CN116789773B - Crude purification method for intracellular expression virus-like particles - Google Patents
Crude purification method for intracellular expression virus-like particles Download PDFInfo
- Publication number
- CN116789773B CN116789773B CN202211519795.8A CN202211519795A CN116789773B CN 116789773 B CN116789773 B CN 116789773B CN 202211519795 A CN202211519795 A CN 202211519795A CN 116789773 B CN116789773 B CN 116789773B
- Authority
- CN
- China
- Prior art keywords
- solution
- target protein
- supernatant
- bacterial liquid
- crude
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000002245 particle Substances 0.000 title claims abstract description 25
- 238000000746 purification Methods 0.000 title claims abstract description 21
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 62
- 239000006228 supernatant Substances 0.000 claims abstract description 50
- 239000007788 liquid Substances 0.000 claims abstract description 48
- 230000001580 bacterial effect Effects 0.000 claims abstract description 43
- 239000002244 precipitate Substances 0.000 claims abstract description 35
- 238000005406 washing Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 56
- 239000000243 solution Substances 0.000 claims description 35
- 239000011780 sodium chloride Substances 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 23
- 239000012452 mother liquor Substances 0.000 claims description 20
- 238000000855 fermentation Methods 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 18
- 239000012460 protein solution Substances 0.000 claims description 18
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 17
- 229920000053 polysorbate 80 Polymers 0.000 claims description 17
- 239000011534 wash buffer Substances 0.000 claims description 15
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 12
- 229920001223 polyethylene glycol Polymers 0.000 claims description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000265 homogenisation Methods 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 7
- 239000008118 PEG 6000 Substances 0.000 claims description 6
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 6
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 6
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000010413 mother solution Substances 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 9
- 239000012501 chromatography medium Substances 0.000 abstract description 4
- 238000011097 chromatography purification Methods 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 238000011068 loading method Methods 0.000 abstract description 2
- 239000002609 medium Substances 0.000 abstract description 2
- 108010042365 Virus-Like Particle Vaccines Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 20
- 238000001962 electrophoresis Methods 0.000 description 16
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 235000019750 Crude protein Nutrition 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 241000320412 Ogataea angusta Species 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 241001052560 Thallis Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 241000700605 Viruses Species 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 241000726445 Viroids Species 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 239000002808 molecular sieve Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 241000701806 Human papillomavirus Species 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 229920002560 Polyethylene Glycol 3000 Polymers 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a crude purification method for intracellular expression of virus-like particles, which comprises the following steps: (1) Preparing a broken bacterial liquid, clarifying the broken bacterial liquid, and collecting a broken bacterial liquid supernatant; (2) adding a precipitant to precipitate the target protein; (3) Washing and redissolving target protein precipitate, and collecting supernatant to obtain crude pure protein liquid. The crude purification method disclosed by the invention is simple and convenient to operate, has a short reaction period, greatly improves the purity of crude and pure protein liquid, reduces the subsequent purification difficulty, prolongs the service life and the loading capacity of the chromatographic medium, and reduces the use cost of high medium in chromatographic purification for scientific research and pharmacy of intracellular virus-like particle vaccines needing purification and biological product enterprises.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical engineering, and particularly relates to a crude and pure method for intracellular expression of virus-like particles.
Background
Virus-like particles (Virus like particle, VLPs for short) are an emerging nanoscale protein assembly, assembled from one or more structural proteins of multiple viruses, with an overall structure similar to that of a viral particle. The VLP has the characteristics of high safety and strong immunogenicity, and is a very promising vaccine. There has been an increasing use in human, veterinary vaccines and drug delivery systems for the last two decades. More than 100 VLPs have been prepared and identified since the 1980 s.
The key to the production of VLP-based biopharmaceuticals is the ability to produce high quality VLPs on a large scale. The preparation of VLP mainly comprises cloning and expressing virus structural genes, selecting host expression systems, purifying, identifying and other links. To ensure that the constructed and synthesized VLPs maintain particle integrity for downstream applications, it is important to select an appropriate purification method.
