CN106177934B - A kind of haemophilus parasuis subunit vaccine and preparation method thereof - Google Patents
A kind of haemophilus parasuis subunit vaccine and preparation method thereof Download PDFInfo
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- CN106177934B CN106177934B CN201610641888.6A CN201610641888A CN106177934B CN 106177934 B CN106177934 B CN 106177934B CN 201610641888 A CN201610641888 A CN 201610641888A CN 106177934 B CN106177934 B CN 106177934B
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- 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
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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
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Abstract
The invention discloses a kind of for preventing the subunit vaccine and preparation method thereof of pig leather Laplace disease.By prokaryotic expression method from one plant of haemophilus parasuis (Haemophilus parasuis,H.parasuis) the outer membrane protein Omp16 of (culture presevation number: CGMCC NO.11145, application for a patent for invention publication No.: CN105524857A) clonal expression with immunogenicity.It is that haemophilus parasuis subunit microspheres vaccine has been made in material coating Omp16 albumen with chitosan, olive oil and sodium alginate using the albumen as antigen.Carry out the mouse of injection and oral immunity respectively with the vaccine, it is rightH.parasuisThe immune protective rate of Serotype 5 attack is respectively up to 80% and 60%.
Description
Technical field
The present invention relates to the preparation technical fields of zoonosis recombinant vaccine.Genetic engineering is used more particularly to a kind of
Method, which obtains outer membrane protein protection antigen from haemophilus parasuis and is made into microspheres vaccine and the vaccine, is preventing secondary pig
Application in haemophilus disease.
Background technique
Haemophilus parasuis (Haemophilus parasuis,H. parasuis) it is a kind of Grain-negative that NAD is relied on
Bacterium belongs to p pestic section hemophilus.As a kind of conditioned pathogen,H. parasuisIn pig emergency, immunosupress
Deng under the conditions of, 2-8 week old pig can be infected, the pig characterized by polyserositis, meningitis, arthritis etc. is caused to remove from office Laplace disease
(Glasser's disease).The disease disease incidence is usually 10%-20%, and the death rate is up to 50% or more.It is wide that pig removes from office Laplace disease
It is general to be present in countries in the world pig farm, the huge economic loss of pig breeding industry is caused, is the significant bacterial disease for influencing pig production
One of.
H. parasuisSerotype is numerous, has identified 15 kinds, Shang Youyue 15-40% can not parting.Different regions stream
Capable predominant serotype is not quite similar, and with 4 types, 5 types are the most popular in China.There is also bright for virulence between different serotypes bacterial strain
Significant difference is different.Clinically, antibacterials and vaccine immunity are the anti-major measures for making the disease.Bacterium is easily led to using antibiotic
Drug resistance, therefore vaccine immunity prevention is still one of main measure.Currently, existingH. parasuisCommercially available vaccine with
Based on full bacterium inactivated vaccine, butH. parasuisCulture relies on NAD, needs to add serum, and slow growth in culture medium, also easily
Pollution microbes, therefore traditional whole-bacterial-vaccine preparation method not can solve growing vaccine demand.Research and development, which have, to intersect
The new generation vaccine of protection has become current prevention and controlH. parasuisUrgent need.Subunit vaccine, which refers to, to be protected
Shield property antigen gene is expressed in protokaryon or eukaryotic, and vaccine is made with gene product-protein or polypeptide, has peace
Quan Xinggao, purity is high, stability are good, and the advantages such as yield height have broad application prospects, and are the important sides of new generation vaccine research and development
One of to.It has reported both at home and abroad at present a large amount ofH. parasuisSubunit vaccine, such as useH. parasuisLipopolysaccharides
(LPS), subunit vaccine made of capsular polysaccharide (CPS) and outer membrane protein (Omp) etc., wherein compared to lipopolysaccharides and pod membrane
Polysaccharide,H. parasuisOmp have stronger immunogenicity.However, there is also certain deficiencies for subunit vaccine, it is main
If immunogenicity is weaker than full vaccine, therefore generally requires to increase adjuvant to improve its immunogenicity, such as in subunit vaccine
The adjuvants such as aluminium hydroxide, white oil.But there are certain side effects, such as aluminium adjuvant vaccine to generate in intramuscular injection for these adjuvants
Granuloma, mineral oil adjuvant cannot be metabolized, and also can generate granuloma, inflammation, the side effects such as fever in injection portion.
