CN103044531A - Purification method of methicillin-resistant staphylococcus aureus (MREA)-resistant vaccine recombinant protein antigen HI2 - Google Patents

Abstract

The invention discloses a purification method of methicillin-resistant staphylococcus aureus (MREA)-resistant vaccine recombinant protein antigen HI2. An HI2 protein is obtained by the recombinant fusion of active function segments of two antigen molecules of the methicillin-resistant staphylococcus aureus H1a and IsdB and the expression of an escherichia coli genetically engineered bacterium. The technologies, i.e. the high-pressure bacterium breaking, the salt precipitation, the GST (glutathione s transferase) affinity chromatography, the PP (propene polymer) digestion, the MMC (mitomycin) chromatography, the gel filtration chromatography, are adopted for the genetically engineered bacterium, so that high-purity methicillin-resistant staphylococcus aureus (MREA)-resistant vaccine recombinant protein antigen HI2 can be obtained. The purification method disclosed by the invention is simple in technology, easy to amplify, and good in repeatability. The obtained target protein is high in purity. The animal experiment proves that an organism can be effectively stimulated to generate the higher humoral immune response and the better immunoprotection.

Description

Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI 2Purification process

Technical field

The invention belongs to field of biological pharmacy, particularly a kind of methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI ₂Purification process.

Background technology

Anti-X-1497 Golden yellow staphylococcus (methicillin-resistant Staphylococcus aureus, MRSA) be the gram positive coccus that can infect any one position of human body, local infection is prolonged not to heal, the systemic infection mortality ratio has become one of the highest pathogenic bacteria of the infection rates such as global ICU ward, burn, war wound up to 20%.

Vancomycin be the treatment MRSA last line of defense, but 1997 with the coming year vancomycin resistance MRSA in succession be separated, make MRSA be about to face the severe challenge that antibiotic-free can be controlled.Therefore, strengthen the study on prevention to the MRSA infection, extremely urgent.Therefore, researching and developing a kind of effective vaccine may be that prevention and control MRSA infect and resistance development effective way.

Bacterial outer membrane albumen has good antigenic activity, and the main target that these outer membrane proteins are attacked as antibody and immunocyte can mediate the most direct effective killing action of bacterium, is to determine that whether immune response has the key factor of protectiveness to human body.IsdB is not only important adventitia rivet albumen of MRSA, adheres to the vital role in mid-term in the MRSA field planting, and it also is MRSA obtains iron from the host a main tool simultaneously.Recombinant vaccine take IsdB as component is carrying out the II clinical trial phase.In addition, the Golden yellow staphylococcus can cause that various diseases one hemolysin (Hla) of humans and animals is a kind of important virulence factor wherein by producing the multiple virulence factors such as hemolysin, Thrombin coagulase, leueocidin.Hla is that a perforation of all expressing on most of clinical separation strains forms toxin.It is relevant to various diseases pneumonia, septicemia sacroiliitis and brain running sore etc.The Hla monomer is 33kDa approximately, and with heptamer form performance function, the 110-150 amino acid of each monomer is drawn close at phospholipid bilayer mutually and formed the hole.Cause subsequently cytolemma to destroy, ion oozes out etc. in the born of the same parents.Its toxicity is very strong, can cause a disease in the very low concentrations situation.The Hla antiserum(antisera) can suppress the staphylococcic cytotoxic effect of Golden yellow, also can reduce the Golden yellow staphylococcus and be attached to probability on the host cell.Make body obtain the provide protection of Kang Golden yellow staphylococcal infections after the Hla immunization.

Hla and IsdB are the intrinsic protein ingredients of MRSA, and its encoding gene has high conservative, are the important candidate antigens of MRSA vaccine.We made up can fine these albumen of removal MRSA to the toxicity of cell, can keep again the two subunit genetic engineering albumen HI of restructuring of these protein antigenicities ₂Have not yet to see for the two subunit genetic engineering albumen HI of (MRSA) restructuring ₂The report of the purification process in the vaccine preparation.

Summary of the invention

Purpose of the present invention provides the two subunit genetic engineering vaccine candidate antigens HI of a kind of methicillin-resistant staphylococcus aureus (MRSA) restructuring ₂Purification process in the preparation.The method technique is simple, the target protein purity that obtains high, the rate of recovery is better.

The present invention has adopted following steps:

Purification process in the two subunit genetic engineering vaccine candidate antigens HI2 preparations of methicillin-resistant staphylococcus aureus restructuring, this antigen HI ₂Nucleotide sequence shown in SEQ ID NO:1, aminoacid sequence is shown in SEQ ID NO:2, its purification process may further comprise the steps:

A) collect from construction expression antigen HI ₂The thalline of colibacillus engineering high density fermentation;

B) adopt the high bacterium, centrifugal that crushes, the ammonium sulfate fractional precipitation, GST affinity chromatography, PP enzyme are cut, and the sequential combination of MMC chromatography, gel filtration chromatography is carried out purifying to the HI2 of preparation, has obtained highly purified rHI2.

Described step B is specific as follows:

1) height crushes bacterium: with the two subunit genetic engineering albumen HI of methicillin-resistant staphylococcus aureus restructuring that collect ₂Coli somatic suspend with the 10-20mM PBS damping fluid mixing of pH7.0-7.5, adopt the broken bacterium of high-pressure homogenization after the precooling, high speed centrifugation is collected supernatant;

2) ammonium sulfate fractional precipitation: under 4 ℃ of agitation conditions, slow adding final concentration is 30% ammonium sulfate in the supernatant, stirs half an hour, and 10000-15000g high speed centrifugation 20 minutes is collected supernatant; The slow adding of continuation final concentration is 40% ammonium sulfate in the supernatant, stirs half an hour, 10000-15000g high speed centrifugation 20 minutes, collecting precipitation;

3) precipitation is redissolved: weighing precipitation weight in wet base, and by weight: volume ratio 1:10 ratio adds the 10-20mM PBS damping fluid of pH7.0-7.5, and stirring and evenly mixing 10-15 minute, 10000-15000g high speed centrifugation 20 minutes was collected supernatant;

4) GST affinity purification: select GST affinity chromatography filler to carry out preliminary purification, use under PBS-Tween80pH7.0-7.5 condition target protein is carried out purifying, Prescission Protease enzyme (PP enzyme) carries out enzyme and cuts wash-out:

5) MMC chromatography purification: the sample that step 4) is collected, pH transfers to 3-5, uses NaAC damping fluid balance layer analysis system and the MMC chromatography column of pH3-5, and employing pH10.5-11 is Na ₂CO ₃Buffer solution for gradient elution;

6) gel permeation chromatography purifying: with the sample of step 5) purifying acquisition, use gel-filtration Superdex chromatography column purifying, adopt 10-20mM PBS balance layer analysis system and the chromatography column of pH7.0-7.5, remove the impurity such as the non-target protein of trace, the separation and purification target protein, the displacement damping fluid.

Step 1) adopts the broken bacterium technology of the 60-80MPa high-pressure homogenization in production or the pilot scale purifying, breaks the bacterium rate greater than 96%, the centrifugal bacterium supernatant that obtains brokenly.

Step 2) sour ammonium sulphur content step precipitation and step 3) are redissolved again.

The described GST affinity purification of step 4), employed filler are GST-Sepharose4B or GST-Sepharose6B or GST-Sepharose FastFlow or GST-Sepharose HP.

The employed Prescission Protease of step 4) enzyme is beneficial to remove the PP enzyme with the GST label.

The described purification process of step 5), the filler of MMC chromatography are Capto ^TMMMC;

The described gel chromatography column of step 6) is Superdex75 or Superdex200 or Superdex HR10/30.

Described antigen prepares by following steps:

1) design forward primer and reverse primer are synthetic to obtain coding HI by pcr amplification or full gene ₂The nucleotide sequence of protein active fragment;

2) nucleotide sequence that step 1) is obtained is cloned into the expression vector establishment recombinant vectors, then this recombinant vectors is converted into Host Strains;

3) Host Strains behind the Induction Transformation is expressed recombinant protein.

The invention effect

The purification process of the present invention that adopts, (the application obviously is HI from expressing the two subunit genetic engineering candidate antigens of methicillin-resistant staphylococcus aureus (MRSA) restructuring ₂) colibacillus engineering in can obtain HI greater than 98% ₂, yield is more than 60%, and whole purge process need not additionally to replace damping fluid again.Through identifying that the inventor makes up the HI of acquisition ₂Molecular weight is about 46kD.

Purification process of the present invention mainly contains ammonium sulfate fractional precipitation, GST purifying, MMC chromatography, molecular sieve and respectively goes on foot SDS-PAGE.

