CN107827986A - Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine - Google Patents
Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine Download PDFInfo
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- CN107827986A CN107827986A CN201710345392.9A CN201710345392A CN107827986A CN 107827986 A CN107827986 A CN 107827986A CN 201710345392 A CN201710345392 A CN 201710345392A CN 107827986 A CN107827986 A CN 107827986A
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- 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
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- 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
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- 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
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- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- 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
- A61K2039/55522—Cytokines; Lymphokines; Interferons
- A61K2039/55527—Interleukins
- A61K2039/55533—IL-2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- 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
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention relates to a kind of preparation method and applications for being used to prevent the fusion protein and the fusion protein of O-shaped foot and mouth disease virus.Specifically, the present invention relates to a kind of fusion protein, the fusion protein includes the polypeptides of molecule adjuvant IL 2, T cell helper antigen epitope polypeptide, 7 sections of antigen epitope polypeptides and killer T cell epitope polypeptide related to O/Mya98 and O/PanAsia type aftosa strain major structural proteins VP1, VP2 and VP3.The invention further relates to the preparation method and applications of the fusion protein.This production of vaccine stable preparation process, it is adapted to large-scale production, experiment shows that this vaccine is safe to use, can effectively prevent the infection of different O-shaped aftosa prevalence strains.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of fusion protein for being used to prevent O-shaped foot and mouth disease virus and this melts
The preparation method and applications of hop protein.Specifically, using gene recombination technology, by T cell helper antigen epitope polypeptide, 7 sections
The antigen epitope polypeptide related to O/Mya98 and O/PanAsia type aftosa strain major structural proteins VP1, VP2 and VP3 with
And killer T cell epitope polypeptide is connected with molecule adjuvant IL-2 polypeptides, carrier is connected into, converts Host Strains, through everfermentation, pure
Change, emulsifying process preparation, obtain pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine and the vaccine pre-
Prevent the application in O-shaped mouth disease virus infection.
Background technology
Aftosa (Foot and mouth disease, FMD) is by foot and mouth disease virus (Foot-and-mouth
Disease Virus, FMDV) acute, the hot, highly contagious disease suffered from altogether of artiodactyl caused by infection, it is international
Animal doctor office (OIE) is classified as A class infectious diseases.Ox, sheep, pig are all very sensitive to the virus, infected animal alimentary canal skin shape mucous membrane,
Mouth, tongue, lip, hoof gap and coronet edge, breast and skin other bubble occurs without hair position and festers, and the sick infectiousness
Extremely strong, spread speed is fast, and Epidemic Scope is wide, and infection rate is high, and the death rate may be up to 50%-70%, livestock breeding industry be caused huge
Big economic loss.With expanding economy, the globalization of trade, international trade also exacerbates the diffusion of aftosa disease, in
State is livestock-raising big country, and it is even more most important to carry out aftosa prevention work.
Hostis Picornaviridae (Picornaviridae), Hostis (Aphthovirus), bag
Containing O, A, c-type (title Europe class), SAT1, SAT2, SAT3 (claiming African type) and 7 kinds of serotypes of Asia I (title Asian type), and it is every
One serotype is divided into a variety of hypotypes again, almost without cross immunity between each serotype.The distribution of different serotypes foot and mouth disease virus
Region is different, and O-shaped and A type foot and mouth disease virus Major Epidemics are in Africa, South America, South Asia and the Far East, and in the mouth of China's prevalence
Aphtovirus is mainly O-shaped and Asia I types, wherein O-shaped aftosa Mya-98 strains (pedigree of Burma 98) at the beginning of 2011 at me
Since state is found, multiple epidemic situation occurs in succession in 13 provinces and cities successively, has caused extensive concern.And PanAsia exists in nineteen ninety
India separates acquisition first, is then passed to Saudi Arabia, and the incoming adjacent periphery country in 1994, China in 1999
Taiwan finds that the pedigree viral infectivity is strong, host range is wide, spread speed is fast, and serious economic damage is brought to livestock-raising
Lose.At present, immunity inoculation is still to prevent the Main Means of aftosa.Preferable vaccine must be provided simultaneously with safe and effective, inexpensive
The features such as facilitating.Though there is the wind such as not thorough, the scattered poison of batch production inactivation in traditional inactivated vaccine with good immunogenicity
Danger, bio-safety hidden danger be present in production, transport and on, and the protection period is shorter.Chemical synthesising peptide vaccine safety is effective, but
Expensive production cost limits its popularization.Gene engineering vaccine has that safe, production cost is low, easy to use and can be simultaneously
The advantages that for various serotype, turn into the focus of FMD vaccine researches.Foot and mouth disease virus particle encodes four kinds of structural proteins：
VP1, VP2, VP3 and VP4, respectively it is located at capsid surface containing 60 copies, VP1, VP2 and VP3, VP4 is located inside capsid.Its
Middle VP1 G-H rings and C-terminal amino acid residue is the main antigen sites of FMDV, can induce body and produces protective immune response
(Brown F, 1995), and study at present more.
Generally, as preferable immunogene, it is necessary to simultaneously comprising purpose antigen B cell epitope and itself or external source T
Cell epitope, it can induce out the body fluid or cell immune response (Nardin EH et al, 2001) of high specific.FMDV infects
Body, which produces neutralizing antibody, needs B cell and T cell to participate in jointly, and T cell is formed in FMDV antigen and risen in immune response
Important function.FMDV VP1G-H rings have the site of inductive formation B cells, also there is the site for exciting and producing Th cells, experiment hair
It is existing, 11 different t cell epitopes in its 41-209 amino acids at least be present.Mixing Th epitopes can excite effectively T it is thin
Born of the same parents' booster action, B cell epitope that can be weak with immunogenicity itself combine to form effective peptide based immunogens.This mixing Th energy
It is derived by effectively virus or bacterial origin immunogens sequence, including measles virus F protein, hepatitis type B virus table
Face antigen, chlamydia trachomatis MOMP etc..Many known Th have shown effectively to strengthen weak immunogene
(U.S.pat.No.5759551).Potent Th epitopes magnitude range is 15-50 amino acid residue
(U.S.pat.No.5759551) special significant sequence may generally, be included with common architectural feature.
IL-2 is interleukin 2 (interleukin-2, IL-2), also known as SCIF, mainly by activating
CD4+T cells and CD8+T cells produce.IL-2 can promote T cell differentiation and maturation and amplification in vitro, promote THC growth,
Stimulate NK cell growths and strengthen its molten cell function, strengthen the effect of B cell and macrophage, be that one kind has biology extensively
The cell factor of activity.IL-2 plays a significant role in specific immune response reaction, by IL-2 and protective antigen gene
Fusion protein is connected and composed, antibody generation and cellular immune level, the immune efficacy that can strengthen subunit vaccine are advantageous.
The content of the invention
Effect of the present invention according to different structure albumen in virus infects, using relevant biological information software to Burma
98 and O-shaped foot and mouth disease virus major structural protein VP1, VP2, VP3 of Pan Asia pedigree hydrophily, antigenicity, plasticity, surface can
And property and Garnier-Robson secondary structure are analyzed, according to epitope B cell and CTL epitope positions and amino acid sequence
Conservative design oligonucleotides fragment, while introduce effective versatility Th cell epitopes and IL-2 as vaccine framework.String
Connection rear clone converts Escherichia coli after entering pRSETA carriers, and through techniques such as everfermentation, purifying, emulsifications, obtaining has preferable be immunized
Pig O/Mya98 and O/PanAsia the type foot-and-mouth disease gene engineering inactivated vaccine of originality.The vaccine prepared using the present invention can have
Effect prevents the infection of O-shaped foot and mouth disease virus.
In the first aspect, the invention provides a kind of fusion protein for being used to prevent O-shaped mouth disease virus infection, it contains
There are 7 sections of antigen tables for screening to obtain to Burma 98 and O-shaped foot and mouth disease virus major structural protein VP1, VP2, the VP3 of Pan Asia pedigree
Position, T cell helper antigen epitope polypeptide, molecule adjuvant IL-2 polypeptides and killer T cell epitope polypeptide." the O-shaped mouth hoof
Epidemic disease poison major structural protein VP1, VP2, VP3 epitope " refers to having the O-shaped foot and mouth disease virus of immunogenicity main
A part of polypeptide in Structural protein VP1, VP2, VP3 or its there is the function equivalent of essentially identical immunogenicity, preferably
Selected from SEQ ID No.8,10,12,14,16,18 and 20 amino acid sequence or its function equivalent.Described fusion protein or
Carrier required for polypeptide or pharmaceutically acceptable salt and expression epitope.Carrier can also include separately encoded each anti-
The sequence of former epitope, series connection can be carried out by genetic engineering method.The vaccine also includes nonimmune active material, is each
The coupling part of polypeptide, the immunogenicity without epitope, also without any adjuvanticity, mainly have purification tag,
Joint peptide, chemical modification part, N-terminal signal peptide and C-terminal polyadenylic acid etc..The pharmaceutically acceptable salt refers to nontoxic
Property, stimulation and allergy, suitable for the salt of human or animal tissues.Inert matter and pharmaceutically acceptable salt are this area
Known to technical staff.
In second aspect, the invention provides a kind of nucleic acid molecule, it encodes the pig described in first aspect present invention
O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine polypeptides.Nucleotide of the present invention can be rna form,
DNA form, by artificial synthesized mode synthetic antigen epitope tandem sequence, connection rear clone is then operated by genetic engineering and entered
Carrier, Escherichia coli are transformed into, by screening, fermenting, the technique such as purifying and obtain pig O/Mya98 and O/PanAsia type aftosa
Genetic engineering inactivated vaccine polypeptide.Conventional molecular biology manipulations can be carried out to the nucleic acid in the present invention, such as：PCR, limit
Property endonuclease digestion processed, connection etc..It is preferred that the nucleotide sequence in the present invention is as shown in SEQ ID No.1.
At the 3rd aspect, the invention provides a kind of carrier, and it contains the nucleic acid molecules described in second aspect of the present invention.
Term " recombinant expression carrier " herein, " expression vector ", are only called " carrier ", can interact be replaced herein sometimes,
Refer to bacterial plasmid commonly used in the art, clay, phasmid, yeast plasmid, plant cell virus, animal virus and other are each
Kind viral vector.The carrier being applicable in the present invention includes but is not limited to：The carrier (prokaryotic expression carrier) of the expression in bacterium,
The carrier (such as pichia vector, Hansenula vectors) of the expression in yeast, the shaft-like disease expressed in insect cell
Poisonous carrier, in mammalian cell expression carrier (vaccinia virus vector, retroviral vector, adenovirus vector
Deng), in plant the plant viral vector of expression and in mammal galactophore expression various carriers.In a word, only
Stable in host cell it can replicate, any plasmid and carrier can use.Preferred expression carrier includes selected marker base
Cause, as the ampicillin resistance gene of bacterium, tetracycline resistance gene, kalamycin resistance gene, streptomycin resistance gene,
Chloramphenicol resistance gene；Neomycin resistance gene, the Zeocin resistant genes of saccharomycete, selection marker the defects of saccharomycete, such as
His, Leu, Trp etc.；The neomycin resistance gene of eukaryotic, Zeocin resistant genes, dihydrofolate reductase gene and glimmering
Photoprotein marker gene etc..In a preferred embodiment, the expression vector is coli expression carrier, art technology
Personnel can utilize a series of technologies such as DNA recombinant techniques, build the DNA sequence dna containing encoding fusion protein of the present invention, suitable
The particular element such as transcription and translation regulating and controlling sequence, promoter and selected marker expression vector.Above-mentioned carrier can use
To convert, transfect suitable host cell, to obtain required fusion protein.
At the 4th aspect, the invention provides a kind of host cell, it is characterised in that described cell is with the present invention
Nucleic acid molecules conversion or transfection described in the third aspect.Host cell can be prokaryotic or eukaryotic, as carefully
Bacterium cell, yeast cells, plant cell, insect cell, mammalian cell etc..Host cell is being converted or transfected containing the present invention
After the gene order of the encoding fusion protein, that is, engineering cell or cell line are formed, available for fusion protein needed for production.
Those skilled in the art can rightly select appropriate carrier, host cell, and it is known how to convert by carrier high-efficiency or
It is transfected into host cell, method therefor includes but is not limited to：Calcium Chloride Method, electroporation are used for bacterial cell, electroporation
It is used for yeast cells with protoplast fusion method, liposome, coprecipitation of calcium phosphate, electro fusion method and microinjection are used
In eukaryotics such as mammalian cells.
At the 5th aspect, the invention provides the method for preparing the present invention one side fusion protein, and it is wrapped
Include following steps：With the fusion protein of the host cell expression one side of the invention of the 4th aspect of the present invention, and separate
Described fusion protein.The engineering cell of acquisition can express required fusion protein by conventional method culture, induction, wrap
Include fermentation process and purifying process.The albumen of above-mentioned expression can in the cell, on cell membrane or be secreted into periplasmic, cell
Outside.As needed, using physics, the chemical and other biological characteristic of fusion protein, isolated and purified.Method
Including but not limited to：Bacterium (ultrasonic wave splits bacterium, infiltration pressure break bacterium) is split, centrifugation, saltouts, molecular sieve chromatography, ion-exchange chromatography, inhales
The point focusing such as attached chromatogram (affinity chromatography, metal chelate chromatography), reverse chromatograms, high performance liquid chromatography, Capillary Electrophoresis, preparative
And conventional denaturation, renaturation process etc., these methods are well-known to those skilled in the art.
At the 6th aspect, the invention provides boar O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivation
Vaccine, it includes the polypeptide and pharmaceutically acceptable carrier described in first aspect present invention.The aftosa vaccine can
Prevent infecting for O-shaped aftosa prevalence strain.Pharmaceutically acceptable carrier of the present invention is immunopotentiator or is immunized
Adjuvant, preferably immunologic adjuvant are import white-oil adjuvant.
At the 7th aspect, the invention provides pig O/Mya98 and the O/PanAsia type described in the 6th aspect of the present invention
The application of foot-and-mouth disease gene engineering inactivated vaccine.In a preferred embodiment of the present invention, by experimental animal with spy
Determine dosage intramuscular injection, be effectively protected experimental animal.
In addition, it is necessary to, it is noted that on the basis of the disclosure of the context of the application, other of the invention have
The aspect of substantive distinguishing features is obvious for the ordinary skill people of this area.In addition, the present invention which also uses disclosure
Document, their entire contents are included to be referred to herein.
Brief description of the drawings
Drawings below is used to illustrate specific embodiments of the present invention, rather than limits what is be defined by the claims
The scope of the invention.
Fig. 1 is the schematic diagram of the expression vector pRSETA-FMDV-IL-2 containing fusion protein encoding gene.
Fig. 2 shows electrophoresis knots of the recombinant expression carrier pRSETA-FMDV-IL-2 after EcoR I+HindIII digestions
Fruit, wherein M are DNA Marker, and swimming lane 1 is plasmid enzyme restriction figure, and swimming lane 2 compares for non-digestion, and swimming lane 3 is empty plasmid.
Fig. 3 is fusion protein encoding gene expression product SDS-PAGE qualification results, and wherein M is molecular weight Marker, from
116.0KDa, 66.2KDa, 45.0KDa, 35.0KDa, 25.0KDa, 18.4KDa, 14.4KDa are up to followed successively by down, and 1 swimming lane is
Induced samples.
Fig. 4 shows Sample Purification on Single Western Blot Blot results, and wherein M is pre-dyed Marker, and 1 is sample, and 2 be the moon
Property control.
Embodiment
Specific test method description described in embodiment is only exemplary description, for elaborating the present invention, but simultaneously
Be not meant to limit the scope of the invention, experimental method as described below, do not illustrate, according to《Molecular Cloning: A Laboratory
Guide》(2002, the third edition, Science Press) methods described progress.
The source of the antigen-4 fusion protein gene of embodiment one
Comprehensive analysis Burma 98 of the present invention and gene order, antigenic structure, the epidemic disease of the O-shaped foot and mouth disease virus of Pan Asia pedigree
Progress is learned, according to its major structural protein VP1, VP2, VP3 amino acid sequence, utilizes relevant biological information software pair
Its hydrophily, antigenicity, plasticity, surface accessibility and secondary structure are analyzed, predict possible B cell antigen epi-position and
T cell antigen epitope, and comprehensive relevant report, so that it is determined that different 4 sections of strain VP1 epitopes, 2 sections of VP2 epitopes,
1 section of VP3 epitopes, while introduce T cell helper antigen epitope dominant antigen epitope and killer T cell dominant antigen table
Position.By all epitopes with being connected again with molecule adjuvant IL-2 after flexible Linker connections, the vaccine general structure is：
IL-2-The-VP1 Epitope1-VP1 Epitope2-VP1 Epitope3-VP1 Epitope4-VP2
Epitope1-VP2 Epitope2 -VP3-Tce
The structure of the coli expression carrier of embodiment two and expression bacterial strain
Designed polypeptide-coding nucleotide in embodiment one is served into the handsome biotech company's synthesis in sea, nucleotides piece
Section both ends have separately designed EcoR I (5 ' end) and HindIII (3 ' end) restriction enzyme site, and synthetic fragment is cloned
Onto pMD18T carriers, sequencing confirms that insertion genetic fragment is consistent with being related to sequence (see sequence table).Recombinant plasmid is ordered
Entitled pMD18T-FMDV-IL-2, digestion processing, coli expression carrier are carried out to plasmid with corresponding restriction enzyme
From the pRSETA plasmids of Invitrogen companies, also using identical restriction enzyme ferment treatment, digestion condition：10 μ L react
System, system is interior to add the μ L of plasmid 2,5 active units of restriction enzyme (New England biolabs), 10 × buffering
The μ L of liquid 1, deionized water polishing, 37.0 DEG C of digestions 1.5 hours.Digestion adds 1 μ L 200mM EDTA terminating reactions after terminating.
Electrophoresis 30 minutes in 1% agarose gel electrophoresis.PRSETA plasmids and purpose fragment are cut under uviol lamp, it is public according to Qiagen
Take charge of gel reclaims kit specification and carry out glue reclaim.According to carrier：The ratio that fragment is 1: 2-3 is by multi-epitope nucleotide fragments
Mix, the μ L of reaction system 15, be attached by T4 DNA ligases with expression vector, 16 DEG C of connections overnight, obtain recombinant plasmid
It is named as pRSETA-FMDV-IL-2 (see Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.
Conversion：PRSETA-FMDV-IL-2 is put and melted on ice, 1mL coupled reaction liquid is added, mixes again, ice-water bath 30
Minute, 42 DEG C 30 seconds, then put back to ice-water bath rapidly 90 seconds, add 1mL LB nutrient solutions, 37.0 DEG C of quiescent cultures 1 are small
When, 4000g low-temperature centrifugations abandon supernatant in 10 seconds, and thalline is resuspended with 200 μ L LB culture mediums, bacterium solution is spread evenly across containing 100
On the LB agar plates of μ L/mL ampicillins, culture 12-16 hours in 37 DEG C of insulating boxs are inverted in, until Clone formation.
Identification：Monoclonal on picking flat board is into LB culture mediums, 37 DEG C, 200rpm concussion and cultivates 12 hours, extracts matter
Grain, double digestion is carried out using restriction enzyme EcoR I and HindIII, the clone that can cut out correspondingly sized fragment is
1100bp or so, it can primarily determine that and carry out determined dna sequence for positive colony (see Fig. 2), positive colony and further verifying it just
True property (see sequence table).
Induced expression：Positive colony is incubated overnight, morning next day is according to 1: 100 switching, 37 DEG C of concussion and cultivates 3 hours
Wait, add 0.5mM IPTG inductions, continue culture 3 hours, prepare sample.The expression of conventional SDS-PAGE testing goals albumen
Situation, specific band can be seen at 41KD molecular weight correctly to clone (see Fig. 3).Take correct clone, amplification culture, SDS-
After PAGE verification tables reach correctly, further confirm that its expresses accuracy using conventional Western-blot (see Fig. 4).Finally sieve
The engineering bacteria of obtained efficient secretory expression fusion protein is selected, is named as pRSETA-FMDV-IL-2/BL21 (DE3, PLys).
Fermentation, purifying and the emulsification of the engineering bacteria of embodiment three
Production strain is inoculated into the LB fluid nutrient mediums that 2mL contains 100 μ L/mL ampicillins by fermentation, 37 DEG C,
12 hours activated spawns of 180rpm concussion and cultivates.The strain activated is accessed into shaking flask, 37 DEG C of concussion trainings with 1: 100 inoculum concentration
Support to OD600=3, can be inoculated with 10% ratio into fermentation tank.Fermentation is semisynthetic medium with culture medium, is matched somebody with somebody with distilled water
System, wherein not containing any antibiotic.Dissolved oxygen and pH value electrode are corrected, opens tank body stirring, revolution 300rpm, tank body exists
Line sterilizes, and when culture-liquid temp in tank is down to 37 DEG C, demarcates pH and dissolved oxygen (OD) zero point.Fermentation temperature is 37.0 DEG C ± 0.1
DEG C, dissolved oxygen control flow feeding when thalline OD600=1.0-1.2 is being cultivated in 20% or so, pH controls after 7.0, inoculation
500mL, add IPTG (final concentration of 0.5mM) induced expression within 1 hour after feed supplement, 6 hour after fermentation of continuous induction terminate, taken
Sample does SDS-PAGE detection expressions.
Purify the thalline of collection, it is mixed with inclusion body washing lotion I (1%Triton X-100,20Mm Tris-cl pH8.0)
Ultrasound, 2000W ultrasonic degradations 1 hour are carried out after outstanding.4 DEG C, 12000rpm is collected by centrifugation inclusion body, and with inclusion body washing lotion II
(1%DOC, 4M urea, 20mM Tris-cl pH8.0) suspension twice ultrasonic washs inclusion body, and secondary low-temperature centrifugation, which is collected, to be forgiven
Body.Inclusion body precipitation 8M urea, 0.3% β-ME, 20mM Tris-cl (pH=8.0) are mixed, are stirred at room temperature 4 hours,
8000rpm low-temperature centrifugations 30 minutes, discard precipitation.Albuminate 1: 100 dilutes, renaturation solution Tris (pH=8.0) buffer body
System, 0.3M arginine is added, 4 DEG C are stirred renaturation 24 hours.Renaturation solution pH=8.0 20mM phosphate buffers, 0.5M chlorinations
Sodium, 20mM imidazoles, affinity column in balance, washed with pH=8.0 20mM phosphate buffers, 0.5M sodium chloride, 0.5M imidazoles
It is de-.Use 1.5M ammonium sulfate, 100mM EDTA, pH=8.5 10mM disodium hydrogen phosphates to balance again and go up hydrophobic chromatography post, releveling,
Eluted with pH=8.5 10mM disodium hydrogen phosphates, produce pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivation epidemic disease
Seedling semi-finished product stoste, carries out whether SDS-PAGE and Western-blot markings calibrating purified product is purpose albumen.
The semi-finished product stoste of purifying is diluted to 200 μ g/mL by emulsification with sterilizing PBS.Take import white oil mineral oil adjuvant
DUOPRIME (pharmaceutical grade) sterilizes 15 minutes through 121 DEG C, standby.In oil phase: aqueous phase=50: 50 ratio is prepared, first by oil phase
Add in emulsion tank, start mixer and be slowly stirred with 80-100r/min speed, be slowly added into aqueous phase, be stirred for after adding
2 minutes, then emulsified 9 minutes with 5500r/min high-speed circulatings, the single-phase vaccine of Water-In-Oil is made.
Example IV pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine safety experiment
Material
Vaccine：Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine, lot number 20150406,
20150408th, 20150410, provided by research and development centre of company.
Experimental animal：8 week old BaIb/c small white mouses please experimental animal purchased from Jinan friend and breed Co., Ltd.28 ages in days health
Three way cross weanling pig is provided by the pharmacy of Guangdong Yongshun.
Method
Security of the vaccine to small white mouse
40 8 week old BaIb/c small white mouses are randomly divided into 3 sets of batches and 1 control group, 10/group.Batch component
Not Pi Xiazhushe 3 different batches pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine, 0.5mL/
Only.Physiological saline white oil emulsion 0.5mL/ is subcutaneously injected only in control group.Continuous Observation 14 days, observe the health status of small white mouse.
Security of the vaccine to piglet
20 first 28 age in days health three way cross weanling pigs are randomly divided into 3 sets of batches and 1 control group, 5/group.
Sets of batches difference posterior auricular muscle meat injects the pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivation epidemic disease of 3 different batches
Seedling, 2mL/ heads.Control group injecting normal saline white oil emulsion 2mL/ is only.Continuous Observation 14 days, observe the health status of piglet.
Result of the test
Safety testing of the vaccine to small white mouse
1 is the results are shown in Table, after being immunized, all test mices do not occur any allergic reaction or poisoning symptom, the state of mind
Well, body temperature, feeding, drinking-water etc. are all gone well, and do not occur obvious local inflammation equivalent damage clinical side reaction, no dead hair
Raw, consistent with control group, it is peace to small white mouse to show pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine
Complete.
Safety testing result of the vaccine of table 1 to small white mouse
| Group | Size of animal | Body temperature | Appetite | Spirit | Health status | The dead quantity |
| 20150406 | 10 | Normally | Normally | Normally | Well | 0 |
| 20150408 | 10 | Normally | Normally | Normally | Well | 0 |
| 20150410 | 10 | Normally | Normally | Normally | Well | 0 |
| Control group | 10 | Normally | Normally | Normally | Well | 0 |
Safety testing of the vaccine to piglet
As a result such as table 2, in the whole observation period, all immune piglet body temperature and appetite are normal, and the state of mind is good, not
There is any clinical anomaly, immune position does not find allergy or inflammation, and no dead generation, sets of batches is consistent with control group,
Show that pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine is safe to weanling pig.
Safety testing result of the vaccine of table 2 to piglet
| Group | Size of animal | Body temperature | Appetite | Spirit | Health status | The dead quantity |
| 20150406 | 5 | Normally | Normally | Normally | Well | 0 |
| 20150408 | 5 | Normally | Normally | Normally | Well | 0 |
| 20150410 | 5 | Normally | Normally | Normally | Well | 0 |
| Control group | 5 | Normally | Normally | Normally | Well | 0 |
The protest test of embodiment five
Material
Vaccine：Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine, lot number 20150406,
20150408th, 20150410, provided by research and development centre of company.
Experimental animal：The 35 age in days health pig 38 without O-shaped foot-and-mouth disease antibody is detected from through suckling mouse neutralization test.
Strain：Poison is attacked to be provided by the pharmacy of Guangdong Yongshun for O/Mya/98 and O/ZheJ/CHA/08 with strain.
Dilute is 10 by PBS (PH7.6-7.8,0.04M)-5、10-6、10-7、10-8, the note 2mL musculi collis per titre
Feeder pig 4 is injected, is observed 10, to be isolated in time after finding morbid pig.After off-test, kind of a half sense of the poison to pig is calculated
Dye amount ID50。
Method
38 pigs are randomly divided into two big groups, malicious group by force of O/Mya/98 and O/ZheJ/CHA/08, per the groups of Zu Zaifen 5,3
Individual immune group, malicious control group and blank group are attacked rather, immune group every group 5, attack 2/group of malicious control group and blank group rather.Exempt from
Epidemic disease group injects the genetic engineering inactivated vaccine of 3 batches, 2mL/ heads respectively, and observed after immunization each group is immunized whether target animals are adopted
Eat it is abnormal, anxious, be short of breath, body temperature fluctuation, the clinical symptoms such as spit out white foams.28 days after exempting from, in addition to blank group, every head
Use 1000ID50O/Mya/98 and O/ZheJ/CHA/08 is injected after malicious ear by force.Poison is attacked after 14 days, observes the clinical response of target animals
And record.
Result of the test
Blank group animal subject is healthy.3 vaccine immunity groups do not occur phenomena such as pig heating, be short of breath after exempting from,
Feeding is normal, and with blank group without significant difference, protective rate is 100% after attacking poison.Attack malicious control group rather all to fall ill, performance
For typical mouth disease virus infection symptom, animal subject is all dead, the results are shown in Table 3.
Pig O/Mya98 and O/PanAsia the type foot-and-mouth disease gene engineering inactivated vaccine protest test of table 3
Claims (7)
1. a kind of fusion protein for being used to prevent O-shaped mouth disease virus infection, its amino acid sequence is as shown in SEQ ID No.2.
2. a kind of nucleic acid molecules, it encodes the fusion protein described in claim 1.
3. the nucleic acid molecules described in claim 2, its nucleotide sequence is as shown in SEQ ID No.1.
4. a kind of carrier, it contains the nucleic acid molecules described in Claims 2 or 3.
5. a kind of host cell, it contains the carrier described in claim 4.
6. a kind of genetic engineering inactivated vaccine for being used to prevent O-shaped mouth disease virus infection, it includes melting described in claim 1
Hop protein and pharmaceutically acceptable carrier.
7. application of the fusion protein in O-shaped foot-and-mouth disease gene engineering inactivated vaccine is prepared described in claim 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110981946A (en) * | 2019-12-30 | 2020-04-10 | 华派生物工程集团有限公司 | Solution for large-scale production of foot-and-mouth disease virus-like particle antigen and purification and assembly method |
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| CN102372766A (en) * | 2011-07-13 | 2012-03-14 | 青岛宝麦德生物医药科技有限公司 | O-type foot-and-mouth disease multi-epitope vaccine with cross immunity protective efficiency |
| CN105820217A (en) * | 2014-03-07 | 2016-08-03 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN105906693A (en) * | 2014-03-07 | 2016-08-31 | 中牧实业股份有限公司 | Synthetic peptide vaccine as well as preparation method and application thereof |
| CN106279431A (en) * | 2016-07-13 | 2017-01-04 | 青岛明勤生物科技有限公司 | A kind of pig circular ring virus subunit inactivated vaccine |
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Patent Citations (4)
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| CN102372766A (en) * | 2011-07-13 | 2012-03-14 | 青岛宝麦德生物医药科技有限公司 | O-type foot-and-mouth disease multi-epitope vaccine with cross immunity protective efficiency |
| CN105820217A (en) * | 2014-03-07 | 2016-08-03 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN105906693A (en) * | 2014-03-07 | 2016-08-31 | 中牧实业股份有限公司 | Synthetic peptide vaccine as well as preparation method and application thereof |
| CN106279431A (en) * | 2016-07-13 | 2017-01-04 | 青岛明勤生物科技有限公司 | A kind of pig circular ring virus subunit inactivated vaccine |
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| CN110981946A (en) * | 2019-12-30 | 2020-04-10 | 华派生物工程集团有限公司 | Solution for large-scale production of foot-and-mouth disease virus-like particle antigen and purification and assembly method |
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