VLPs are classified according to the presence of non-enveloped structures on the surface of the viral capsid, and can be classified into non-enveloped VLPs and enveloped VLPs. In contrast, enveloped VLP structures are more complex and more challenging in vaccine production. CN109689862a discloses a purification method of EV71 virus-like particles, which comprises the steps of collecting thalli through centrifugation, breaking cells through high-pressure homogenate, precipitating ammonium sulfate, standing overnight, redissolving, ultrafiltering, ion exchange chromatography, molecular sieve chromatography, hydroxylapatite chromatography and other purification steps to obtain a target product; CN103255163B discloses an EV71 virus-like particle, a preparation method and application thereof, the preparation method of the crude pure protein liquid comprises the steps of washing fermentation thalli, crushing fermentation thalli, clarifying, ultrafiltering, precipitating virus particles, standing for 2h, centrifuging and taking the supernatant of the second time to obtain the crude pure protein liquid. Yuqin et al used the protein purification method for studying the immune effect of human papillomavirus 58-type viroid particles recombinantly expressed by Hansenula, as follows: thallus disruption, ammonium sulfate precipitation, dialysis overnight, centrifugation, affinity chromatography (Yuqin, etc., immune effect of human papillomavirus 58-type viroid particles recombinantly expressed by Hansenula polymorpha [ J ] J journal of biological products of China, 2013, 26 (12): 1697-1704).
Although the above purification methods are all final to obtain the target product with excellent yield and performance, the crude and pure protein solution often contains more impurity proteins, nucleic acids and pigments before chromatographic purification. CN104673760B discloses a purification method of prokaryotic cell expression viroid particle, which comprises the steps of homogenate crushing, ammonium sulfate precipitation treatment, stirring for 30 minutes at room temperature, re-dissolving, molecular sieve chromatography, hydroxylapatite chromatography purification, ultrafiltration concentration and heparin affinity chromatography purification in sequence, wherein the purity of target protein is only 60% as found by performing SDS-PAGE and Western blot detection on crude pure protein solution obtained by re-dissolving. When the crude pure protein solution containing more impurities is subjected to the subsequent chromatography step, the chromatography ligand is adsorbed by a large amount of impurity proteins, so that the load of the chromatography column on virus-like particles is obviously reduced, and the more the impurities are adsorbed on a medium, the more the impurities are collected by the chromatography target protein, so that the difficulty of subsequent chromatography purification is greatly increased. Meanwhile, the impurity content of the sample can also reduce the service life of the chromatographic column and increase the cost of the chromatographic medium.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a crude purification method of intracellular expression virus-like particles, which is simple and convenient to operate, short in reaction period, and convenient for scientific research and industrial production and use, and can greatly improve the purity of crude and pure protein liquid of the intracellular expression virus-like particles, reduce the difficulty of subsequent chromatographic purification and prolong the service life of chromatographic media.
The invention provides a crude purification method of intracellular expression virus-like particles, which specifically comprises the following steps:
(1) Preparing a broken bacterial liquid: and (3) suspending the fermentation bacteria in a homogenizing and crushing buffer solution, homogenizing and crushing cells under high pressure to obtain a crushing bacterial solution.
The pH of the homogenate crushing buffer solution is 8-9.2, and the components are 50-200mM Tris-HCl and 2-20mM EDTA-Na 2 0.01-1% Tween-80 or 20-100mM PB,2-20mM EDTA-Na 2 ,0.01-1%Tween-80。
Wherein PB is phosphate buffer solution%NaH 2 PO 4 &Na 2 HPO 4 ）。
(2) Clarifying: centrifuging the crushed bacterial liquid for 10-60min, and collecting the supernatant of the crushed bacterial liquid.
(3) Precipitating the target protein: and (3) respectively adding 2-6M NaCl mother liquor and 20% -60% PEG mother liquor into the supernatant of the crushed bacterial liquid slowly under the stirring state until the final concentration of NaCl is 0.5-1M and the final concentration of PEG is 3-10%, stirring uniformly, immediately centrifuging for 10-30min, removing the supernatant, and collecting target protein precipitate.
(4) Washing the target protein: adding a washing buffer solution into the target protein precipitate, stirring until the mixture is uniform, centrifuging for 10-30min, and collecting the target protein precipitate again.
The washing buffer solution is a mixed solution of a homogenizing and crushing buffer solution, 2-6M NaCl mother solution and 20-60% PEG mother solution, wherein the final concentration of NaCl and PEG in the mixed solution is the same as that of the NaCl and PEG in the step (3).
(5) Redissolving the target protein: adding a redissolving buffer solution into the target protein precipitate obtained in the step (4), stirring until the mixture is uniform, centrifuging for 10-60min, and collecting supernatant to obtain crude pure protein solution.
The pH value of the redissolving buffer solution is 7-8, and the components are as follows: 50-200mM Tris-HCl,0.01-1% Tween-80 or 20-100mM PB,0.01-1% Tween-80.
Wherein the volumes of the washing buffer solution and the re-dissolving buffer solution are the same as the volumes of the supernatant of the broken bacterial liquid in the step (2).