Summary of the invention
The purpose of the present invention is to provide a kind of haemophilus parasuis subunit vaccines and preparation method thereof, overcome existing skill
Art defect, i.e., existing haemophilus parasuis subunit vaccine antigenic is weaker, is helped using traditional subunit vaccine adjuvant such as aluminium
Agent, mineral oil adjuvant etc. are also easy to produce the side effects such as inflammation, granuloma, therefore the using effect of existing haemophilus parasuis vaccine
The deficiencies of undesirable.
The invention is realized by the following technical scheme:
Applicant fromH. parasuis(culture presevation number: CGMCC NO.11145, application for a patent for invention are public for LY02 plants of bacterium
Cloth number: CN105524857A) in expanded to obtain a kind of outer membrane protein omp16 genetic fragment with round pcr, nucleotide sequence is such as
Shown in table SEQ ID NO.1, sequence length 486bp, the gene encodes 162 amino acid, sequence such as list SEQ ID NO.2
It is shown.
The omp16 genetic fragment of acquisition is purified, digestion handles rear clone to pET-28a(+), and outer membrane can be expressed by constructing
The recombinant plasmid pET- of albumen Omp16hps-omp16.The plasmid is converted into e. coli bl21 (DE3), is obtained under IPTG induction
Obtained the outer membrane protein Omp16 by the expression of recombinant plasmid.The great expression outer membrane protein, and by it through the affine column purification of nickel and
Renaturation.
A kind of haemophilus parasuis subunit vaccine, the vaccine include amino acid sequence as shown in SEQ ID NO.2
Adjuvant haemophilus parasuis outer membrane protein Omp16 antigen and be made of olive oil, chitosan and sodium alginate；Each component
Volume parts are as follows: 8-9 parts of the Omp16 antigen protein of 200 mg/ml, 2% 9-11 parts of (m/v) sodium alginate soln, olive oil 28-
30 parts, 1%(m/v) 19-21 parts of chitosan solution, 1-2 parts of Tween80,1-2 parts of Span80,8%(m/v) CaCl2 90-110
Part.
Preferably, the volume parts of the vaccine each component are as follows: 8.8 parts of the Omp16 antigen protein of 200 mg/ml, 2%
(m/v) 10 parts of sodium alginate soln, 28.8 parts of olive oil, 1%(m/v) 20 parts of chitosan solution, 1.2 parts of Tween80, Span80
1.2 parts, 8%(m/v) CaCl2100 parts.
Preparation method are as follows:
(1) it by the haemophilus parasuis outer membrane protein Omp16 of 200 mg/ml, is added in 2% m/v sodium alginate soln,
Tween80 is added, olive oil and Span-80 are added after being stirred, stirring keeps its fully emulsified；
(2) the slow drop of above-mentioned emulsification is entered into 8%(m/v) CaCl2In solution, stir 30 minutes, crosslinking curing forms micro-
Ball；
(3) cured microballoon is washed with sodium acetate buffer to precipitate；
(4) with 10 parts of sodium acetate buffer suspension microballoons, and be added into 1%(m/v) chitosan solution in, stirring 30
Minute；
(5) it is centrifugated microballoon, is washed with sodium acetate buffer；
(6) up to microspheres vaccine after pellet frozen is dry.
The Tween80 be 6%(v/v) Tween80.
The Span-80 be 4%(v/v) Span-80.
The sodium acetate buffer is 0.1 mol/ sodium acetate buffer.
