CN109021115A - A kind of pig circular ring virus trivalent subunit vaccine - Google Patents
A kind of pig circular ring virus trivalent subunit vaccine Download PDFInfo
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- CN109021115A CN109021115A CN201810905436.3A CN201810905436A CN109021115A CN 109021115 A CN109021115 A CN 109021115A CN 201810905436 A CN201810905436 A CN 201810905436A CN 109021115 A CN109021115 A CN 109021115A
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- circular ring
- ring virus
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- pig circular
- subunit vaccine
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Classifications
-
- 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
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—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 and application of pig circular ring virus trivalent subunit vaccine.The vaccine is using the porcine circovirus 2 type and 1 type Cap protein B cell of 3 type Cap protein of pig circular ring virus and screening and t cell epitope as vaccine frame structure, it is connected by flexible linker, Escherichia coli are converted after being cloned into pRSETB carrier, through techniques such as everfermentation, purifying, preparations, the pig circular ring virus trivalent subunit vaccine with Desirable immunogenic is obtained.Animal experiments show that pig circular ring virus trivalent subunit vaccine not only good security, but also effective humoral immunity and cell immune response can be excited.
Description
Technical field
The invention belongs to biotechnology genetic engineering fields, relate generally to a kind of pig circular ring virus trivalent subunit vaccine system
It is standby with application.Specifically, using gene recombination technology, by the porcine circovirus 2 type of 3 type Cap protein and screening of pig circular ring virus
It connects with 1 type Cap protein B cell and t cell epitope and is cloned into carrier, host strain is converted, through everfermentation, purifying, preparation etc.
Technique obtains a kind of pig circular ring virus trivalent subunit vaccine and the vaccine and is preventing a variety of diseases caused by pig circular ring virus
In application.
Background technique
Pig circular ring virus (Porcine circoviruses, PCV) is the cricoid DNA virus of sub-thread, and genome length is about
It is one of the smallest animal DNA virus for 1.7kb long.Have determined the PCV there are two types of type, i.e. 1 type of circovirus (PCV1)
With circovurus type 2 (PCV2).PCV1 was used as a kind of pollutants identification in 1974 in PK cell culture for the first time, to pig
It is only not pathogenic.PCV2 was reported for the first time in 1998, can cause the pig circular ring virus correlation disease of pig in the clinical setting
Sick (Porcine circovirus associated diseases, PCVAD) mainly causes piglet multisystem failure comprehensive
Sign, pneumonia, pigskin inflammation and nephrosis close disease and breeding difficulty eventually, are mainly shown as breathing, uropoiesis, enteron aisle, lymph, angiocarpy, mind
Through, the dysfunction of propagating system and skin, it is to influence one of the most destructive disease of live pig industry, and in the whole world
Heavy economic losses is caused in range.
Have found within 2016 a kind of new pig circular ring virus, 3 type of pig circular ring virus (PCV3).The event of first record is sent out
Life is in the pigs in 2 to 3 week of the U.S. and 9 to 10 weeks, the heart pathology symptom that these pigs have multisystem inflammation and do not assign a cause for an illness,
Clinical manifestation is infection piglet syntexis, arthroncus, respiratory disease and sow breeding difficulty etc..It is comprehensive in pigskin inflammation and nephrosis
The sow of simulator sickness (PDNS) and PCV3 is also detected that from the mummy fetus miscarried in these sows.In addition, being deposited at 48 parts
PCV3 is found in 45 parts in the PDNS tissue samples of shelves, and is collected by real-time PCR (q PCR) from respiratory disorder case
271 parts of samples in 34 parts in detect PCV3.In China, PCV3 is detected in the piggy of respiratory disease and fever,
These piglets are also negative to PCV2, Porcine Reproductive and Respiratory Syndrome virus and aujeszky's disease virus.In addition, Brazil, wave
The epidemiological study of the country such as orchid, Germany, South Korea, Thailand, Britain has also discovered 3 type of pig circular ring virus.PCV3 can be never
It detects and obtains in same porcine tissue, the PCV3 positive test symbol in stillborn foetus and semen sample shows that PCV3 has vertical transmission
Risk.
Genomic sequence analysis finds that PCV3 genome includes 2000 bases, has genome similar with PCV1 and PCV2
Structure, two genes of main code Cap and Rep.Cap gene encodes 214 amino acid residues, 19~20 few compared with PCV2, this
Outside, PCV2 is compared with duck circovirus, and similitude is only 36~37%.
Summary of the invention
The present invention is thin with the shared conservative Cap protein of the different popular strains of 3 type of pig circular ring virus, the B of porcine circovirus 2 type
The t cell epitope of 1 type of born of the same parents' epitope and pig circular ring virus is as vaccine construct, after expression in escherichia coli, by protein purification,
The techniques such as preparation obtain the pig circular ring virus trivalent subunit vaccine with Desirable immunogenic.After this vaccine immunity target animals
It can induce effective humoral immunity and cell immune response.
One of the objects of the present invention is to provide a kind of new pig circular ring virus 2s that can excite humoral immunity, cellular immunity
Malicious trivalent subunit vaccine polypeptide and its vaccine composition, vaccine composition can be used for preventing the related disease of pig circular ring virus initiation
Disease；The second object of the present invention is the provision of building and the preparation method of the pig circular ring virus trivalent subunit vaccine；This
The third purpose of invention is the provision of the engineering strain that can express the pig circular ring virus trivalent subunit vaccine；This
The fourth purpose of invention is the provision of the preparation method of the pig circular ring virus trivalent subunit vaccine；The purpose of the present invention it
Five are the provision of purposes of the circovirus trivalent subunit vaccine in prevention Porcine circovirus desease.
In a first aspect, the present invention provides a kind of recombinant porcine circovirus trivalent subunit vaccine polypeptides for prevention
And combinations thereof.The B of its shared conservative Cap protein for containing the different popular strains of 3 type of pig circular ring virus, porcine circovirus 2 type
The t cell epitope of 1 type of cell epitope and pig circular ring virus.B cell epitope, t cell epitope refer to the main of immunogenicity
A part of polypeptide or its equivalent with essentially identical immunogenicity function in nucleocapsid protein.Series connection can pass through heredity
Engineering method or artificial synthesized progress, in addition to anti-containing main nucleocapsid protein in recombinant porcine circovirus trivalent subunit vaccine
Former epitope also includes nonimmune active material.The nonimmune active material is the coupling part of each polypeptide, does not have nucleocapsid
Proteantigen immunogenicity does not have any adjuvanticity yet, mainly there is joint peptide, chemical modification part, N-terminal signal peptide and C
Hold polyadenylic acid etc..The pharmaceutically acceptable salt refers to non-toxic, stimulation and allergy, is suitable for human or animal's group
The salt knitted.Inert matter and pharmaceutically acceptable salt are well known to those skilled in the art.
It is preferred that the main core described in the recombinant porcine circovirus trivalent subunit vaccine polypeptide of first aspect present invention
Capsid protein antigen and its epitope sequences are respectively selected from the shared conservative Cap protein, pig of the different popular strains of 3 type of pig circular ring virus
The t cell epitope of 1 type of B cell epitope and pig circular ring virus of circovurus type 2.
In addition preferably, the amino acid sequence of recombinant porcine circovirus trivalent subunit vaccine polypeptide described in first aspect present invention
It arranges as follows:
(1) shared conservative Cap protein (the SEQ ID No.4) of the different popular strains of 3 type of pig circular ring virus:
(2) 1 amino acid sequence of B cell epitope of porcine circovirus 2 type is Be1 (SEQ ID No.6):
VDMMRFNINDFLPPGGGSNP；
(3) 2 amino acid sequence of B cell epitope of porcine circovirus 2 type is Be2 (SEQ ID No.8):
VPFEYYRIRKVKVEFWPCSPITQGDRGV；
(4) 3 amino acid sequence of B cell epitope of porcine circovirus 2 type is Be3 (SEQ ID No.10):
(5) 4 amino acid sequence of B cell epitope of porcine circovirus 2 type is Be4 (SEQ ID No.12):
TMYVQFREFNLKDPPLNP；
(6) 1 amino acid sequence of t cell epitope of 1 type of pig circular ring virus is Te1 (SEQ ID No.14):
PYLAHPAFRNRYRWRRKTGIFNGSGEFVLTIK
(7) 2 amino acid sequence of t cell epitope of 1 type of pig circular ring virus is Te2 (SEQ ID No.16):
(8) 3 amino acid sequence of t cell epitope of 1 type of pig circular ring virus is Te3 (SEQ ID No.18):
(9) 4 amino acid sequence of t cell epitope of 1 type of pig circular ring virus is Te4 (SEQ ID No.120):
Considered based on vaccine challenge mechanism and vaccine biochemical characteristic itself, more preferably peptide fragment combination time
Sequence are as follows: PCV3 Cap-PCV2 Be1-PCV2 Be2-PCV2 Be3-PCV2 Be4-PCV1 Te1-PCV1 Te2-PCV1 Te3-
PCV1 Te4。
Therefore, recombinant porcine circovirus trivalent subunit vaccine polypeptid acid sequence is as follows:
In second aspect, the present invention provides a kind of nucleic acid molecules, encode recombination described in first aspect present invention
Pig circular ring virus trivalent subunit vaccine polypeptide.Nucleotide of the present invention can be rna form, DNA form, by artificial synthesized
Mode synthesizes more epitope tandem sequences, then enters carrier by genetic engineering operation connection rear clone, is transformed into large intestine bar
Bacterium, screening, obtain recombinant porcine circovirus trivalent subunit vaccine polypeptide at fermentation after purification.It in the present invention can be to the core
Acid carries out conventional molecular biology manipulations, such as: PCR, digestion with restriction enzyme, connection, 5 ' end of nucleic acid design and 3 ' ends
Restriction enzyme site is added.It is preferred that the nucleotide sequence in the present invention is as follows:
Note: being restriction enzyme site in box.
In the third aspect, the present invention provides a kind of carriers, in addition to containing coding weight described in second aspect of the present invention
Group pig circular ring virus trivalent subunit vaccine nucleic acid molecule, also containing with the operable connection of the nucleotide sequence, in protokaryon
Expression control element needed for cell expresses (transcription and translation).Most basic expression control element includes promoter, transcribes eventually
Only son, enhancer, selected marker etc., these controlling elements are known in the art.Preferred pRSETB is as mesh in the present invention
Gene expression vector.
In fourth aspect, the present invention provides a kind of host cells, contain carrier described in third aspect present invention.Place
Chief cell it is inverted or transfection containing it is of the present invention coding albumen gene order, and after through detection have good heredity
After expression stability, recombinant porcine circovirus trivalent subunit vaccine antigen needed for can be used for fermentation expression production.This hair
Preferred e. coli bl21 (DE3, Plys) protein expression host strain as a purpose in bright.
At the 5th aspect, the present invention provides a kind of preparation method of recombinant porcine circovirus trivalent subunit vaccine,
The following steps are included: engineering bacterium fermentation expresses recombinant porcine circovirus trivalent subunit vaccine polypeptide, by slightly purifying and finely
Purifying process and subsequent with seedling technique, recombinant porcine circovirus trivalent subunit vaccine required for obtaining.It is directed to
Method includes but is not limited to bacterial cell disruption, inclusion body washing, centrifugation, denaturation, affinity chromatography, hydrophobic chromatography, anion exchange
Chromatography, reverse-phase chromatography, renaturation, emulsification etc..
At the 6th aspect, the present invention provides one kind for preventing recombinant porcine circovirus trivalent subunit vaccine, wraps
Include polypeptide described in first aspect present invention and pharmaceutically acceptable carrier.The recombinant porcine circovirus trivalent subunit
Vaccine can prevent syndrome caused by pig circular ring virus 2.Pharmaceutically acceptable carrier of the present invention be immunopotentiator or
Immunologic adjuvant, preferably immunologic adjuvant are W/O/W adjuvant.
At the 7th aspect, the present invention provides answering for recombinant porcine circovirus trivalent subunit vaccine described in the 6th aspect
With.Vaccine can certain effective dose intramuscular injection, intradermal or inoculated with subcutaneous injections animal, preferably intramuscular injection can generate foot
Enough effective humoral immunities and cell immune response (see embodiment five, six, seven, eight, nine, ten, 11,12), stimulate IgG's
It generates, induction IFN γ generates, while providing antiviral activity, reduces lesion reaction, protects animal attacking from pig circular ring virus
It hits.In addition, in embodiments of the invention, by carrying out laboratory safety test to vaccine, showing of the present invention
Recombinant porcine circovirus trivalent subunit vaccine is safe (see example IV).
In addition, it is necessary to which, it is noted that based on the disclosure in the context of this application, other of the invention have
The aspect of substantive distinguishing features is obvious for those of ordinary skill in the art.In addition, the present invention which also uses disclosure
Document, their entire contents are included in be referred to herein.
Detailed description of the invention
Following drawings is for illustrating specific embodiments of the present invention, rather than limits and be defined by the claims
The scope of the invention.Fig. 1 recombinant porcine circovirus trivalent subunit vaccine expression plasmid pRSETB-PCV (3/2/1) structure figures；Figure
2pRSETB-PCV (3/2/1) vector plasmid cleavage map, wherein swimming lane 1 is DNA marker, and molecular weight is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, swimming lane 2 are blank control, and swimming lane 3 is complete plasmid, and swimming lane 4 is
Digested plasmid；Fig. 3 SDS-PAGE detection figure, wherein swimming lane 1 is not induce control sample, and swimming lane 2 is albumen Marker, from up to
Under be followed successively by 175KDa, 80KDa, 58KDa, 46KDa, 30KDa, 25KDa, 17KDa, 7KDa, swimming lane 3 is induced samples, arrow
Meaning is the destination protein of expression；Fig. 4 Western-blot detection figure, wherein swimming lane 1 is pre-dyed marker, from top to bottom successively
For 99KDa, 70KDa, 43KDa, 31KDa, 24KDa, 15KDa, swimming lane 2 is negative control, and swimming lane 3 is purpose albumen.Fig. 5 is hair
Ferment sample SDS-PAGE figure: wherein swimming lane 1 be albumen Marker, be followed successively by from top to bottom 175KDa, 80KDa, 58KDa,
46KDa, 30KDa, 25KDa, 17KDa, 7KDa, swimming lane 2 are not induce control, and swimming lane 3 is positive control, and swimming lane 4 is that fermentation lures
Lead sample；Fig. 6 is fermented sample Western-blot detection figure, and wherein swimming lane 1 is pre-dyed marker, is followed successively by from top to bottom
99KDa, 70KDa, 43KDa, 31KDa, 24KDa, 15KDa, swimming lane 2 are negative control, and swimming lane 3 is positive control lane, swimming lane 4
For the purification of samples that ferments.Fig. 7 is that ELISA method detects vaccine PCV1 IgG antibody result；Fig. 8 is that ELISA method detects vaccine
PCV2；Fig. 9 is that ELISA method detects vaccine PCV3 IgG antibody result；IFN-gamma detection knot in Figure 10 immune serum
Fruit.
Specific embodiment
Specific test method description as described in the examples is only exemplary description, for elaborating the present invention, but simultaneously
It is not meant to limit the scope of the invention.
The mentality of designing of one recombinant porcine circovirus trivalent subunit vaccine albumen of embodiment
The present invention guards Cap protein, 2 porcine circovirus according to different the shared of popular strain of current 3 type of pig circular ring virus
The t cell epitope of 1 type of B cell epitope and pig circular ring virus of type is analyzed.By designed epitope series connection after in Escherichia coli table
It reaches, through techniques such as everfermentation, purifying, preparations, obtains the recombinant porcine circovirus trivalent subunit epidemic disease with Desirable immunogenic
Seedling.Prepared by the method vaccine can effectively prevent syndrome caused by pig circular ring virus.Comprehensive analysis country pig annulus
Viral 3 types, 2 types, 1 type epidemic strain genome sequence, antigenic structure, Advance of Epidemiological Research, to recombinant porcine circovirus three
Design is optimized in valence subunit vaccine, the final shared conservative Cap protein for determining the different popular strains of 3 type of pig circular ring virus
One section, 4 sections of PCV2 B cell epitope, 4 sections of PCV1 T cell antigen epitope will form the skeleton knot of vaccine after the series connection of all epitopes
Structure.The overall structure of the vaccine are as follows: PCV3 Cap-PCV2 Be1-PCV2 Be2-PCV2 Be3-PCV2 Be4-PCV1 Te1-
PCV1 Te2-PCV1 Te3-PCV1 Te4。
The building of two coli expression carrier of embodiment and expression bacterial strain
Designed polypeptide-coding nucleotide is served the handsome biotech company in sea by 1 to be synthesized, nucleotide fragments both ends point
BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site are not devised, are cloned into respectively after this segment is synthesized
On pMD18T carrier, sequencing confirms that insertion genetic fragment is consistent with implementation sequence (see sequence table).Recombinant plasmid is distinguished
It is named as pMD18T-PCV (3/2/1).Plasmid is subjected to digestion processing with corresponding restriction enzyme, Bacillus coli expression carries
Body selects the pRSETB plasmid of Invitrogen company, also uses identical restriction enzyme enzymatic treatment, digestion condition: 10 μ l are anti-
System is answered, 2 μ l plasmids are added in system, restriction enzyme is 5 active units (New England Biolabs), is added
10 × buffer, 1 μ l, deionized water polishing, 37 DEG C digestion 1.5 hours.1 μ l 200mM EDTA is added after digestion to terminate instead
It answers.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.By 2.85kb pRSETB plasmid and 1614bp PCV under ultraviolet lamp
(3/2/1) segment is cut, and carries out glue recycling according to Qiagen company gel reclaims kit specification.According to carrier: segment 1: 2
~3 ratio individually mixes multi-epitope nucleotide fragments with expression vector, and 15 μ l of reaction system is carried out by T4 DNA ligase
Connection, 16 DEG C of connections overnight, obtain recombinant plasmid and are named as pRSETB-PCV (3/2/1), (see Fig. 1), transformed competence colibacillus large intestine
Bacillus BL21 (DE3) pLysS.
2 conversions: pRSETB-PCV (3/2/1) being set and is melted on ice, and 2 μ l connection reaction solutions are added, mix again, ice-water bath
30 minutes, 42 DEG C 30 seconds, then put back to rapidly ice bath 1.5 minutes, be added 1mL LB culture solution, 37 DEG C, stationary culture 1 hour,
Thallus is resuspended with 200 μ lLB culture mediums in 10 seconds abandoning supernatants of 4000g low-temperature centrifugation；Bacterium solution is spread evenly across containing 100 μ g/
It on the LB agar plate of mL ampicillin, is inverted in 37 DEG C of insulating boxs and cultivates 12~16 hours, until Clone formation.
3 identifications: the monoclonal on picking plate is into LB culture medium, 37 DEG C, 200rpm shake culture 12 hours, extracts matter
Grain carries out double digestion using restriction endonuclease BamH I and HindIII respectively, can cut out corresponding pig circular ring virus trivalent subunit epidemic disease
The clone of seedling gene size segment, 1614bp can primarily determine as positive colony (see Fig. 2)；Positive colony carries out DNA sequence dna survey
It is fixed further to verify its correctness (see sequence table).
4 inducing expressions.Positive colony is incubated overnight, morning next day is added after culture 3 hours by 1: 100 switching
0.1mM IPTG continues culture 4 hours, prepares sample；Conventional SDS-PAGE testing goal protein expression situation --- in 65KD
(see Fig. 3), seeing specific band is correct clone；Correct clone, amplification culture are taken, SDS-PAGE is confirmed after expressing correctly,
Further confirm that it expresses accuracy using conventional Western-blot (see Fig. 4)；It, can after above-mentioned building and evaluation program
The foundation of original species word bank is carried out using the positive colony selected as engineering bacteria, strain names pRSETB-PCV (3/2/1)/BL21
(DE3, Plys).
Fermentation, purifying and the preparation of three engineering bacteria of embodiment
1 fermentation takes production strain pRSETB-PCV (3/2/1)/BL21 (DE3, Plys), is inoculated in 2mL LB Liquid Culture
(contain 100 μ g/mL ampicillins) in base, 37 DEG C, 12 hours activated spawns of 200rpm shaken cultivation.Again with 1: 100 inoculation
Amount access shaking flask, 37 DEG C of shaken cultivations to OD600=3 can be inoculated in 10% ratio into fermentor.Fermentation culture medium is hemizygous
At culture medium, prepared with distilled water.Dissolved oxygen and pH value electrode are corrected, tank body stirring is opened, revolution 300rpm, tank body goes out online
Bacterium demarcates pH and dissolved oxygen (OD) zero point when culture-liquid temp in tank is down to 37.0 DEG C.Fermentation temperature is 37.0 ± 0.1 DEG C, molten
Oxygen control flow feeding 500mL when cultivating thallus OD600=1.0~1.2 after 7.0, inoculation in 40% or so, pH control, is mended
1 hour addition IPTG (final concentration of 0.2mM) inducing expression, 5 hour post-fermentations of continuous induction terminate after material, and SDS- is in sampling
PAGE identifies expression (see Fig. 5).
The thallus that 2 purifying will be collected into, with occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0)
Ultrasound is carried out after suspension, 2000W ultrasound cracks 1 hour.4 DEG C, 12000rpm is collected by centrifugation occlusion body, and with occlusion body washing lotion II
(1%DOC, 4M urea, 20mMTris-cl PH8.0) suspension twice ultrasonic washs occlusion body, and secondary low-temperature centrifugation collection includes
Body.Occlusion body precipitating 8M urea, 0.3% β-ME, 20mM Tris-cl (pH=8.00) are mixed, are stirred at room temperature 4 hours,
8000rpm low-temperature centrifugation 30min, discards precipitating.Albuminate 1: 100 dilutes, renaturation solution Tris (PH8.0) buffer system,
Be added 0.3M arginine, 4 DEG C stirring renaturation 24 hours.The 20mM phosphate buffer of renaturation solution pH=8.0,0.5M sodium chloride,
20mM imidazoles, affinity column in balance, with the 20mM phosphate buffer of pH=8.0,0.5M sodium chloride, the elution of 0.5M imidazoles；
Up to recombinant porcine circovirus trivalent subunit vaccine semi-finished product stoste, take semi-finished product stoste do western-blot identification (see
Fig. 6).
The PBS that the semi-finished product of purifying sterilize is diluted to 300 μ g/mL by 3 preparations, and 206 oil adjuvants pass through 121 DEG C, sterilizing
It is 15 minutes, spare.In adjuvant: vaccine=1: 1 ratio is prepared, and the speed of 80~100r/min is slowly stirred, and mixes sterile point
It can be used to be immunized after dress.
Example IV recombinant porcine circovirus trivalent subunit vaccine safety testing
1 material
1.1 vaccines: recombinant porcine circovirus trivalent subunit vaccine is provided by the bright diligent biological research and development centre in Qingdao, and lot number is
201701、201702、201703。
1.2 experimental animals: 18-22g Balb/C small white mouse is purchased from Beijing China Fukang.20~30 age in days sodium selenites
(ELISA method detects PCV1, PCV2, PCV3 negative antibody, and PCR method detects PCV1, PCV2, PCV3 antigen negative) is by Guangdong
Yongshun pharmacy provides.
2 methods
Safety of 2.1 vaccines to small white mouse
18-22g Balb/C small white mouse, every batch of vaccine immunity 5, totally three batches, are immunized 15 small white mouses, and side is immunized
Method are as follows: every subcutaneous injection 0.5ml concurrently sets 2 blank controls, is observed continuously 10 days, observes the health status of small white mouse.
Safety of 2.2 vaccines to piglet
20~30 age in days sodium selenites of selection, recombinant porcine circovirus trivalent subunit vaccine, every batch of 5, totally three
15 piglets are immunized in batch.Immunization method are as follows: every pig posterior auricular muscle meat injects 2ml, while setting up blank control 5, and clinic is seen
It examines 14 days.
3 results
Safety testing of 3.1 vaccines to small white mouse
As a result such as table 1, after being immunized, the appetite of all mouse, spirit and health status are without exception, with blank control group one
Cause, it is no dead to occur, it is seen that recombinant porcine circovirus trivalent subunit vaccine be to small white mouse it is safe, be shown in Table 1.
Safety testing result of 1 vaccine of table to small white mouse
| Group | Size of animal | Body temperature | Appetite | Spirit | Health status | The dead quantity |
| 201701 | 5 | Normally | Normally | Normally | It is in a good state of health | 0 |
| 201702 | 5 | Normally | Normally | Normally | It is in a good state of health | 0 |
| 201703 | 5 | Normally | Normally | Normally | It is in a good state of health | 0 |
| Control | 2 | Normally | Normally | Normally | It is in a good state of health | 0 |
Safety testing of 3.2 vaccines to piglet
As a result such as table 2, in entire 14 days experimental observation phases, all immune piglets, body temperature, spirit and appetite are normal,
There is not any clinical abnormal phenomenon, after the test, 15 piglets are strong to live.5 piglets of blank control group also without appoint
What adverse reaction.This explanation, recombinant porcine circovirus trivalent subunit vaccine is safe to piglet.
Safety testing result of 2 vaccine of table to piglet
| Group | Size of animal | Body temperature | Appetite | Spirit | Unusual condition | The dead quantity |
| 201701 | 5 | Normally | Normally | Normally | Nothing | 0 |
| 201702 | 5 | Normally | Normally | Normally | Nothing | 0 |
| 201703 | 5 | Normally | Normally | Normally | Nothing | 0 |
| Control | 5 | Normally | Normally | Normally | Nothing | 0 |
Embodiment quintet pig circular ring virus trivalent subunit vaccine potency test animal packet, immune, evaluation method
1 experimental animal
(ELISA method detects PCV1, PCV2, PCV3 negative antibody, PCR method detection to 20~30 age in days sodium selenites
PCV1, PCV2, PCV3 antigen negative), test pig environment before starting to test adapts to one week.
2 vaccines
Recombinant porcine circovirus trivalent subunit vaccine is provided by the bright diligent biological research and development centre in Qingdao, lot number 201701,
201702、201703。
3 experimental designs and method
3.1 with 20~30 age in days sodium selenite 45, (ELISA method detects PCV1, PCV2, PCV3 negative antibody, the sides PCR
Method detects PCV1, PCV2, PCV3 antigen negative), it is divided into 3 groups, first group is Immunization group 30, each batch vaccine immunity
10 pigs, the 2nd group is attacked poison group 10 to be nonimmune, and the 3rd group is blank control group (nonimmune, non-to attack poison) 5.0 day after immune,
7 days, 14 days, 21 days, 28 days take a blood sample to experimental animal spare.5 pigs were selected at random to each batch immune group in 28 days after immune,
Every pig through collunarium and intramuscular injection be inoculated with GD-SG-09 plants of 2ml porcine circovirus 2 type (viral level be 1.0 ×
107.0TCID50/ ml), remaining 5 pigs of every batch of are inoculated with 3 type of pig circular ring virus, and every through pig collunarium and intramuscular injection circovirus
(viral level is 1.0 × 10 to 3 SD-HZ-17 plants of types5.0TCID50/ml).The feeding of piglet, drinking-water, essence are tested in observation after attacking poison
Mind, excrement, death condition detect anus temperature and record.Dissect is carried out to dead piglet.It attacks after poison 28, all piglets of slaughter take
Inguinal lymph nodes and lymphonodi mesenterici carry out pathological section detection.
3.2 antibody titers and IFN γ detection acquisition immune group and control group 0 day, 7 days, 14 days, 21 days, 28 after immune
It serum detects detection and the IFN γ Concentration Testing for carrying out immune antiboidy IgG by ELISA method；
3.3 anus temperature attack poison and a few days ago record anus temperature daily within 14 days to after attacking poison；
3.5 attack malicious protective rate
3.5.1 morbidity standard clinical observation results abnormity continues to exceed three days or anus temperature continuous three days sentence more than 40 DEG C
For morbidity, death is judged to fall ill, and the visible obvious pathology lesion of dissect is judged to fall ill.
3.5.2 it is unprotect that criterion experiment pig, which attacks malicious protective index less than 80%, has guarantor more than or equal to 80%
Shield, then calculate test pig as follows and attack malicious protective rate: experiment pig attacks malicious protective rate=have protection test pig number/test pig total
Number.
Six serum of embodiment evaluation: IgG antibody titre detection
With ELISA method to 0 day after experimental animal vaccine immunity, 7 days, 14 days, 21 days, 28 days serum antibody IgG into
Row detection.PCV1, PCV2 and PCV3 IgG changing rule are shown in Fig. 7, Fig. 8, Fig. 9.Three batches of vaccines, 0 day, 7 days nonreactives after immune
Physical examination goes out.It can detecte low-level antibody within 14 days after immune.28 days after immune, it can detecte in peripheral blood and contain
Compared with PCV1, PCV2 and PCV3 antibody titer of high titre, compared to it is non-exempt from it is non-attack control group, difference is significant (p < 0.05).
Animal IFN γ Concentration Testing is immunized in embodiment seven
0 day, 7 days, 14 days, 21 days, 28 days experimental animal serum after conventional method acquisition is immune, according to goat-anti pig IFN γ
ELISA detection kit specification carries out IFN γ Concentration Testing to the serum of acquisition.The results show that after vaccine immunity, with exempting from
The epidemic disease time increases, and IFN γ concentration persistently rises, and 28 days concentration is higher after being immunized；Rather than exempt from the non-test pig IFN γ for attacking control group
Concentration remains lower concentration (see Figure 10).
Embodiment eight attacks clinical observation and anus temperature measurement after poison
Self tapping poison a few days ago changes to 14 days measurement anus temperature to attacking after poison.GD-SG-09 plants of porcine circovirus 2 type immune to attack
Poison organizes all immune animals and increases phenomenon without body temperature.It is non-to exempt to attack malicious control group and have detection in 2 object temperature continuous three days normal, it is non-to exempt from
Attacking malicious control group to have 4 experimental animal body temperature is more than 40 DEG C, and have 3 continuous three days more than 40 DEG C.
All experimental animals of vaccine immunity group do not occur apparent clinical symptoms during entirely attacking poison, and nonimmune to attack poison right
Appetite stimulator, apathetic, the activity clinical apparent symptom such as bad are gradually showed since attacking 3 after poison days according to group.
Nine lymph node pathology of embodiment slice
Be sliced as the result is shown: all immune animals of GD-SG-09 plants of Immunization groups of porcine circovirus 2 type have no obvious leaching
Fawn on pathology lesion；It is non-exempt to attack malicious control group have the visible apparent lymph node pathology lesion of 4 experimental animals.Pig circular ring virus 3
The immune animal of SD-HZ-17 plants of type Immunization group 1 sees obvious lymph node pathology lesion；It is non-exempt to attack malicious control group have 5 experiments
The visible apparent lymph node pathology lesion of animal.
12 vaccine protective rate of embodiment determines
Animal challenge test result is summarized and is shown in Table 3, whole experiment process occurs without piglet death.According to morbidity and determine
Standard, GD-SG-09 plants of porcine circovirus 2 type of the protective rate that the vaccine immunity group experimental animal of three batches is directed to is 5/
5, being for SD-HZ-17 plants of 3 type of pig circular ring virus of protective rates is respectively 5/5,4/5,5/5, and two non-to exempt to attack poison control component
It Wei not 4/5,5/5 morbidity.
Table 3 attacks malicious challenge test result and judgement
Claims (6)
1. a kind of pig circular ring virus trivalent subunit vaccine fusion protein, amino acid sequence is SEQ ID No.2.
2. a kind of nucleic acid molecules, encode claim 1 fusion protein, specific nucleotides sequence is classified as SEQ ID
No.1。
3. a kind of carrier contains nucleic acid molecules as claimed in claim 2.
4. a kind of host cell contains carrier as claimed in claim 3.
5. a kind of for preventing the vaccine of porcine circovirus associated diseases, it includes fusion protein described in claim 1 and
Pharmaceutically acceptable carrier.
6. fusion protein described in claim 1 is preparing the application in pig circular ring virus trivalent subunit vaccine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114409741A (en) * | 2021-12-02 | 2022-04-29 | 吉林大学 | PCV2, PCV3 and PCV4 triplet subunit vaccine and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101151329B1 (en) * | 2004-12-06 | 2012-07-19 | 주식회사 코미팜 | Recombinant protein, vaccine and prevention method for pmws using the same |
| JP2013188211A (en) * | 1997-12-11 | 2013-09-26 | Univ Of Saskatchewan | Postweaning multisystemic wasting syndrome virus from pig |
| 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 |
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
-
2018
- 2018-08-06 CN CN201810905436.3A patent/CN109021115B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013188211A (en) * | 1997-12-11 | 2013-09-26 | Univ Of Saskatchewan | Postweaning multisystemic wasting syndrome virus from pig |
| KR101151329B1 (en) * | 2004-12-06 | 2012-07-19 | 주식회사 코미팜 | Recombinant protein, vaccine and prevention method for pmws using the same |
| 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 |
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
Non-Patent Citations (2)
| Title |
|---|
| DEBIN TIAN等: "Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus", 《J GEN VIROL》 * |
| 宋瑞雪等: "猪圆环病毒2型Cap蛋白B细胞表位的预测及鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114409741A (en) * | 2021-12-02 | 2022-04-29 | 吉林大学 | PCV2, PCV3 and PCV4 triplet subunit vaccine and preparation method and application thereof |
| CN114409741B (en) * | 2021-12-02 | 2023-04-21 | 吉林大学 | PCV2, PCV3 and PCV4 triple subunit vaccine, and preparation method and application thereof |
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