04-09 | The Trustees Of Princeton University | Phosphohistidine mimetics and antibodies to same |
| WO2016018562A1 (en) * | 2014-07-31 | 2016-02-04 | Salk Institute For Biological Studies | Generation and use of polyclonal and monoclonal antibodies specific for 3-phosphohistidine |
| CN106008361A (en) * | 2015-07-01 | 2016-10-12 | 北京维德维康生物技术有限公司 | Albendazole artificial antigen and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| JUNG-MIN KEE等: "A Second-Generation Phosphohistidine Analog for Production of Phosphohistidine Antibodies", 《ORGANIC LETTERS》 * |
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Application publication date: 20181120 |