CN102260651A - Method for purifying virus-like particle protein and use thereof - Google Patents
Method for purifying virus-like particle protein and use thereof Download PDFInfo
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- CN102260651A CN102260651A CN 201110171728 CN201110171728A CN102260651A CN 102260651 A CN102260651 A CN 102260651A CN 201110171728 CN201110171728 CN 201110171728 CN 201110171728 A CN201110171728 A CN 201110171728A CN 102260651 A CN102260651 A CN 102260651A
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Abstract
The invention belongs to the field of biotechnology, and particularly relates to a method for purifying a virus-like particle protein and use thereof. The high-efficiency method for purifying the virus-like particle protein is constructed on the basis of a low-pH buffer system, and comprises: placing a sample containing virus-like particles in the low-pH buffer system which provides a strong acid buffer environment to denature an absolute major part of other proteins and to break the absolute major part of the other proteins into pieces, and performing further acidification to improve the impurity of the protein to about 80 percent; removing the denatured proteins and segments by simple centrifugation and further separation and further chromatography; and thus obtaining the purified virus-like particle protein. The invention also discloses the use of the method in the purification of Q beta bacteriophage virus-like particle protein, Q beta-2alpha alpha bacteriophage virus-like particle protein and HK-97 bacteriophage virus-like particle protein. The purifying method is simple, low in cost and high in purification efficiency and has a very bright application prospect.
Description
Invention field
The invention belongs to biological technical field, relate to a kind of method and application of adopting low pH buffer system purified virus sample granule protein.
Background of invention
Therapeutic vaccine at chronic disease is one of focus of present vaccine research, and these diseases comprise: chronic viral infection, anaphylactic disease, tumour, diabetes, hypertension and alzheimer's disease etc.Different with common preventative vaccine is: therapeutic vaccine at be autoantigen or ectophylaxination tolerance antigen, therefore, therapeutic vaccine will be brought into play therapeutic action must at first break immunological tolerance, produces autoantibody or id reaction T cell at specific antigen.Since therapeutic vaccine institute at target antigen generally be small peptide, antigenicity a little less than, must be with after suitable carriers combines, competence exertion effect under the help of immunological adjuvant, therefore, selection suitable carriers and adjuvant be the key of therapeutic vaccine success often.
Carrier such as KLH, Toxoid,tetanus etc. commonly used at present, render a service generally not strong, must use adjuvant, though and adjuvant such as freund's adjuvant usefulness commonly used are stronger, but can not be applied to human body, though the aluminum adjuvant can be applicable to human body, immunizing potency is very weak, and exists as untoward reactions such as cerebral arteriosclerosis, aluminium salt local accumulation.Therefore, ideal therapeutic vaccine carrier should under the situation of not using immunological adjuvant, just can effectively be broken the immunological tolerance of body with after target antigen combines, and produces high titre antibody, the performance therapeutic action.
The virus-like particle class ideal vaccine carrier that comes to this.Virus-like particle (VLP) is the hollow shell structure that does not contain viral nucleic acid that is assembled into by viral capsid proteins, profile and immunogenicity with virus, can induce the reaction of body generation based on humoral immunization, thereby produce at bonded antibody with it, owing to do not have the nucleic acid component of virus, therefore avoided the risk of virus replication propagation.Virus-like particle is formed by the capsid protein monomer polymerization of a large amount of repeating structures, can be with specific b cells surface receptor (BCR) extensively cross-linked and activate the B cell, carry out angtigen presentation with T cell dependent/non-dependent (TI) and T cell dependency (TD) dual mode, and can make target antigen high-sequential and multiple be illustrated in its surface as exogenous t cell epitope, help breaking through the autoimmunization tolerance, body immune system there is powerful hormesis, so the use of virus-like particle more and more widely.Prepared multiple phage virus-like particle at present both at home and abroad, as Q phagus beta virus-like particle, human papillomavirus virus-like particles (HPV), MS2 virus-like particle etc.Switzerland Cytos company is that carrier has been developed a series of therapeutic vaccines with Q phagus beta virus-like particle, comprise alzheimer's disease AD vaccine (CAD106), Nicotine vaccine (NIC002), allergic asthma vaccine (CYT003-QbG10) etc., the step-down vaccine (CYT006-AngQb) at Angiotensin II of its development wherein, finished IIa phase clinical experiment, finished IIb phase clinical experiment at the vaccine (CYT003-QbG10) of allergia nose's conjunctivitis development.And the main vaccine composition of the vaccine of cervical cancer Cervarix (TM) that has gone on the market of Belgian GlaxoSmithKline PLC company (GSK) development is exactly HPV-16 and HPV-18.These virus-like particles have shown that powerful immunogenicity and people use security afterwards.