More specifically, the preparation method of the broken bacterial liquid comprises the following steps:
(a) Suspending the fermentation strain in homogenate crushing buffer solution to make the final concentration of the fermentation strain body be 5% -25%;
(b) Disrupting the cells 1 time, preferably 200bar, at a pre-cooling temperature of 4-8deg.C and a homogenization pressure of 100-300 bar;
(c) Circularly crushing cells for 5-10 times under the high-pressure homogenizing pressure of 1300-1500bar until the cell crushing rate reaches 75-95%, so as to obtain crushed bacterial liquid, wherein the optimal crushing conditions are as follows: the mixture was subjected to cyclic crushing for 6 times at 7℃and a homogenization pressure of 1500 bar.
The temperature of the centrifugal force is 2-8 ℃, and the centrifugal force is 10000-30000 Xg.
In the step (3), the PEG solution is any one of PEG2000 solution, PEG3000 solution, PEG4000 solution, PEG6000 solution, PEG8000 solution or PEG10000 solution.
The intracellular expression virus-like particle of the present invention is a norovirus-like particle.
The crude pure protein liquid prepared by the invention can be refined by adopting a conventional refining method (such as ion exchange chromatography, molecular sieve chromatography and the like) during industrial application, thereby obtaining refined intracellular expression virus-like particles.
The beneficial effects of the invention are as follows:
1. the method adopts a specific homogenate crushing buffer solution, a washing buffer solution and a re-dissolving buffer solution, and is matched with a specific process step to crush, precipitate, wash and re-dissolve target proteins, so that most cell fragments, host proteins and host DNA are removed in a short time, and the purity of the target proteins of the crude pure protein solution can reach 91 percent at most.
2. Compared with the traditional process, the method of the invention greatly saves the reaction period, improves the production efficiency, simultaneously avoids the degradation of the target protein during the incubation period, and further improves the yield of the target protein.
3. The method is simple, convenient and rapid, greatly reduces the difficulty of subsequent further purification, prolongs the service life of the chromatographic medium, reduces the production cost, and is suitable for scientific research and industrial production.
Drawings
Fig. 1: non-reducing SDS-PAGE gel analysis of the product of example 1:
wherein, lane 1 is the result of the centrifugal supernatant electrophoresis of the broken bacterial liquid, lane 2 is the result of the centrifugal supernatant electrophoresis after the precipitation of the target protein, and lane 3 is the result of the crude pure protein liquid electrophoresis.
Fig. 2: non-reducing SDS-PAGE gel analysis of the product of example 2:
wherein, lane 1 is the result of the centrifugal supernatant electrophoresis of the broken bacterial liquid, lane 2 is the result of the centrifugal supernatant electrophoresis after the precipitation of the target protein, and lane 3 is the result of the crude pure protein liquid electrophoresis.
Fig. 3: non-reducing SDS-PAGE gel analysis of the product of example 3:
wherein, lane 1 is the result of the centrifugal supernatant electrophoresis of the broken bacterial liquid, lane 2 is the result of the centrifugal supernatant electrophoresis after the precipitation of the target protein, and lane 3 is the result of the crude pure protein liquid electrophoresis.
Fig. 4: non-reducing SDS-PAGE gel analysis of the product of example 4:
wherein, lane 1 is the result of the centrifugal supernatant electrophoresis of the broken bacterial liquid, lane 2 is the result of the centrifugal supernatant electrophoresis after the precipitation of the target protein, and lane 3 is the result of the crude pure protein liquid electrophoresis.
Fig. 5: non-reducing SDS-PAGE gel analysis of the product of comparative example 1:
lane 1 shows the result of the centrifugation supernatant of the disrupted strain solution, lane 2 shows the result of the electrophoresis of the centrifugation supernatant after precipitation of the target protein, and lane 3 shows the result of the electrophoresis of the crude pure protein solution.
Detailed Description
To further verify the effect of the present technical solution, experiments were conducted according to the following purification process, while the description was made by way of comparative example, and finally SDS-PAGE gel electrophoresis analysis was performed on the centrifuged supernatant and crude pure protein solution of the bacterial solutions of each example and comparative example under non-reducing conditions.
The non-reducing SDS-PAGE gel electrophoresis analysis steps are as follows:
taking 30 mu L of a Sample to be detected, adding 10 mu L of Sample Buffer, uniformly suspending by using a suspension device, boiling for 5min, centrifuging, taking 10 mu L of supernatant for Sample loading electrophoresis, taking down gel after electrophoresis is finished, carrying out coomassie brilliant blue dyeing and decoloring, and scanning and analyzing the target protein content of the Sample to be detected by using gel imaging system software.
