CN108823245A - A kind of purification process of virus-like particle - Google Patents
A kind of purification process of virus-like particle Download PDFInfo
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- CN108823245A CN108823245A CN201810618077.3A CN201810618077A CN108823245A CN 108823245 A CN108823245 A CN 108823245A CN 201810618077 A CN201810618077 A CN 201810618077A CN 108823245 A CN108823245 A CN 108823245A
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Abstract
The present invention relates to field of immunology more particularly to a kind of purification process of virus-like particle.This method successively chromatographs the liquid containing virus-like particle through sucrose concentration, gel chromatography, ceramic hydroxyapatite.Ceramic hydroxyapatite has not yet to see the purifying for virus-like particle.And the ceramic hydroxy-apatite of present invention application shortens purification step, successful purification goes out the virus-like particle for having similar morphology feature with natural viral, and purification process yield is higher, and purified product purity is good.
Description
The application be the applying date be 2015.06.01, entitled " a kind of purification process of virus-like particle ", application
Number for 201510291894.9 invention divisional application.
Technical field
The present invention relates to field of immunology more particularly to a kind of purification process of virus-like particle.
Background technique
Vaccines classes common at present have genetic engineering subunit vaccine, DNA vaccination, virus sample particle vaccines, attenuated live
Vaccine.Wherein, virus-like particle (virus-like particles, VLPs) is one or more structures containing certain virus
The hollow bead of albumen cannot be replicated independently without viral nucleic acid.Since VLPs is similar with natural viral form, largely
On maintain the natural structure of albumen, and VLP antigenic determinant and epitope and really virus does not have an essential distinction, therefore can be with
Induction excites stronger cellular immunity and humoral immunity.Also, since VLPs does not contain virus gene genome nucleic acid, there is phase
To higher safety.Currently, VLPs vaccine is considered most potential vaccine.
Although virus-like particle and live virus have a similar morphological structure, the property of virus-like particle protein may be with
Live virus is variant, since the space structure that virus-like particle is assembled into is that ability forms it into regular dodecahedron structure mesh
It is preceding to be also not very clear, and its size is close with baculoviral size.This just gives the purifying in virus-like particle preparation process
Bring many difficulties.
Existing result of study shows that conventional ion-exchange chromatography is selected to be difficult to separate destination protein and foreign protein
Come, the purifying of VLPs is usually required by multiple steps.However, one step of every increase will be treated in purification process
Purified causes certain loss, and step is more, and yield is lower.And the generation of virus-like particle passes through insect or yeast table mostly
It is obtained up to system, if way of purification is improper, then is difficult to obtain virus-like particle, this undoubtedly limits the wide of virus sample particle vaccines
General application.
Hydroxyapatite be by calcium and phosphate at compound.In ceramic hydroxyapatite (CHT), phosphate ion and band
The protein of positive electricity is combined with ionic bond, is had ion exchange property, can be washed by NaCl concentration gradient or sodium phosphate concentration gradient
It is de-, Ca therein2+Ion is combined in a manner of metal-chelating with the free carboxyl group of negatively charged protein, the combination to NaCl not
Sensitivity, can be by phosphoric acid, na concn gradient elution.Hydroxyapatite is divided into 2 seed types because of ceramming process difference：I type and II type.
Existing result of study thinks that ceramic hydroxyapatite I type has bigger reservation to protein, has to acidic protein bigger
Dynamic carrying capacity, therefore, ceramic hydroxyapatite I type be mainly used for purifying most of protein (molecular weight generally 100kd with
Under)；Ceramic hydroxyapatite II type is since aperture is big compared with I type, thus to the large molecular weight proteins such as antibody and part recombinant vaccine
The dynamic carrying capacity of matter is higher, and almost without reserve to HSA, thus ceramic hydroxyapatite II type is more suitable for the purifying of antibody,
II type has bigger reservation to nucleic acid simultaneously, can offer an explanation the DNA of the various higher structures such as single, double chain, supercoil, thus
It is suitble to purification of nucleic acid but current it is believed that ceramic hydroxyapatite loss amount in purification process is excessively high, therefore, there is no will make pottery
Ceramic hydroxyl apatite is applied to the report of virus-like particle.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of purification process of virus-like particle.The party
The method rate of recovery is high, virus-like particle purity obtained is big.
The purification process of virus-like particle provided by the invention, includes the following steps：By the liquid containing virus-like particle
Successively chromatographed through sucrose concentration, gel chromatography, ceramic hydroxyapatite.
Since ceramic hydroxyapatite has good capture power and selectivity, it is indissociable that other media can be separated
The various difficult separation of comparison, therefore the close biomolecule of Nature comparison can greatly reduce purification step using CHT purifying,
And products therefrom purity is higher.Nevertheless, existing result of study shows the method purifying chromatographed using ceramic hydroxyapatite
Sample loss amount is higher, and yield is lower, and therefore, ceramic hydroxyapatite chromatography is generally unsuitable for purifying, and concentration is low, content is few
Substance.And virus-like particle usually passes through fungi or insect cell expression generates, yield is usually lower, therefore at present not yet
There is the purifying that ceramic hydroxyapatite chromatography is used for virus-like particle.The present invention using CHT chromatograph, by virus-like particle with
CHT specific adsorption removes most of high molecular weight protein and impurity protein through elution.In an embodiment of the present invention, ceramic hydroxyl
The filler of base apatite chromatography is CHT I type.
In an embodiment of the present invention, the equilibrium liquid of ceramic hydroxyapatite chromatography is PBS buffer solution；Wherein NaCl's is dense
Degree is 0.1mol/L~0.5mol/L；PH value is 7.0~8.5.
In some embodiments, ceramic hydroxyapatite chromatography equilibrium liquid in NaCl concentration be 0.2mol/L~
0.5mol/L, pH value 8.5.
Preferably, the concentration of NaCl is 0.5mol/L in the equilibrium liquid of ceramic hydroxyapatite chromatography
In further embodiments, ceramic hydroxyapatite chromatography equilibrium liquid in NaCl concentration be 0.1mol/L~
0.5mol/L, pH value 8.0.
Preferably, the concentration of NaCl is 0.4mol/L in the equilibrium liquid of ceramic hydroxyapatite chromatography.
In an embodiment of the present invention, the eluent of ceramic hydroxyapatite chromatography is PBS buffer solution；Wherein NaCl's is dense
Degree is 1mol/L；PH value is 7.0~8.5.
In some embodiments, the pH value of the eluent of ceramic hydroxyapatite chromatography is 8.0.
PBS buffer solution, i.e. phosphate buffered saline solution, including NaCl, KCl, Na2HPO4And KH2PO4.The present invention uses
PBS buffer solution in, the concentration of KCl is 2.7mmol/L, Na2HPO4Concentration be 10mmol/L, KH2PO4Concentration be
2mmol/L.Experiments indicate that KCl, Na in fixed PBS buffer solution2HPO4And KH2PO4Concentration, and adjust therein
The concentration of NaCl balances chromatographic column with the PBS of low NaCl concentration, elutes virus-like particle energy with the PBS of high NaCl concentration
Enough reach good purification effect, and can guarantee yield.Experiment shows the method being supplied to using the present invention, with ceramic hydroxyl
The yield of the method purified virus sample particle of base apatite chromatography is 86.31%, and purity can be promoted to by 60%~70%
95%.
In an embodiment of the present invention, the filler of gel chromatography is superdex-500.
In gel chromatography step, collects void volume and flow through peak.After gel chromatography, some small molecules can be removed
Albumen.
In an embodiment of the present invention, gel chromatography equilibrium liquid is PBS buffer solution；Wherein the concentration of NaCl is 0.2mol/L
~0.5mol/L；PH value is 7.0~8.5.
In some embodiments, the concentration of NaCl is 0.5mol/L, pH value 8.5 in the equilibrium liquid of gel chromatography.
The main purpose of sucrose concentration is to remove lipid and some foreign proteins in order to which destination protein to be enriched with.Sucrose
Concentration and centrifugation speed can to concentration result have an impact.
In an embodiment of the present invention, sucrose, which is concentrated, is specially：By the liquid containing virus-like particle and contain sucrose
PBS buffer solution mixing, 27500rpm are centrifuged 3~4 hours；The mass fraction of sucrose is 30% in PBS buffer solution containing sucrose.
In the PBS buffer solution that sucrose concentration uses, the concentration of KCl is 2.7mmol/L, Na2HPO4Concentration be 10mmol/
L, KH2PO4Concentration be 2mmol/L, the concentration of NaCl is 137mmol/L.
It is concentrated through sucrose, 10~50 times of sample concentration.In some embodiments, 20 times are concentrated through sucrose concentrating sample.
Purification process provided by the invention is used for CVA16 virus-like particle.
Preferably, purifying CVA16 virus-like particle, the concentration of NaCl is in the equilibrium liquid of ceramic hydroxyapatite chromatography
0.1mol/L~0.5mol/L, pH value 8.5.
Preferably, CVA16 virus-like particle is purified, the concentration of NaCl is in the equilibrium liquid of ceramic hydroxyapatite chromatography
0.4mol/L。
In an embodiment of the present invention, the preparation method of the liquid containing virus-like particle is：With the base containing CVA16 mesh
The baculovirus infection insect cell of cause is cultivated to 30%~40% cell and lesion occurs；Cell is harvested by centrifugation, is frozen repeatedly
After melting, the liquid containing virus-like particle is made through filtering in centrifugation removal residue.
Purification step can be effectively reduced using method provided by the invention purifying CVA16 virus-virus, it is sick after purified
The purity of malicious sample particle is not less than 95%, and total recovery is not less than 31%.
The present invention also provides a kind of preparation methods of CVA16 virus-like particle, include the following steps：
Step 1：The P1 segment of CVA16 virus and 3CD segment are built into pFastBAcdual carrier, with Escherichia coli
The recombination of DH10BAC swivel base, obtains the expression vector containing CVA16 target gene；
Step 2：With the expression vector transfection insect cell containing CVA16 target gene, culture, passage are contained
The baculoviral of CVA16 target gene；
Step 3：With the baculovirus infection insect cell containing CVA16 target gene, cultivate to 30%~40% it is thin
There is lesion in born of the same parents；Cell is harvested by centrifugation, after multigelation, centrifugation removal residue is filtered to be made and contains virus-like particle
Liquid；
Step 4：By the liquid containing virus-like particle successively through sucrose concentration, gel chromatography, ceramic hydroxyapatite layer
Analysis；CVA16 virus-like particle is made.
The nucleotide sequence of the P1 segment of CVA16 virus such as SEQ ID NO:Shown in 1, it is built into pFastBAcdual carrier
The restriction enzyme site used is Not I and Hind III.
The 3CD segment of CVA16 virus such as SEQ ID NO:Shown in 2, it is built into the digestion of pFastBAcdual carrier use
Site is NCo I and Xho I.
In embodiment provided by the invention, insect cell is SF9 cell.
In some embodiments, the time cultivated in step 2 is 3~4 days；The number of passage is 2 times.
In some embodiments, insect cell is activated in step 3, and specific method is：The culture of SF9 cell line is shaken in 1L
In bottle, by 1：3 ratios point kind, culture are inoculated in WAVE bioreactor and cultivate for 4 days.
In some embodiments, the inoculum concentration of the baculovirus infection insect cell in step 3 containing CVA16 target gene
MOI is 0.5~1.
In some embodiments, the temperature cultivated in step 3 is 27 DEG C, and the time is 72h~96h.
In some embodiments, the number of multigelation is 3~4 times in step 3.
In some embodiments, filtering uses 0.45 μm of filter membrane in step 3.
In some embodiments, the filler of ceramic hydroxyapatite chromatography is CHT I type.
In some embodiments, the equilibrium liquid of ceramic hydroxyapatite chromatography is PBS buffer solution；Wherein the concentration of NaCl is
0.1mol/L~0.5mol/L；PH value is 7.0~8.5.
In some embodiments, ceramic hydroxyapatite chromatography equilibrium liquid in NaCl concentration be 0.2mol/L~
0.5mol/L, pH value 8.5.
Preferably, the concentration of NaCl is 0.4mol/L in the equilibrium liquid of ceramic hydroxyapatite chromatography
In some embodiments, the eluent of ceramic hydroxyapatite chromatography is PBS buffer solution；Wherein the concentration of NaCl is
1mol/L；PH value is 7.0~8.5.
In some embodiments, the pH value of the eluent of ceramic hydroxyapatite chromatography is 8.4.
In some embodiments, the filler of gel chromatography is superdex-500.
In some embodiments, gel chromatography equilibrium liquid is PBS buffer solution；Wherein the concentration of NaCl be 0.2mol/L~
0.5mol/L；PH value is 7.0~8.5.
In some embodiments, the concentration of NaCl is 0.4mol/L, pH value 7.4 in the equilibrium liquid of gel chromatography.
In an embodiment of the present invention, sucrose, which is concentrated, is specially：By the liquid containing virus-like particle and contain sucrose
PBS buffer solution mixing, 27500rpm are centrifuged 3~4 hours；The mass fraction of sucrose is 30% in PBS buffer solution containing sucrose.
In the PBS buffer solution that sucrose concentration uses, the concentration of KCl is 2.7mmol/L, Na2HPO4Concentration be 10mmol/
L, KH2PO4Concentration be 2mmol/L, the concentration of NaCl is 137mmol/L.
It is concentrated through sucrose, 10~50 times of sample concentration.In some embodiments, 20 times are concentrated through sucrose concentrating sample.
The present invention also provides a kind of CVA16 virus sample particle vaccines, including made from preparation method provided by the invention
CVA16 virus-like particle and adjuvant.
In some embodiments, adjuvant is Al (OH)3Adjuvant.
Preferably, virus-like particle and Al (OH)3The mass ratio of adjuvant is (0.5~1): (250~500).
Preferably, the immunizing dose of CVA16 virus sample particle vaccines provided by the invention is 0.5 μ g/ times~1 μ g/ times.
Purification process provided by the invention being capable of effective purified virus sample particle, removal cell fragment and non-purpose egg
White, therefore, vaccine obtained can have good quality in this way.
The present invention provides a kind of purification process of virus-like particle, this method by the liquid containing virus-like particle successively
Through sucrose concentration, gel chromatography, ceramic hydroxyapatite chromatography.Ceramic hydroxyapatite has not yet to see for virus-like particle
Purifying,.Method provided by the invention shortens purification step, and successful purification goes out virus-like particle, and purification process yield is higher,
Purified product purity is good.
Detailed description of the invention
3 ceramic hydroxyapatite chromatographic analysis figure of Fig. 1 embodiment；
The virus-like particle that Fig. 2 efficient liquid phase chromatographic analysis embodiment 3 purifies.
Specific embodiment
The present invention provides a kind of purification process of virus-like particle, those skilled in the art can use for reference present disclosure,
It is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications carry out those skilled in the art
Say it is it will be apparent that they are considered as being included in the present invention.Method and application of the invention has passed through preferred embodiment
Be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to methods herein and application into
Row change or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.
Below with reference to embodiment, the present invention is further explained：
The preparation of liquid of the embodiment 1 containing CVA16 virus-like particle
One, preparation and reorganization baculoviral
CVA16 epidemic strain is needed, the genbank accession number of gene chemical synthesis full-length genome, full-length genome is：AF177911.1.
Design primer expands P1 segment and 3CD segment, and restriction enzyme site is added.
Wherein, P1 fragment sequence such as SEQ ID NO:Shown in 1, restriction enzyme site is Not I and Hind III., amplimer
Sequence is：
Upstream 5 ' [GCGGCCGCGCCACCATGGGCTCGCAGGTC] 3 '；Such as SEQ ID NO:Shown in 3；
Downstream 5 ' [AAGCTTTTACAATGTTGTGATCTTGTCCC] 3 ' such as SEQ ID NO:Shown in 4；
3CD fragment sequence such as SEQ ID NO:Shown in 2, restriction enzyme site be Xho I and Sph I and, amplimer sequence is：
5 ' d of upstream [CCGCTCGAGGCCACCATGGGACCCT] 3 ' such as SEQ ID NO:Shown in 5；
Downstream 5 ' [ACATGCATGCTCAGAAGAGTTCCAGCCAGTTA] 3 ' such as SEQ ID NO:Shown in 6.
Using gene chemical synthesis full-length genome as template, respectively by PCR amplification, target gene is recycled, respectively by target gene
It is building up in pFastBAcdual double promoter expression vector, building obtains pFastBAcdual-CVA16 (P1)-CVA16
(3CD).PFastBAcdual-CVA16 (P1)-CVA16 (3CD) and DH10a are recombinated, obtain recon, extracted rod granule it
Afterwards, rod granule is identified by primer PCR of M13.
The program of PCR is：
The upstream primer sequence of M13 is 5 ' d [GTTTTCCCAGTCACGAC] 3 ',
Downstream primer sequence is 5 ' d [CAGGAAACAGCTATGAC] 3 '
It will test correct rod granule and transfect SF9 insect cell by Invitrogen specification, insect cell number 6 orifice plates are each
Hole is 9 × 105It is a, transfection reagent (CellfectinI) μ L：Rod granule μ g is 2:8.The cell of transfection goes out through cultivating 3~4 days cells
Existing lesion, it is the rod-shaped poison that pretends to be sick of P1 that 1000rpm, which is centrifuged 10min harvest cell conditioned medium,.2 SF9 cell amplification viruses of persistent infection
For P3 baculoviral
Two：Baculovirus titers measurement
It harvests and stablizes growth period SF9 insect cell, prepare cell suspension in SFX culture medium, mix completely, 5 × 105Carefully
Born of the same parents/ml, are added 2ml cell suspension in each hole of 6 orifice plates, and 27 DEG C of incubators are incubated overnight.Virus stock solution used is diluted with culture medium, quasi-
Standby 5 gradient dilutions (such as 10-3~10-7) viral suspension 1ml, discard archaeocyte culture solution, sequentially add 6 orifice plates corresponding aperture
In, a cell negative control is arranged in every block of plate, and 27 DEG C of incubators are incubated for 4-5 hours.It draws cell upper layer viral suspension to discard, gently
The light 1ml fresh culture that is added is washed, and is discarded cleaning solution, is then slowly added into the nutrient agar sugar culture-medium containing dimethyl diaminophenazine chloride
3ml (containing 0.8 ﹪ low melting-point agarose, with the dilution of 1.5 × blank cultures, pre-balance is to 36-38 DEG C) covering cell, stands
30 minutes, 27 DEG C of incubator cultures 7~10 days are put into after agarose solidifies completely.Determine virus titer., the results showed that, disease
The titre of poison is 5 × 108PFU/ml。
Three, the virus sample particle vaccines technological process of production
Cell culture：The cell recovery of three liquid nitrogen cryopreservations is inoculated into a 1L culture bottle by resuscitative efforts library SF9 cell
In, after culture 3~4 days, according to 1：4 ratios are inoculated into 4 1L culture bottles, and so passage is inoculated into WAVE bioreactor
In.Reach 2 × 10 in cell density6A/L~3 × 106Baculoviral of the inoculation containing coding CVA16 target gene when a/L.
Virus inoculation：Culture cell density reaches 2 × 106A/L~3 × 106Inoculation contains coding CVA16 purpose when a/L
The baculoviral of gene.Virus inoculation amount MOI is 0.5~1, and 27 DEG C of cultivation temperature, incubation time is 72h~96h.
Harvest cell：When 30%~40% or so lesions occurs in cell, continuous flow concentrating cells suspension is harvested by centrifugation thin
Born of the same parents.Freeze thawing 3~4 times, centrifugation removal cell residue.0.45 μm of membrane filtration harvest includes the supernatant of CVA16 virus-like particle.
The purifying of 2 CVA16 virus-like particle of embodiment
Harvest the insect cell of the baculovirus infection containing CVA16 target gene, freeze thawing 3-4 times, centrifugation removal cell
Residue.0.45um membrane filtration harvests supernatant.
Sucrose concentrating virus sample particle：Supernatant is placed in 30% sucrose PBS solution, and 27,500rpm, ultracentrifugation 3 hours, sample
Product are concentrated 20 times,
Gel permeation chromatography：Superdex-500 medium carries out viral purification, and the PBS that balancing liquid pH value is 7.0 is buffered
Solution, sodium chloride salt concentration are 0.4mol/L, collect void volume and flow through peak.
Ceramic hydroxyapatite chromatography：Ceramic hydroxyapatite (CHTI type), the PBS buffer solution of balancing liquid pH7.0,
Sodium chloride salt concentration is 0.4mol/L.The PBS buffer solution of eluting liquid pH7.0, sodium chloride salt concentration are 1mol/L.
The purifying of 3 CVA16 virus-like particle of embodiment
Harvest the insect cell of the baculovirus infection containing CVA16 target gene, freeze thawing 3-4 times, centrifugation removal cell
Residue.0.45um membrane filtration harvests supernatant.
Sucrose concentrating virus sample particle：Supernatant is placed in 30% sucrose PBS solution, and 27,500rpm, ultracentrifugation 3 hours, sample
Product are concentrated 20 times,
Gel permeation chromatography：Superdex-500 medium carries out viral purification, and the PBS that balancing liquid pH value is 7.5 is buffered
Solution, sodium chloride salt concentration are 0.4mol/L, collect void volume and flow through peak.
Ceramic hydroxyapatite chromatography：Ceramic hydroxyapatite (CHTI type), the PBS buffer solution of balancing liquid pH7.5,
Sodium chloride salt concentration is 0.4mol/L.The PBS buffer solution of eluting liquid pH7.5, sodium chloride salt concentration are 1mol/L.(as schemed
1)
The identification of 4 CVA16 virus-like particle of embodiment
Electronic Speculum：Sample after the purifying of embodiment 2~3, sampling Electronic Speculum observation are observed through Electronic Speculum, the CVA16 after purifying
Virus-like particle and CVA16 virus morphological feature having the same.
The above result shows that the present invention successfully prepares and is purified into CVA16 virus-like particle.
The quality of CVA16 virus-like particle is identified：
Performance liquid chromatographic column analyzes sample purity：HPLC-TSKRG5000, the PBS buffer solution of pH7.0~8.5, chlorine
Change sodium salt concentration is 0.2mol/L~0.5mol/L.(such as Fig. 2)
ELISA detects the content of virus-like particle in sample in 2~3 purification process of embodiment.
It is as shown in table 1 to the testing result of embodiment 2：
In 1 embodiment of table, 2 purification process in sample virus-like particle content
Similarly to the testing result of embodiment 3.
The preparation of 5 CVA16 virus sample particle vaccines of embodiment
The CVA16 virus-like particle of embodiment 2 after purification and aluminum hydroxide adjuvant are adsorbed.And molten and PBS buffer solution
In, wherein virus-like particle and Al (OH)3The mass ratio of adjuvant is 1 μ g：500μg
The preparation of 6 CVA16 virus sample particle vaccines of embodiment
The CVA16 virus-like particle of embodiment 3 after purification and aluminum hydroxide adjuvant are adsorbed.And molten and PBS buffer solution
In, wherein virus-like particle and Al (OH)3The mass ratio of adjuvant is 1 μ g：500μg
7 vaccine purity detecting of embodiment
According to《Chinese Pharmacopoeia (the 3rd edition)》And《Products in China regulation》Measure vaccine finished product prepared by embodiment 5~6
Appearance, sterility test, DNA content, protein content etc..It the results are shown in Table 2：
2 vaccine assay result of table
| Project | Embodiment 5 | Embodiment 6 |
| Protein content | <120ug/ dosage | <120ug/ dosage |
| Appearance | Transparency liquid | Transparency liquid |
| Discrimination test | CVA16 antigen | CVA16 antigen |
| Endotoxin | <100EU/ dosage | <100EU/ dosage |
| Sterility test | It is negative | It is negative |
| pH | 7.0~7.2 | 7.0~7.2 |
| Undue toxicity | Pass through | Pass through |
| Effect | Pass through | Pass through |
Embodiment 8：Potency determination
It prepares：Two batches CVA16 virus-like particle after purification is adsorbed with aluminum hydroxide adjuvant respectively.And molten and PBS is buffered
In solution, wherein virus-like particle and AL (OH)3The mass ratio of adjuvant is 1ug：0.5ug
Mouse immune：6-8 weeks female ICR mouse is taken, rear 21st day booster immunization immune for the first time.After 7 days, eyeball
Blood is taken, 4 DEG C are collected by centrifugation supernatant, and each peritoneal immunity dosage is 1 μ g EV71 virus-like particle protein.
Titration：After 56 DEG C of serum to be detected are inactivated 30 minutes, not contain the MEM culture medium 2 of serum3~215
It is added after gradient dilution and contains 100TCID50Virus, 37 DEG C of incubators react 1h.RD cell (1 × 10 is added4Cells/well) 37 DEG C incubate
Case is incubated for 7 days, observes CPE, calculating antibody potency.It the results are shown in Table 3：
3 vaccine valence result of table
| Embodiment 2 | Embodiment 3 |
| 1：256 | 1：128 |
| 1：512 | 1：512 |
| 1：512 | 1：256 |
| 1：256 | 1：256 |
| 1：512 | 1：512 |
The results show that the vaccine that the present invention is supplied to has good and stable potency.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Sequence table
<110>Changchun BCHT Biotechnology Co.；Jilin University
<120>A kind of purification process of virus-like particle
<130> MP1809429F
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2586
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<213>Artificial sequence (Artificial Sequence)
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atgggctcgc aggtctctac acaaaggtcg ggctctcacg aaaacagcaa ctcggcttct 60
gaaggcagca ctatcaacta cactactatc aactactaca aggacgccta cgctgccagc 120
gctggtaggc aggacatgtc acaagatccc aagaagttca ccgaccctgt tatggatgtg 180
atccacgaga tggctccccc tttgaagtct cctagcgccg aagcctgcgg ctactcggac 240
agagtggccc agctgaccat cggaaactcc actatcacca ctcaagaggc tgccaacatc 300
gtgatcgctt acggcgagtg gccagaatac tgtccggaca ccgatgccac tgctgtcgac 360
aagcccacca ggcctgatgt gtctgtcaac agattcttca cactggacac caagtcatgg 420
gccaaggatt cgaagggatg gtactggaag ttccccgacg tgctcaccga ggttggtgtg 480
ttcggccaga acgctcaatt ccactacttg taccgctctg gcttctgcgt ccacgttcag 540
tgtaacgcca gcaagttcca ccaaggagct ctgctcgtgg ccgtcctgcc agagtacgtc 600
ctcggtacaa tcgctggtgg caccggcaac gaaaactccc acccaccata cgctacaacc 660
cagccaggac aagtcggtgc tgttctgact cacccttacg tgctcgacgc cggcatccca 720
ttgtctcagc tgacagtctg cccccaccaa tggatcaacc tgaggactaa caactgtgct 780
acaatcatcg ttccatacat gaacaccgtg ccgttcgaca gcgccctgaa ccactgcaac 840
ttcggattgc tggtggtccc agttgtgccg ctcgatttca acgccggtgc tacttcagag 900
atcccaatca cagttaccat cgccccgatg tgtgctgaat tcgccggcct gcgtcaggct 960
gtcaagcaag gaatccccac tgagttgaag cctggtacaa accagttcct gactacagac 1020
gatggcgtga gcgctcccat cctccctgga ttccacccca ccccccctat ccacatccct 1080
ggcgaggtcc acaacctctt ggaaatctgt agggtcgaga ctatcttgga agttaacaac 1140
ctgaagacaa acgagaccac tccaatgcag agattgtgct tcccggtttc cgtgcaatct 1200
aagaccggcg aactgtgtgc tgctttccgt gctgacccag gaagagatgg accctggcag 1260
tcgactatcc tcggtcaatt gtgccgttac tacacccagt ggtcaggctc gctcgaagtg 1320
actttcatgt tcgccggatc attcatggct acaggcaaga tgttgatcgc ttacacccca 1380
ccaggtggta acgtccctgc tgaccgcatc accgctatgc tgggtactca cgtcatctgg 1440
gatttcggct tgcagtcctc tgttacactg gtcgttccct ggatcagcaa cacccactac 1500
cgtgctcacg ctagagctgg atacttcgac tactacacaa ccggtatcat cacaatctgg 1560
taccagacca actacgtggt ccccatcgga gctcctacta cagcctacat cgtggctctg 1620
gctgccgctc aagacaactt caccatgaag ctctgcaagg acaccgagga tatcgaacag 1680
actgccaaca tccaaggtga tcctatcgct gacatgatcg atcagaccgt gaacaaccaa 1740
gtcaaccgtt ccctgactgc cctccaggtg ttgcccactg ccgctgacac agaggctagc 1800
tcacaccgcc tcggcacagg agttgtgcct gccttgcagg ccgctgaaac cggtgcctcg 1860
tccaacgctt ccgacaagaa cctgatcgag acccgttgcg tcctcaacca ccactctact 1920
caggaaacag ccatcggaaa cttcttctcc cgcgctggtc tggtgtctat catcactatg 1980
cccaccactg gcacccagaa cactgacgga tacgtcaact gggacatcga tctcatgggc 2040
tacgctcaat tgcgtcgcaa gtgtgagctg ttcacttaca tgcgtttcga cgccgaattc 2100
acattcgtcg ttgctaagcc caacggcgag ctcgttcctc agctgctcca atacatgtac 2160
gtgccacctg gagctccaaa gcctaccagc cgcgactcat tcgcctggca gactgctaca 2220
aacccttcag tcttcgttaa gatgactgat ccaccggccc aggtgtcggt cccattcatg 2280
tcgccggctt ccgcctacca atggttctac gacggttacc ctaccttcgg cgagcacctc 2340
caggctaacg acttggatta cggccaatgc ccaaacaaca tgatgggaac cttctccatc 2400
aggacagtgg gtaccgaaaa gtctccccac agcatcactc tgagggtgta catgagaatc 2460
aagcacgtca gagcttggat tccaaggccg ctcagaaacc agccatactt gttcaagacc 2520
aaccccaact acaagggaaa cgacatcaag tgtaccagca caagcaggga caagatcaca 2580
acattg 2586
<210> 2
<211> 1941
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atgggaccct cgctggattt cgctctctca ctcctccgtc gtaacatcag gcaggttcaa 60
acagaccaag gccacttcac tatgctcgga gtgcgtgacc gcctcgctat cttgccaagg 120
cactcccagc cgggcaagac tatctgggtg gagcacaagc tgatcaacgt cctcgacgcc 180
gttgagttgg tggatgaaca aggagttaac ctggagctca ctttggtgac actggacacc 240
aacgaaaagt tccgtgatgt caccaagttc atccctgaga caatcaccgg tgctagcgac 300
gccaccctgg ttatcaacac tgaacacatg ccctcaatgt tcgtgcctgt cggcgatgtg 360
gtccagtacg gattcctgaa cctctcaggc aagccaactc accgcacaat gatgtacaac 420
ttcccgacaa aggctggcca gtgcggtggc gttgtgacct cggtcggcaa gatcatcgga 480
atccacatcg gtggtaacgg taggcaaggc ttctgtgctg gtctgaagag aggctacttc 540
gccagcgagc agggagaaat ccaatggatg aagccaaaca aggagactgg caggctgaac 600
atcaacggac caactagaac aaagctcgaa ccgtcagtct tccacgacgt tttcgagggt 660
aacaaggaac ccgctgtgtt gacctcgaag gaccctaggc tggaggtcga tttcgaacag 720
gccttgttct ccaagtacgt tggcaacacc ctgcacgagc ccgacgaata cgtgactcaa 780
gctgccctgc actacgctaa ccagctgaag caactcgata tcaacatcaa caagatgtct 840
atggaggaag cctgctacgg tactgagtac ctcgaagcca tcgacttgca cacatctgct 900
ggttacccat acagcgccct cggcgtgaag aagcgtgaca tcttagatcc gatcactcgc 960
gacaccacta agatgaagtt ctacatggac aagtacggct tggatctgcc atacagcacc 1020
tacgtgaagg acgagttgcg ttcactggat aagatcaaga agggcaagtc gcgcttgatc 1080
gaggcctcct ctctgaacga ctctgtctac ctccgtatga cattcggaca cctgtacgag 1140
accttccacg ctaacccagg aaccgtgact ggttccgctg tcggctgtaa ccctgacgtc 1200
ttctggtcta agctgcccat cctgctccct ggatcactct tcgctttcga ctactcgggt 1260
tacgatgcca gcctctcacc cgtgtggttc cgtgctttgg aggtcgttct gcgcgaaatc 1320
ggctactcgg aggaagccgt ctccctcatc gagggaatca accacactca ccacgtctac 1380
aggaacaaga catactgcgt tttgggcgga atgccttcgg gatgttccgg tacctctatc 1440
ttcaacagca tgatcaacaa catcatcatc agaacattgc tgatcaagac cttcaagggt 1500
atcgacctcg atgagttgaa catggttgct tacggcgacg atgtgctggc ctcctacccc 1560
ttccctatcg actgctctga gctcgctaag accggaaagg aatacggttt gacaatgacc 1620
ccagccgata agtctccgtg tttcaacgaa gtgacttggg aaaacgctac attcctgaag 1680
aggggtttcc tccccgacca ccagttccct ttcctgatcc acccaaccat gccgatgaga 1740
gagatccacg aatctatcag gtggaccaag gacgctagaa acactcaaga tcacgtccgt 1800
agcctctgcc tcttggcctg gcacaacggc aaggaggaat acgagaagtt cgtcagcacc 1860
atccgctcag ttcccatcgg taaggctctg gctatcccta acttcgagaa cttgcgtcgt 1920
aactggctgg aactcttctg a 1941
<210> 3
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gcggccgcgc caccatgggc tcgcaggtc 29
<210> 4
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
aagcttttac aatgttgtga tcttgtccc 29
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ccgctcgagg ccaccatggg accct 25
<210> 6
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
acatgcatgc tcagaagagt tccagccagt ta 32
Claims (3)
1. a kind of preparation method of CVA16 virus-like particle, which is characterized in that include the following steps：
Step 1：The P1 segment of CVA16 virus and 3CD segment are built into pFastBAcdual carrier, with Escherichia coli
The recombination of DH10BAC swivel base, obtains the expression vector containing CVA16 target gene；
Step 2：With the expression vector transfection insect cell containing CVA16 target gene, culture, passage are contained
The baculoviral of CVA16 target gene；
Step 3：With the baculovirus infection insect cell containing CVA16 target gene, cultivate to 30%~40% it is thin
There is lesion in born of the same parents；Cell is harvested by centrifugation, after multigelation, centrifugation removal residue is filtered to be made and contains virus-like particle
Liquid；
Step 4：By the liquid containing virus-like particle successively through sucrose concentration, gel chromatography, ceramic hydroxyapatite layer
Analysis；CVA16 virus-like particle is made.
2. virus-like particle made from preparation method as described in claim 1.
3. a kind of CVA16 virus sample particle vaccines, which is characterized in that including disease made from preparation method as described in claim 1
Malicious sample particle and adjuvant.
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| 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 |
| CN114008067A (en) * | 2019-07-04 | 2022-02-01 | 株式会社钟化 | Method for purifying virus or virus-like particle |
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| CN106754765B (en) * | 2016-12-20 | 2020-07-17 | 吉林大学 | Newcastle disease virus-like particle, preparation method and application thereof |
| CN108524929B (en) * | 2017-03-06 | 2019-05-07 | 广州瑞贝斯药业有限公司 | A kind of production method of rabies vacciness |
| CN113637054B (en) * | 2021-08-30 | 2023-10-03 | 雪莱(武汉)生物医药技术有限公司 | Purification method and application of recombinant Sendai virus-like particles |
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