HI by above-mentioned art breading different batches ₂Make 12%SDS-PAGE, present the simple target protein band, molecular weight is about 46kD.The analysis of HPLC C3 post presents single peak, and purity is more than 99%.Iso-electric point is about pH7.1.Different HI ₂Peptide fingerprinting is composed each peak-to-peak number average and is consistent, and all fluctuations in ± 10Sec of the retention time at each peak illustrate the peptide mapping favorable reproducibility.HI behind the purifying ₂With aluminum hydroxide adjuvant or the common injecting immune BalB/C of aluminum phosphate adjuvant mouse, find HI ₂Be significantly higher than negative control group (PBS group) (P＜0.01) with the IgG level in the immunological adjuvant group serum, prove the HI that uses inventor's purification process to obtain ₂But the effective stimulus body produces higher immunne response.Use MRSA international standard strain 252(available from ATCC) infect, find HI ₂Be 15% with immunological adjuvant group infection rate, negative control group (PBS group) infection rate is 90%, calculates HI ₂The protection ratio that anti-MRSA infects is 83.3%, shows that this protein fragments has good immunogenicity and immune protective, can be used as the candidate antigens of methicillin-resistant Staphylococcus recombination engineered vaccine.

Description of drawings

Fig. 1 is as a result figure of ammonium sulfate precipitation SDS-PAGE, among the figure, and swimming lane 1: standard protein; Swimming lane 2:30% ammonium sulfate supernatant; Swimming lane 3:30% ammonium sulfate precipitation; Swimming lane 4:40% ammonium sulfate precipitation supernatant; Swimming lane 5:40% ammonium sulfate precipitation;

Fig. 2 is HI ₂Protein G ST affinity chromatography and PP enzyme are cut as a result figure of SDS-PAGE, among the figure, and swimming lane 1: standard protein; Swimming lane 2:GST cuts sample in conjunction with the PP enzyme; Swimming lane 3:GST cuts rear PBS washing sample 1 in conjunction with the PP enzyme; Swimming lane 4:GST cuts rear PBS washing 2 in conjunction with the PP enzyme; Swimming lane 5:GST is in conjunction with front sample; Swimming lane 6:GST wears sample in conjunction with stream; Swimming lane 7:GST is in conjunction with sample;

Fig. 3 is HI ₂Albumen MMC tomographic map;

Fig. 4 is HI ₂Protein S uperder200 tomographic map;

Fig. 5 is MMC and Superdex200 chromatography purification design sketch, among the figure, and swimming lane 1: standard protein; Swimming lane 2:GST is affine, and the PP enzyme is cut rear sample; Sample behind the swimming lane 3:MMC chromatography; Sample behind the swimming lane 4:Superdex200 chromatography.

Embodiment

Below in conjunction with embodiment the present invention is described in detail:

Embodiment one antigen HI ₂Preparation

(1) methicillin-resistant staphylococcus aureus ClfA, IsdB, mSEC gene cloning

1. methicillin-resistant staphylococcus aureus MRSA252 (deriving from ATCC) (preservation of pharmacology teaching and research room of Third Military Medical University)

2. take out the methicillin-resistant staphylococcus aureus WHO-2 bacterial strain of preserving in the liquid nitrogen container and coat on the WHO-2 special solid substratum, in 37 ℃, overnight incubation.Genome extraction agent box extracting MRSA genome.

3. adopt PCR method from increase the respectively encoding gene of IsdB, Hla of MRSA genome.

1) design of primers synthetic following (underscore shows the restriction enzyme site base sequence)

Gene order and design of primers principle according to GenBank announces design corresponding primer, introduce restriction enzyme site.

2) pcr amplification of goal gene:

Take the MRSA genomic dna as template, P1 and P2, P3 and P4, P5 and P6 primer increase respectively IsdB, Hla, HI ₂Gene, adopt following PCR system and program:

Template DNA 1 μ l; 10 * PCR damping fluid (magnesium chloride containing), 5 μ l; DNTPs(10mmol/L) 4 μ l; Each 1 μ l of upstream and downstream primer (0.025mmol/L); Taq archaeal dna polymerase (5u/ μ l) 0.5 μ l adds deionized water to final volume 50 μ l.

With the reaction system mixing, after the centrifugal treating, add 30 μ l paraffin oils.94 ℃ of denaturations 5 minutes, 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 1min seconds, 72 ℃ are extended 1min second, 35 circulations, 72 ℃ were extended 10 minutes fully.React and get 1 μ l reaction product after complete, 1.2% agarose gel electrophoresis detects the PCR effect.

3) recovery of PCR product (reclaiming test kit is Time Inc. available from sky, Beijing, presses the operation of test kit working instructions)

(1) records 1.0% sepharose;

(2) pcr amplification product is added in the electrophoresis loading hole stop electrophoresis when indicator migrates to the appropriate location;

(3) under UV-lamp, separate the gel that contains the purpose fragment, move into 1.5ml EP pipe;

(4) add DNA binding buffer liquid, 65 ℃ of water-baths are dissolved gel fully and are kept pH value of solution between 5.0～6.0;

(5) sol solutions is moved into separator tube, centrifugal 1 minute of 12000g discards the liquid in the collection tube;

(6) add 500 μ l lavation buffer solutions, centrifugal 1 minute of 12000g discards the liquid in the collection tube;

(7) repeating step (6);

(8) 12000g is centrifugal 1 minute, and another clean 1.5ml EP of separator tube dislocation manages, and adds the TE damping fluid of 20 μ l, 65 ℃

Hatched 10 minutes, centrifugal 1 minute of 12000g gets 2 μ l electrophoresis, and the UVP UV scanning checks recovering effect.Pcr amplification HI ₂The gene result respectively as shown in Figure 1.

4.PCR the clone of product

1) ligation

Reclaiming production concentration according to PCR is respectively 1: 2～10 principle by external source fragment and carrier mole ratio with the pGEX-6p-2 carrier, designs the ligation system as follows:

The purpose fragment connects:

Reclaim fragment (200ng/ μ l)	1μl
		pMD18-T(50ng/μl)	1μl
ddH 2O	3μl
		Connect solution	5μl
Cumulative volume	10μl

16 ℃ connect 3 hours.

2) screening, the evaluation of the conversion of connection product and recon

(1) preparation (CaCl of bacillus coli DH 5 alpha competence bacteria ₂Method)

The aseptic inoculation ring dips-70 ℃ of frozen DH5 α conservation liquid, and the trilinear method streak inoculation is dull and stereotyped in LB, cultivates 12～16 hours for 37 ℃.

The single colony inoculation of picking is in 2ml LB nutrient solution, and 37 ℃ of shaking tables were cultivated 12～16 hours.

With the DH5 α of incubated overnight in 1% ratio transferred species to the LB nutrient solution, 37 ℃ of shaking tables are cultured to OD ₆₀₀Be 0.2～0.4 o'clock, 800g collected bacterium in centrifugal 5 minutes.

The 0.1M CaCl that adds the 1ml precooling ₂Resuspended precipitation, ice-water bath 3 hours.Centrifugal 5 minutes of 4 ℃ of 800g abandon supernatant.The 0.1M CaCl that adds 100 μ l precoolings ₂Suspend and precipitate, ice-water bath 1 hour, for subsequent use.

(2) contain the Amp of X-Gal, IPTG ⁺The preparation of LB flat board

The front fusing of LB solid medium treats that temperature is down to about 50 ℃ that to add Amp be 100mg/L to final concentration, and the mixing hypsokinesis is to dull and stereotyped, natural coagulation.Use and got Amp in front 2～3 hours ⁺LB is dull and stereotyped, adds 40 μ l X-Gal (20mg/ml), 5 μ l IPTG (200mg/ml), with the coating of L rod evenly, places 37 ℃ of incubators for subsequent use.

(3) connecting product transforms

Get respectively simultaneously three each 100 μ l of pipe competence bacteria liquid, the first pipe adds the ligation product, and the second pipe adds contrast inserting paragraph (control insert) DNA and connects product, as positive control, the 3rd pipe does not add foreign DNA, as negative control, and ice-water bath 60 minutes.42 ℃ of water-bath heat-shockeds 90 seconds were placed rapidly ice-water bath 1～2 minute.Every pipe adds 700 μ l LB nutrient solutions, and 37 ℃ of shaking tables were cultivated 1 hour.Mixing precipitated after each pipe centrifugal 1 minute with 800g, suction were abandoned 400 μ l supernatants, respectively got 50 μ l coating Amp ⁺LB is dull and stereotyped, 37 ℃ of incubator overnight incubation.

(4) screening of purpose recon and evaluation

1. picking connection product transforms good indigo plant, the hickie bacterium colony of dull and stereotyped upper separation and inoculates respectively Amp ⁺LB is dull and stereotyped, cultivates 12～16 hours for 37 ℃.Transferred species is in Amp ⁺In the LB nutrient solution, 37 ℃ of shaking table overnight incubation.

2. plasmid DNA extracting (using Omega company plasmid extraction test kit) is the blue hickie plasmid of extracting respectively

Get bacterium liquid and be sub-packed in the 1.5ml centrifuge tube, centrifugal 3 minutes of 12000g leaves and takes precipitation.

