CN105131110A - Immunoaffinity chromatography column and purification method for purifying Bordetella pertussis Prn protein - Google Patents
Immunoaffinity chromatography column and purification method for purifying Bordetella pertussis Prn protein Download PDFInfo
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- CN105131110A CN105131110A CN201510473326.0A CN201510473326A CN105131110A CN 105131110 A CN105131110 A CN 105131110A CN 201510473326 A CN201510473326 A CN 201510473326A CN 105131110 A CN105131110 A CN 105131110A
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Abstract
The present invention relates to an immunoaffinity chromatography column and a purification method for purifying Bordetella pertussis Prn protein. According to the present invention, Prn protein is adopted as antigen to immunize rabbits to prepare polyclonal antibody, and through experimental optimization, the polyclonal antibody is used to prepare the immunoaffinity chromatography column for purifying the Prn protein, wherein the immunoaffinity chromatography column can specifically purify the Prn protein, has the less introduced impurities, and provides the novel method for Prn protein purification.
Description
Technical field
The present invention relates to a kind of immune affinity chromatographic column and method for purifying proteins of purifying pertussis bacillus Prn albumen.
Background technology
Whooping cough is a kind of Acute respiratory infectious disease caused by Bordetella pertussis infection, the infant of main infection less than 6 years old.The most economical effective means of prevention for this disease is vaccinate.Whole cell pertussis vaccine development success in 1914, the great function played in the morbidity of Control and prevention Whooping cough, but this vaccine side reaction is larger, is resisted by some developed countries.The eighties, Japanese invention acellular pertussis vaccine, which removes the toxicant causing side reaction, has remained the antigenicity of immanoprotection action, promoted the progress of vaccinology.
The principle active component of acellular pertussis vaccine is Toxins, pertussis (PT), filamentous hemagglutinin (FHA), adhesin (Prn), agglutinogen (AGG) etc.Composition in China's vaccine mainly PT, FHA.
Prn is a kind of agglutinogen of non-cilium, has stronger immunogenicity, it can be used as the important component of pertussis vaccine at Some Enterprises that is American-European and China.Prn protein yield is little, and the purifying for it has a lot of method.The Affi-gelblue that utilizes had adsorbs, and uses high eluting salt, through chromatofocusing after dialysis.The method is non-specific adsorption, and foreign protein is more, and introduces more salt.Present most of enterprise first utilizes ammonium sulfate more precipitation for this albumen, carrys out concentrated target protein, and recycling chromatography column filters, and uses high eluting salt.The method complex steps, consuming time more, non-specific adsorption, introduces foreign protein and pollutes.
Summary of the invention
The shortcoming that present method exists in purifying Prn albumen for prior art, a kind of immune affinity chromatographic column and method for purifying proteins of purifying pertussis bacillus Prn albumen are provided, using Prn albumen as antigen, immunize rabbit prepares polyclonal antibody, optimize by experiment, utilize the how anti-affine immunochromatography post having prepared purifying Prn albumen, this immunochromatography post can specificity purifying Prn albumen, and it is few to introduce impurity, for the purifying of Prn albumen provides novel method.
According to a first aspect of the invention, a kind of polyclonal antibody of purifying pertussis bacillus Prn albumen is taken a blood sample after immune animal by bordetella pertussis Prn albumen, and after being separated antiserum(antisera), purifying obtains.
Described animal is rabbit.
According to a second aspect of the invention, a kind of immune affinity chromatographic column of purifying pertussis bacillus Prn albumen, forms by solid phase carrier with the polyclonal antibody of the purifying pertussis bacillus Prn albumen of its coupling.
Described solid phase carrier is Mierocrystalline cellulose, dextrane gel, polyacrylamide gel, sintered glass, sepharose or ultragel ACA22.
Described carrier is the Sepharose4B of CNBr activation.
According to a third aspect of the present invention, the preparation method of the immune affinity chromatographic column of purifying pertussis bacillus Prn albumen, comprises the steps:
Take a blood sample after 1st step, Prn protein immune animal, after being separated antiserum(antisera), purifying obtains polyclonal antibody;
2nd step, by support-activated, carry out coupling with polyclonal antibody, obtain immune affinity chromatographic column.