Immunization test is carried out in mouse with the haemophilus parasuis subunit vaccine of preparation, the results show that note
It penetrates immune and oral immunity mouse and malicious protective rate is attacked respectively up to 80% and 60% to haemophilus parasuis LY02.
Advantages of the present invention:
(1) a conservative outer membrane protein of haemophilus parasuis has been screened as antigen, which has stronger exempt from
Epidemic focus；
(2) biodegradable materials such as sodium alginate, chitosan, olive oil have been used in adjuvant, in injecting immune and
It ensure that safety in oral immunity, also generation without side-effects；
(3) subunit vaccine is prepared into microballoon, injecting immune, which can be used, can also be used oral immunity, especially oral to exempt from
Epidemic disease, it is easy to use, save manpower and time.
In an embodiment of the present invention, applicant provide fromH. parasuisLY02 plants of (culture presevation number: CGMCC of bacterium
NO. 11145, application for a patent for invention publication No.: CN105524857A) the novel Outer membrane protein antigen omp16 of clonal expression and with should
Albumen prepares the detailed process of subunit vaccine.
Detailed description of the invention
The SDS-PAGE electrophoresis of Fig. 1 recombinant antigen protein Omp16 prepared by the present invention after purification.M. protein molecule
Amount standard；PET-28a 1. (+) empty vector control bacterial strain；2. pET- containing plasmid vectorHps-omp16 expression bacterium.
The Western blot qualification figure of Fig. 2 haemophilus parasuis Omp16 albumen.
Fig. 3 microspheres vaccine prepared by the present invention observes figure under the microscope.
Fig. 4 the present invention used in vector plasmid pET-28a(+) map.
Specific embodiment
Main material and reagent of the present invention
Carrier pET-28a(+) and e. coli bl21 (DE3) be purchased from Merck Bioisystech Co., Ltd；The secondary bloodthirsty bar of pig
LY02 plants of bacterium (culture presevation number: CGMCC NO.11145, application for a patent for invention publication No.: CN105524857A) is by Fujian Province
Preventive Veterinary Medicine and Vet Biotechnology key lab separating and preserving；Bacterial genomes extracts kit, PCR Mix, limitation
Property endonucleaseEcoRI andHindIII, T4 DNA ligase etc. is purchased from precious biological (Dalian) Bioisystech Co., Ltd；
PCR product QIAquick Gel Extraction Kit, extraction of plasmid DNA kit, DNA Ago-Gel QIAquick Gel Extraction Kit, low molecular weight protein standard,
Bag filter, nickel agar column (NTA), chitosan, sodium alginate, olive oil, SDS-PAGE kit and ELISA reaction plate etc. are purchased from
Shanghai Sheng Gong bioengineering Co., Ltd.BCA protein determination kit is purchased from the green skies Bioisystech Co., Ltd in Shanghai；TSA,
TSB and NAD is purchased from OXOID company；Goat-anti pig IgG, goat anti-mouse igg are public purchased from Bo Aosen Bioisystech Co., Ltd (Beijing)
Department.
Embodiment oneH. parasuisThe clone of omp16 gene and the expression in e. coli bl21 (DE3)
1, design of primers and synthesis are by designing a pair of omp16 amplimer, going to omp16 bioinformatic analysis
In addition to its signal peptide moiety, i.e. 8 amino acid sequences of N-terminal；Primer amplification fragment length is 162 total 486bp of amino acid；
omp16-up 5‘-cgggaattc(underscore part is gcgcctgtaggtgaaac -3 'EcoRI restriction enzyme site), omp16-
dn 5’- cccaagctt(underscore part is ttagaaacgataggac -3 'HindIII digestion site) primer given birth to by Shanghai
The synthesis of work bioengineering Co., Ltd.
、H. parasuisRecovery and genome take freeze-drying saveH. parasuisStrain, scribing line connect
Kind sets 37 DEG C, 5% CO in TSA solid plate2Incubator overnight incubation.The activated bacterial colony inoculation of second day picking
In TSB culture medium, 37 DEG C of 10 h of shaking table shaken cultivation.It is extracted with bacterial genomes extracts kit and pays haemophilus suis gene
Group DNA.
3, it the PCR amplification of omp16 gene and identifies using the haemophilus parasuis genomic DNA of extraction as template, establishes
PCR reaction system are as follows: 2 × PCR Mix 10 μ l, primer omp16- up (10 μM) 1 μ l, primer omp16- dn (10 μ
M) 1 μ l, with dd H2O is supplemented to 20 μ l.PCR response procedures are as follows: 95 DEG C of 5 min of denaturation, subsequently into " 94 DEG C of denaturation 40
The circulation of s, 56 DEG C of annealing 40 s, 72 DEG C of 1 min ", totally 30 times；Then 72 DEG C of 5 min of extension；Last 4 DEG C terminate instead
It answers.5 μ l reaction products are taken to make agarose gel analysis.
, recombinant plasmid pET-Hps-omp16 building
The PCR product for taking 100 μ l amplification omp16 gene, is purified with PCR QIAquick Gel Extraction Kit.It will after purification
Omp16 DNA and carrier pET-28a (+) are usedEcoRI andHindIII carries out double digestion, the method is as follows: takes 2 1.5 ml respectively
Centrifuge tube prepares endonuclease reaction by table 1:
The endonuclease reaction pipe of above-mentioned carrier and target gene is set into 37 DEG C of 2 h of incubation.Digestion is produced respectively after reaction
Object carries out agargel electrophoresis and the recycling of DNA Ago-Gel.
It is attached after endonuclease reaction and conversion reaction.Prepare coupled reaction system in 1.5 ml centrifuge tubes: 10
2 μ l of × T4 DNA connection buffer, 2 μ l, the omp16 gene DNA of pET-28a (+) (50 ng/ μ l) of the recycling of digestion
(0.2 pM/ μ l) 1 μ l, T4 DNA ligase (5 U/ μ l) 1 μ l, ddH2O is supplemented to 20 μ l.Centrifuge tube is set into 16 DEG C of companies
Take over night.It takes 10 μ l connection products to convert bacillus coli DH 5 alpha, is coated with the plate of LB containing kanamycins.37 DEG C were cultivated 12 as a child,
Picking colony carries out plasmid identification.Plasmid is extracted with extraction of plasmid DNA kit, and (square with double digestion method identification recombinant plasmid
Method is same as above), positive recombinant plasmid is converted into e. coli bl21 (DE3), obtains Omp16 protein expression bacterium.
5, expression and identification of the recombinant protein Omp16 in e. coli bl21 (DE3)
(1) it takes one expression bacterium single colonie of kalamycin resistance plate, is inoculated with the LB bacterium solution containing kanamycins, 37 DEG C, 200
R/m concussion is incubated for 12 hours, and 1 ml bacterium solution is taken to extract Plasmid DNA.By the Plasmid DNA PCR method of extraction and it is double it is restricted in
Identification is cut in enzyme cutting.The system and reaction condition of PCR method and digestion identification are the same as step 3 and 4.Positive recombination is accredited as by above-mentioned
Plasmid serves the sequencing identification of Hai Shenggong bioengineering Co., Ltd.Sequencing is accredited as positive recombinant plasmid, by its host cell
BL21 (DE3) is inoculated with LB liquid medium (the 100 μ g/ ml containing kanamycins), and 37 DEG C of 200r/m shake cultures to OD600 are
IPTG to final concentration of 1mM is added after 0.6, is continued shake culture and is induced 2-3h.
(2) SDS-PAGE analyzes the expression of recombinant protein
Bacterium solution 1 ml, 12000 r/m 2 min of centrifugation after taking induction, take precipitating, add after precipitating is resuspended with 300 μ l PBS
Enter 100 μ 4 × loading of l buffer.Later, referring to " Molecular Cloning:A Laboratory guide (third edition) " (J. Pehanorm Brooker,
Deng work, Huang Peitang is waited and is translated, Science Press) carry out SDS-PAGE electrophoretic analysis.
(3) Western blot identifies recombinant protein
After SDS-PAGE electrophoresis, transferring film reaction is carried out.It cuts and is dimensioned slightly smaller than the pvdf membrane 1 of gel and opens and filter paper
6, pvdf membrane is impregnated to 1 min in methyl alcohol, filter paper is soaked in electricity and turns 10 min in buffer.According to " filter paper-pvdf membrane-
After the sequence of gel-filter paper " successively sequences, drive away bubble, with 50 min of voltage transfer of 12-15V.Transferring film finishes, with 1 ×
PBST is washed pvdf membrane 3 times (5 min every time), and film is put into 5% skimmed milk, and 4 DEG C of closings are overnight.Confining liquid is outwelled, with 1 ×
PBST is put into diluted with 5% skimmed milk 1:500 after washing pvdf membrane 3 times (5 min every time)H. parasuisInfect pig health
In multiple serum, it is incubated at room temperature 2 h.Pvdf membrane is taken out into new plate, is put after washing 3 times (10 min every time) with 1 × PBST
Enter in the 5% goat-anti pig IgG antibody of the diluted HRP label of skimmed milk 1:5000, is incubated at room temperature 1 h.Pvdf membrane is taken out, l is used
× PBST washs 3 times (10 min every time) and is reacted afterwards with ECL chemiluminescence colour reagent box, then the tabletting in darkroom carries out
Development, fixing.
6, the great expression of recombinant protein Omp16, purifying and renaturation
Western blot is accredited as to the host strain BL21 (DE3) of expression recombination Omp16 protein positive, inducing expression
500 ml bacterium solutions.After induction, 4 DEG C, 5000 r/m are centrifuged 10 min and collect thallus.The cracking buffering of opportunistic pathogen body 1/10 is added
Liquid sufficiently suspends.Under condition of ice bath, with ultrasonic disruption cell (condition are as follows: 300 W of power works 3 seconds, interval 10 seconds, altogether
200 times).12000 r/m are centrifuged 10 min, abandon supernatant, and precipitating is added 5 ml PBS and washs 1 time.12000 r/m centrifugation 20
min.6 ml 8M Urea Lysis liquid are added, 4 DEG C stand overnight.12000 r/m are centrifuged 20 min, take supernatant.By forgiving for dissolution
Body protein solution is purified with NTA column, and method is referring to kit specification.The recombinant protein Omp16 of purifying is put into dialysis
Bag, respectively in urea containing 6M, 4M urea, 2M urea and not urea-containing PBS, sets 4 DEG C of refrigerators, and each 2h that dialyses carries out renaturation.
After renaturation, with the recombination Omp16 protein concentration of BCA kit measurement renaturation.Method is referring to kit specification.
The preparation of two haemophilus parasuis Omp16 subunit microspheres vaccine of embodiment
1, emulsification-ionic cross-linking prepares microspheres vaccine.Omp16 recombinant protein after purifying, renaturation is dissolved in 8.8
It is configured to concentration in the PBS of ml pH8.0 to be mixed after 200 mg/ml solution with 2% sodium alginate soln of 10 ml, is added
1.2 ml Tween 80, are thoroughly mixed.1.2 ml of addition 28.8 ml of olive oil, Span-80 in mixed liquor, 1500
R/min stirs 20 min, fully emulsified；100 ml, 8% CaCl is added in emulsion droplet2In solution, 1200 r/min stirring 30
Min, crosslinking curing；Cured microsphere suspensions are transferred in centrifuge tube, 1500g is centrifuged 10 min, discards supernatant liquid, microballoon
Precipitating is washed 3 times with 0.1 mol/L sodium acetate buffer (pH4.5) of 10ml, is suspended with same buffer；Take microsphere suspension 5
Ml is added to the chitosan solution (NaAc containing 0.1mol/L, pH5.0) of 20 ml, 1%(m/v) into beaker, continues stirring 10
min；1200g is centrifuged 10 min and separates microballoon, is washed 3 times with buffer, precipitates freeze-dried rear up to microspheres vaccine.
2, the mode of appearance observation and particle size determination of microballoon.The microballoon of preparation is coated on glass slide, it is micro- with being inverted
The mode of appearance of sem observation microballoon, and measure the diameter of 20 microballoons.As the result is shown the diameter of microballoon (0.1-5) μm it
Between, average diameter is (2.5 ± 0.5) μm.
3, the entrapment efficiency determination of subunit vaccine microballoon.The protein content of solution before being first coated with using BCA kit measurement, packet
After being moved to end, 1.0 ml microsphere suspensions are taken, 12000g is centrifuged 15 min, with the protein content in BCA kit measurement supernatant, calculates
Encapsulation rate [ protein content × 100% in encapsulation rate=(protein content in protein content-supernatant of solution)/solution ].As the result is shown originally
The microspheres vaccine encapsulation rate of invention preparation is 85.3%.
The potency test of three haemophilus parasuis subunit vaccine of embodiment
1, mouse immuning test.Take 50 8-10 week old cleaning grade kunming mices (purchased from Medical University Of Fujian's zoopery
Center), it is divided into 5 groups, every group 10.Each group immune programme such as table 2.The each group of injecting immune is immunized twice altogether, is spaced 14 days；Mouthful
It is 3 times immune to take stomach-filling group, every minor tick 14 days.
After last time is 14 days immune, mouse orbit venous blood is acquired, separates serum, -20 DEG C save backup.
Serum specific antibody detection.Using present invention purifying, the Omp16 albumen of renaturation, it is coated with elisa plate, establishes inspection
It surveysH. parasuisThe indirect ELISA method of antibody.It is diluted with coating buffer (0.05 mol/L carbonate buffer solution, pH9.6)
Elisa plate (200 hole ng/50 μ l/) is added after Omp16 albumen, is incubated overnight at 4 DEG C.Coating buffer is discarded, is buffered with PBST
Liquid washs 3 times.Then the BSA that 50 μ l 0.5% are added in every hole is washed 3 times in 37 DEG C of 1 h of closing with PBST buffer.It will be upper
After 300 times of the PBS dilution of mice serum for stating acquisition, take 100 μ l that ELISA reacting hole is added, while setting up positive control and yin
Property control.37 DEG C of 30 min of reaction.It is washed 3 times with PBST, the goat anti-mouse igg of the diluted HRP label of addition 1:10000,37
DEG C reaction 1h.After PBST is washed 3 times, every hole is added 50 μ l TMB solution and carries out 20 min of chromogenic reaction, and 50 μ l H are added2SO4
After (1.25 mol/L) terminates reaction, elisa plate is put into microplate reader measurement OD450 value.Antibody level testing result is shown in Table 3.
The results show that injection group and oral immunity group mice serum are anti-H. parasuisS/N value, that is, antibody level be significantly higher than assistant
Agent group and PBS control group (p < 0.05), and injection group antibody level is higher than oral group.
Mouse protest test
(1) measurement of toadstool LD50 is attacked.Choose Longyan separation strains haemophilus parasuis LY02 plants of (Serotype 5, culture presevation
Number: CGMCC NO.11145, application for a patent for invention publication No.: CN105524857A) it is used as and attacks toxic bacterial strain.Before attacking poison, first use
Karber's method measures LY02 plants of median lethal doses (LD50), and (Yuan Anwen waits haemophilus parasuis Screening of Media and mouse half
Lethal dose measures the Shanghai animal and veterinary and communicates, and 2011,02:8-9.), the method is as follows: preliminary experiment is carried out first: being chosen and experiment
For mouse with several of the about 18g cleaning grade kunming mice of batch, the minimum for measuring 100% dead mouse attacks toxic dose and the death rate
It respectively as the maximum dose level (Dm) and lowest dose level (Dn) of formal test is respectively 2.34 × 10 for 0 maximum tolerated dose9
CFU and 1.71 × 109CFU.Formal test: taking kunming mice 60, and random point 6 groups, every group 10.According to preliminary result,
LY02 bacterium solution is diluted to the bacterium solution of different gradient concentrations, intraperitoneal inoculation mouse, every 200 μ l of mouse inoculation with sterile PBS.
It after attacking poison, is observed continuously 2 weeks, meter record dead mouse situation.According to Kou Shi formula calculate obtain the bacterial strain LD50 be 2.0 ×
109 CFU。
(2) challenge test.15 days after last time is immune, challenge test is carried out.Every mouse peritoneal injection 0.2
Ml bacterial content is 2 × LD50(i.e. 4.0 × 109CFU)H. parasuisLY02 bacterium solution.Observation 10 days counts each group mouse
Death condition.As a result (table 4) is shown, the immune protective rate of injecting immune group and oral immunity group is respectively 80% and 60%, and
PBS control group 1 and PBS control group 2 are all dead, and adjuvant control group 1 and 2 protective rate of adjuvant control group are only 20% and 10%.
The safety testing of example IV haemophilus parasuis subunit vaccine
20 about 18g health kunming mices are taken, are divided into 2 groups, every group 10, with preparationH. parasuisSubunit is micro-
Ball vaccine carries out injection and oral immunity respectively.Injection group is only subcutaneously injected by 500 μ g/, is taken orally group and is used stomach-filling mode,
Every 1 mg of stomach-filling.Observed after immunization 10 days.The results show that two groups of mouse are no different paradoxical reaction, the state of mind and healthy shape
Condition is good.Each group mouse injection site is without abnormal responses such as granuloma, inflammation.
Nucleotide or amino acid sequence table
<110>Agricultural University Of South China, Longyan School
<120>a kind of haemophilus parasuis subunit vaccine and preparation method thereof
<130> 100
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 465
<212> DNA
<213> Haemophilus parasuis
<400> 1
gcgcctgtag gtgaaacttt tacaggtggg ggaattggta tggatgttac aactacaaaa 60
tataaaaatg gtgatttgca aggtaagcag gcaataggca taaactttac tttagactat 120
gcaacagatt atggtaggaa tttaattgga ataattgatg gtaaagcaaa attaggaagt 180
agcaatattt ttcatgatat caaacaaaaa tcccaatata gtattggata tgctcaagga 240
taccgtctcc taccagatat attaccatac gttaaattaa attatagtat tagtaaaata 300
tcggattttg gtagtttcaa agggattgga tatgcgattg gtataaaata tgcaatctct 360
aacgatattg aagtaggttt agaatactca gaaaataatt tgaaacgaaa tggtacaaaa 420
ttaaagggaa atgctattgg aaccaatctg tcctatcgtt tctaa 465
<110>Agricultural University Of South China, Longyan School
<120>a kind of haemophilus parasuis subunit vaccine and preparation method thereof
<130> 100
<160> 1
<170> PatentIn version 3.5
<210> 2
<211> 154
<212> PRT
<213> Haemophilus parasuis
<400> 1
Ala Pro Val Gly Glu Thr Phe Thr Gly Gly Gly Ile Gly Met Asp Val
1 5 10 15
Thr Thr Thr Lys Tyr Lys Asn Gly Asp Leu Gln Gly Lys Gln Ala Ile
20 25 30
Gly Ile Asn Phe Thr Leu Asp Tyr Ala Thr Asp Tyr Gly Arg Asn Leu
35 40 45
Ile Gly Ile Ile Asp Gly Lys Ala Lys Leu Gly Ser Ser Asn Ile Phe
50 55 60
His Asp Ile Lys Gln Lys Ser Gln Tyr Ser Ile Gly Tyr Ala Gln Gly
65 70 75 80
Tyr Arg Leu Leu Pro Asp Ile Leu Pro Tyr Val Lys Leu Asn Tyr Ser
85 90 95
Ile Ser Lys Ile Ser Asp Phe Gly Ser Phe Lys Gly Ile Gly Tyr Ala
100 105 110
Ile Gly Ile Lys Tyr Ala Ile Ser Asn Asp Ile Glu Val Gly Leu Glu
115 120 125
Tyr Ser Glu Asn Asn Leu Lys Arg Asn Gly Thr Lys Leu Lys Gly Asn
130 135 140
Ala Ile Gly Thr Asn Leu Ser Tyr Arg Phe
145 150
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<400> 3
cgggaattcg cgcctgtagg tgaaac 26
<210> 4
<211> 25
<212> DNA
<213>artificial sequence
<400> 4
cccaagcttt tagaaacgat aggac 25
Claims (4)
1. a kind of haemophilus parasuis subunit vaccine, it is characterised in that: the vaccine includes amino acid sequence such as SEQ ID
Haemophilus parasuis outer membrane protein Omp16 antigen and the assistant being made of olive oil, chitosan and sodium alginate shown in NO.2
Agent；The volume parts of each component are as follows: 8-9 parts of the Omp16 antigen protein of 200 mg/ml, 2% 9-11 parts of m/v sodium alginate soln,
28-30 parts of olive oil, 1% 19-21 parts of m/v chitosan solution, 1-2 parts of Tween80,1-2 parts of Span80,8% m/v CaCl2
90-110 parts；
The vaccine preparation method, comprising the following steps:
(1) it by haemophilus parasuis outer membrane protein Omp16, is added in sodium alginate soln, Tween80 is added, is stirred
After add olive oil and Span-80, stirring keeps its fully emulsified；
(2) the slow drop of above-mentioned emulsion is entered into CaCl2It in solution, stirs 30 minutes, crosslinking curing forms microballoon；
(3) cured microballoon is washed with sodium acetate buffer to precipitate；
(4) it with 10 parts of sodium acetate buffer suspension microballoons, and in the chitosan solution being added into, stirs 30 minutes；
(5) it is centrifugated microballoon, is washed with sodium acetate buffer；
(6) up to microspheres vaccine after pellet frozen is dry.
2. haemophilus parasuis subunit vaccine according to claim 1, it is characterised in that: the body of the vaccine each component
Product number are as follows: 8.8 parts of the Omp16 antigen protein of 200 mg/ml, 2% 10 parts of m/v sodium alginate soln, 28.8 parts of olive oil,
1% 20 parts of m/v chitosan solution, 1.2 parts of Tween80,1.2 parts of Span80,8% m/v CaCl2100 parts.
3. haemophilus parasuis subunit vaccine according to claim 1, it is characterised in that: the vaccine is microballoon, partial size
It 0.1-5 μm, 2.5 ± 0.5 μm of average grain diameter, is immunized using injection or oral way.
4. haemophilus parasuis microspheres vaccine according to claim 1 is in the drug of preparation prevention haemophilus parasuis infection
In application.
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Non-Patent Citations (4)
| Title |
|---|
| Omp16-based vaccine encapsulated by alginate-chitosan microspheres provides significant protection against Haemophilus parasuis in mice;Zheng，X.T.等;《Vaccine》;20170207;第35卷;第1417-1423页 |
| WP_010787112.1;Genbank;《Genbank》;20130526;序列部分 |
| 副猪嗜血杆菌不同血清型外膜蛋白的免疫原性的研究;方夏云;《中国优秀硕士学位论文全文数据库 农业科技辑》;20100715(第7期);摘要 |
| 壳聚糖-海藻酸钠包被的副溶血弧菌外膜蛋白K微球疫苗的制备及其口服免疫效果;李梅芳 等;《中国生物制品学杂志》;20140531;第27卷(第5期);第1.2-1.4节，第2.1节，第2.7节，第605页，讨论部分 |
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