CN105535963A - EV71 subunit vaccine of mixed adjuvant and preparation method thereof - Google Patents
EV71 subunit vaccine of mixed adjuvant and preparation method thereof Download PDFInfo
- Publication number
- CN105535963A CN105535963A CN201511023413.2A CN201511023413A CN105535963A CN 105535963 A CN105535963 A CN 105535963A CN 201511023413 A CN201511023413 A CN 201511023413A CN 105535963 A CN105535963 A CN 105535963A
- Authority
- CN
- China
- Prior art keywords
- adjuvant
- protein
- albumen
- inclusion body
- carbamide
- 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.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an EV71 subunit vaccine of a mixed adjuvant and a preparation method thereof. According to the vaccine, C4 sub-type EV71 virus VP1 protein is used as an antigen, and aluminum hydroxide and monophosphoryl lipid A are used as a mixed adjuvant. VP1 protein which is expressed and purified by escherichia coli and does not have any label is used as an antigen; the prepared protein is combined with an aluminum adjuvant under a denaturing condition, is subjected to detergence determination, and is mixed with an MPLA adjuvant to immune mice; and the prepared polyvalent antibody can be used for specifically neutralizing the EV71 virus to generate a neutral protective antibody having a titer nearly 1:128. Compared with an EV71 inactivated vaccine in current clinical tests, the human enterovirus 71 type subunit vaccine has the characteristics of low cost, simple operation, high safety, easy large-scale production and the like in the production process. The vaccine can generate relatively good immunogenicity in a human body, has a relatively high titer of the neutralizing antibody, and is an alternative vaccine with potential clinical application value.
Description
Technical field
The present invention relates to biomedical sector, be specifically related to a kind of EV71 subunit vaccine mixing adjuvant and preparation method thereof.
Technical background
Hand-foot-mouth disease is generated heat and occurs by positions such as hands, foot, oral cavities the common pediatric disease of one that erythra, ulcer and herpetic pharyngitis are principal character, small number of patients also can cause the mortality complication such as myocarditis, pulmonary edema and encephalitis, the multiple infant being born in less than 5 years old.Cause pathogen mainly enterovirns type 71 and the coxsackie virus A 16-type of hand-foot-mouth disease.
Research shows, EV71 is main relevant to serious symptom hand-foot-mouth disease, and it has addicted to neurotoxicity and causes the complication such as aseptic isolator, meningoencephalitis and myocarditis further, and severe patient may be disabled or lethal.Since EV71 was separated first in 1969 from the Patients With Central Nervous Diseases stool sample in California, the hand-foot-mouth disease that EV71 infection worldwide repeatedly occurred is popular, the EV71 frequently having broken out high death nearly decades in TaiWan, China, Malaysia, Vietnam and China's Mainland infects, serious harm publilc health safety.
EV71 belongs to picornavirus, its genome is made up of the single-stranded positive RNA of 7408 nucleotide, to encode 2194 amino acid whose polyproteins, this polyprotein can be further hydrolyzed to P1, P2, P3 tri-precursor proteins, P1 precursor protein coding VP1, VP2, VP3, VP4 tetra-structural protein, these four albumen constitute the ion shell of virus jointly, wherein VP1 directly determines the antigenicity of virus, and there is the genetic diversity completely corresponding with virus serotype, therefore extensively paid attention in the genome and vaccine development of EV71.
Also do not have specific medicament for EV71 at present, therefore the development of vaccine is just extremely urgent.Because the basic researchs such as the nosetiology to EV71, infection mechanism, mechanism of causing a disease and epidemiology are done not enough, EV71 Strain easily morphs in epidemiological process, and does not have good animal model, and this just considerably increases the difficulty of vaccine research and development.The vaccine of current EV71 mainly contains inactivated vaccine, attenuated live vaccine, viruslike particle vaccine, subunit vaccine, transgenic oral vaccine and synthetic peptide vaccine, although inactivated virus vaccine has been in third experimental stage, but it need checking in safety, other these vaccines are all in research and development and clinical trial stage, the product of real listing is not also seen, this just needs the troop putting into vaccine development of our more great dynamics.
The subunit vaccine of EV71 is mainly using VP1 as first-selected target, the antigen that correlational study shows to produce good neutrality antibody is the VP1 albumen that obtains of insect cell expression purification mainly, and the VP1 albumen of escherichia coli expression is tired lower as the general neutrality antibody of antigen, but it is high that the albumen of the VP1 of escherichia coli expression has expression efficiency, production cost is low and be easy to the feature of large-scale production, and the albumen obtained from escherichia coli has higher safety as vaccine, be easy to be accepted, therefore the subunit vaccine research obtained from escherichia coli expression also becomes more and more to be favored.The less immunogenic of subunit vaccine self, suitable adjuvant must be selected to strengthen the immune effect of vaccine, the adjuvant mainly aluminium adjuvant of current people, but aluminium adjuvant mainly stimulates body to produce Th2 immunoreation, body effectively cannot be stimulated to produce Th1 immunoreation, cause producing good immunogenic response.
Summary of the invention
The object of the invention is to overcome in the generation of existing EV71 subunit vaccine low defect of tiring with protection antibody; a kind of EV71 subunit vaccine preparation method of mixing adjuvant is provided; be antigen with the VP1 albumen of the not tape label of EV71; albumen elder generation and aluminium adjuvant combine, after washing, be mixed with into EV71 subunit vaccine again with MPLA adjuvant.
In order to realize object of the present invention, the present invention adopts following technical scheme:
Mix a human enterovirus 71 subunit vaccine for adjuvant, comprise the VP1 albumen of EV71 and the mixing adjuvant of aluminum and MPLA coupling.
Wherein, the VP1 nucleotide sequence of EV71 is as shown in SEQIDNO.1, and the VP1 protein amino acid sequence of EV71 is as shown in SEQIDNO.2.
The VP1 albumen of described EV71 is preferably recombinant expressed in escherichia coli and through ultrasonic, washing, degeneration, the albumen of expressing not with any unnecessary sequence (as His-Tag sequence), and is obtain through different isolation and purification methods under Denaturing.
Described subunit vaccine more preferably VP1 albumen combines with aluminium adjuvant under urea-denatured condition, is mixed to get after rinsing liquid washing with MPLA adjuvant.
The preferred aluminium hydroxide of aluminium adjuvant used or aluminum phosphate.
The human enterovirus 71 subunit vaccine of described mixing adjuvant, the weight ratio of VP1 albumen and aluminum and MPLA adjuvant is preferably 1-50:1-100:1-10; Preferred 20-30:40-60:2-8 further; Particularly preferably 25:50:5.
The preparation method of enterovirns type 71 subunit vaccine of the present invention, comprises following steps:
(1) the VP1 sequence clone shown in SEQIDNO.1 is entered pET22b expression vector establishment and become pET22b-VP1 recombinant expression carrier, plasmid is imported e. coli bl21 trxB (DE3) and carry out abduction delivering, the bacterium liquid obtained is through ultrasonic centrifugal acquisition VP1 inclusion body protein;
(2) inclusion body protein is through repeatedly washing with centrifugal containing the carbamide of variable concentrations and the lavation buffer solution of surfactant, final with the solubilize inclusion body containing high concentration urea; The inclusion body protein dissolved exchanges purification through strong anion under carbamide existent condition can obtain the higher albumen of purity, further with obtaining purity after gel filtration chromatography more than 95% antigen VP1 albumen;
(3) the degeneration VP1 albumen that after purification, carbamide dissolves is combined with aluminium adjuvant, repeatedly washs drift and removes denaturant, obtain the mixture of aluminium adjuvant and antigen protein, mixed by this mixture be finally prepared into EV71 subunit vaccine with MPLA adjuvant with physiological buffer.
The weight ratio of VP1 albumen and aluminum and MPLA adjuvant is preferably 1-50:1-100:1-10; Preferred 20-30:40-60:2-8 further; Particularly preferably 25:50:5.
Wherein, described step (2) preferably includes:
1. the washing of VP1 inclusion body protein:
Inclusion body lavation buffer solution 1 condition of ice bath obtained is washed 2-3 time, be 1 ~ 1.5h at every turn, after centrifugal supernatant discarded, lavation buffer solution 2 condition of ice bath washing 2-3 time is added subsequently in precipitation, be 1 ~ 1.5h at every turn, after centrifugal supernatant discarded, add lavation buffer solution 3, condition of ice bath washing 1 ~ 1.5h, centrifugally remove supernatant, finally by inclusion body ultra-pure water ice bath washing 1 ~ 1.5h, centrifugally remove supernatant, again use ultra-pure water rinse, the albumen finally obtained is the VP1 inclusion body protein after washing, by inclusion body protein with dissolving buffer solution, insoluble matter is slightly ultrasonic, harvested by centrifugation supernatant is the inclusion body protein after carbamide dissolving, wherein, lavation buffer solution 1 formula is: 20mmol/LTris-HClpH8.0,0.15mol/LNaCl, 2mol/L carbamide, 1%TritonX-100,5mmol/LEDTA, lavation buffer solution 2 formula is: 20mmol/LTris-HClpH8.0,0.15mol/LNaCl, 4mol/L carbamide, 1%TritonX-100,5mmol/LEDTA, lavation buffer solution 3 formula is: 20mmol/LTris-HClpH8.0,0.15mol/LNaCl, 2mol/L carbamide, 1%TritonX-100,5mmol/LEDTA, 2% NaTDC, dissolving buffer formulation is: 20mmol/LTris-HClpH8.0,8mol/L carbamide, 70mmol/L beta-mercaptoethanol,
2. the anion exchange purification of inclusion body protein:
Install the strong anion exchange column through soda acid rinsing or weak anion exchange column in advance, ion-exchange purification is carried out to the albumen after solubilization of inclusion bodies; First use a large amount of ultrapure water pillars, then use level pad, until baseline balance, by slow for 25ml protein sample loading, again use Equilibration buffer wash pillar, until baseline balance, wherein do not collect protein stream and wear liquid, albumen almost all combines; By sample elution buffer 1 eluting after balance, the destination protein obtained after purification, purity of protein is more than 95%; Wherein, described equalizing and buffering formula of liquid is: 20mmol/LTris-HClpH8.0,8mol/L carbamide, 70mmol/L beta-mercaptoethanol, elution buffer 1 formula is: 20mmol/LTris-HClpH8.0,8mol/L carbamide, 70mmol/L beta-mercaptoethanol, 80mmol/LNaCl;
3. the SepharoseCL-4B gel filtration chromatography of VP1 inclusion body protein:
The embedding of PEG20000 bag filter is carried out to the sample after ion-exchange purification concentrated, the about 1.2mg/ml of sample concentration after concentrated, the about 25ml of volume, carry out SepharoseCL-4B gel filtration chromatography to concentrating sample, concrete steps comprise: first use ultrapure water pillar, then use level pad, until baseline balance, by slow for the sample after ion-exchange purification loading, use Equilibration buffer wash pillar, collect corresponding eluting peak.
Described step (3) preferably includes: the first and aluminum hydroxide adjuvant combination by the VP1 protein solution of purification, obtain the antigen protein of aluminum hydroxide adjuvant parcel, again parcel albumen is added the mixing of MPLA adjuvant, obtain the human enterovirus 71 subunit vaccine of described mixing adjuvant; Wherein, VP1 albumen and aluminum and MPLA adjuvant is preferably 1-50:1-100:1-10; Preferred 20-30:40-60:2-8 further; Particularly preferably 25:50:5.
Beneficial effect:
The present invention have selected aluminium adjuvant and MPLA (monophosphoryl lipid A) two kinds of adjuvants mix as immunological adjuvant, MPLA can effectively stimulate body to produce Th1 immunoreation, have selected the VP1 albumen without any unnecessary sequence of escherichia coli expression as antigen simultaneously, good immunogenic response is created in vivo after immune mouse, the serum obtained has higher Neutralizing titer, and this is that the development of EV71 subunit vaccine is laid a good foundation.
VP1 sequence provided by the invention is through company's full genome synthesis optimizing, concrete sequence is shown in SEQIDNO.1, the VP1 nucleotide sequence obtained is cloned into pET22b expression vector establishment and becomes pET22b-VP1 recombinant expression carrier, plasmid is imported e. coli bl21 trxB (DE3) and carry out abduction delivering, the bacterium liquid obtained is through ultrasonic centrifugal acquisition inclusion body protein.Inclusion body protein is through the carbamide of variable concentrations and the repeatedly washing of surfactant and centrifugal, and the carbamide of final high concentration dissolves inclusion body.Dissolve inclusion body protein under carbamide existent condition through strong anion exchange purification can obtain the higher albumen of purity, further by the antigen protein purity obtained after gel filtration chromatography more than 95%.The Denatured protein that after purification, carbamide dissolves is combined with aluminium adjuvant, repeatedly washs drift and removes denaturant, obtain the mixture of aluminium adjuvant and antigen protein, mixed by this mixture be finally prepared into EV71 subunit vaccine with MPLA adjuvant with physiological buffer.
VP1 albumen provided by the invention is without any unnecessary sequence, also there is no the report of the associated protein vaccine of not tape label at present both at home and abroad, compare the protein purification of other tape label, there is the advantages such as preparation process is simple, low price, and the adjuvant used is aluminium adjuvant and MPLA adjuvant, the antigen protein coherent detection index obtained all meets vaccine for man standard.
Inclusion body protein of the present invention, through the urea washes of low concentration and middle concentration, wherein also adds TritonX-100 and NaTDC, eliminates the foreign protein of most Host Strains in washing process, for subsequent purification provides conveniently.
The antigen protein of purification of the present invention is under carbamide and mercaptoethanol existent condition, now protein structure is opened completely, it is consistent that conformation almost becomes, epitope also fully exposes, when exchanging purification with strong anion, albumen to be much all adsorbed onto on pillar and can to elute with the salt ion of low concentration, this albumen degree of polymerization just further demonstrating now is very low, it may be even much monomeric form, aluminium adjuvant and MPLA mixing adjuvant immunity are selected, this just greatly strengthen immunogenicity and the immunoreactivity of antigen protein, the antibody titer obtained and NAT are all higher.
The subunit vaccine of human enterovirus 71 provided by the invention, compare the EV71 inactivated vaccine of current clinical trial, it is low that this vaccine has cost in process of production, simple to operate, safety is high, is easy to the features such as large-scale production, and this vaccine can produce good immunogenicity in vivo, the NAT obtained is higher, is therefore the alternative vaccine with potential clinical value.
Accompanying drawing explanation
Fig. 1 is pET22b-VP1 recombinant vector digestion verification
1.DNA standard molecular weight; Before 2.pET22b-VP1 enzyme action; 3.pET22b-VP1NdeI single endonuclease digestion; 4.pET22b-VP1NdeI and SalI double digestion.
Fig. 2 is the protein induced expression of VP1
1. protein standard molecular weight; 2. before induction; 3. after induction; 4. broken supernatant; 5. broken precipitation.
Fig. 3 is that VP1 protein ion exchanges purification and gel filtration chromatography
1. protein standard molecular weight; 2. after inclusion body carbamide dissolves; 3. ion-exchange purification; 4. gel filtration chromatography.
Fig. 4 is that ELISA detects IgG antibody titre
Fig. 5 is that microneutralization detects NAT
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention will be further described.
Embodiment 1.
The synthesis of 1.VP1 gene and the structure of recombinant vector pET22b-VP1
The EV71 Strain deriving from C4 hypotype obtains from Institut Pasteur of Shanghai, extract test kit (Tian Gen biochemical technology company limited) with viral RNA and extract acquisition viral RNA, the cDNA of virus is obtained with Reverse Transcription box reverse transcription, the complete sequence of design primer segmentation amplicon virus, fragment sequence enzyme action is connected into complete sequence, serve the order-checking of Hai Ying fine horse Bioisystech Co., Ltd, the sequence finally recording VP1 is shown in SEQIDNO.1, aminoacid sequence is shown in SEQIDNO.2, and this full length cDNA sequence has also repacked out the new virus having infection ability by vitro transcription, therefore confirm that cDNA sequence is all complete and function is not also lost.
The VP1 sequence obtained with above-mentioned order-checking is standard, at the complete genome sequence of calm and peaceful Bioisystech Co., Ltd of Sino-U.S. synthesis VP1, because pET22bNdeI end exists start codon to start the synthesis of albumen, therefore with the addition of NdeI restriction enzyme site at 5 ' end when VP1 design, SalI restriction enzyme site is added at 3 ' end, and with the addition of termination codon at 3 ' end, make the VP1 albumen of expression not with any unnecessary sequence, take pET22b as skeleton plasmid, pET22b and VP1 is used NdeI and SalI double digestion respectively, and system is as follows:
Mixed gently by mixture, collected by centrifugation, at the bottom of pipe, proceeds to 80 DEG C of water-bath 20min after 37 DEG C of water-bath 3h.Get 5 μ l double digestion products and carry out 1.0% agarose gel electrophoresis, all the other digestion products are used for agarose gel electrophoresis and reclaim, and measure by multi-functional microplate reader the concentration that purification reclaims product.Product fragment after purification reclaims connects with T4 ligase (TaKaRa) 16 DEG C spends the night, and linked system is as follows:
Spend the night in 16 DEG C of connections, connect product conversion 100 μ lE.coliDH5a, overnight incubation, when by the time cloning easy picking, whether bacterium colony PCR checking is positive colony, and the positive colony of checking extracts plasmid, with NdeI, the mono-double digestion of SalI verifies whether successful connection (Fig. 1), and result shows that recombinant vector pET22b-VP1 successfully constructs.
2. recombinant vector pET22b-VP1 transforms and expresses bacterial strain inducing expression
By above-mentioned recombinant vector Transformed E .coliBL21trxB (DE3) competent cell successfully constructed, bacterium colony PCR qualification and enzyme action qualification are carried out, to positive colony abduction delivering to bacterium colony simultaneously.Picking list bacterium colony proceeds to 100ml with 1:50 (v/v) and cultivates, to OD containing continuing in the LB culture medium of ampicillin (50 μ g/ml) after 37 DEG C of incubated overnight
600about 0.6, get 1ml bacterium liquid in 1.5ml centrifuge tube, the centrifugal 5min of 12000r/min, abandons supernatant, and adding IPTG in remaining bacterium liquid to final concentration is 0.5mmol/L, 37 DEG C of abduction delivering 5h, and the bacterium liquid got after 0.3ml induction does same treatment.By bacterium liquid after abduction delivering in 10000rpm, 4 DEG C of centrifugal 10min, collect thalline, with the resuspended thalline of PBS buffer of 10ml100mmol/LpH7.0, get the broken liquid of 100 μ l after ultrasonication centrifugal, as fragmentation precipitation cleer and peaceful in fragmentation, thalline before simultaneously getting induction and after induction, adds sample-loading buffer, carries out SDS-PAGE electrophoretic analysis (Fig. 2), result shows that the albumen of expressing is all inclusion body protein, and destination protein expression is approximately 100mg/L.
3.VP1 the washing of inclusion body protein
To the bacterium liquid ultrasonication of 500ml abduction delivering, harvested by centrifugation inclusion body protein, by the inclusion body 2mol/L low urea washing obtained, lavation buffer solution 1 is 25ml (20mmol/LTris-HClpH8.0, 0.15mol/LNaCl, 2mol/L carbamide, 1%TritonX-100, 5mmol/LEDTA), condition of ice bath has been washed twice, each is 1.5h, after centrifugal supernatant discarded, 25ml (20mmol/LTris-HClpH8.0 is added subsequently in precipitation, 0.15mol/LNaCl, 4mol/L carbamide, 1%TritonX-100, 5mmol/LEDTA) lavation buffer solution 2, condition of ice bath has been washed twice, each is 1.5h, after centrifugal supernatant discarded, add 25ml lavation buffer solution 3 (20mmol/LTris-HClpH8.0, 0.15mol/LNaCl, 2mol/L carbamide, 1%TritonX-100, 5mmol/LEDTA, 2% NaTDC), condition of ice bath washing 1.5h, centrifugally remove supernatant, finally by inclusion body ultra-pure water ice bath washing 1.5h, centrifugally remove supernatant, again use ultra-pure water rinse, the albumen finally obtained is the inclusion body protein after washing, inclusion body protein 25ml is dissolved buffer (20mmol/LTris-HClpH8.0, 8mol/L carbamide, 70mmol/L beta-mercaptoethanol) dissolve, insoluble matter is slightly ultrasonic, harvested by centrifugation supernatant is the inclusion body protein after carbamide dissolving.
4.VP1 the anion exchange purification of inclusion body protein
The strong anion installed in advance through soda acid rinsing exchanges filler or weak anion-exchange, anion exchange purification is carried out to the albumen after solubilization of inclusion bodies, wherein above-mentioned albumen volume 25ml, the about 3mg/ml of concentration, ion-exchange purification amount of filler is greatly about about 50ml.First use a large amount of ultrapure water pillars, use level pad (20mmol/LTris-HClpH8.0 again, 8mol/L carbamide, 70mmol/L beta-mercaptoethanol), until baseline balance, by slow for 25ml protein sample loading, again use Equilibration buffer wash pillar, until baseline balance, wherein do not collect protein stream and wear liquid, albumen almost all combines.By the sample elution buffer (20mmol/LTris-HClpH8.0 after balance, 8mol/L carbamide, 70mmol/L beta-mercaptoethanol, 80mmol/LNaCl) eluting, the destination protein obtained after purification is shown in (Fig. 3), purity of protein is more than 95%, with this eluent by after albumen stripping equilibria, again with the elution buffer (20mmol/LTris-HClpH8.0 that salt ionic concentration is slightly high, 8mol/L carbamide, 70mmol/L beta-mercaptoethanol, 130mmol/LNaCl) eluting, although destination protein content is higher after electrophoresis, but purity of protein reduces a lot, and all loading albumen almost all elutes, rinse with the NaCl of 1mol/L again, almost there is no albumen, therefore the albumen of 80mmol/LNaCl concentration eluting is decided to be results albumen the most at last, obtain the volume of 75ml, protein concentration is approximately 0.5mg/ml.
The SepharoseCL-4B gel filtration chromatography of 5.VP1 inclusion body protein
Install the SepharoseCL-4B filler (Yuan Ye bio tech ltd, Shanghai) through soda acid rinsing in advance, the embedding of PEG20000 bag filter is carried out to the sample after ion-exchange purification concentrated, the about 1.2mg/ml of sample concentration after concentrated, the about 25ml of volume, carries out gel filtration chromatography analysis to concentrating sample.First a large amount of ultrapure water pillars is used, use level pad (20mmol/LTris-HClpH8.0,8mol/L carbamide, 70mmol/L beta-mercaptoethanol) again, until baseline balance, by slow for the sample after ion-exchange purification loading, use Equilibration buffer wash pillar, collect corresponding eluting peak, the about 50ml of final elution volume, the about 0.5mg/ml of protein concentration, carries out SDS-PAGE electrophoretic analysis, the results are shown in (Fig. 3).
Endotoxin content after 6.VP1 inclusion body protein washing purification detects
Endotoxin detection has been carried out to the protein sample that each step is collected, tachypleus amebocyte lysate endotoxin detection kit is purchased from company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City, owing to all containing the carbamide of high concentration in the sample of our purification, and the detection of carbamide induced by endotoxin test kit has interference, therefore we have carried out diluting groping to sample, just response rate R value is close to 100% for the sample of result display dilution 2000 times, and the sample detection reliable results of this multiple dilutions is accurate.
Concrete operations are as follows: first the standard substance of 10EU/ml are diluted to 1EU/ml, 0.5EU/ml, 0.25EU/ml, 0.1EU/ml respectively, the additional negative blank without bacterial endotoxin water, experimental group sample is determined as: 1. loading diluted sample 2000 times, the about 3mg/ml of protein concentration before dilution, 2. the concentrated rear diluted sample 2000 times of ion-exchange purification, the about 1.2mg/ml of protein concentration, 3. the concentrated rear diluted sample 2000 times of gel filtration chromatography, the about 0.5mg/ml of protein concentration, 4. the standard substance endotoxin of loading diluted sample 2000 times of+0.5EU/ml, first tachypleus amebocyte lysate 37 DEG C of water-bath 10min are added according to operating instruction, add chromogenic substrate 37 DEG C of water-bath 6min again, add even diazotizing reagent 1 respectively, 2, 3, numerical value is measured 545 times in λ after leaving standstill 5min, the results are shown in Table 1: after conversion, in loading sample, endotoxin content is close to 900EU/ml, endotoxin is electronegative, so endotoxin content rises after anion exchange purification, be approximately 1000EU/ml, and endotoxin content is less than 100EU/ml after gel filtration chromatography, meet States Pharmacopoeia specifications standard.
Table 1 endotoxin assay
Host's residual protein after 7.VP1 inclusion body protein washing purification detects
The detection of host's residual protein has been carried out to the protein sample that each step is collected, host's residual protein detection kit is purchased from Cygnus company, because sample is all prepared under high concentration urea existence condition, and carbamide has interference to test kit detection, first carried out carbamide interference to sample to grope, result shows that the sample solution of dilution more than 20 times does not exist interference to the detection of host's residual protein.
Concrete operation step is as follows: host protein standard substance 0ng/ml, 1ng/ml, 3ng/ml, 12ng/ml, 40ng/ml and 100ng/ml are added respectively 96 orifice plates being coated with residual protein antibody, experimental group sample is determined as follows: 1. ultrasonication bacterium liquid supernatant dilutes 10 times, 10
2doubly, 10
3doubly, 10
4doubly, BCA method measures protein concentration and is respectively 400 μ g/ml, 40 μ g/ml, 4 μ g/ml, 0.4 μ g/ml, and these are as positive control, 2. loading albumen dilution 10
2doubly, 10
3doubly, 10
4doubly, protein concentration is respectively 30 μ g/ml, 3 μ g/ml, 0.3 μ g/ml, 3. the concentrated rear diluted sample 10 of ion-exchange purification
2doubly, 10
3doubly, 10
4doubly, protein concentration is respectively 12 μ g/ml, 1.2 μ g/ml, 0.12 μ g/ml, 4. gel filtration chromatography concentrating sample dilution 10
2doubly, 10
3doubly, 10
4doubly, protein concentration is respectively 5 μ g/ml, 0.5 μ g/ml, 0.05 μ g/ml, 5. the urea buffer solution of the protein standard substance+0.8mol/L of 40ng/ml, 6. the urea buffer solution of the protein standard substance+0.4mol/L of 40ng/ml.96 orifice plates are added by also corresponding for these samples, add the escherichia coli host protein antibodies of horseradish peroxidase labelling, mixing leaves standstill, add nitrite ion and stop buffer, absorption value is measured 450 times at λ, testing result is in table 2: after converting, the ultrasonic bacterium liquid residual protein of positive control approximates greatly 35 μ g/ml, be significantly less than the protein concentration that BCA method records, may be less due to the antibody for these albumen in test kit, host's residual protein of loading albumen is approximately 800ng/ml, and the sample after purification almost can't detect foreign protein, experimental result meets standards of pharmacopoeia.
Table 2 host residual protein assay
Embodiment 2
1. immune adjuvants and the mixing of VP1 antigen protein
Collect the elder generation of the protein sample after gel permeation chromatography post and aluminum hydroxide adjuvant combination, protein sample place solution is (20mmol/LTris-HClpH8.0, 8mol/L carbamide, 70mmol/L beta-mercaptoethanol), protein concentration is approximately 0.5mg/ml, volume is approximately 200 μ l, aluminum hydroxide adjuvant consumption is 200 μ g, 30min is left standstill in mixed at room temperature, middle with the mixing of rifle head, 3000rpm is centrifugal, remove supernatant, with the rinsing of 10mmol/LpH7.0HEPES buffer several times, remove supernatant, obtain the antigen protein of aluminum hydroxide adjuvant parcel, be immunogen 1., again parcel albumen is added 20 μ gMPLA (Invivogen) adjuvants mixing, add to 200 μ l with 10mmol/LpH7.0HEPES buffer, as mice immunogen 2., by the protein sample dialysis lyophilizing after gel filtration chromatography, obtain 100 μ g protein samples and 3. 20 μ gMPLA adjuvants are mixed into immunogen, with inactivation of viruses immunogen, formula is as follows: in EV71 virus liquid, add the formaldehyde that final concentration is 0.2%, 37 DEG C of deactivations 16 hours, then add final concentration be 0.2% sodium pyrosulfite stop deactivation, immunizing dose is every mice 100 μ l10
7as immunogen 4. the inactivation of viruses amount of TCID50/ml and the aluminum hydroxide adjuvant mixing of 50 μ g, fill volume to 200 μ l with 10mmol/LpH7.0HEPES, 5. and only immune aluminum hydroxide adjuvant matched group is 6. to add normal mouse matched group in addition.
2. immune adjuvants and VP1 antigen protein mix rear immune mouse and antiserum titre detection
Choose 24 BALB/c mouse, be divided into 6 groups at random, often organize 4, experimental group comprises the antigen injection (antigen is 100 μ g/) of 3 kinds of different adjuvant mixing in 8, the inactivation of viruses immune group of aluminum hydroxide adjuvant mixing is positive controls, all the other aluminum hydroxide adjuvant groups and normal mouse group are insidious matched group, and mice adopts subcutaneous multi-point injection.
(1) first time inoculation:
Inject after the antigen prepared and adjuvant mixing respectively in Mice Body, the antigen injection amount of every mice is 100 μ g, within after exempting from 14 days, carries out eyeball blood sampling to mice one, 4 DEG C of centrifugal 10min of 3000rpm, isolate serum ,-80 DEG C frozen treats that ELISA detects (IgG detection).
(2) second time inoculation: exempts to carry out second time inoculation after two weeks, and immunization method dosage is the same.After exempting from two weeks two, carry out eyeball blood sampling to mice, by the blood collected at 4 DEG C of centrifugal 10min of 3000rpm, isolate serum ,-80 DEG C frozen treats that ELISA detects (IgG detection).Carry out eyeball blood sampling to mice respectively after exempting from three weeks and surrounding two, method is the same, isolates serum, and-80 DEG C frozen treats that ELISA detects (IgG detection).
ELISA antiserum titre detects:
One, diluted sample: sample PBS doubling dilution, arranges feminine gender simultaneously, positive, blank.
Two, bag quilt: get soluble antigen and add coating buffer dissolving, add the antigen coated mixed liquor of VP1 in 50 μ l/ holes, final concentration is 1 μ g/ hole, hatches 2 hours or 4 DEG C of overnight incubation for 37 DEG C.
Three, wash plate: get rid of liquid, get one piece of clean gauze, the liquid in plate hole is patted dry only.Wash plate three times with washing trigger, firmly clappers, the liquid in plate hole is clapped.
Four, close: add 200 μ l/ hole confining liquids in ELISA Plate hole, hatch one hour for 37 DEG C, get rid of after hatching end on the clean gauze of confining liquid in plate hole and the liquid in plank is patted dry only.
Five, primary antibodie: by the serum-dilution collected to 1:100 join the first hole in ELISA Plate (the first hole adds the diluent of 100 μ l1:100, with confining liquid dilution, after add the confining liquid of 50 μ l), rear doubling dilution, hatches 1.5h for 37 DEG C.
Six, two resist: (resist two and be diluted to 1:5000-1:8000, dilute with confining liquid) resists be diluted to working concentration two, is added in hole, hatches one hour for 37 DEG C with 50 μ l/ holes.
Seven, wash plate: get rid of liquid, get one piece of clean gauze, the liquid in plate hole is patted dry only.Wash plate three times with washing trigger, more firmly have the final say, the liquid in plate hole is clapped.
Eight, colour developing and reading: TMB develop the color, and are added in plate, hatch 25min for 25 DEG C, add the substrate 50 μ l/ hole of preparation 1mol/LHCl50 μ l/ hole cessation reaction and open microplate reader, adjust to OD
450reading, and the data that record is relevant, negative value is almost less than 0.06, with numerical value be greater than 0.2 for positive value, antibody titer detects as (Fig. 4), as seen from Figure 4, the ELISA antibody titer of aluminium hydroxide+VP1 immune group, aluminium hydroxide+MPLA+VP1 immune group, MPLA+VP1 immune group is relatively, many about 1:70000 greatly; Aluminium hydroxide+inactivation of viruses immune group ELSIA tires relatively low, many about 1:512 greatly; Normal mouse matched group and an adding aluminum hydroxide vehicle control group are tired and are only had 1:16.
3. the NAT of immune adjuvants and VP1 antigen group serum detects
The blood serum sample of above-mentioned 9 kinds after ELSIA detects, get the higher serum of antibody titer be used for doing in and protection test, in and Protection step as follows:
1) virus dilution: virus is diluted to 100TCID in sterile chamber with DMEM cell culture fluid
50/ 50 μ l.
2) serum-dilution: above-mentioned 6 serum are marked A, B, C, D, E, F respectively, what in sterile tube, add 0.35ml contains 10% dual anti-cell culture fluid, add the blood serum sample of 0.05ml again, mixing, 56 DEG C, 30min deactivation, be the dilute serum of 1:8, then be mixed with 1:16,1:32,1:64,1:128,1:256,1:512 and 1:1024 dilute serum gradient with containing 1% dual anti-cell culture fluid.
3) in 96 orifice plates, every hole adds 50 μ l dilute serums.
4) in every hole, add 50 μ l concentration is 100TCID
50the virus dilution liquid of/50 μ l.
5) mix, put into 37 DEG C of CO
22h is hatched in incubator.
6) add the every hole of Vero cell suspension 100 μ l (for going down to posterity 4-5 for cell after recovery) being in exponential phase, concentration of cell suspension is 2 × 10
5individual/ml, puts into 37 DEG C of CO
2cultivation is hatched in incubator.
7) use inverted microscope to observe CPE and record every day, when complete pathological changes appears in virus control wells, judge final result (about 5-7d), result of the test is as (Fig. 5).As seen from Figure 5, the NAT of aluminium hydroxide+inactivation of viruses immune group is the highest, greatly about 1:256; Aluminium hydroxide+MPLA+VP1 immune group is taken second place, and is approximately 1:128; MPLA+VP1 immune group NAT is approximately 1:64; Other immune group neutralizing antibody antibody titer is within 1:8, and therefore, relative VP1 subunit vaccine, aluminium hydroxide and MPLA mixing adjuvant immunity group are optimal case.
Claims (10)
1. mix a human enterovirus 71 subunit vaccine for adjuvant, it is characterized in that comprising the VP1 albumen of EV71 and the mixing adjuvant of aluminum and MPLA coupling.
2. the human enterovirus 71 subunit vaccine of mixing adjuvant as claimed in claim 1, it is characterized in that the coding nucleotide sequence of the VP1 albumen of EV71 is as shown in SEQIDNO.1, the VP1 protein amino acid sequence of EV71 is as shown in SEQIDNO.2.
3. the human enterovirus 71 subunit vaccine of mixing adjuvant as claimed in claim 1, is characterized in that the VP1 albumen of described EV71 is recombinant expressed in escherichia coli and obtains through ultrasonic, washing, degeneration.
4. the human enterovirus 71 subunit vaccine of mixing adjuvant as claimed in claim 1, is characterized in that the VP1 albumen of expressing is not with any unnecessary sequence, and obtains through corresponding ion-exchange purification and molecular sieve purification under Denaturing.
5. the human enterovirus 71 subunit vaccine of mixing adjuvant as claimed in claim 1, is characterized in that described subunit vaccine is that VP1 albumen combines with aluminium adjuvant under urea-denatured condition, is mixed to get after rinsing liquid washing with MPLA adjuvant.
6. the human enterovirus 71 subunit vaccine of mixing adjuvant as claimed in claim 1, is characterized in that aluminium adjuvant used is aluminium hydroxide or aluminum phosphate.
7. the human enterovirus 71 subunit vaccine of the mixing adjuvant according to any one of claim 1 ~ 6, it is characterized in that the weight ratio of VP1 albumen and aluminum and MPLA adjuvant be 1 ?50:1 ?100:1 ?10; Preferred 25:50:5.
8. the preparation method of enterovirns type 71 subunit vaccine as claimed in claim 1, is characterized in that comprising following steps:
(1) the VP1 sequence clone shown in SEQIDNO.1 is entered pET22b expression vector establishment become pET22b ?VP1 recombinant expression carrier, plasmid is imported e. coli bl21 trxB (DE3) and carry out abduction delivering, the bacterium liquid obtained is through ultrasonic centrifugal acquisition VP1 inclusion body protein;
(2) inclusion body protein is through repeatedly washing with centrifugal containing the carbamide of variable concentrations and the lavation buffer solution of surfactant, final with the solubilize inclusion body containing high concentration urea; The inclusion body protein dissolved exchanges purification through strong anion under carbamide existent condition can obtain the higher VP1 albumen of purity, further with obtaining the antigen VP1 albumen of purity more than 95% after gel filtration chromatography;
(3) the degeneration VP1 albumen that after purification, carbamide dissolves is combined with aluminium adjuvant, repeatedly washs drift and removes denaturant, obtain the mixture of aluminium adjuvant and antigen protein, mixed by this mixture be finally prepared into EV71 subunit vaccine with MPLA adjuvant with physiological buffer.
9. preparation method according to claim 8, is characterized in that described step (2) comprising:
1. the washing of VP1 inclusion body protein:
Inclusion body lavation buffer solution 1 condition of ice bath obtained is washed 2 ?3 times, be 1 ~ 1.5h at every turn, after centrifugal supernatant discarded, add in precipitation subsequently lavation buffer solution 2 condition of ice bath wash 2 ?3 times, be 1 ~ 1.5h at every turn, after centrifugal supernatant discarded, add lavation buffer solution 3, condition of ice bath washing 1 ~ 1.5h, centrifugally remove supernatant, finally by inclusion body ultra-pure water ice bath washing 1 ~ 1.5h, centrifugally remove supernatant, again use ultra-pure water rinse, the albumen finally obtained is the VP1 inclusion body protein after washing, by inclusion body protein with dissolving buffer solution, insoluble matter is slightly ultrasonic, harvested by centrifugation supernatant is the inclusion body protein after carbamide dissolving, wherein, lavation buffer solution 1 formula is: 20mmol/LTris ?HClpH8.0,0.15mol/LNaCl, 2mol/L carbamide, 1%TritonX ?100,5mmol/LEDTA, lavation buffer solution 2 formula is: 20mmol/LTris ?HClpH8.0,0.15mol/LNaCl, 4mol/L carbamide, 1%TritonX ?100,5mmol/LEDTA, lavation buffer solution 3 formula is: 20mmol/LTris ?HClpH8.0,0.15mol/LNaCl, 2mol/L carbamide, 1%TritonX ?100,5mmol/LEDTA, 2% NaTDC, dissolving buffer formulation is: 20mmol/LTris ?HClpH8.0,8mol/L carbamide, 70mmol/L β ?mercaptoethanol,
2. the anion exchange purification of inclusion body protein:
Install the strong anion exchange column through soda acid rinsing or weak anion exchange column in advance, strong anion carried out to the albumen after solubilization of inclusion bodies and exchanges purification; First use a large amount of ultrapure water pillars, then use level pad, until baseline balance, by slow for 25ml protein sample loading, again use Equilibration buffer wash pillar, until baseline balance, wherein do not collect protein stream and wear liquid, albumen almost all combines; By sample elution buffer 1 eluting after balance, the destination protein obtained after purification, purity of protein is more than 95%; Wherein, described equalizing and buffering formula of liquid is: 20mmol/LTris-HClpH8.0,8mol/L carbamide, 70mmol/L beta-mercaptoethanol, elution buffer 1 formula is: 20mmol/LTris-HClpH8.0,8mol/L carbamide, 70mmol/L beta-mercaptoethanol, 80mmol/LNaCl;
3. VP1 inclusion body protein SepharoseCL ?4B gel filtration chromatography:
The embedding of PEG20000 bag filter is carried out to the sample after ion-exchange purification concentrated, the about 1.2mg/ml of sample concentration after concentrated, the about 25ml of volume, to concentrating sample carry out SepharoseCL ?4B gel filtration chromatography, concrete steps comprise: first use ultrapure water pillar, then use level pad, until baseline balance, by slow for the sample after ion-exchange purification loading, use Equilibration buffer wash pillar, collect corresponding eluting peak.
10. preparation method according to claim 8, it is characterized in that described step (3) comprising: the first and aluminum hydroxide adjuvant combination by the VP1 protein solution of purification, obtain the antigen protein of aluminum hydroxide adjuvant parcel, again parcel albumen is added the mixing of MPLA adjuvant, obtain the human enterovirus 71 subunit vaccine of described mixing adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511023413.2A CN105535963A (en) | 2015-12-31 | 2015-12-31 | EV71 subunit vaccine of mixed adjuvant and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511023413.2A CN105535963A (en) | 2015-12-31 | 2015-12-31 | EV71 subunit vaccine of mixed adjuvant and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105535963A true CN105535963A (en) | 2016-05-04 |
Family
ID=55815837
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511023413.2A Pending CN105535963A (en) | 2015-12-31 | 2015-12-31 | EV71 subunit vaccine of mixed adjuvant and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105535963A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907776A (en) * | 2016-05-20 | 2016-08-31 | 金福赛（北京）生物科技有限公司 | Subunit vaccine capable of inducing immune response to porcine reproductie and respiratou syndrome virus |
| 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 |
| CN111000991A (en) * | 2019-12-20 | 2020-04-14 | 浙江普康生物技术股份有限公司 | Novel coxsackievirus A group 6 type recombinant subunit protein vaccine and preparation method thereof |
| CN113773371A (en) * | 2021-09-16 | 2021-12-10 | 北京凯悦宁医药科技有限公司 | VP1 protein, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337289A (en) * | 2011-09-30 | 2012-02-01 | 中国疾病预防控制中心病毒病预防控制所 | Prokaryotic expression of enterovirus 71 type VP1 (virus protein 1) and vaccine containing VP1 |
-
2015
- 2015-12-31 CN CN201511023413.2A patent/CN105535963A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337289A (en) * | 2011-09-30 | 2012-02-01 | 中国疾病预防控制中心病毒病预防控制所 | Prokaryotic expression of enterovirus 71 type VP1 (virus protein 1) and vaccine containing VP1 |
Non-Patent Citations (4)
| Title |
|---|
| MEGAN K. L. MACLEOD等: "Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells", 《PNAS》 * |
| SANDRA L. GIANNINI等: "Enhanced humoral and memory B cellular immunity using HPV16、18 L1 VLP vaccine formulated with the MPL、aluminium salt combination (AS04) compared to aluminium salt only", 《VACCINE》 * |
| SE-HEE MOON等: "Evaluation of hyaluronic acid-based combination adjuvant containing monophosphoryl lipid A and aluminum salt for hepatitis B vaccine", 《VACCINE》 * |
| 李晓楠等: "EV71型vP1蛋白表达、纯化及免疫原性的初步评价", 《免疫学杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907776A (en) * | 2016-05-20 | 2016-08-31 | 金福赛（北京）生物科技有限公司 | Subunit vaccine capable of inducing immune response to porcine reproductie and respiratou syndrome virus |
| CN105907776B (en) * | 2016-05-20 | 2019-10-29 | 金福赛（北京）生物科技有限公司 | The subunit vaccine of the immune response for pig blue-ear disease poison can be induced |
| 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 |
| CN111000991A (en) * | 2019-12-20 | 2020-04-14 | 浙江普康生物技术股份有限公司 | Novel coxsackievirus A group 6 type recombinant subunit protein vaccine and preparation method thereof |
| CN111000991B (en) * | 2019-12-20 | 2023-04-21 | 浙江普康生物技术股份有限公司 | Coxsackie virus A group 6 recombinant subunit protein vaccine and preparation method thereof |
| CN113773371A (en) * | 2021-09-16 | 2021-12-10 | 北京凯悦宁医药科技有限公司 | VP1 protein, and preparation method and application thereof |
| CN113773371B (en) * | 2021-09-16 | 2024-06-18 | 北京凯悦宁医药科技有限公司 | VP1 protein, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104404074B (en) | The preparation method of hoof-and-mouth disease virus capsid protein series connection coexpression and virus-like particle | |
| CN102864157B (en) | Immune protective antigen of haemophilus parasuis | |
| CN105535963A (en) | EV71 subunit vaccine of mixed adjuvant and preparation method thereof | |
| CN103585625A (en) | Porcine epidemic diarrhea recombinant baculovirus gene engineering subunit vaccine, preparation method and application thereof | |
| CN104873967A (en) | O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof | |
| CN105418738A (en) | A-type antigen polypeptide, fusion antigen polypeptide and vaccine of foot and mouth disease virus | |
| CN105198970A (en) | VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein | |
| CN104211784B (en) | It is used to prepare the protein and method of Hepatitis E virus sample particle | |
| CN102816246B (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| JP2010538619A (en) | Malva mosaic virus and virus-like particles and their use | |
| CN102058881B (en) | Gene recombinant vaccine for preventing enterovirus 71 infection and preparation method thereof | |
| CN106554965B (en) | It is used to prepare the nucleic acid construct and method of EV71 virus-like particle | |
| Ou et al. | Construction, expression and immunogenicity of a novel anti-hypertension angiotensin II vaccine based on hepatitis A virus-like particle | |
| DK2899267T3 (en) | Method for in vitro preparation of rotavirus double-layered virus-like particles | |
| CN104436187A (en) | Vaccine for expressing rabbit hemorrhagic disease virus VP60 protein | |
| CN1470285A (en) | Polypeptide vaccine of anti Asiatic I virus of foot-and-mouth disease and its preparing method | |
| CN105267988A (en) | Novel echinococosis granulosis vaccine with CPG DNA (deoxyribonucleic acid) immune adjuvants | |
| Chen et al. | Development and evaluation of nucleoprotein-based rapid detection test for Siniperca chuatsi rhabdovirus | |
| CN105267989A (en) | Novel water-soluble echinococosis granulosis vaccine with immune adjuvants | |
| CN106397546B (en) | Artificial recombinant antigen of O-type foot-and-mouth disease virus, preparation and application thereof | |
| CN102242132B (en) | Enterovirus 71 capsid protein 1 antigen, preparation method and immunodetection reagent | |
| Zhang et al. | Efficient capture and detection of Zika virion by polyclonal antibody against prokaryotic recombinant envelope protein | |
| CN101955950A (en) | Method for preparing recombinant fibrin-related antigen of egg drop syndrome viruses | |
| CN108619508A (en) | A kind of season influenza-RSV- epidemic meningitis-pneumococcus combined vaccine based on recombinant vector albumen | |
| CN113773371B (en) | VP1 protein, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160504 |
|
| RJ01 | Rejection of invention patent application after publication |