According to a fourth aspect of the present invention, a kind of purification process of purifying pertussis bacillus Prn albumen, comprises the steps:
1st step, by the culture supernatant of Whooping cough bacterial strain removing FHA and PT albumen, then send in ultra-filtration membrane and concentrate, then concentrated solution is centrifugal, collection supernatant liquor;
2nd step, by supernatant liquor send into described in purifying pertussis bacillus Prn albumen immune affinity chromatographic column in, carry out loading;
After 3rd step, end of the sample, then carry out wash-out with elutriant, obtain purifying protein.
In the 1st described step, the molecular weight cut-off of ultra-filtration membrane is 30kD; Carry out centrifugal before, need with 50mMTris-HCl, pH=7.5 solution carry out redissolution process.
In the 2nd described step, before by supernatant liquor loading, need first to balance chromatography column, balance liquid used is the solution of 50mMTris-HCl, pH=7.5.
In the 2nd described step, the volume of supernatant liquor is 4 times of column volume.
In the 3rd described step, elution process is first with the eluant solution foreign protein that 50mMTris-HCl, pH of 5 times of column volumes are 7.5, then is 3.5 damping fluids with 50mMTris-HCl, pH, wash-out Prn albumen.
Invention effect: one, the immune affinity chromatographic column of the how anti-preparation of the present invention's application can efficiently purifying Prn albumen; Two, apply the immune affinity chromatographic column purifying Prn albumen of how anti-preparation, can shorten the operating time, reduce purifying cost, reduce the pollution introducing impurity, purifying protein is purer.
Accompanying drawing explanation
Fig. 1, adult rabbits immunity Prn albumen Serum Antibody Detection is tired and serum diluting multiple graph of a relation;
Polyclonal antibody SDS-PAGE electrophorogram after Fig. 2, purifying; Wherein, 1 swimming lane is sample, and 2 swimming lanes are marker;
Fig. 3, SDS-PAGE electrophorogram to sample different in Prn protein purification procedures in supernatant; Wherein, 1 swimming lane is sample before loading, and 2 swimming lanes were post effluent liquid, and 3 swimming lanes are elutriants, and 4 swimming lanes are standard P rn albumen.
Embodiment
Below by embodiment, the present invention is described in further detail.But it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1 polyclonal antibody preparation
1, immune rabbit
Get Prn albumen prepared by production plant, survey protein content, with adjuvant by 1:1(V/V) mix, shake one hour on the oscillator, rear ultrasonic (power is 350W, working hour 2s, intermittent time 10s, temperature is arranged on 25 DEG C) emulsification, ultrasonic complete to emulsification, the immunogen that emulsification is good is dripped one to be dropped in the water of precooling, and indiffusion in 30 minutes is that emulsification is complete.Select SPF adult rabbits as immune animal.Ear edge vein exploitating blood before each immunity.Twice immunization interval is 2 weeks.First time Freund's complete adjuvant, albumen dosage be 75 μ g/ only, total dose 100 μ l/ only, selects 4 ~ 6 subcutaneous injections in rabbit backbone both sides; Second time Freund's incomplete adjuvant, same immunizing antigen dosage and method for the first time; Third time, immunization method was with second time immunity; Booster immunization, method is as second time immunization method.
2, immunize rabbit serum antibody measures
By the rabbit anteserum gathered before each immunity, detect serum titer with indirect ELISA method, establish negative control simultaneously, indirect ELISA program is as follows:
Bag quilt: dilute Prn albumen with the PBS of pH value 7.2, make its concentration be 4 μ g/mL, 100 μ l/ holes join in enzyme plate, and after 4 DEG C of overnight incubation, PBS ' T washes 5 times, dry residual liquid on plate.Close: every hole adds 200 μ l confining liquids, and incubated at room 1h, washes plate with same method.Primary antibodie: every hole adds the rabbit anteserum to be checked that 100 μ l have diluted, and (extent of dilution is 1:10
2, 1:10
3, 1:10
4and 1:10
5four extent of dilution), incubated at room 1h, washes plate with same method.Two resist: every hole adds the goat anti-rabbit igg (1:4000) that 100 μ lHRP mark, and incubated at room 1h, washes plate with same method.Substrate develops the color: join in elisa plate by the TMB newly prepared with 100 μ l/ holes, hatch 15min under room temperature condition.Stop: every hole adds 50 μ l3MH
2sO
4, vibrate gently, by microplate reader at OD
450nmtime reading.
With EXCEL data preparation and analytical data.As Fig. 1, rabbit three is exempted from rear serum diluting multiple and reaches 10
5, meet needed for Anti-TNF-α preparation.
3, the preparation of anti-Prn protein polyclone antibody and purifying
After booster immunization the 4th day, get whole blood, obtain serum, slowly added by serum in equivalent saturated ammonium sulphate (stirring of limit edged), 4 DEG C are stirred 2 hours, and 12000rpm10min is centrifugal, abandon supernatant, PBS is resuspended, adds in the dialysis tubing handled well, dialysed overnight (4 DEG C) by the IgG that slightly carries resuspended for PBS.
The protein sample of having dialysed centrifugal 1000rpm, 5min, supernatant uses 0.45 μm of frit again.Utilize precious biotechnology (Dalian) company limited of ProteinGResin() resist is further purified more.First four in ProteinGResin is steamed current to the greatest extent, add appropriate PBS and to make in pillar effluent liquid OD280 light absorption value close to 0; Add sample (repeatedly 5 times), add PBS wash-out foreign protein to effluent liquid OD280 value close to 0, with bufferB wash-out object IgG, effluent liquid Tris-HCl is adjusted pH to 7 ~ 8, and the effluent liquid that OD value is less than 0.3 discards.If Fig. 2 is polyclonal SDS-PAGE electrophorogram after purifying.
The preparation of the immune affinity chromatographic column of embodiment 2 purifying Prn albumen and working method
1, polyclonal antibody and the coupling of Sepharose4B gel
Centrifugal acquisition 5mlSepharose4B(GE) dry glue, with 5gCNBr activation, activate complete, respectively with the water for injection of 25ml, the acetone of 5ml respectively wash one time, then use 25ml sodium hydrogen carbonate solution (0.2M, pH8.9) to wash five times, for subsequent use.
The polyclonal antibody that purifying obtains is dialysed in sodium hydrogen carbonate solution (0.2M, pH8.9), surveys concentration, the gel mixing coupling of 10mg and activation, 4 DEG C of overnight incubation.Coupling liquid 2000rpm gets precipitation in centrifugal 5 minutes, and collect supernatant and calculate Conjugate ratio, Conjugate ratio is 92%.By 20ml sodium hydrogen carbonate solution (0.2M, pH8.9) resuspended precipitation, use 20ml sodium hydrogen carbonate solution (0.2M, pH8.9), sodium bicarbonate-sodium-chlor mixed solution (0.2M sodium bicarbonate, 0.5M sodium-chlor, pH8.9) successively, sodium hydrogen carbonate solution (0.2M, pH8.9) washes one time.4 hours are closed with thanomin (50mM, pH7.5) solution 4 DEG C.San Bian Installed post is washed for subsequent use with PBS.
2, the method for separation and purification PRN albumen
(1) sample pre-treatments
The bacterial strain used is Whooping cough CS strain, and sample bacterium liquid bacteria concentration is 300 × 10
8individual/more than ml, hemagglutination activity reaches more than 1:128.
Collect the effluent liquid after purified FHA and the PT albumen of microbial culture supernatant, by tangential flow systems (Quixstand Middle hollow fiber membrane system, GE), dress 30KD hollow fiber column (UFP-30-E-4X2MA, GE) filtering and concentrating, to 1/10th of original volume, with 50mMTris-HCl, pH=7.5 redissolves, centrifugal 10 minutes of 10000rpm, collects supernatant.
(2) purifying of Prn albumen
4 DEG C of immune affinity chromatographic columns preserved are taken out, balances chromatography column with 10 times of volume 50mMTris-HCl, pH=7.5.To be purified 4 times of column volume samples are added (1ml/min) in pillar, the complete use of application of sample 5 times of volume 50mMTris-HCl, pH=7.5, wash-out foreign protein (2ml/min), with 50mMTris-HCl, pH=3.5 damping fluid, eluted protein, rapid adjust pH to 7.5.Analyze with SDS-PAGE.Chromatography column is finished using with 10 times of volume PBS balance chromatography columns, is stored in 4 DEG C of next times reusable.
As Fig. 3, band 1 is the sample before loading, and band 2 is the foreign protein of wash-out, and band 3 is the target protein of wash-out, and band 4 is standard P rn albumen, and can find out that protein purification effect is better, purity of protein more than 95%, the rate of recovery is more than 90%.
Claims (10)
1. a polyclonal antibody for purifying pertussis bacillus Prn albumen, is characterized in that, is to be taken a blood sample after immune animal by bordetella pertussis Prn albumen, and after being separated antiserum(antisera), purifying obtains.
2. the polyclonal antibody of purifying pertussis bacillus Prn albumen according to claim 1, is characterized in that: described animal is rabbit.
3. an immune affinity chromatographic column for purifying pertussis bacillus Prn albumen, is characterized in that: form by solid phase carrier with the polyclonal antibody of the purifying pertussis bacillus Prn albumen according to claim 1 of its coupling.
4. the immune affinity chromatographic column of purifying pertussis bacillus Prn albumen according to claim 3, is characterized in that: described solid phase carrier is Mierocrystalline cellulose, dextrane gel, polyacrylamide gel, sintered glass, sepharose or ultragel ACA22.
5. the immune affinity chromatographic column of purifying pertussis bacillus Prn albumen according to claim 3, is characterized in that: described solid phase carrier is the Sepharose4B of CNBr activation.
6. the preparation method of the immune affinity chromatographic column of purifying pertussis bacillus Prn albumen according to claim 3, is characterized in that: take a blood sample after comprising the steps: the 1st step, Prn protein immune animal, and after being separated antiserum(antisera), purifying obtains polyclonal antibody; 2nd step, by support-activated, carry out coupling with polyclonal antibody, obtain immune affinity chromatographic column.
7. a purification process for purifying pertussis bacillus Prn albumen, is characterized in that: comprise the steps:
1st step, by the culture supernatant of Whooping cough bacterial strain removing FHA and PT albumen, then send in ultra-filtration membrane and concentrate, then concentrated solution is centrifugal, collection supernatant liquor;
2nd step, supernatant liquor to be sent in the immune affinity chromatographic column of purifying pertussis bacillus Prn albumen according to claim 3, carry out loading;
After 3rd step, end of the sample, then carry out wash-out with elutriant, obtain purifying protein.
8. the purification process of purifying pertussis bacillus Prn albumen according to claim 7, is characterized in that: in the 1st described step, the molecular weight cut-off of ultra-filtration membrane is 30kD; Carry out centrifugal before, need with 50mMTris-HCl, pH=7.5 solution carry out redissolution process.
9. the purification process of purifying pertussis bacillus Prn albumen according to claim 7, is characterized in that: in the 2nd described step, before by supernatant liquor loading, need first to balance chromatography column, balance liquid used is the solution of 50mMTris-HCl, pH=7.5; The volume of supernatant liquor is 4 times of column volume.
10. the purification process of purifying pertussis bacillus Prn albumen according to claim 7, it is characterized in that: in the 3rd described step, elution process is first with the eluant solution foreign protein that 50mMTris-HCl, pH of 5 times of column volumes are 7.5, be 3.5 damping fluids with 50mMTris-HCl, pH again, wash-out Prn albumen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106496328A (en) * | 2016-11-22 | 2017-03-15 | 浙江达美生物技术有限公司 | The preparation method of hyaluronic acid binding protein polyclonal antibody |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
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| CN1696698A (en) * | 2005-05-19 | 2005-11-16 | 中国农业大学 | Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit |
| CN101074442A (en) * | 2007-04-29 | 2007-11-21 | 中国药品生物制品检定所 | Recombinant expression and use for pertussis vaccine protective antigen |
| CN102584958A (en) * | 2012-02-08 | 2012-07-18 | 北京天坛生物制品股份有限公司 | Purification method for 69KD outer membrane protein of pertussis bacillus |
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Patent Citations (3)
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| CN1696698A (en) * | 2005-05-19 | 2005-11-16 | 中国农业大学 | Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit |
| CN101074442A (en) * | 2007-04-29 | 2007-11-21 | 中国药品生物制品检定所 | Recombinant expression and use for pertussis vaccine protective antigen |
| CN102584958A (en) * | 2012-02-08 | 2012-07-18 | 北京天坛生物制品股份有限公司 | Purification method for 69KD outer membrane protein of pertussis bacillus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496328A (en) * | 2016-11-22 | 2017-03-15 | 浙江达美生物技术有限公司 | The preparation method of hyaluronic acid binding protein polyclonal antibody |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
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