Every pipe adds 100 μ l solution I and suspends, and mixing fully vibrates.

Add 100 μ l solution II, soft mixing, ice-water bath 5 minutes.

Add 250 μ l solution III, the mixing that gently shakes, room temperature was placed 10 minutes.

Centrifugal 10 minutes of 4 ℃, 12000g move to supernatant in the separator tube.

Centrifugal 1 minute of 12000g topples over the waste liquid in the collection tube.

Add 500 μ l lavation buffer solutions in separator tube, the same centrifugal and discard waste liquid in the collection tube.

Repeating step 7.

Centrifugal 1 minute of 12000g volatilizees ethanol fully.

Separator tube is placed another clean EP pipe and adds a certain amount of TE damping fluid, 65 ℃ of water-baths 5 minutes, centrifugal 1 minute of 12000g.

Get a certain amount of elutriant and carry out electrophoresis, all the other place-20 ℃ to save backup.

3. enzyme is cut evaluation: respectively locus coeruleus plasmid DNA and hickie plasmid DNA are carried out double digestion.(restriction enzyme is available from Dalian TaKaRa company)

The locus coeruleus plasmid DNA	1μl
		BamHI 2	1μl
10 * damping fluid (K)	1μl
		ddH 2O	7μl
Cumulative volume	10μl

Recombinant plasmid dna	5μl
		NdeI or NcoI	0.5μl
XhoI	0.5μl
		10 * damping fluid (K)	1μl
ddH 2O	3μl
		Cumulative volume	10μl

Behind the mixing, 37 ℃ of water-baths 4 hours.

5.PCR the sequential analysis of product

The positive bacterial strain that transforms of TA clone is delivered to company, extract according to a conventional method plasmid, adopt the terminal cessation method of two deoxidations, Insert Fragment is carried out sequencing.

(2) structure of recombinant gene expression plasmid and efficient expression engineering and screening

1. the structure of recombinant plasmid

To contain the pGEX-6p-2 carrier of goal gene and expression vector pGEX-6p-2 (available from U.S. Novagen company, preserve this chamber) double digestion, enzyme is cut product after 1.0% agarose electrophoresis, purpose fragment glue reclaim purifying, connect with ligase enzyme, transform bacillus coli DH 5 alpha, extract plasmid, double digestion, 1.0% agarose gel electrophoresis is identified.

Relevant operation concrete steps are as follows:

1.1 plasmid DNA extracting (use Omega company plasmid extraction test kit, press the operation of test kit working instructions)

(1) separates good bacterium colony transferred species on the picking flat board in being with corresponding antibiotic LB nutrient solution, 37 ℃ of shaking table overnight incubation;

(2) get bacterium liquid and be sub-packed in the 1.5mL centrifuge tube, centrifugal 3 minutes of 12000g leaves and takes precipitation;

(3) every pipe adds the suspension of 100 μ L solution I, and mixing fully vibrates;

(4) add 100 μ L solution II, soft mixing, ice-water bath 5 minutes;

(5) add 250 μ L solution III, the mixing that gently shakes, room temperature was placed 10 minutes;

Centrifugal 10 minutes of (6) 4 ℃, 12000g move to supernatant in the separator tube;

(7) 12000g is centrifugal 1 minute, topples over the waste liquid in the collection tube;

(8) add 500 μ L lavation buffer solutions in separator tube, the same centrifugal and discard waste liquid in the collection tube, repeated washing once;

(9) 12000g is centrifugal 1 minute, and ethanol is volatilized fully;

(10) separator tube is placed another clean EP pipe and add a certain amount of TE damping fluid, 65 ℃ of water-baths 5 minutes, centrifugal 1 minute of 12000g;

(11) get a certain amount of elutriant and carry out electrophoresis, all the other place-20 ℃ to save backup.

1.2 agarose gel electrophoresis:

1.0% sepharose, 1 * TAE damping fluid, 120-150mA, electrophoresis 20-40 minute.

50 * TAE storage liquid prescription: 2.0mol/L Tris alkali, 1.0mol/L NaAc, 0.1mol/LNa ₂EDTA; Regulate pH8.3 with Glacial acetic acid.

1.3 the endonuclease reaction of plasmid DNA:

1 μ g plasmid DNA

1 μ l10 * K damping fluid (seeing Shanghai living worker company product description)

1 μ l restriction enzyme Nco I/XhoI or NdeI/XhoI(10u/ μ l)

Mend to 10 μ l with distilled water

Mixed rear 37 ℃ of incubation 1-2 hours.

1.4 the target DNA of agarose electrophoresis glue reclaims purifying:

Under ultraviolet lamp, observe and downcut the target DNA electrophoresis band on the sepharose, move into 1.5mL EP pipe.

Add Omega company glue and reclaim the DNA binding buffer liquid of test kit, 65 ℃ of water-baths are dissolved gel fully and are kept pH value of solution between 5.0 ~ 6.0.Sol solutions is moved into separator tube, and centrifugal 1 minute of 12000g discards the liquid in the collection tube.

Add supporting lavation buffer solution, centrifugal 1 minute of 12000g discards the liquid in the collection tube.Repeated washing 1 time.

Centrifugal 1 minute of 12000g, another clean 1.5mL EP pipe of separator tube dislocation, the TE damping fluid of adding certain volume was hatched 10 minutes for 65 ℃, and centrifugal 1 minute of 12000g gets a certain amount of electrophoresis, and the UVP ultraviolet scanner detects and reclaims purification effect.

1.5 ligation (using Shanghai to give birth to worker company dna ligation kit)

By the concentration of UV spectrophotometer measuring target DNA fragment and carrier segments, be generally the principle of 1:2～10 according to external source fragment and carrier mole ratio, design ligation system is as follows:

Target DNA 1 μ l; Plasmid vector 1～2 μ l; Connect solution 5 μ l; DdH ₂O2～3 μ l, cumulative volume 10 μ l.22 ℃ connect 12-16 hour.

1.6 the preparation (CaCl of competence bacteria ₂Method)

(1) the aseptic inoculation ring dips-70 ℃ of frozen bacterium conservation liquid, and the trilinear method streak inoculation is dull and stereotyped in LB, cultivates 12～16h for 37 ℃.

(2) the single colony inoculation of picking is in 2mL LB nutrient solution, and 37 ℃ of shaking tables are cultivated 12～16h.

(3) with the DH5a of incubated overnight in 1% ratio transferred species to the LB nutrient solution, 37 ℃ of shaking tables are cultured to OD ₆₀₀To 0.2～0.4 o'clock, 8000g collected bacterium in centrifugal 5 minutes.

(4) the 0.1mol/L CaCl of adding 1ml precooling ₂Resuspended precipitation, ice-water bath 3 hours.Centrifugal 5 minutes of 4 ℃ of 8000g abandon supernatant.The 0.1mol/L CaCl that adds 100 μ l precoolings ₂Suspend and precipitate, ice-water bath 1 hour, for subsequent use.

1.7 connecting product transforms

(1) gets competence bacteria liquid 100 μ l, add the ligation product; Ice-water bath 60 minutes, 42 ℃ of water-bath heat-shocked 100s placed rapidly ice-water bath 1～2 minute.

(2) add 100 μ l LB nutrient solutions, 37 ℃ of shaking tables were cultivated 1 hour.

(3) with 8000g centrifugal 10 minutes, inhale and abandon mixing precipitation behind the 100 μ l supernatants, respectively get 50 μ l spread plates, 37 ℃ of incubator overnight incubation.

1.8 the Screening and Identification of positive engineering bacteria

(1) picking transforms dull and stereotyped single bacterium colony cultivation

(2) extracting plasmid (method is the same)

(3) double digestion is identified (method is the same)

2. efficient expressed fusion protein engineering bacteria induces and protein expression

The recombinant bacterium identified of learning from else's experience is inoculated in the LB nutrient solution that 10ml contains Amp, and 37 ℃ of shaking tables once activate 10 hours.The recombinant bacterial strain of once activation is contained in the LB nutrient solution of Amp in 1L in 1% ratio transferred species, and 37 ℃ of shaking tables carried out re-activation 5 hours.The bacterium of ratio in 10% after with re-activation is inoculated in the 10L fermentor tank, and substratum is TB, ferments, and 30 ℃ are cultured to the bacterium logarithmic phase with IPTG abduction delivering 4 hours, and SDS-PAGE detects the expression of fusion rotein.

Embodiment two: height crushes bacterium, centrifugal

The colibacillus engineering of the two subunit genetic engineering albumen HI2 of expression solubility methicillin-resistant staphylococcus aureus restructuring that the applicant is made up, by high density fermentation, the target protein expression rate is 13%, centrifugal collection thalline is for subsequent use.

2, thalline 200-500g is with PBS (10-20mM, pH7.0-7.5) damping fluid, by weight: volume ratio 1:10 ratio mixing suspends, 4 ℃ of precoolings.

3, high pressure homogenizer: use distilled water flushing high pressure homogenizer pipeline, the cold cycle system open be chilled in advance 1-4 ℃ for subsequent use.

4, the suspension bacteria liquid of precooling adds high pressure homogenizer, and pressure maintains the broken bacterium of 60-80Mpa 3-5 time, gets brokenly the capable violet staining of bacterium liquid smear, under the oily mirror under each visual field not broken bacterium individual to be considered as brokenly bacterium complete less than 1-2.

5, high speed centrifugation: the centrifugal barrel of packing into of the liquid behind the broken bacterium, 4 ℃, 10,000-15,000 centrifugal 15-30min collects supernatant for subsequent use.

Embodiment three: ammonium sulfate fractional precipitation, redissolution:

1, under 4 ℃ of agitation conditions, slowly add final concentration in the supernatant and be 30% ammonium sulfate, stir more than half an hour, the 10000-15000g high speed centrifugation was collected supernatant more than 20 minutes; Continue slowly to add final concentration in the supernatant and be 40% ammonium sulfate, stir more than half an hour, the 10000-15000g high speed centrifugation is more than 20 minutes, collecting precipitation;

2, precipitation is redissolved: weighing precipitation weight in wet base, and by weight: volume ratio 1:10 ratio adds PBS (10-20mM, pH7.0-7.5) 0.5%Tween80 damping fluid, and stirring and evenly mixing 10-15 minute, the 10000-15000g high speed centrifugation was collected supernatant more than 20 minutes.Sample electrophoresis result after the redissolution shown in Fig. 1-5, HI after the ammonium sulfate fractional precipitation is redissolved ₂Purity of protein reaches about 35%.

Embodiment four: the GST affinity purification:

1, select GST affinity chromatography filler to carry out preliminary purification, the affine filler of GST is one of GST-Sepharose4B, GST-Sepharose6B, GST-Sepharose FastFlow, GST-Sepharose HP, and the broken every 100g amount of filler of bacterium thalline weight in wet base is 100ml.

2, GST combination: select GST affinity chromatography filler to carry out preliminary purification, the supernatant of collecting among the embodiment two is added the Glutathione Sepharose4B gel beads (beads) that is used in conjunction with gst fusion protein, 4 ℃ in conjunction with more than the 3h, and cohesive process adopts the method for vertical rotary or stirring to promote the combination of albumen and beads.

3.Prescission the Protease enzyme is cut target protein and GST label are separated, obtain HI ₂Target protein.

Behind 3-5 volume of above-mentioned protein-bonded beads employing redissolution liquid washing, adopt PBS to continue to wash 3-5 volume, remove the foreign protein of not being combined with beads.Then the PreScission protease(PP enzyme that adds certain volume in the beads), after 4 ℃ of vertical rotary enzymes were cut about 3h, with PBS washing 3 times, 4 ℃ of preservations were used for follow-up polishing purification.Simultaneously, respectively get 10 μ L sample denaturing treatment after, loading 5 μ L carry out the SDS-PAGE protein electrophoresis.Electrophoresis result is shown in Fig. 2 swimming lane 2-2,3,4, and just pure through GST, the purity of target protein is further mentioned, and reaches about 85%, still remains further purifying, removes trace impurity.

Embodiment five: the MMC chromatography purification

The sample that embodiment three collects, adopt HAC that pH is transferred to 3-5, use NaAC damping fluid (10-20mM, pH3-5) balance layer analysis system and MMC chromatography column, until electricity lead with the UV Exponential Stability after loading, till adopting buffer A to wash post behind the end of the sample no longer to descend to UV280, adopt buffer B(20-50mM Na this moment ₂CO _3,PH10.5-11) gradient elution and collect each elution samples is preserved and is carried out the SDS-PAGE electrophoresis for 4 ℃ and identifies.Tomographic map as shown in Figure 3, in conjunction with electrophoresis result (Fig. 5 swimming lane 3), the peak that occurs about pH10.5 is mainly target protein, purity reaches more than 90%.Thus, collect the sample retention at this peak for subsequent use at 4 ℃.

Embodiment six: the gel permeation chromatography purifying:

Use gel-filtration Superdex7526/60 prepackage chromatography column purifying, adopt PBS (10-20mM, pH7-7.5) balance layer analysis system and chromatography column, until electricity lead with the UV Exponential Stability after loading.The sample loading 5-10ml that embodiment four collects collects each elution samples, preserves and carries out the SDS-PAGE electrophoresis for 4 ℃ and identify.Tomographic map is shown in 4, and in conjunction with electrophoresis result (Fig. 5 swimming lane 4), the peak that occurs in the 200-240ml scope is target protein, and purity reaches more than 98%.Thus, the sample of collecting this peak obtains final target protein, i.e. the antigen of purifying.

Although the present invention discloses as above with preferred embodiment; so it is not to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the invention; when can doing a little change and improvement, so the present invention's protection domain is as the criterion when looking the claim person of defining.

Claims (9)

1. a methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI ₂Purification process, it is characterized in that, may further comprise the steps:

A) collect from construction expression antigen HI ₂The thalline of colibacillus engineering high density fermentation;

B) adopt the high bacterium, centrifugal that crushes, the ammonium sulfate fractional precipitation, GST affinity chromatography, PP enzyme are cut, and the sequential combination of MMC chromatography, gel filtration chromatography is carried out purifying to the HI2 of preparation.

2. purification process according to claim 1 is characterized in that, step B is as follows:

1) height crushes bacterium: the coli somatic of the two subunit genetic engineering albumen HI2 of methicillin-resistant staphylococcus aureus restructuring that the collect 10-20mM PBS damping fluid mixing with pH7.0-7.5 is suspended, adopt the broken bacterium of high-pressure homogenization after the precooling, high speed centrifugation is collected supernatant;

2) ammonium sulfate fractional precipitation: under 4 ℃ of agitation conditions, slow adding final concentration is 30% ammonium sulfate in the supernatant, stirs half an hour, and 10000-15000g high speed centrifugation 20 minutes is collected supernatant; The slow adding of continuation final concentration is 40% ammonium sulfate in the supernatant, stirs half an hour, 10000-15000g high speed centrifugation 20 minutes, collecting precipitation;

3) precipitation is redissolved: weighing precipitation weight in wet base, and by weight: volume ratio 1:10 ratio adds the 10-20mM PBS damping fluid of pH7.0-7.5, and stirring and evenly mixing 10-15 minute, 10000-15000g high speed centrifugation 20 minutes was collected supernatant;

4) GST affinity purification: select GST affinity chromatography filler to carry out preliminary purification, use under the PBS-Tween80 pH7.0-7.5 condition target protein is carried out purifying, Prescission Protease enzyme carries out enzyme and cuts wash-out:

5) MMC chromatography purification: the sample that step 4) is collected, pH transfers to 3-5, uses NaAC damping fluid balance layer analysis system and the MMC chromatography column of pH3-5, and employing pH10.5-11 is Na ₂CO ₃Buffer solution for gradient elution;

6) gel permeation chromatography purifying: with the sample of step 5) purifying acquisition, use gel-filtration Superdex chromatography column purifying, adopt 10-20mM PBS balance layer analysis system and the chromatography column of pH7.0-7.5, remove the impurity such as the non-target protein of trace, the separation and purification target protein, the displacement damping fluid.

3. described purification process according to claim 1 is characterized in that: step 1) adopt produce or the pilot scale purifying in the broken bacterium technology of 60-80MPa high-pressure homogenization, the centrifugal bacterium supernatant that obtains brokenly.

4. purification process according to claim 1 is characterized in that: step 2) ammonium sulfate fractional precipitation and step 3) redissolve again.

5. purification process according to claim 1, it is characterized in that: the described GST affinity purification of step 4), employed filler are GST-Sepharose 4B or GST-Sepharose 6B or GST-Sepharose FastFlow or GST-Sepharose HP.

6. purification process according to claim 1, it is characterized in that: the employed Prescission Protease of step 4) enzyme is with the GST label.

7. purification process according to claim 1, it is characterized in that: the described purification process of step 5), the filler of MMC chromatography are Capto ^TMMMC;

8. purification process according to claim 1, it is characterized in that: the described gel chromatography column of step 6) is Superdex75 or Superdex200 or Superdex HR10/30.

9. such as each described purification process of claim 1 to 8, it is characterized in that, described antigen prepares by following steps:

1) design forward primer and reverse primer are synthetic to obtain coding HI by pcr amplification or full gene ₂The nucleotide sequence of protein active fragment;

2) nucleotide sequence that step 1) is obtained is cloned into the expression vector establishment recombinant vectors, then this recombinant vectors is converted into Host Strains;

3) Host Strains behind the Induction Transformation is expressed recombinant protein.

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