Along with the needs to proteinic research and production, separation and purification more shows its importance as Genetic engineering downstream procession in recent years.According to statistics, the separation and purification cost of gene engineering product accounts for the 60-80% of its overall cost, is a step the most consuming time in the protein preparation process, that cost is maximum.Virus-like particle faces such problem equally as proteinic a kind of.Present purification process commonly used substantially all is applicable to the purifying of virus-like particle, but need design and explore at the feature and the preparation cost problem of each virus-like particle.Switzerland Cytos company is at Q β virus-like particle, set up the technology of its purifying, its main purification process is: tangential flow (TFF) is centrifugal, anion-exchange chromatography (AIX), endotoxin removal (HAPA), hydrophobic interaction chromatography (HIC) and gel permeation chromatography (SEC).Though through obtaining the virus-like particle of 99% above purity after above 5 steps, its cost cost is huge.
Do not see the report of relevant a kind of efficient, virus-like particle purification process that cost is low up to now as yet.
Summary of the invention
Purpose of the present invention is intended to overcome the defective of prior art, satisfies the needs to virus-like particle, and a kind of method of efficient, purified virus sample granule protein that cost is low is provided.
The present invention is achieved through the following technical solutions:
The method of this sample of purified virus efficiently granule protein is based upon on the low pH buffer system.This low pH buffer system can be cushioned constituting, formic acid/acetate/dihydrogen phosphate-formate/acetate/monohydric phosphate, formic acid-formate buffering, acetate-acetate buffering by citric acid-Citrate trianion buffering.The sample that will contain virus-like particle directly places low pH buffer system or places low pH buffer system indirectly by dialysis tubing, and the volume of buffer system is 20-500 a times of sample volume.The pH value of this low pH buffer system is between 2.4-4.0, and this strongly-acid buffer environment can make most foreign protein sex change and fragmentation, and after a step acidifying, albumen just can reach the purity about 80%.Metaprotein and fragment are removed with the further chromatography that separates by simply centrifugal (10, centrifugal 10 minutes of 000g), the virus-like particle protein behind the acquisition purifying is identified its purity by SDS-PAGE.This method can be used for the precursor of purifying Q phagus beta virus-like particle protein, Q β-2aa phage virus-like particle protein, HK-97 phage virus-like particle protein wild-type and the precursor and the ripe body of ripe body, the chimeric precursor of HK-97 phage virus-like particle protein and ripe body and HK-97 phage virus-like particle protein mutant.
Compared with prior art, the present invention has the following advantages:
1, the ion-exchange of the present invention and routine, hydroxyapatite, density gradient centrifugation etc. are separated method and are compared, and have characteristics simple to operate, with low cost.
2, the present invention can carry out purifying to multiple virus-like particle protein, and very high purification efficiency is arranged, and lays a good foundation for enlarging to produce.
Description of drawings
Fig. 1: the SDS-PAGE electrophoresis purity contrast of Q β-2aa phage virus-like particle behind serial purifying.M is albumen Marker, 1 swimming lane is a bacterium cracking supernatant behind the abduction delivering, 2 swimming lanes are sample behind the ammonium sulfate precipitation, 3 swimming lanes are supernatant after the acidifying, 4 swimming lanes are sample behind the hydrophobic chromatography, and 5 swimming lanes are sample after the gel-filtration, and 6 swimming lanes are that purifying concentrates the back sample, the about 14KDa of target protein size, last purity is about 95%.
Fig. 2: different pH values (pH2.4, pH3.8, pH8.07) under the condition, Q β-2aa virus-like particle circular dichroism spectrum result.
Fig. 3: with the Q β-2aa phage virus-like particle of transmission electron microscope observing behind serial purifying (comprising acidifying).Virus-like particle transmission electron microscope diagram shows that Q β-2aa virus-like particle can be assembled into spheroidal particle voluntarily, and diameter is approximately 30nm.
Fig. 4: sample after the sample (0min) after the acidifying of HK-97 virus-like particle, the neutralization (5min, 10min, 30min, 60min 2hrs) carries out the SDS-PAGE electrophoresis, identifying virus sample particle-precursors, intermediate and ripe body and purity thereof.
Fig. 5: with form and the size after the transmission electron microscope observing HK-97 acidifying maturation.The similar sphere of form of ripe body HK-97, tenui-exinous, the about 66nm of diameter.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and embodiment, but be not restriction the present invention.
The acidifying of case study on implementation 1 Q β-2aa phage virus-like particle
The acidifying separating step of Q β-2aa phage virus-like particle is as follows:
1) citric acid-Citrate trianion acidifying damping fluid of the 40mM of preparation pH3.6, its prescription is: C
6H
8O
7H
2O 8.42g (1L), C
6H
5Na
3O
72H
2O 11.75g (1L), the former with the latter accurately mixes with 2: 1 volumes under the room temperature;
2) dissolved behind the ammonium sulfate preliminary purification is precipitated in the dialysis tubing (14,000 molecular retention sizes) of packing into, put into the citric acid-Citrate trianion acidifying damping fluid of step 1), 4 ℃ of dialysis were changed liquid once every 6 hours, totally 2 times;
3) 12, centrifugal 10 minutes of 000rpm discards precipitation, repeated centrifugation once, supernatant is with the PBS of pH7.4 dilution, obtains the sample after the acidifying.Get supernatant after the bacterium cracking, the ammonium sulfate sample behind the sample and gel-filtration that slightly gets sample behind sample after product, the acidifying, the hydrophobic interaction chromatography respectively, carry out the SDS-PAGE electrophoresis, the SDS-PAGE electrophoresis is the purity of sample (Fig. 1) more separately.
Secondary structure after case study on implementation 2 Q β-2aa virus-like particle acidifying
Q β-2aa phage virus-like particle is at three kinds of different pH value (pH2.4, pH3.8, pH8.07) purifying obtains down, after subsequent purification, its secondary structure is carried out the analysis of circular dichroism spectrometry, find that Q β-2aa virus-like particle has all shown highly consistent secondary structure similarity (Fig. 2).
Form after case study on implementation 3 Q β-2aa-VLP acidifying
Make through the Electronic Speculum of Q β-2aa phage virus-like particle that purifying is good under pH3.8 and the pH8.07 condition and drip sheet (copper mesh, the uranyl acetate negative staining), under transmission electron microscope, carry out the observation of form and general structure, find structure unanimity under both Electronic Speculum, all about 30nm of diameter (Fig. 3).
The acidifying maturation of case study on implementation 4 HK-97VLP
The precursor PII-HK97 of HK97 phage virus-like particle could be ripe external need inducing through peracidity, and acidifying also can be used as its good means of purification as itself sophisticated step so.The acidifying of PII-HK97 virus-like particle precursor induces step to be: 1.5ml is diluted in 150ml acidifying damping fluid (pH3.8 with 30mg/ml PII-HK97 virus-like particle precursor, 250mMKCl, 50mM Citrate) in 1 hour, neutralization buffer (the pH8.3 that adds 1/6 volume subsequently, 1M Tris-HCl) pH of extremely total system is more than 7.0, room temperature was placed 2 hours, can place afterwards 4 ℃ 24 hours.Get the sample (0min) after the acidifying, neutralization back sample (5min, 10min, 30min, 60min 2hrs) carries out SDS-PAGE electrophoresis identifying virus sample particle-precursors, intermediate and ripe body and purity (Fig. 4) thereof, ripe body HK-97 form of transmission electron microscope observing and size (Fig. 5).
Claims (3)
1. the method for a purified virus sample granule protein is characterized in that it may further comprise the steps:
1) secure ph is in the low pH buffer system of 2.4-4.0;
2) sample that will contain virus-like particle directly places the low pH buffer system of step 1) or places low pH buffer system indirectly by dialysis tubing, and 4 ℃ of dialysis were changed liquid once every 6 hours, totally 2 times;
3) 10, centrifugal 10 minutes of 000rpm discards precipitation, repeated centrifugation once, supernatant is with the PBS of pH7.4 dilution, obtains the sample after the acidifying.
2. the method for a kind of purified virus sample granule protein according to claim 1 is characterized in that described low pH buffer system, can be by following buffering to constituting:
(a) citric acid-Citrate trianion buffering is right;
(b) acetate-acetate buffering is right;
(c) formic acid-formate buffering is right;
(d) formic acid/acetate/dihydrogen phosphate-formate/acetate/monohydric phosphate buffering is right.
3. the method for a kind of purified virus sample granule protein according to claim 1 is characterized in that its application at purifying Q phagus beta virus-like particle protein, Q β-2aa phage virus-like particle protein and HK-97 phage virus-like particle protein.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105339495A (en) * | 2013-07-11 | 2016-02-17 | 宝生物工程株式会社 | Method for manufacturing non-enveloped virus |
| US10023846B2 (en) | 2014-07-10 | 2018-07-17 | Takara Bio Inc. | Production method for non-enveloped virus particles |
| US10167320B2 (en) | 2012-06-22 | 2019-01-01 | Takeda Vaccines, Inc. | Purification of virus like particles |
| US10172930B2 (en) | 2007-03-14 | 2019-01-08 | Takeda Vaccines, Inc. | Virus like particle purification |
| US10415020B2 (en) | 2015-01-09 | 2019-09-17 | Takara Bio Inc. | Method for producing non-enveloped viral particles |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009080781A1 (en) * | 2007-12-21 | 2009-07-02 | Cytos Biotechnology Ag | Process for clarifying cell homogenates |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009080781A1 (en) * | 2007-12-21 | 2009-07-02 | Cytos Biotechnology Ag | Process for clarifying cell homogenates |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10172930B2 (en) | 2007-03-14 | 2019-01-08 | Takeda Vaccines, Inc. | Virus like particle purification |
| US10792353B2 (en) | 2007-03-14 | 2020-10-06 | Takeda Vaccines, Inc. | Virus like particle purification |
| US10167320B2 (en) | 2012-06-22 | 2019-01-01 | Takeda Vaccines, Inc. | Purification of virus like particles |
| AU2013277959B2 (en) * | 2012-06-22 | 2019-05-16 | Takeda Vaccines, Inc. | Purification of virus like particles |
| US11091519B2 (en) | 2012-06-22 | 2021-08-17 | Takeda Vaccines, Inc. | Purification of virus like particles |
| CN105339495A (en) * | 2013-07-11 | 2016-02-17 | 宝生物工程株式会社 | Method for manufacturing non-enveloped virus |
| US10072250B2 (en) | 2013-07-11 | 2018-09-11 | Takara Bio Inc. | Method for manufacturing non-enveloped virus |
| US10023846B2 (en) | 2014-07-10 | 2018-07-17 | Takara Bio Inc. | Production method for non-enveloped virus particles |
| US10415020B2 (en) | 2015-01-09 | 2019-09-17 | Takara Bio Inc. | Method for producing non-enveloped viral particles |
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Application publication date: 20111130 |