Example 1
(1) 50g of Hansenula polymorpha nori expressing cell subtype I was added to the homogenization crushing buffer (50 mM PB,6mM EDTA-Na) 2 0.05% Tween-80, pH 9.0), the final volume is fixed to 400mL, the final concentration of the fermentation body mass is 12.5%, and the fermentation body mass is stirred until the fermentation body mass is uniformly mixed. At a pre-cooling temperature of 7 ℃,high-pressure homogenizing 1 time at 200bar to break up the cells; after the bacterial liquid is sufficiently cooled, homogenizing and breaking the walls for 6 times under the homogenizing pressure of 1500bar, and obtaining the broken bacterial liquid.
(2) The disrupted strain solution was centrifuged at 16000 Xg at 2℃for 30min, and the cell debris and other precipitate were discarded to collect 380mL of the disrupted strain solution supernatant.
(3) Under the stirring state, respectively slowly adding 5M NaCl mother liquor and 50% PEG6000 mother liquor into the supernatant of the broken bacterial liquid to make the final concentration of NaCl in the mixed solution be 0.65M and the final concentration of PEG6000 be 5%, stirring until uniformly mixed, immediately centrifuging for 30min at 6 ℃ under 18000 Xg condition, removing supernatant and collecting target protein precipitate.
(4) Adding 5M NaCl mother liquor and 50% PEG6000 mother liquor into the homogenate crushing buffer solution to make the final concentration of NaCl in the mixed solution be 0.65M and the final concentration of PEG6000 be 5% so as to obtain the washing buffer solution.
380mL of washing buffer solution is taken and added into the target protein precipitate, the mixture is stirred until the mixture is uniform, the mixture is centrifuged for 30min at 8 ℃ and 18000 Xg, the supernatant is removed, and the target protein precipitate is collected.
(5) Adding 380mL of redissolving buffer solution (50 mM PB,0.05% Tween-80, pH 7.5) into the target protein precipitate obtained in the step (4), stirring the precipitate, uniformly suspending the precipitate, centrifuging for 60min at 8 ℃ under the condition of 18000 Xg, and collecting supernatant to obtain crude pure protein solution.
As can be seen from FIG. 1, the crude protein solution of example 1 had an electrophoretic purity of 18.8% (lane 3) and a crude protein solution had an electrophoretic purity of 18.8% (lane 1).
Example 2
(1) 50g of Hansenula polymorpha nori expressing cell subtype II was added to the homogenization crushing buffer (100 mM Tris-HCl,5mM EDTA-Na) 2 0.03% Tween-80, pH 8.9), and the final volume is fixed to 500mL, so that the final concentration of the mass of the fermentation thalli is 10%, and stirring until the fermentation thalli are uniformly mixed. Homogenizing at a pre-cooling temperature of 5 ℃ for 1 time by using a high-pressure homogenizer at a pressure of 200bar to crush the bacterial cells; after the bacterial liquid is sufficiently cooledAnd then, carrying out repeated homogenization and wall breaking for 6 times under the homogenization pressure of 1500bar to obtain the broken bacterial liquid.
(2) The disrupted strain solution was centrifuged at 12000 Xg at 8℃for 50 minutes, and the cell debris and other sediments were discarded to collect 450mL of a disrupted strain solution supernatant.
(3) Slowly adding 5.5M NaCl mother liquor and 60% PEG4000 mother liquor into the supernatant of the crushed bacterial liquid under stirring to obtain a mixed solution with NaCl concentration of 1M and PEG4000 concentration of 9%, stirring to uniformly mix, immediately centrifuging at 6deg.C and 18000 Xg for 30min, removing the supernatant, and collecting target protein precipitate;
(4) To the homogenization and disruption buffer solution, 5.5M NaCl mother liquor and 60% PEG4000 mother liquor were added so that the final concentration of NaCl in the mixed solution was 1M and the final concentration of PEG4000 was 9%, to prepare a washing buffer solution.
450mL of washing buffer solution is taken and added into the target protein precipitate, the mixture is stirred until the mixture is uniform, the mixture is centrifuged for 20min at 8 ℃ and 18000 Xg, the supernatant is removed, and the target protein precipitate is collected;
(5) Adding 450mL of redissolving buffer solution (50 mM Tris-HCl,0.05% Tween-80, pH 7.5) into the target protein precipitate obtained in the step (4), stirring the precipitate, uniformly suspending the precipitate, centrifuging for 30min at 8 ℃ and 18000 Xg, and collecting supernatant to obtain crude pure protein solution.
As can be seen from FIG. 2, the crude protein solution of example 2 had an electrophoretic purity of 30.5% (lane 1) and a crude protein solution had an electrophoretic purity of 90.5% (lane 3).
Example 3
(1) 50g of Hansenula polymorpha nori expressing cell subtype III was added to the homogenization crushing buffer (150 mM Tris-HCl, 10mM EDTA-Na) 2 0.06% Tween-80, pH 9.2), and the final volume is fixed to 500mL, so that the final concentration of the fermentation thallus is 12.5%, and the fermentation thallus is stirred until the fermentation thallus is uniformly mixed. Homogenizing at a pre-cooling temperature of 5 ℃ for 1 time by using a high-pressure homogenizer at a pressure of 200bar to crush the bacterial cells; after the bacterial liquid is sufficiently cooled, homogenizing and breaking the walls for 6 times under the homogenizing pressure of 1500bar, and obtaining the broken bacterial liquid.
(2) The disrupted broth was centrifuged at 30000 Xg at 8℃for 10min, and the cell debris and other sediments were discarded, and 460mL of the disrupted broth supernatant was collected.
(3) Under the stirring state, respectively slowly adding the NaCl mother liquor with the concentration of 4M and the PEG8000 mother liquor with the concentration of 40% into the supernatant of the broken bacterial liquid to enable the final concentration of NaCl in the mixed solution to be 0.6M and the final concentration of PEG8000 to be 5%, stirring until the materials are uniformly mixed, immediately centrifuging for 30min at the temperature of 6 ℃ under the condition of 18000 Xg, removing the supernatant, and collecting target protein precipitate.
(4) Adding NaCl mother liquor with the concentration of 4M and PEG8000 mother liquor with the concentration of 40% into the homogenate crushing buffer solution, so that the final concentration of NaCl in the mixed solution is 0.6M and the final concentration of PEG8000 is 5%, and obtaining the washing buffer solution.
460mL of washing buffer solution is taken and added into the target protein precipitate, the mixture is stirred until the mixture is uniform, the mixture is centrifuged for 20min at 8 ℃ and 18000 Xg, the supernatant is removed, and the target protein precipitate is collected.
(5) And (3) adding 460mL of redissolving buffer solution (50 mM Tris-HCl, 0.03% Tween-80 and pH 8.0) into the target protein precipitate obtained in the step (4), stirring the precipitate, uniformly suspending the precipitate, centrifuging for 30min at 8 ℃ and 15000 Xg, and collecting supernatant to obtain crude pure protein solution.
As can be seen from FIG. 3, the crude protein solution of example 3 had an electrophoretic purity of 45.7% (lane 1) and 86% (lane 3).
Example 4
(1) 50g of Hansenula polymorpha nori expressing cell subtype IV was added to the homogenization crushing buffer (200 mM Tris-HCl, 10mM EDTA-Na) 2 The method comprises the steps of carrying out a first treatment on the surface of the 0.02% Tween-80, pH 8.5), and the final volume is fixed to 250mL, so that the final concentration of the fermentation thallus is 20%, and stirring until the fermentation thallus is uniformly mixed. Homogenizing at a pre-cooling temperature of 7deg.C under high pressure with a high pressure homogenizer at 200bar for 1 time to break up the thallus; after the bacterial liquid is sufficiently cooled, the bacterial liquid is circularly homogenized and broken for 7 times under the homogenizing pressure of 1500bar, and the broken bacterial liquid is prepared.
(2) The disrupted broth was centrifuged at 10000 Xg at 2℃for 60min, and the cell debris and other sediments were discarded to collect 220mL of the disrupted broth supernatant.
(3) Under the stirring state, respectively slowly adding 5M NaCl mother liquor and 30% PEG10000 mother liquor into the supernatant of the broken bacterial liquid to make the final concentration of NaCl in the mixed solution be 0.5M and the final concentration of PEG10000 be 4%, stirring until uniformly mixed, immediately centrifuging for 30min at 6 ℃ under 20000 Xg condition, removing supernatant and collecting target protein precipitate.
(4) Adding 5M NaCl mother liquor and 30% PEG10000 mother liquor into homogenate breaking buffer solution to make the final concentration of NaCl in the mixed solution be 0.5M and final concentration of PEG10000 be 4% so as to obtain the invented washing buffer solution.
220mL of washing buffer solution is taken and added into the target protein precipitate, the mixture is stirred until the mixture is uniform, the mixture is centrifuged for 20min at 8 ℃ and 2000 Xg, the supernatant is removed, and the target protein precipitate is collected.
(5) Adding 220mL of redissolving buffer solution (50 mM Tris-HCl,0.05% Tween-80, pH 7.0) into the target protein precipitate obtained in the step (4), stirring the precipitate, uniformly suspending the precipitate, centrifuging for 10min at 6 ℃ and 20000 Xg, and collecting supernatant to obtain crude pure protein solution.
As can be seen from FIG. 4, the crude protein solution of example 4 had an electrophoretic purity of 91.1% (lane 3) with the crushed bacterial cell solution of 46.9% (lane 1).
Comparative example 1
(1) Crushing: the Hansenula polymorpha nod expression bacterial subtype I is resuspended in homogenate disruption buffer (20 mmol/L NaH) 2 PO 4 ，2mmol/L EDTA-Na 2 400mmol/L NaCl,2 mM PMSF,0.1%Tween-80, pH 7.4), and crushing the cells 6 times under the condition of 1400bar pressure by using a high-pressure homogenizer to obtain a crushed bacterial liquid.
(2) Clarifying: the crushed bacterial liquid was poured into a centrifuge tube, centrifuged at 15000 Xg for 50min, and the supernatant was collected.
(3) Precipitation of virus-like particles: ammonium sulfate was added to the supernatant collected above to a concentration of 26% ammonium sulfate, and left overnight.
(4) And (3) re-dissolving: the above-mentioned materials are mixedThe supernatant was decanted from the pellet, the pellet was centrifuged at 10000rpm for 50min, the pellet was collected and added to reconstitution buffer (20 mmol/L NaH) 2 PO 4 0.06mol/L NaCl, pH 7.4), stirring for 40min, centrifuging at 10000rpm for 50min, and collecting redissolved supernatant.
(5) Ultrafiltration: and (3) ultrafiltering the collected redissolved supernatant with a 300KD membrane package by adopting 50mmol/L Tris (pH 8.0) to remove small molecular substances, and collecting ultrafiltrate to obtain crude pure protein liquid.
As can be seen from FIG. 5, comparative example 1 shows that the electrophoretic purity of the crushed bacterial cells from the heart was 16.5% (lane 1), and that of the crude protein from the heart was 68.5% (lane 3).
In conclusion, the purity of the target protein in the crude and pure protein liquid obtained by the crude and pure method is obviously improved compared with that before purification, and can reach more than 90% at most, while the purity of the target protein in the crude and pure protein liquid by the traditional purification method can only reach about 60. The method is simple, convenient and rapid, has obvious advantages, is more beneficial to scientific research and industrial production and use, and has better economic value and application prospect.
Claims (4)
1. A method for the crude purification of intracellular expression of norovirus-like particles, comprising the steps of:
(1) Preparing a broken bacterial liquid: suspending the fermentation bacteria in homogenizing and crushing buffer solution, homogenizing and crushing cells under high pressure to obtain crushed bacteria solution;
the pH value of the homogenizing and crushing buffer solution is 8.5-9.2, and the components are 100-200mM Tris-HCl, 5-10mM EDTA-Na 2 0.02-0.06% Tween-80 or pH9.0, 50mM PB,6mM EDTA-Na 2 ，0.05%Tween-80；
Wherein PB is phosphate buffer solution, and the components are NaH 2 PO 4 And Na (Na) 2 HPO 4 ；
(2) Clarifying: centrifuging the crushed bacterial liquid for 10-60min, and collecting the supernatant of the crushed bacterial liquid;
(3) Precipitating the target protein: slowly adding 4-5.5M NaCl mother liquor and 30-60% PEG mother liquor into the supernatant of the crushed bacterial liquid respectively under stirring until the final concentration of NaCl is 0.5-1M and the final concentration of PEG is 4-9%, stirring uniformly, immediately centrifuging for 10-30min, removing the supernatant, and collecting target protein precipitate;
the PEG mother solution is any one of PEG4000 solution, PEG6000 solution, PEG8000 solution or PEG10000 solution;
(4) Washing the target protein: adding a washing buffer solution into the target protein precipitate, stirring until the mixture is uniform, centrifuging for 10-30min, and collecting the target protein precipitate again;
the washing buffer solution is a mixed solution of homogenizing and crushing buffer solution, 4-5.5M NaCl mother solution and 30-60% PEG mother solution, wherein the final concentration of NaCl and PEG in the mixed solution is the same as the final concentration of NaCl and PEG in the step (3);
(5) Redissolving the target protein: adding a redissolving buffer solution into the target protein precipitate obtained in the step (4), stirring until the mixture is uniform, centrifuging for 10-60min, and collecting supernatant to obtain crude pure protein solution;
the pH value of the redissolving buffer solution is 7-8, and the components are as follows: 50mM Tris-HCl,0.03-0.05% Tween-80 or pH7.5, comprising the following components: 50mM PB,0.05%Tween-80.
2. The method for purifying a crude product of intracellular expression norovirus-like particles according to claim 1, wherein the step of preparing the disrupted bacterial liquid according to step (1) is:
(a) Suspending the fermentation strain in homogenate crushing buffer solution to make the final concentration of the fermentation strain body be 5% -25%;
(b) Crushing cells for 1 time under the homogenization pressure of 100-300bar at the precooling temperature of 4-8 ℃;
(c) Circularly crushing cells for 5-10 times under the high-pressure homogenizing pressure of 1300-1500bar to obtain crushed bacterial liquid.
3. The method of claim 1, wherein the washing buffer and reconstitution buffer are the same volume as the disrupted bacterial supernatant of step (2).
4. The method for the crude purification of intracellular expression norovirus-like particles according to claim 1, characterized in that the centrifugation is at a temperature of 2-8 ℃ and a centrifugal force of 10000-30000 xg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211519795.8A CN116789773B (en) | 2022-11-30 | 2022-11-30 | Crude purification method for intracellular expression virus-like particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211519795.8A CN116789773B (en) | 2022-11-30 | 2022-11-30 | Crude purification method for intracellular expression virus-like particles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116789773A CN116789773A (en) | 2023-09-22 |
| CN116789773B true CN116789773B (en) | 2024-02-20 |
Family
ID=88033279
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211519795.8A Active CN116789773B (en) | 2022-11-30 | 2022-11-30 | Crude purification method for intracellular expression virus-like particles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116789773B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1272538A (en) * | 1999-11-17 | 2000-11-08 | 北京东康龙病毒生物技术工程研究中心 | Quick high-effective separation and purification method of virus related to rocombinant adenovirus and its application |
| CN101023103A (en) * | 2004-09-21 | 2007-08-22 | 赛托斯生物技术公司 | Virus-like particles comprising a fusion protein of the coat protein of ap205 and an antigenic polypeptide |
| CN101330927A (en) * | 2005-11-08 | 2008-12-24 | 诺华疫苗和诊断有限公司 | Manufacture of vaccines that contain both hepatitis b virus surface antigen and surfactant |
| CN102512671A (en) * | 2011-12-16 | 2012-06-27 | 厦门大学 | Anti-foot-and-mouth disease type O virus-like particle vaccine and preparation method thereof |
| RU2012117012A (en) * | 2012-04-27 | 2013-11-10 | Закрытое акционерное общество научно-производственная компания "Комбиотех" | RECOMBINANT VACCINE FOR PREVENTION OF VIRAL HEPATITIS E IN ANIMALS |
| JP2015017066A (en) * | 2013-07-12 | 2015-01-29 | 株式会社Ｕｍｎファーマ | Purification method of virus-like particles |
| CN104583393A (en) * | 2012-06-22 | 2015-04-29 | 武田疫苗股份有限公司 | Purification of virus like particles |
| CN104611358A (en) * | 2015-02-17 | 2015-05-13 | 武汉生物制品研究所有限责任公司 | Method for preparing Norovirus virus-like particles in escherichia coli |
| CN108330145A (en) * | 2017-12-11 | 2018-07-27 | 南京赛威信生物医药有限公司 | The production method of recombination hepatitis B surface antigen |
| CN109680026A (en) * | 2019-02-28 | 2019-04-26 | 深圳鑫泰康生物科技有限公司 | The purifying of recombinant C A16 virus-like particle, the application in vaccine and vaccine |
| CN112806435A (en) * | 2020-12-28 | 2021-05-18 | 青岛今墨堂生物技术有限公司 | Preparation method of milk containing anti-new coronavirus antibody |
-
2022
- 2022-11-30 CN CN202211519795.8A patent/CN116789773B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1272538A (en) * | 1999-11-17 | 2000-11-08 | 北京东康龙病毒生物技术工程研究中心 | Quick high-effective separation and purification method of virus related to rocombinant adenovirus and its application |
| CN101023103A (en) * | 2004-09-21 | 2007-08-22 | 赛托斯生物技术公司 | Virus-like particles comprising a fusion protein of the coat protein of ap205 and an antigenic polypeptide |
| CN101330927A (en) * | 2005-11-08 | 2008-12-24 | 诺华疫苗和诊断有限公司 | Manufacture of vaccines that contain both hepatitis b virus surface antigen and surfactant |
| CN102512671A (en) * | 2011-12-16 | 2012-06-27 | 厦门大学 | Anti-foot-and-mouth disease type O virus-like particle vaccine and preparation method thereof |
| RU2012117012A (en) * | 2012-04-27 | 2013-11-10 | Закрытое акционерное общество научно-производственная компания "Комбиотех" | RECOMBINANT VACCINE FOR PREVENTION OF VIRAL HEPATITIS E IN ANIMALS |
| CN104583393A (en) * | 2012-06-22 | 2015-04-29 | 武田疫苗股份有限公司 | Purification of virus like particles |
| JP2015017066A (en) * | 2013-07-12 | 2015-01-29 | 株式会社Ｕｍｎファーマ | Purification method of virus-like particles |
| CN104611358A (en) * | 2015-02-17 | 2015-05-13 | 武汉生物制品研究所有限责任公司 | Method for preparing Norovirus virus-like particles in escherichia coli |
| CN108330145A (en) * | 2017-12-11 | 2018-07-27 | 南京赛威信生物医药有限公司 | The production method of recombination hepatitis B surface antigen |
| CN109680026A (en) * | 2019-02-28 | 2019-04-26 | 深圳鑫泰康生物科技有限公司 | The purifying of recombinant C A16 virus-like particle, the application in vaccine and vaccine |
| CN112806435A (en) * | 2020-12-28 | 2021-05-18 | 青岛今墨堂生物技术有限公司 | Preparation method of milk containing anti-new coronavirus antibody |
Non-Patent Citations (5)
| Title |
|---|
| Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing;Maria Zahid;《Vaccine》;20150612;第3739-3745页 * |
| Utilização da levedura como sistema de produção de uma proteína da cápside de um novo norovirus canino;Dias Lopes;《minho大学硕士论文》;全文 * |
| Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen;Lünsdorf等;《Microbial Cell Factories》;全文 * |
| 蓝舌病1型病毒灭活疫苗绵羊免疫试验;廖德芳;苗海生;李乐;寇美玲;高林;李华春;;中国动物检疫(07);全文 * |
| 重组诺如病毒P颗粒的表达纯化及免疫原性;金浩;付璐;史晶莹;吴丛梅;吴慧;殷玉和;;吉林大学学报(理学版);20170726(04);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116789773A (en) | 2023-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111249456A (en) | Purification method of rabies virus inactivated vaccine | |
| CN116590346B (en) | Lentiviral vector purification method | |
| WO2018119746A1 (en) | Purification of recombinant ev71 virus-like particle and method for preparing vaccine thereof | |
| CN105420201A (en) | Method for removing Vero cell DNA in vaccine | |
| CN116789773B (en) | Crude purification method for intracellular expression virus-like particles | |
| CN105601735B (en) | A kind of intravenous Giant cell human immunoglobulin and preparation method thereof | |
| US6268492B1 (en) | Methods for purifying non-chromosomal nucleic acid molecules from cells | |
| CN114457132A (en) | Method for preparing starch and non-thermal denatured protein powder by using rice as raw material | |
| JPH051280B2 (en) | ||
| CN114645024B (en) | Method for reducing cell protein and DNA residues in rabies virus products | |
| CN105907729A (en) | Method for preparing human papilloma virus (HPV) L1 protein virus-like particles (VLP) | |
| CN110339351A (en) | The stabilizer that a kind of preparation method of veterinary rabies inactivated vaccine and the vaccine include | |
| CN113603804B (en) | Refining method of streptococcus pneumoniae capsular polysaccharide | |
| CN106905442B (en) | Preparation method of micromolecular beta-1, 3-glucan for improving immunity of hepatitis patients | |
| CN115491311A (en) | Method for crushing cell wall of pichia pastoris and application thereof | |
| CN111575249B (en) | Purification method of novel coronavirus Vero cell inactivated vaccine virus liquid | |
| CN112226418B (en) | Recombinant adeno-associated virus purification method | |
| CN103333938A (en) | Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine | |
| CN114107389A (en) | Recombinant adenovirus expressing African swine fever virus B602L-B646L protein and construction method thereof | |
| CN115261341B (en) | Method for clarifying oncolytic vaccinia virus harvest liquid | |
| CN109666067A (en) | A kind of recombinant virus sample particle and its preparation method and application | |
| CN111166873B (en) | Crude and pure process for recombinant hansenula polymorpha expressed hand-foot-and-mouth disease vaccine antigen, vaccine stock solution and preparation method thereof | |
| CN116768985B (en) | Method for effectively purifying virus-like particles | |
| CN114015659B (en) | Purification method of rabies vaccine | |
| CN117653723A (en) | Purification method and application of recombinant canine distemper virus vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |