CN109053896A - A kind of pig circular ring virus bivalent genetic engineering vaccine - Google Patents
A kind of pig circular ring virus bivalent genetic engineering vaccine Download PDFInfo
- Publication number
- CN109053896A CN109053896A CN201810717823.4A CN201810717823A CN109053896A CN 109053896 A CN109053896 A CN 109053896A CN 201810717823 A CN201810717823 A CN 201810717823A CN 109053896 A CN109053896 A CN 109053896A
- Authority
- CN
- China
- Prior art keywords
- thr
- leu
- arg
- circular ring
- vaccine
- 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.)
- Granted
Links
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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial 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
-
- 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 preparations of pig circular ring virus bivalent genetic engineering vaccine.The vaccine includes PCV1 structural proteins Cap protein subunit, PCV3 structural proteins Cap protein, pig interleukin -5 and purification tag.This vaccine preparation process stabilizing is suitble to large-scale production.Animal experiment shows that pig circular ring virus bivalent genetic engineering vaccine of the present invention has good safety, and animal body can be induced to generate stronger immune response, effectively prevents the infection of pig circular ring virus 1 type and 3 types.
Description
Technical field
The invention belongs to biotechnology genetic engineering fields, are related to a kind of for preventing 1 type of pig circular ring virus and pig annulus
The fusion protein of viral 3 types.Specifically, using gene recombination technology, by 1 type Cap protein subunit of pig circular ring virus, pig annulus
Viral 3 type Cap proteins are connected with molecule adjuvant IL-5, and are cloned into carrier, host strain are converted, through everfermentation, purifying, emulsification etc.
Technique obtains a kind of pig circular ring virus bivalent genetic engineering vaccine and the vaccine in prevention 1 type of pig circular ring virus and pig circular ring virus 2
Application in malicious 3 types.
Background technique
Pig circular ring virus (Porcine circovirus, PCV) belongs to circovirus section Circovirus, is that a kind of annular is single
Chain DNA virus.PCV Genome Size is about 1.7kb, and Cap protein is the exclusive architecture albumen of PCV, and related (Yankee is immunized to it
Gift etc., 2017).Traditionally PCV is divided to for two kinds of serotypes of PCV-1 and PCV-2, two kinds of serotypes infection can cause breeding to hinder
Hinder, causes sow to return feelings rate and increase, produce the mummification of fetus, miscarriage and stillbirth and the weak son of production etc., although clinically caused by PCV-2
Breeding difficulty is more serious, but PCV-1 pollution rate in normal swinery and pig source cell is high, brings to clinical prevention and greatly chooses
War.
American scholar Palinski in 2016 etc., which is reported in, has found a kind of novel pig circle in the swinery with PDNS
Circovirus virus is named as 3 type of pig circular ring virus (PCV-3).PCV-3 genome includes 2000 bases, is had and PCV-1 and PCV-2
Similar genome structure, two genes of main code Cap and Rep.From PCV-3 since U.S.'s discovery, China, South Korea, wave
The countries and regions such as blue, Brazil and Italy also report the prevalence of the virus in succession, and detection is found on a large scale, which can be with
It is detected in 8 provinces in China and 1 municipality directly under the Central Government, clinical manifestation goes out the typical pigskin inflammation nephrotic syndrome lesion in part, packet
Include necrotizing angitis, ephritis, granulomatous adenolymphitis, bronchus interstitial pneumonia.
IL-5 (Interleukin-2, IL-5) once be referred to as Bcell growth factor, t cell replacing factor, be by
A kind of faintly acid glycoprotein containing N- acetylgalactosamine residues that the second hypotype of THC generates after the stimulation such as PPD, has
The effect for promoting B cell differentiation to generate with growth, induced eosinophil's differentiation, inducing cytotoxic T-cell (CTL), is one
Kind has the lymphokine of multiple biological function.
It there is no domestic or international registration PCV-3 vaccine and PCV-1 and PCV-3 bivalent vaccine at present.
Summary of the invention
Effect of the present invention according to structural proteins in PCV virus infection selects PCV-1 Cap protein subunit and PCV-
3 Cap proteins are cloned into pRSETB load by connecting again with IL-5 after flexible Linker connection as vaccine frame structure
Escherichia coli are converted after body, through techniques such as everfermentation, purifying, emulsifications, obtain the pig circular ring virus two with Desirable immunogenic
Bivalent gene engineered vaccine.Prepared by the method vaccine can effectively prevent 3 type of 1 type of pig circular ring virus and pig circular ring virus
Infection.
Preferably, PCV-1 Cap protein of the present invention subunit, amino acid sequence selected from SEQ ID No.4 or its
Function equivalent.
Preferably, PCV-3 Cap protein of the present invention, amino acid sequence or its function selected from SEQ ID No. 6 etc.
Valence object.
Preferably, IL-5 of the present invention, amino acid sequence or its function equivalent selected from SEQ ID No. 8.
Carrier required for fusion protein of the present invention also includes pharmaceutically acceptable salt and expresses.
The vaccine also includes nonimmune active material, and the coupling part of as each polypeptide is exempted from without epitope
Epidemic focus does not have any adjuvanticity yet, mainly there is purification tag, joint peptide, chemical modification part etc..
Pig circular ring virus bivalent genetic engineering vaccine provided by the present invention has good safety, clinically there is higher exempt from
Epidemic focus, the high-level specific antibody of stimulation animal body generation, effective prevention 1 type of pig circular ring virus and 3 type of pig circular ring virus
Infection.
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 is the schematic diagram of the expression vector pRSETB-PCV-IL-5 containing fusion protein encoding gene.
Fig. 2 shows electrophoresis result of the recombinant expression carrier pRSETB-PCV-IL-5 after I+Hind of BamH, III digestion,
Wherein swimming lane 1 is DNA Marker, and swimming lane 2 is non-digestion control, and swimming lane 3 is plasmid enzyme restriction figure, and swimming lane 4 is empty plasmid.
Fig. 3 is fusion protein encoding gene expression product SDS-PAGE qualification result, and wherein swimming lane 1 is molecular weight
Marker, is followed successively by 97.4KD, 66.2KD, 43KD, 31KD, 22.0KD, 14.4KD from top to bottom, and swimming lane 2 is negative control, swimming
Road 3 is induction purification of samples.
Fig. 4 is Sample Purification on Single Western Blot Blot results, and wherein swimming lane 1 is pre-dyed Marker, from top to bottom successively
For 97.4KD, 66.2KD, 43KD, 31KD, 22.0KD, 14.4KD, swimming lane 2 is negative control, and swimming lane 3 is sample.
Fig. 5 be immune small white mouse ELISA detect serum specific antibody as a result, wherein (- ◆ -) be 20171107 groups；(-
■ -) it is 20171108 groups；(- ▲ -) it is 20171109 groups；(- × -) it is blank control group.
Specific embodiment
Specific test method description as described in the examples is only exemplary description, for the present invention to be elaborated, but simultaneously
It is not meant to limit the scope of the invention, experimental method as described below does not illustrate, according to " Molecular Cloning: A Laboratory
Guide " progress of (2002, the third edition, Science Press) the method.
The source of one antigen-4 fusion protein gene of embodiment
Gene order, antigenic structure, the prevalence of comprehensive analysis domestic and international 1 type of pig circular ring virus and 3 type Major Epidemic strains of the present invention
Disease learns progress, hydrophilic to its using relevant bioinformatics software according to the amino acid sequence of its structural proteins Cap protein
Property, antigenicity, plasticity, surface accessibility and secondary structure are analyzed, and predict possible B cell antigen epi-position and T cell
Epitope, and comprehensive relevant report, so that it is determined that PCV-1 and PCV-3 Cap protein subunit.It is connected by flexible Linker
It connects again with IL-5 after forming vaccine skeleton structure, the vaccine overall structure are as follows:
PCV1 Cap - PCV3 Cap - IL-5 - Tag
The building of two coli expression carrier of embodiment and expression bacterial strain
Polypeptide-coding nucleotide designed in embodiment one is served into the handsome biotech company's synthesis in sea, nucleotide fragments two
End has separately designed BamH I (5 ' end) and Hind III (3 ' end) restriction enzyme site, and synthetic segment is cloned into
On pMD18T carrier, sequencing confirms that insertion genetic fragment is consistent with sequence is related to (see sequence table).Recombinant plasmid is named
For pMD18T-PCV-IL-5, digestion processing is carried out to plasmid with corresponding restriction enzyme, coli expression carrier is selected
The pRSETB plasmid of Invitrogen company also uses identical restriction enzyme enzymatic treatment, digestion condition: 10 μ L reactants
System, system is interior to be added 2 μ L of plasmid, 5 active units of restriction enzyme (New England biolabs), 10 × buffer 1
μ L, deionized water polishing, 37.0 DEG C digestion 1.5 hours.1 μ L 200mM EDTA is added after digestion and terminates reaction.In 1% fine jade
Electrophoresis 30 minutes in sepharose electrophoresis.PRSETB plasmid and target fragment are cut under ultraviolet lamp, it is solidifying according to QIAGEN company
Plastic recovery kit specification carries out glue recycling.According to carrier: segment is that the ratio of 1:2~3 carries nucleotide fragments and expression
Body mixing, 15 μ L of reaction system are attached by T4 DNA ligase, and 16 DEG C of connections overnight, obtain recombinant plasmid and are named as
PRSETB-PCV-IL-5(is shown in Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.
Conversion: pRSETB-PCV-IL-5 being set and is melted on ice, and 1mL connection reaction solution is added, mixes again, ice-water bath 30
Minute, 42 DEG C 30 seconds, then put back to rapidly ice-water bath 90 seconds, 1mL LB culture solution be added, 37.0 DEG C of stationary cultures 1 are small
When, 10 seconds abandoning supernatants of 4000g low-temperature centrifugation are resuspended thallus with 200 μ L LB culture mediums, bacterium solution are spread evenly across containing 100
It on the LB agar plate of μ L/mL ampicillin, is inverted in 37 DEG C of insulating boxs and cultivates 12~16 hours, until clone's shape
At.
Identification: 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 enzyme BamH I and Hind III, and the clone that can cut out correspondingly sized segment is 1500bp
Left and right can primarily determine as positive colony (see figure 2), positive colony carry out determined dna sequence further verify its correctness (see
Sequence table).
Inducing expression: positive colony is incubated overnight, and morning next day transfers according to 1:100,37 DEG C shake culture 3 hours
It waits, 0.5mM IPTG induction is added, continues culture 3 hours, prepares sample.The expression feelings of conventional SDS-PAGE testing goal albumen
Condition, it is correct clone's (see figure 3) that specific band can be seen at about 55KD molecular weight.Take correct clone, amplification culture, SDS-
After PAGE confirms that expression is correct, further confirm that it expresses accuracy (see figure 4) using conventional Western Blot.Finally screen
The engineering bacteria of obtained efficient secretory expression fusion protein is named as pRSETB-PCV-IL-5/BL21 (DE3, PLysS).
Fermentation, purifying and the emulsification of three engineering bacteria of embodiment
Production strain is inoculated into 2mL and contained in the LB liquid medium of 100 μ L/mL ampicillins by fermentation, and 37 DEG C,
12 hours activated spawns of 180rpm shake culture.Activated strain is accessed into shaking flask, 37 DEG C of concussion trainings with the inoculum concentration of 1:100
It supports to OD600=3, can be inoculated in 10% ratio into fermentor.Fermentation is semisynthetic medium with culture medium, is prepared with distilled water,
Any antibiotic is not contained wherein.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 DEG C.Fermentation temperature is 37.0 DEG C ± 0.1 DEG C, molten
Oxygen control flow feeding 500mL, feed supplement when cultivating thallus OD600=1.0~1.2 after 7.0, inoculation in 20% or so, pH control
1 hour final concentration of 0.5mM of addition IPTG(afterwards) inducing expression, 6 hour post-fermentations of continuous induction terminate, and SDS- is in sampling
PAGE detects expression.
Purifying is by the thallus of collection, with inclusion body washing lotion I (1% Triton X-100,20Mm Tris-cl pH 8.0)
Ultrasound is carried out after suspension, 2000W ultrasound cracks 1 hour.4 DEG C, 12000rpm is collected by centrifugation inclusion body, and with inclusion body washing lotion II
(1% DOC, 4M urea, 20mM Tris-cl pH 8.0) suspension twice ultrasonic washs inclusion body, and secondary low-temperature centrifugation collects packet
Contain body.Inclusion body precipitating 8M urea, 0.3% Tris-cl(pH=8.0 β-ME, 20mM) it mixes, it is stirred at room temperature 4 hours,
8000rpm low-temperature centrifugation 30 minutes, discard precipitating.Albuminate 1:100 dilution, renaturation solution Tris(pH=8.0) buffer body
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 chlorination
Sodium, 20mM imidazoles, affinity column in balance, with the 20mM phosphate buffer of pH=8.0,0.5M sodium chloride, 0.5M imidazoles is washed
It is de-.Upper hydrophobic chromatography column is balanced with the 10mM disodium hydrogen phosphate of 1.5M ammonium sulfate, 100mM EDTA, pH=8.5 again, rebalancing is used
The 10mM disodium hydrogen phosphate elution of pH=8.5 carries out SDS- to get pig circular ring virus bivalent genetic engineering vaccine semi-finished product stoste
PAGE the and Western Blot marking examines and determine whether purified product is purpose albumen.
The semi-finished product stoste of purifying is diluted to 200 μ g/mL with sterilizing PBS by emulsification.Take import white oil mineral oil adjuvant
DUOPRIME (pharmaceutical grade) sterilizes 15 minutes through 121 DEG C, spare.In oily phase: water phase=50:50 ratio is prepared, and is first added oil
Enter in emulsion tank, starts blender and be slowly stirred with the speed of 80~100r/min, be slowly added into water phase, be stirred for 2 after adding
Minute, then with 5500r/min high-speed circulating emulsification 9 minutes, the single-phase vaccine of Water-In-Oil is made.According to current edition " Chinese veterinary drug
Allusion quotation " annex progress steriling test, viscosimetric analysis, Stability Determination qualification, it is placed in 2~8 DEG C and saves backup.
Example IV pig circular ring virus bivalent genetic engineering vaccine safety experiment
Material
Vaccine: pig circular ring virus bivalent genetic engineering vaccine, lot number 20171107,20171108,20171109 are researched and developed by company
Center provides.
Experimental animal: 8 week old BALB/c small white mouses please experimental animal purchased from Jinan friend and breed Co., Ltd.28 ages in days are strong
Health three way cross weanling pig is provided by the pharmacy of Guangdong Yongshun.
Method
Safety of the vaccine to small white mouse
40 8 week old BALB/c small white mouses are randomly divided into 3 sets of batches and 1 control group, 10/group.Sets of batches distinguishes skin
The pig circular ring virus bivalent genetic engineering vaccine of 3 different batches of lower injection, 0.5mL/ is only.Physiological saline is subcutaneously injected in control group
White oil emulsion 0.5mL/ is only.It is observed continuously 14 days, records the health status of small white mouse.
Safety of the vaccine to piglet
20 28 age in days health three way cross weanling pigs are randomly divided into 3 sets of batches and 1 control group, 5/group.Batch
Group difference posterior auricular muscle meat injects the pig circular ring virus bivalent genetic engineering vaccine of 3 different batches, 2mL/ head.Control group injection life
Manage salt water white oil emulsion 2mL/ only.It is observed continuously 14 days, records the health status of piglet.
Test result
Safety testing of the vaccine to small white mouse
It the results are shown in Table 1, after being immunized, three test group small white mouses 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 the clinical side reactions such as obvious local inflammation, it is no it is dead occur, and it is right
It is consistent according to group, show that pig circular ring virus bivalent genetic engineering vaccine is safe to small white mouse.
Safety testing result of 1 vaccine of table to small white mouse
| Group | Size of animal | Body temperature | Appetite | Spirit | Health status | Inflammatory reaction | The dead quantity |
| 20171107 | 10 | Normally | Normally | Normally | Well | Nothing | 0 |
| 20171108 | 10 | Normally | Normally | Normally | Well | Nothing | 0 |
| 20171109 | 10 | Normally | Normally | Normally | Well | Nothing | 0 |
| Control group | 10 | Normally | Normally | Normally | Well | Nothing | 0 |
Safety testing of the vaccine to piglet
As a result such as table 2, in the entire observation period, the piglet body temperature and appetite of all immune groups are normal, and the state of mind is good, does not go out
What incumbent clinical abnormal phenomenon, immune position do not find allergy or inflammatory reaction, and no dead generation is consistent with control group, shows
Pig circular ring virus bivalent genetic engineering vaccine is safe to weanling pig.
Safety testing result of 2 vaccine of table to piglet
| Group | Size of animal | Body temperature | Appetite | Spirit | Health status | Inflammatory reaction | The dead quantity |
| 20171107 | 5 | Normally | Normally | Normally | Well | Nothing | 0 |
| 20171108 | 5 | Normally | Normally | Normally | Well | Nothing | 0 |
| 20171109 | 5 | Normally | Normally | Normally | Well | Nothing | 0 |
| Control group | 5 | Normally | Normally | Normally | Well | Nothing | 0 |
Antibody level detection after five pig circular ring virus bivalent genetic engineering vaccine of embodiment is immune
Vaccine: pig circular ring virus bivalent genetic engineering vaccine, lot number 20171107,20171108,20171109 are researched and developed by company
Center provides.
Experimental animal: 8 week old BALB/c small white mouses please experimental animal purchased from Jinan friend and breed Co., Ltd.
Method
20 8 week old BALB/c small white mouses are randomly divided into 4 groups, 3 vaccine immunity groups and 1 blank control group, 5/group.It presses
Test small white mouse is immunized according to grouping situation, subcutaneous injection 0.2ml/ is only.Respectively 7 before exempting from and after exempting from, 14,21,28,
35, docking in 42,49,56 days is taken a blood sample, and separation serum is used for antibody test.
The recombinant protein antigen of purifying is diluted to 1 μ g/mL with the CBS(pH9.6 of 50mmol/L), is added in ELISA Plate,
100 holes μ L/, 4 DEG C of standing coatings are overnight；Remove liquid, with PBST(contain 0.05% Tween-20, pH7.4) board-washing three times, be added
Confining liquid (PBST containing 5% small horse serum), 100 holes μ L/, 37 DEG C are closed 1 hour；Board-washing (contains 5% three times, with serum dilution
The PBST of small horse serum) by measuring samples 1:50,1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400 times
Dilution is added ELISA Plate, 100 holes μ L/, while setting negative control, and 37 DEG C are incubated for 1 hour；Three times, 1:5000 dilution is added in board-washing
HRP mark sheep anti-mouse igg, 100 holes μ L/, 37 DEG C be incubated for 1 hour；Board-washing four times, 100 hole μ L/ of tmb substrate is added, is protected from light aobvious
Color 15 minutes；2M H is added2SO4Reaction is terminated, detects absorbance value (680 microplate reader of BIORAD) under 450nm wavelength.
Test result:
Such as Fig. 5,14 days after exempting from, start to detect specific antibody in the mouse serum of three batch vaccine immunity groups, then
Antibody level constantly increases, and 35 days after exempting from peak, and then stablizes decline, is continued until off-test, and three are criticized
Without significant difference between secondary vaccine group, antibody is not detected in blank control group.
Six Immunoprotection test of embodiment
Vaccine: pig circular ring virus bivalent genetic engineering vaccine, lot number 20171107,20171108,20171109 are researched and developed by company
Center provides.
Experimental animal: 28 age in days health three way cross weanling pigs are provided by the pharmacy of Guangdong Yongshun.
Method
40 28 age in days piglets are randomly divided into 8 groups, 5/group.Test piglet is immunized according to grouping situation, wherein 1 group
20171107 batches of vaccines are injected with 5 groups of posterior auricular muscle meat, 1ml/ is only；2 groups and 6 groups of posterior auricular muscle meat 20171108 batches of vaccines of injection,
1ml/ is only；3 groups and 7 groups of posterior auricular muscle meat 20171109 batches of vaccines of injection, 1ml/ is only；4 groups and 8 groups are control group.The 35th day after exempting from
1~4 group is carried out attacking poison using PCV1 BJ-1,5~8 groups are carried out attacking poison using PCV3-China/GD2016.Observation and note
The morbidity and death condition of record test piglet.Attack the state of mind, clinical symptoms, dead feelings of continuous 14 days observation swinerys after poison
Condition counts disease incidence and calculates protective rate.
Protective rate=(attack malicious control group disease incidence-vaccine immunity group disease incidence)/attack malicious control group disease incidence ] × 100%.
Test result:
As a result such as table 3, poison, the morbidity of 20171107 vaccine groups one, no death, protective rate 80% are attacked using PCV1 strain；
20171108 groups and 20171109 groups without morbidity without death, protective rate 100%；Control group five is only fallen ill, a death.It adopts
Poison is attacked with PCV3 strain, 20171107 groups and 20171108 groups without morbidity without death, protective rate 100%；20171109 groups one
Morbidity, no death, protective rate 80%；Control group five is only fallen ill, three death.
Vaccine effectiveness of the 3 pig circular ring virus bivalent genetic engineering vaccine of table to piglet
Sequence table
<110>Qingdao Ming Qin Biotechnology Co., Ltd
<120>a kind of pig circular ring virus bivalent genetic engineering vaccine
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1473
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctgccgctgc cgtttcagta ttaccgcatt cgtaaagcaa aatatgaatt ctacccgcgc 60
gatccgatca ccagcaatga acgtggcgtc ggttctacgg tggttattct ggatgcaaac 120
tttgtgaccc cgtccacgaa tctggcttat gacccgtaca ttaactatag ctctcgccat 180
accatccgtc agccgtttac gtaccacagc cgctatttca ccccgaaacc ggaactggat 240
aaaacgattg actggttcca tccgaacaat aaacgtaatc aactgtggct gcatctgaac 300
acccacacga atgtggaaca caccggtctg ggtggttctg gtttacttag agaacggact 360
tgtaacgaat ccaaacttct ttggtgccgt agaagtctgt cattccagtt ttttccggga 420
cataaatgct ccaaagcagt gctccccatt gaacggtggg gtcatatgtg ttgagccatg 480
gggtgggtct ggagaaaaag aagaggcttt gtcctgggtg agcgctggta gttcccgcca 540
gaattggttt gggggtgaag taacggctgt gttttttttt agaagtcata actttacgag 600
tggaactttc cgcataaggg tcgtcttgga gccaagtgtt tgtggtccag gcgccgtcta 660
gatctatggc tgtgtgcccg aacatagttt ttgtttgctg agctggagaa attacagggc 720
tgagtgtaac tttcatcttt agtatcttat aatattcaaa gctaatcgca gtttcccatt 780
cgtttaggcg ggtaatgaag tggttggcgt gccacggctt gttattctga ggggttccaa 840
cggaaatgac gttcatggtg gagtatttct ttgtgtagta tgtgccagct gtgggcctcc 900
taatgaatag ttttcttctg acatagcgcc ttctgtggcg tcgtcgtctc cttgggcggg 960
gtcttcttct gaatatagct ctgtgtctca tttggttctg gtatgagaat gcttctgcat 1020
ttgagtctgc taggtcttgg agctgcctac gttagtgcca ttgctgtaga aaataccatg 1080
aatagactgg tggcagagac cttgacactg ctctccattc atcgaactct gctgataggc 1140
gatgggaact tgatgatttc aactcctgta catacaaatc accaactatg cattgaagaa 1200
gtctttcagg gaatagacac gttgaagaat caaactgcac gaggggatgc cgtggaaaaa 1260
ctattccaaa acttgtcttt aataaaagaa tatatagacc gccaaaaaaa aaattgtgga 1320
ggggaaagat ggagagtaac gcaattcctg gactacttgc aagtttttct tggtgtgata 1380
aataccgagt ggacaatgga aagttaacta gaacaaaaac tcatctcaga agaggatctg 1440
aatagcgccg tcgaccatca tcatcatcat cat 1473
<210> 2
<211> 487
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Leu Pro Leu Pro Phe Gln Tyr Tyr Arg Ile Arg Lys Ala Lys Tyr Glu
1 5 10 15
Phe Tyr Pro Arg Asp Pro Ile Thr Ser Asn Glu Arg Gly Val Gly Ser
20 25 30
Thr Val Val Ile Leu Asp Ala Asn Phe Val Thr Pro Ser Thr Asn Leu
35 40 45
Ala Tyr Asp Pro Tyr Ile Asn Tyr Ser Ser Arg His Thr Ile Arg Gln
50 55 60
Pro Phe Thr Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Glu Leu Asp
65 70 75 80
Lys Thr Ile Asp Trp Phe His Pro Asn Asn Lys Arg Asn Gln Leu Trp
85 90 95
Leu His Leu Asn Thr His Thr Asn Val Glu His Thr Gly Leu Gly Gly
100 105 110
Ser Gly Met Arg His Arg Ala Ile Phe Arg Arg Arg Pro Arg Pro Arg
115 120 125
Arg Arg Arg Arg His Arg Arg Arg Tyr Val Arg Arg Lys Leu Phe Ile
130 135 140
Arg Arg Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met
145 150 155 160
Asn Val Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala
165 170 175
Asn His Phe Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe
180 185 190
Glu Tyr Tyr Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile
195 200 205
Ser Pro Ala Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp
210 215 220
Leu Asp Gly Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr
225 230 235 240
Ala Glu Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser
245 250 255
Arg Tyr Phe Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His
260 265 270
Pro Gly Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn
275 280 285
Thr Tyr Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr
290 295 300
Val Pro Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val
305 310 315 320
Trp Ile Arg Tyr Lys Ser Val Leu Gly Ser Gly Met Arg Met Leu Leu
325 330 335
His Leu Ser Leu Leu Gly Leu Gly Ala Ala Tyr Val Ser Ala Ile Ala
340 345 350
Val Glu Asn Thr Met Asn Arg Leu Val Ala Glu Thr Leu Thr Leu Leu
355 360 365
Ser Ile His Arg Thr Leu Leu Ile Gly Asp Gly Asn Leu Met Ile Ser
370 375 380
Thr Pro Val His Thr Asn His Gln Leu Cys Ile Glu Glu Val Phe Gln
385 390 395 400
Gly Ile Asp Thr Leu Lys Asn Gln Thr Ala Arg Gly Asp Ala Val Glu
405 410 415
Lys Leu Phe Gln Asn Leu Ser Leu Ile Lys Glu Tyr Ile Asp Arg Gln
420 425 430
Lys Lys Asn Cys Gly Gly Glu Arg Trp Arg Val Thr Gln Phe Leu Asp
435 440 445
Tyr Leu Gln Val Phe Leu Gly Val Ile Asn Thr Glu Trp Thr Met Glu
450 455 460
Ser Leu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val
465 470 475 480
Asp His His His His His His
485
<210> 3
<211> 333
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctgccgctgc cgtttcagta ttaccgcatt cgtaaagcaa aatatgaatt ctacccgcgc 60
gatccgatca ccagcaatga acgtggcgtc ggttctacgg tggttattct ggatgcaaac 120
tttgtgaccc cgtccacgaa tctggcttat gacccgtaca ttaactatag ctctcgccat 180
accatccgtc agccgtttac gtaccacagc cgctatttca ccccgaaacc ggaactggat 240
aaaacgattg actggttcca tccgaacaat aaacgtaatc aactgtggct gcatctgaac 300
acccacacga atgtggaaca caccggtctg ggt 333
<210> 4
<211> 111
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Leu Pro Leu Pro Phe Gln Tyr Tyr Arg Ile Arg Lys Ala Lys Tyr Glu
1 5 10 15
Phe Tyr Pro Arg Asp Pro Ile Thr Ser Asn Glu Arg Gly Val Gly Ser
20 25 30
Thr Val Val Ile Leu Asp Ala Asn Phe Val Thr Pro Ser Thr Asn Leu
35 40 45
Ala Tyr Asp Pro Tyr Ile Asn Tyr Ser Ser Arg His Thr Ile Arg Gln
50 55 60
Pro Phe Thr Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Glu Leu Asp
65 70 75 80
Lys Thr Ile Asp Trp Phe His Pro Asn Asn Lys Arg Asn Gln Leu Trp
85 90 95
Leu His Leu Asn Thr His Thr Asn Val Glu His Thr Gly Leu Gly
100 105 110
<210> 5
<211> 651
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ttacttagag aacggacttg taacgaatcc aaacttcttt ggtgccgtag aagtctgtca 60
ttccagtttt ttccgggaca taaatgctcc aaagcagtgc tccccattga acggtggggt 120
catatgtgtt gagccatggg gtgggtctgg agaaaaagaa gaggctttgt cctgggtgag 180
cgctggtagt tcccgccaga attggtttgg gggtgaagta acggctgtgt ttttttttag 240
aagtcataac tttacgagtg gaactttccg cataagggtc gtcttggagc caagtgtttg 300
tggtccaggc gccgtctaga tctatggctg tgtgcccgaa catagttttt gtttgctgag 360
ctggagaaat tacagggctg agtgtaactt tcatctttag tatcttataa tattcaaagc 420
taatcgcagt ttcccattcg tttaggcggg taatgaagtg gttggcgtgc cacggcttgt 480
tattctgagg ggttccaacg gaaatgacgt tcatggtgga gtatttcttt gtgtagtatg 540
tgccagctgt gggcctccta atgaatagtt ttcttctgac atagcgcctt ctgtggcgtc 600
gtcgtctcct tgggcggggt cttcttctga atatagctct gtgtctcatt t 651
<210> 6
<211> 214
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Met Arg His Arg Ala Ile Phe Arg Arg Arg Pro Arg Pro Arg Arg Arg
1 5 10 15
Arg Arg His Arg Arg Arg Tyr Val Arg Arg Lys Leu Phe Ile Arg Arg
20 25 30
Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val
35 40 45
Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His
50 55 60
Phe Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr
65 70 75 80
Tyr Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro
85 90 95
Ala Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp
100 105 110
Gly Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu
115 120 125
Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr
130 135 140
Phe Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly
145 150 155 160
Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr
165 170 175
Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro
180 185 190
Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile
195 200 205
Arg Tyr Lys Ser Val Leu
210
<210> 7
<211> 405
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atgagaatgc ttctgcattt gagtctgcta ggtcttggag ctgcctacgt tagtgccatt 60
gctgtagaaa ataccatgaa tagactggtg gcagagacct tgacactgct ctccattcat 120
cgaactctgc tgataggcga tgggaacttg atgatttcaa ctcctgtaca tacaaatcac 180
caactatgca ttgaagaagt ctttcaggga atagacacgt tgaagaatca aactgcacga 240
ggggatgccg tggaaaaact attccaaaac ttgtctttaa taaaagaata tatagaccgc 300
caaaaaaaaa attgtggagg ggaaagatgg agagtaacgc aattcctgga ctacttgcaa 360
gtttttcttg gtgtgataaa taccgagtgg acaatggaaa gttaa 405
<210> 8
<211> 134
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Met Arg Met Leu Leu His Leu Ser Leu Leu Gly Leu Gly Ala Ala Tyr
1 5 10 15
Val Ser Ala Ile Ala Val Glu Asn Thr Met Asn Arg Leu Val Ala Glu
20 25 30
Thr Leu Thr Leu Leu Ser Ile His Arg Thr Leu Leu Ile Gly Asp Gly
35 40 45
Asn Leu Met Ile Ser Thr Pro Val His Thr Asn His Gln Leu Cys Ile
50 55 60
Glu Glu Val Phe Gln Gly Ile Asp Thr Leu Lys Asn Gln Thr Ala Arg
65 70 75 80
Gly Asp Ala Val Glu Lys Leu Phe Gln Asn Leu Ser Leu Ile Lys Glu
85 90 95
Tyr Ile Asp Arg Gln Lys Lys Asn Cys Gly Gly Glu Arg Trp Arg Val
100 105 110
Thr Gln Phe Leu Asp Tyr Leu Gln Val Phe Leu Gly Val Ile Asn Thr
115 120 125
Glu Trp Thr Met Glu Ser
130
<210> 9
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ctagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat 60
catcat 66
<210> 10
<211> 22
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Leu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp
1 5 10 15
His His His His His His
20
Claims (5)
1. a kind of fusion protein, amino acid sequence is as shown in SEQ ID No.2.
2. a kind of nucleic acid molecules encode fusion protein described in claim 1, nucleotide sequence is as shown in 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 3 type of 1 type of pig circular ring virus and pig circular ring virus comprising egg described in claim 1
White and pharmaceutically acceptable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810717823.4A CN109053896B (en) | 2018-07-03 | 2018-07-03 | Porcine circovirus bivalent gene engineering vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810717823.4A CN109053896B (en) | 2018-07-03 | 2018-07-03 | Porcine circovirus bivalent gene engineering vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109053896A true CN109053896A (en) | 2018-12-21 |
| CN109053896B CN109053896B (en) | 2021-08-17 |
Family
ID=64819145
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810717823.4A Active CN109053896B (en) | 2018-07-03 | 2018-07-03 | Porcine circovirus bivalent gene engineering vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109053896B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020206452A1 (en) * | 2019-04-04 | 2020-10-08 | Boehringer Ingelheim Animal Health USA Inc. | Porcine circovirus type 3 (pcv3) vaccines, and production and uses thereof |
| RU2813989C2 (en) * | 2019-04-04 | 2024-02-21 | Бёрингер Ингельхайм Ветмедика Гмбх. | Porcine circovirus type 3 (pcv3) vaccines, production and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2017066772A1 (en) * | 2015-10-16 | 2017-04-20 | Kansas State University Research Foundation | Porcine circovirus type 3 immunogenic compositions and methods of making and using the same |
| CN107854688A (en) * | 2017-11-06 | 2018-03-30 | 陕西诺威利华生物科技有限公司 | Porcine circovirus 2 type and the type bivalent inactivated vaccine of pig circular ring virus 3 and preparation method thereof |
-
2018
- 2018-07-03 CN CN201810717823.4A patent/CN109053896B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017066772A1 (en) * | 2015-10-16 | 2017-04-20 | Kansas State University Research Foundation | Porcine circovirus type 3 immunogenic compositions and methods of making and 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 |
| CN107854688A (en) * | 2017-11-06 | 2018-03-30 | 陕西诺威利华生物科技有限公司 | Porcine circovirus 2 type and the type bivalent inactivated vaccine of pig circular ring virus 3 and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| AEN19720.1: "capsid protein [Porcine circovirus 1]", 《GENE BANK》 * |
| APA21932.1: "capsid protein [Porcine circovirus 3]", 《GENE BANK》 * |
| NP_999370.1: "interleukin-5 precursor [Sus scrofa]", 《GENEN BANK》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020206452A1 (en) * | 2019-04-04 | 2020-10-08 | Boehringer Ingelheim Animal Health USA Inc. | Porcine circovirus type 3 (pcv3) vaccines, and production and uses thereof |
| CN114222579A (en) * | 2019-04-04 | 2022-03-22 | 勃林格殷格翰动物保健美国有限公司 | Porcine circovirus type 3 (PCV3) vaccine and production and use thereof |
| US11701419B2 (en) | 2019-04-04 | 2023-07-18 | Boehringer Ingelheim Animal Health USA Inc. | Porcine Circovirus Type 3 (PCV3) vaccines, and production and uses thereof |
| US11896659B2 (en) | 2019-04-04 | 2024-02-13 | Boehringer Ingelheim Animal Health USA Inc. | Porcine Circovirus Type 3 (PCV3) vaccines, and production and uses thereof |
| RU2813989C2 (en) * | 2019-04-04 | 2024-02-21 | Бёрингер Ингельхайм Ветмедика Гмбх. | Porcine circovirus type 3 (pcv3) vaccines, production and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109053896B (en) | 2021-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
| CN108159409A (en) | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application | |
| CN110317278B (en) | Fusion protein of SVV and FMDV, encoding gene, expression vector, cell line, engineering bacterium, vaccine and application thereof | |
| CN101624422B (en) | Schistosoma japonicum recombinant multi-epitope antigens, method for expressing and purifying same and application thereof | |
| CN101955545A (en) | Multi-target recombination gene and application of protein thereof in preventing and treating infection of helicobacter pylori | |
| CN106279431B (en) | A kind of pig circular ring virus subunit inactivated vaccine | |
| CN104628865B (en) | A kind of pseudo- mad dog epitope polypeptide recombinant vaccine | |
| CN102816246B (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN102210860B (en) | Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof | |
| CN103450355B (en) | recombinant bovine alpha interferon and application thereof | |
| CN109627316A (en) | Recombinant protein and the application of 2 gene of grass carp IFN-γ and its coding | |
| CN105906717A (en) | Preparation method and application of Brucella multi-epitope fusion protein vaccine | |
| CN102406929B (en) | Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine | |
| CN112812193A (en) | Recombinant protein vaccine of norovirus GII.4 type and enterovirus 71 type | |
| CN109053896A (en) | A kind of pig circular ring virus bivalent genetic engineering vaccine | |
| CN112142851B (en) | Subunit fusion protein tG on rabies virus surface and preparation method and application thereof | |
| CN110358741A (en) | A kind of recombinant baculovirus and the preparation method and application thereof for expressing pig Senecan virus VP 2 gene | |
| CN106397602B (en) | A kind of reinforced chicken Marek's disease protein engineering vaccine of molecule adjuvant | |
| CN107827986B (en) | Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine | |
| CN109021115B (en) | Porcine circovirus trivalent subunit vaccine | |
| CN103992408A (en) | Preparation of blue ear disease protein engineering vaccine | |
| CN113861277A (en) | Bovine rotavirus recombinant VP8 protein and application thereof | |
| CN109517044B (en) | Porcine epidemic diarrhea virus genetic engineering antigen and antibody | |
| CN104497148B (en) | A kind of preparation for recombinating ubiquitination blue-ear disease vaccine | |
| CN110066827A (en) | Recombinant baculovirus transfer vector, recombinant baculovirus and the preparation method and application of the protein gene of gB containing porcine pseudorabies virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |