CN107224575B - Composition of dairy cow staphylococcus aureus mastitis subunit vaccine and preparation method and application thereof - Google Patents
Composition of dairy cow staphylococcus aureus mastitis subunit vaccine and preparation method and application thereof Download PDFInfo
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- CN107224575B CN107224575B CN201710128121.8A CN201710128121A CN107224575B CN 107224575 B CN107224575 B CN 107224575B CN 201710128121 A CN201710128121 A CN 201710128121A CN 107224575 B CN107224575 B CN 107224575B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/085—Staphylococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
Abstract
The invention discloses a composition of a dairy cow staphylococcus aureus mastitis subunit vaccine, a preparation method and application thereof, wherein the composition comprises α -hemolysin protein, β -hemolysin protein, PV β 2-S protein and a pharmaceutically acceptable adjuvant or β 0-hemolysin protein, β 1-hemolysin protein, PV β 5-F protein and a pharmaceutically acceptable adjuvant, and the preparation method comprises the following steps of 1) preparing β 3-hemolysin protein, β 4-hemolysin protein, PV L-F protein and PV L-S protein respectively, 2) preparing α -hemolysin protein, β -hemolysin protein, PV L-F protein or α -hemolysin protein, PV β -hemolysin protein and PV L-S protein prepared in 1) into an antigen liquid, 3) preparing thimerosal according to the volume of the prepared composition, adding the prepared thimerosal into 2) the antigen liquid to prepare an aqueous phase, 4) emulsifying the prepared thimerosal with a VG phase according to a volume ratio of an organism to generate a cross-emulsion reaction, and generating an antibody according to an ISA 55.
Description
Technical field
The present invention relates to subunit vaccine compositions of a kind of staphylococcus aureus mastitis in dairy cows and preparation method thereof
And application.Belong to live vaccine technical field.
Background technology
Mastitis for milk cows (Mastitis) is one of most common infectious diseases of adult dairy cattle, mainly cow mammary gland group
It knits by a kind of inflammation caused by after microorganism infection, mostly occurs in the postpartum breastfeeding phase, which is widely present in all over the world,
It is to cause one of Dairy Products Industry Implementing economic loss disease the most serious for milk cow common disease, frequently-occurring disease.Cause mastitis for milk cows
Pathogenic microorganism is about more than 150 kind, and mainly based on staphylococcus aureus, Escherichia coli, Streptococcusagalactiae, these three are thin
Microbial mastitis for milk cows accounts for 90% of total incidence or more, wherein being especially most with staphylococcus aureus.
The treatment of current mastitis for milk cows is mainly antibiosis extract for treating.Antibiotic has more than 50 for treating mastitis for milk cows
Year history, served in the prevention of mastitis for milk cows it is certain, but due to long-term single, large dosage of, not science
Using antibiotic, sensitive bacteria elimination is caused, drug tolerant bacteria gradually accounts for mainstream, especially staphylococcus aureus
Resistance problems getting worse results in antibiosis extract for treating and produces little effect；On the other hand, with the improvement of living standards, in milk
Antibiotic residue is a very serious health problem.
There is good foreground with vaccine control mastitis for milk cows, first, vaccine can prevent milk cow infection pathogen and
Cause mammitis；Secondly, vaccine helps to reduce the severity of intramammary infection, controls Subclinical mammitis；Third uses
There is no antibiotic residue problems in milk for vaccine control mammitis；Be finally it is easy to operate, it is low-cost.Currently, it develops
Successful vaccine is seldom, and most of has one to the prevention of mammitis in production practice for weak malicious live vaccine or inactivated vaccine
It is set for using, however as the development of extensive intensive culture, manually causing weak bacterial strain, there is homologous recombination, itself virulence to return by force
Etc. potentially possible, and inactivated vaccine there is also dosages the deficiencies of big, inactivation is not thorough, to improve the safety of traditional vaccine
With immune protective efficiency, efficient, cheap new generation vaccine is developed particularly important.
Staphylococcus aureus (Staphylococcus aureus, abbreviation SA) is also referred to as " S. aureus L-forms " that a kind of leather is blue
The coccus of the Albert'stain Albert positive, 0.8 μm of diameter are arranged in thyrsiform under the microscope, and can generate golden yellow pigment, because
This and gain the name.S. aureus L-forms are one of the main pathogenic fungis for causing chronic/recessive mastitis for milk cows, can be with strong influence milk
Yield and quality brings huge economic loss to dairy industry.
Alpha hemolysin (α-hemolysin), also known as α-toxin are a kind of exotoxins secreted by S. aureus L-forms, are that it is main
One of virulence factor has good immunogenicity.Alpha hemolysin belongs to the beta-barrel structure bacteriotoxin family of hollow shape,
Relative molecular mass is 33,200, is made of 297 amino acid, structural gene hla.Alpha hemolysin is to a variety of mammals
It is the cell membrane hydrophobic region that lps molecule is inserted into red blood cell that red blood cell, which has haemocylolysis, mechanism, forms micropore, destroys film
Integrality, cause cell dissolution.By the 35th hyte Histidine mutations of alpha hemolysin for after alanine, mutant just loses haemolysis
Activity does not have toxicity, but still retains intact immunogenicity, can be used directly to immune animal, be S. aureus L-forms subunit epidemic disease
One of important candidate albumen of seedling.
β hemolysins (also known as β-toxin) are exactly one of its toxin, it has the characteristics such as leucocytotoxicity, hemolytic activity.β
The single chain polypeptide that hemolysin is made of 330 amino acid, molecular weight are 37~39kDa, and isoelectric point (pI) is higher than 9.β haemolysis
Element is a kind of neural esterase of magnesium dependence, has phospholipase C (Phospholipase C, PLC) activity, can decompose phosphoglycerol
Choline, β hemolysins rely on this enzymatic activity, the phospholipid bilayer of hydrolysis composition cell membrane, to destroy the complete of cell membrane
Property, lead to cell cracking, causes haemolysis.After β hemolysins are carried out rite-directed mutagenesis, mutant just loses hemolytic activity, does not have
It is toxic, but still retain intact immunogenicity, it can be used directly to immune animal, be the important time of S. aureus L-forms subunit vaccine
One of sortilin.
Panton-valentine leukocidins (panton-valentine leukocidin, PVL) are by golden yellow
One of extracellular toxin that staphylococcus generates and one of its main virulence factor.PVL is by Van deleld in 1894
It finds, and was separated it from hemolysin by Panton and Valentine first in 1932.PVL is by two kinds of albumen
Matter forms, i.e., PVL-S albumen and PVL-F albumen, molecular weight are respectively 34kDa and 33kDa, and between F protein and S protein
Amino acid sequence homology has 36%.PVL belongs to film drilling toxin family, can induce PMNs necrosis or adjusts and die, is PVL-S first
It is combined with the receptor of the specific high-affinity on PMNs cell membranes, secondly PVL-F formation dimers in combination, then successively
PVL-S and PVL-F ties platform, eventually forms the heteromers of a cyclic structure.This heteromers internal diameter is 3nm, outer diameter 9nm, is divided
The molecular proportion of son amount about 200kDa, PVL-S contained therein and PVL-F are 1:1, the heteromers of this cyclic structure are inserted in
On PMNs cell membranes, the film perforation of a diameter about 2nm is formed, other PVL molecules enter cell by the hole, and online
Duct is established on plastochondria outer membrane, to destroy the interior environment of mitochondria, and it is white to activate caspase 9, caspase 3 and release to kill
Cytokine C, inducing cell tune are died.Therefore, PVL is one of important candidate albumen of S. aureus L-forms subunit vaccine, but works as PVL-S
With PVL-F all in the presence of, be likely to result in cytotoxicity, mouse safety experiment has been done it in this laboratory, as a result with expection
It is identical, when the vaccine simultaneously containing PVL-S albumen and PVL-F albumen is immunized, mouse just death in 3-4 days after immune, still
The PVL-S albumen or PVL-F albumen of only immune same dose, in 14 days tracked after immune, mouse is all normal, does not go out incumbent
What is abnormal, so, in the selection of subunit vaccine, it is only capable of the candidate egg for selecting one of which albumen as subunit vaccine
In vain.
This laboratory has detached more than 40 kinds of staphylococcus aureus from all parts of the country, and has carried out analysis to its exotoxin and ground
Study carefully, first, find that a kind of exotoxin is not only secreted in staphylococcus aureus of the same race strain, may secrete 2 kinds, 3 kinds it is even more a variety of
Exotoxin；Second is that finding that the exotoxin level of the isolated staphylococcus aureus secretion in different regions is not complete one
Sample, the mainly alpha hemolysin of some secretions, the mainly β hemolysins of some secretions.Therefore, using a kind of single exotoxin egg
It is white not satisfactory as the not only possible effect when protecting infection of the milk cow from staphylococcus aureus of subunit vaccine, also not
The immune protective effect of wide spectrum can be provided.
Invention content
The technical problem to be solved in the present invention：One is to provide a kind of mastitis for milk cows as caused by staphylococcus aureus
Subunit vaccine composition and preparation method thereof；Second is that single ectotoxic subunit vaccine is overcome to protect milk cow from golden yellow
The problem of broad spectrum activity difference in terms of color staphy lococcus infection；Third, overcoming existing Attenuate vaccine and inactivated vaccine that may be present homologous heavy
Group, itself virulence return the potential risks such as strong.
The present invention provides a kind of staphylococcus aureus mastitis in dairy cows subunit vaccine composition, the composition includes
Alpha hemolysin albumen, beta hemolysin albumen, PVL-S albumen and pharmaceutically acceptable adjuvant or alpha hemolysin albumen, β-are molten
Sanguinin albumen, PVL-F albumen and pharmaceutically acceptable adjuvant.
In technical scheme of the present invention, it is preferable that the encoding gene of the PVL-S albumen is as shown in SEQ ID NO.1.
In technical scheme of the present invention, it is preferable that the encoding gene of the PVL-F albumen is as shown in SEQ ID NO.2.
In technical scheme of the present invention, it is preferable that the alpha hemolysin albumen be treated forfeiture hemolytic activity but
Retain the albumen of immunogenicity, the processing method has albumen inactivation and gene mutation processing.Preferably, the alpha hemolysin egg
It is the albumen lost hemolytic activity but retain immunogenicity handled by gene mutation in vain.
In technical scheme of the present invention, it is preferable that the beta hemolysin albumen be treated forfeiture hemolytic activity but
Retain the albumen of immunogenicity, the processing method has albumen inactivation and gene mutation processing.Preferably, the beta hemolysin egg
It is the albumen lost hemolytic activity but retain immunogenicity by mutation processing in vain.
In technical scheme of the present invention, it is preferable that the alpha hemolysin albumen, beta hemolysin albumen, PVL-S albumen according to
Equal mass ratioes mixing.
In technical scheme of the present invention, it is preferable that the alpha hemolysin albumen, beta hemolysin albumen, PVL-F albumen according to
Equal mass ratioes mixing.
In technical scheme of the present invention, it is preferable that the alpha hemolysin albumen, beta hemolysin albumen, PVL-S albumen are
100 parts of μ g/.
In technical scheme of the present invention, it is preferable that the alpha hemolysin albumen, beta hemolysin albumen, PVL-F albumen are
100 parts of μ g/.
In technical scheme of the present invention, it is preferable that the pharmaceutically acceptable adjuvant can be aqueous adjuvants (such as aluminium glue
Adjuvant etc.), or oil-in-water adjuvant (such as white oil), or W/O/W adjuvant (such as ISA 206V), it is excellent
Selection of land, the pharmaceutically acceptable adjuvant are ISA201VG adjuvants.
In technical scheme of the present invention, it is preferable that the composition also contains preservative.
In technical scheme of the present invention, it is preferable that the preservative is thimerosal, and the content of the thimerosal is 2 μ g/
Head part.
The present invention also provides a kind of methods preparing the subunit vaccine composition, the described method comprises the following steps：
1) alpha hemolysin albumen, beta hemolysin albumen, PVL-F albumen, PVL-S albumen are prepared respectively；2) by step 1) according to etc. matter
Alpha hemolysin albumen, beta hemolysin albumen, the PVL-F albumen that amount ratio is mixed with, or according to the α-for waiting mass ratioes to be mixed with
Haemolysis fibroin, beta hemolysin albumen, PVL-S albumen are prepared into composition antigen liquid；3) according to the combination prepared in step 2)
The volume of object antigen liquid gets out thimerosal, and ready thimerosal is added in step 2) antigen liquid and is prepared into water phase；
4) by the water phase and ISA 201VG adjuvants according to volume ratio 46:55 mixing and emulsifyings.Preferably, it is emulsified described in step 4)
Temperature is 32-35 DEG C.
It is newborn for preventing and treating milk cow staphylococcus aureus in preparation that the present invention provides a kind of composition again
Application in the vaccine of room inflammation.
Compared with prior art, the present invention clearly provides a kind of novel milk cow staphylococcus aureus breast for the first time
Scorching subunit vaccine composition and its preparation method and application.The composition does not contain nucleic acid first, will not lead to homologous recombination
The potential dangers such as strong are returned with itself virulence；Body can be induced and generate specific antibody, cellular immunity, inducing immunological memory can be promoted
With cause extensive immune response, generate the reaction of good cross protection, thus the vaccine more safety and stability, prepare it is simple,
Using convenient, cheap and time saving and energy saving.Secondly, the composition is due to containing there are many staphylotoxin,
Different regions can be secreted with different ectotoxic staphylococcus aureuses good neutralization, reaches protection milk cow and exempts from
It is infected by the different ectotoxic staphylococcus aureuses of secretion；Finally, the composition is than subunit prepared by single exotoxin
Vaccine is protecting milk cow stronger from the ability in terms of infection of staphylococcus aureus.
Description of the drawings
Fig. 1 shows agarose gel electrophoresis PCR amplification PVL-F, PVL-S results.PVL-F gene PCRs are as a result, size is about
906bp；PVL-S genes PCR is as a result, size is about 849bp；M：DNA molecular amount standard DL2,000.
Fig. 2 indicates pET28a-PVL-F, PVL-S digestion verification results.pET28a-PVL-F：Plasmid pET28a-PVL-F
XhoI and Nde I digestion qualification results, endonuclease bamhi size are 5,369bp and 904bp；pET28a-PVL-S：Plasmid pET28a-
PVL-F XhoI and Nde I digestion qualification results, endonuclease bamhi size are 5,369bp and 849bp；M：DNA molecular amount standard
DL5,000。
Fig. 3 indicates PVL-F albumen and PVL-S protein purification results.
Fig. 4 a indicate the bioactivity result of alpha hemolysin in immune rear composition；
Fig. 4 b indicate the bioactivity result of beta hemolysin in immune rear composition；
Fig. 4 c indicate the bioactivity result of PVL-F in immune rear composition；
Fig. 4 d indicate the bioactivity result of PVL-S in immune rear composition.
Challenge viral dosage result after Fig. 5 is immune.
Specific implementation mode
Below with reference to drawings and examples, the present invention will be further described, and the embodiment of the present invention is merely to illustrate this
The technical solution of invention, and the non-limiting present invention.
The source list of reagent and drug of the present invention is as follows：
Chemical reagent and all commercial product of biological reagent；
Preservative thimerosal is purchased from Life Sciences；
ISA201VG is purchased from match BIC Corp of France.
Embodiment 1：It is prepared by alpha hemolysin albumen, beta hemolysin albumen, PVL-S albumen, PVL-F albumen
1.1：It is prepared by alpha hemolysin albumen
The alpha hemolysin of mutation is prepared with reference to the patent of invention for the Patent No. 201610068723.4 that the applicant submits
Albumen.
1.2：It is prepared by beta hemolysin albumen
The beta hemolysin of mutation is prepared with reference to the patent of invention for the Patent No. 201610068816.7 that the applicant submits
Albumen.
1.3：It is prepared by PVL-S albumen, PVL-F albumen
1.3.1 the structure of expression vector pET28a-PVL-S and pET28a-PVL-F (two kinds of carrier construction methods are identical)
Using the milk cow staphylococcus aureus gene group being clinically separated as template, PCR is carried out, primer is as shown in the table,
The results are shown in Figure 1：PVL-F gene PCRs are as a result, size is about 906bp；PVL-S genes PCR is as a result, size is about
849bp；It is all in the same size with expection.
1.3.1.1 sample-adding system is (50 μ l):
1.3.1.2PCR amplification program:
1.3.1.3 glue recycles DNA fragmentation:
(1) reaction solution of step 1.3.1.2 is subjected to 0.8% agarose gel electrophoresis (110V 30min)；
(2) in the UV lamp, gel extraction DNA fragmentation is in 1.5ml EP pipes；
(3) it is added 500 μ l PC buffer in the 1.5ml EP pipes into step (2), 50 DEG C, water-bath 10min；
(4) solution in step (3) is moved into adsorption column center, stands 2min, centrifugation, 12,000rpm, 30s；
(5) waste liquid is abandoned, 600 μ l PW buffer are added to adsorption column center, stand 3min, 12,000rpm, 30s；
(6) step (5) is repeated；
(7) suction attached column centrifuges, 12,000rpm, 1min；
(8) 30 μ l ddH are added to adsorption column center2O stands 3min, centrifuges (12,000rpm, 2min)；
(9) collection step (8) DNA sample carries out electrophoresis.
1.3.1.4 double digestion reaction (50 μ l systems)：
It is loaded according to above-mentioned system in 1.5ml EP pipes, mixing, the two 50 μ l reaction solutions is then placed in 37
In DEG C thermostat water bath, water-bath 3h.
1.3.1.5 glue recycles DNA fragmentation：
(1) reaction solution of step 1.3.1.4 is subjected to 0.8% agarose gel electrophoresis (110V 30min)；
(2) in the UV lamp, gel extraction DNA fragmentation is in 1.5ml EP pipes；
(3) it is added 500 μ l PC buffer in the 1.5ml EP pipes into step (2), 50 DEG C, water-bath 10min；
(4) solution in step (3) is moved into adsorption column center, stands 2min, centrifugation, 12,000rpm, 30s；
(5) waste liquid is abandoned, 600 μ l PW buffer are added to adsorption column center, stand 3min, 12,000rpm, 30s；
(6) step (5) is repeated；
(7) suction attached column centrifuges, 12,000rpm, 1min；
(8) 30 μ l ddH are added to adsorption column center2O stands 3min, centrifuges (12,000rpm, 2min)；
(9) collection step (8) DNA sample carries out electrophoresis.
1.3.1.6 connection reaction (10 μ l systems)：
It is loaded according to above-mentioned system in 1.5ml EP pipes, mixing, above-mentioned reaction solution is then placed in 16 DEG C, water-bath
It takes out after 16h, 65 DEG C, is inactivated after water-bath 15min, by 4 DEG C of preservations of sample.
1.3.1.7 transformation experiment：
(1) the connection reaction solution for taking out step 1.3.1.6, adds 100 μ l E.coli DH5 α competent cells thereto,
Mixing；
(2) ice bath 30min；
(3) 42 DEG C, water-bath 100s；
(4) ice bath 2min；
(5) it takes out, is added 600 μ l LB liquid mediums into EP pipes, 37 DEG C, water-bath 1h；
(6) sample cell is taken out, is centrifuged (8,000rpm, 2min), 600 μ l are removed, thalline is resuspended in 100 μ l LB of residue；
(7) it takes bacterium solution to be plated in LK tablets (a concentration of 50 μ g/ml of Kan), LK tablets is placed in biochemical constant incubator
In, 37 DEG C of culture 12h.
1.3.1.8 recombinant plasmid extracts and digestion identification：
(1) from picking monoclonal to 3ml LK fluid nutrient mediums in conversion tablet, 37 DEG C, 260rpm shakes bacterium and stays overnight；
(2) it takes in 1ml bacterium solutions to 1.5ml EP pipes, centrifuges (12,000rpm, 2min), abandon supernatant；
(3) 250 μ l P1buffer are added in the EP pipes into step (2), thalline is resuspended；
(4) 250 μ l P2buffer are added into step (3) solution, mild mixing stands 2min；
(5) 350 μ l P3buffer, mild mixing are added into step (4) solution；
(6) it by step (5) solution, centrifuges (12,000rpm, 10min)；
(7) supernatant solution in step (6) is moved into adsorption column center, centrifuged (8,000g, 30s)；
(8) waste liquid is abandoned, 500 μ l wash buffer are added to adsorption column center, centrifuge (9,000g, 30s)；
(9) step (8) is repeated；
(10) suction attached column centrifugation (9,000g, 1min)；
(11) 30 μ l Elution buffer are added to adsorption column, stand 2min, centrifuge (12,000rpm, 2min)；
(12) collection step (11) DNA sample carries out electrophoresis；
(13) as shown in step 1.3.1.4, digestion identification is carried out to the plasmid extracted, then carries out 0.8% agarose
Gel electrophoresis.
(14) recombinant plasmid digestion qualification result is as shown in Figure 2：pET28a-PVL-F：Plasmid pET28a-PVL-F XhoI
With Nde I digestion qualification results, endonuclease bamhi size is 5,369bp and 904bp；pET28a-PVL-S：Plasmid pET28a-PVL-
F XhoI and Nde I digestion qualification results, endonuclease bamhi size are 5,369bp and 849bp.
1.3.2 e. coli bl21 is converted
It draws 1 μ l plasmids to be added in 100 μ l BL21 competent cells, ice bath 30min；
42 DEG C of heat shock 90s；
Ice bath 2min；
The LB culture solutions of 900 μ l non-resistants are added in super-clean bench；
37 DEG C of 180rpm shake 1h；
100 μ l bacterium solutions card-coating that resistance LB tablets are drawn, 37 DEG C are incubated overnight.
1.3.3 a large amount of induced expressions
Choose bacterium：In picking monoclonal to 50ml cards that resistance LB culture solutions, 37 DEG C are incubated overnight；
Switching：By 1:100 ratios transfer bacterium solution to 500ml cards that resistance LB culture solutions, shake 3.5L, 37 DEG C of 220rpm trainings altogether
Support 2-2.5h to OD600It is worth 0.6；
Induction：Bacterium solution OD600After being worth 0.6, be added 500 μ l IPTG (1M) to the final concentration of 1mmol/L of IPTG, 37 DEG C
220rpm Fiber differentiations 4h；
Microorganism collection：Bacterium solution 6,000rpm centrifuge 10min, collect thalline；Clean thalline with 40ml PBS, 6,000rpm from
Heart 10min collects thalline, is placed in -20 DEG C of preservations；
1.3.4PVL-S albumen or PVL-F protein purifications (two kinds of protein purification procedures are identical)
(1) bacterial cell disruption：Lysate (8ml/g weight in wet bases) (50mM NaH are added in obtained thalline into 1.3.32PO4
(pH 8.0), 500mM NaCl, 20mM imidazoles；Membrane filtration using 0.8 μm), with the piping and druming of 50mL syringes uniformly to nothing
Granular fungus block；Thalline sample is poured into cell homogeneous instrument sample cell, outlet is prepared to collect sample with beaker；With sample
90% outflow outlet is that the sample that outlet beaker is collected is refunded sample cell by a cycle after the completion of one recycles, and is continued
Repeat 5 cycles.
(2) complete sample will be crushed in step (1) to be dispensed into 250mL Beckman centrifuge tubes, 12,000rpm, 4 DEG C
30min is centrifuged, Supernatant samples are used as loading sample after crossing 0.8 μm of film, reserve 80 μ L samples and are detected for SDS-PAGE；
(3) column equilibration：With 2~3 column volume of ultrapure water balance (CV), the ethyl alcohol for being discharged 20% preserves liquid；Then with cracking
Liquid balances 2~3 column volumes (CV), 5mL/min.Control pressure is less than 0.5MPa.
(4) loading：2mL/min carries out loading, and collection flows through liquid (Flowthrough), and 80 μ L is taken to be examined for SDS-PAGE
It surveys.
(5) 1 is rinsed：With (the washing buffer 1 of cleaning buffer solution component 1：50mM NaH2PO4(pH 7.4), 500mM
NaCl, 20mM imidazoles, 0.1%TritonX-114, the membrane filtration using 0.8 μm) column is washed, 5mL/min rinses 80 cylinders
Product.
(6) 2 are rinsed：With (the elution buffer1 of elution buffer component 1 of 10 times of column volumes (CV)：50mM
NaH2PO4(pH 7.4), 500mM NaCl, the membrane filtration using 0.8 μm) column is washed, reduce the residual of Triton X-114.
(7) it elutes：50% elution buffer component, 2 (50%Elution buffer 2：50mM NaH2PO4(pH 7.4),
500mM NaCl, 250mM imidazoles, the membrane filtration using 0.8 μm) elution destination protein, until baseline washes flat, speed 5ml/min,
It collects, 80 μ L is taken to be analyzed for SDS-PAGE after mixing；100% de- buffer solution, 2 component cleans pillar (100%Elution
buffer 2：50mM NaH2PO4(pH 7.4), 500mM NaCl, 500mM imidazoles, the membrane filtration using 0.8 μm), until baseline
Flat, 5mL/min is washed, is collected, 80 μ L is taken to be analyzed for SDS-PAGE after mixing.
(8) HiPrep Desalting desalting columns column equilibration：With 2~3 column volume of ultrapure water balance (CV), it is discharged 20%
Ethyl alcohol preserves liquid；Then elution buffer component 1 is used to balance 3-4 column volumes (CV), speed 10mL/min.
(9) loading：Speed 10mL/min is injected (inject), and maximum injection (inject) amount is 13mL.
(10) it collects：After stopping loading, into load patterns, 10mL/min, UV rise to 1mAU and start to collect, i.e. albumen
Sample starts appearance, and 10ml/ pipes are collected, and waits for that UV is down to 5mAU hereinafter, stopping collecting.
(11) it balances：Flow velocity 10ml/min balances 2-3CV.
(12) 7.10~7.12 are recycled, until completion of the sample.
(13) aseptic filtration：At 4 DEG C, 12,000rpm centrifugation 15min collect supernatant, move to biology the protein solution of collection
Safety cabinet, crosses the low syringe needle filter of 0.2 μm of protein binding rate, and filtering is placed on -80 DEG C of refrigerators and preserves.
(14) purification result is as shown in Figure 3：The purity of PVL-S albumen and PVL-F albumen after purification can reach 90%
More than.
Embodiment 2：Composition prepares and (is illustrated for preparing 1ml/ parts)
The consumptive material for preparing vaccine and material used all need it is pre- first pass through aseptic process, preparation process be in Biohazard Safety Equipment or
Other can ensure to complete in whole preparation process all sterile instrument or environment.
(1) it is needed according to experiment, the amount of each ingredient of calculation composition solution keeps alpha hemolysin albumen, β-in composition molten
Sanguinin albumen, PVL-S albumen or alpha hemolysin albumen, beta hemolysin albumen, PVL-F final concentration of protein are 100 μ g/ml, are made
The final concentration of 2 μ g/ml of thimerosal in composition, then load weighted recombinant protein is mixed with load weighted thimerosal and is used as water
Phase is 46 according to water phase and adjuvant ISA201VG volume ratios:55, measure adjuvant；
(2) measured water phase and adjuvant are placed in thermostat water bath and are heated to 33 DEG C ± 1 DEG C, it will after temperature stabilization
Antigen is added in adjuvant pipe, and oscillator shakes 10min and carries out pre-emulsification；
(3) the complete vaccine of pre-emulsification is positioned in the beaker for filling with ice, is fixed on the supersonic cell anticipated
It is emulsified on broken instrument；
(4) after emulsifying, emulsifying effectiveness is observed：Part vaccine is taken to be placed in centrifuge tube, 3,000rpm centrifugation 15min,
Vaccine is not stratified for qualification；
(5) the qualified vaccine of detection is dispensed into 15ml centrifuge tubes, is marked, and sealed membrane sealing is placed in 4 DEG C of preservations.
Embodiment 3：Mouse immune challenge viral dosage
3.1：Following vaccine and composition are prepared according to the method for embodiment 2
3.2：Immunization experiment
The Balb/c female mices for buying 80 20g or so, are classified as 8 groups, every group 10, one of which is as a contrast
PBS is immunized in group, is in addition used as immune group for 7 groups, the 7 kinds of compositions prepared in 3.1 are immunized respectively；It is inhaled with 1ml syringes when immune
1ml vaccines are taken, are then sterilized to mouse hind leg muscle with 75% cotton ball soaked in alcohol, the inserting needle in the middle part of muscle masses, left and right back leg is respectively noted
Penetrate 50 μ l vaccines, 100 μ l vaccines of co-injection.
Two exempt within 14 days after one exempting from, two exempt from after carry out within 7 days three and exempt from, and before exempting from one, two exempt from before, three exempt from before and three exempt from
14 days acquisition serum afterwards, detects antibody titer.
Bioactivity result is as shown in Fig. 4 a, Fig. 4 b, Fig. 4 c, Fig. 4 d, wherein the composition (composition containing alpha hemolysin
1, composition 5, composition 6, composition 7) in the relative potency of alpha hemolysin can reach 544,000 or more before attacking poison,
But the relative potency of composition 6 is minimum in 4 compositions, is 544,000, the relative potency of other 3 compositions reaches
928,000 or more, this (survival rate of composition 7 10%) higher than the survival rate of composition 6 also consistent with malicious result is attacked；Contain β-
The relative potency of beta hemolysin in the composition (composition 2, composition 5, composition 6, composition 7) of hemolysin is before attacking poison
77,778 or more can be reached；PVL-F's in composition (composition 3, composition 6, composition 7) containing PVL-F is opposite
Potency can reach 80,000 or more before attacking poison；In composition (composition 4, composition 6, composition 7) containing PVL-S
The relative potency of PVL-S can reach 80,000 or more before attacking poison.
3:3：Challenge viral dosage (three exempt to carry out within 14 days attacking poison afterwards)
(1) picking SA single bacterium colonies (bacterial strain is the CQ339 bacterial strains that this laboratory preserves) are in 5ml liquid broths
In, 220rpm, 37 DEG C shake bacterium stay overnight；
(2) overnight microbionation will be shaken in 100ml fresh liquid broth bouillons by centesimal volume (1ml)
In, 220rpm, 37 DEG C shake bacterium stay overnight；
(3) 100ml bacterium solutions are encased in 500ml centrifugal bottles, 8,000rpm centrifugation 10min suck culture medium, thalline
It is resuspended, is repeated the above steps 3 times with 100ml PBS, mixing finally is resuspended in all thalline 5ml PBS；
(4) it is counted after bacterium solution being done 10,000 times of dilutions.According to the concentration of bacterium solution, original bacteria liquid is diluted to 1.5 ×
108CFU/ml(2M LD50)；
(5) it uses 1ml syringes to draw bacterium solution, tail vein injection, injection volume is carried out according to the corresponding mouse of each concentration bacterium
For 200 μ l/20g mouse.
(6) survival state of mouse is observed continuously, records the death time of each mouse：It is each in the morning, afternoon and evening to check once.
(7) the results are shown in Figure 5 for mouse survival rate after attacking poison, and 500h internal reference groups mouse is all dead after attacking poison；With
Within the 987.5h of track, 7 groups of immune group survival rates are all higher than 50%, wherein the survival rate of composition 1- compositions 4 exists
The survival rate of 50%-60%, composition 5 reach 70%, composition 6 is deposited than the high 20%-30% of composition 1- compositions 4
Motility rate reaches 80%, and the survival rate of composition 7 reaches 90%, all the significantly larger than survival rate of composition 1- compositions 4, also compares
The survival rate of composition 5 wants high, and the survival rate of composition 7 is higher than the survival rate of composition 6 by 10%, this is molten with α-in composition
Sanguinin bioactivity result is consistent, and (potency of the alpha hemolysin in alpha hemolysin potency ratio composition 6 in composition 7 is high
One thinner ratio)；This illustrates that the exotoxin of one-component prepares the immune protective effect of vaccine and can not show a candle to a variety of exotoxins and prepare
The immune protective effect of vaccine.
Embodiment 4：ELISA antibody titers detect
(1) it is coated with：With the detection albumen (alpha hemolysis of coating buffer (50mM carbonate buffer solutions, pH 9.5) dilution purifying
Fibroin, beta hemolysin albumen, PVL-S albumen or PVL-F albumen) to 0.5 μ g/ml, 100 μ l are added per hole on ELISA Plate,
4 DEG C of refrigerators are stood overnight after sealed membrane is sealed；
(2) it washs：After refrigerator takes out ELISA Plate, it is put into board-washing machine and washs, cleaning solution PBST；
(3) it closes：200 μ l confining liquids (5% defatted milk), 37 DEG C of incubation 2h after sealed membrane is sealed are added per hole；
(4) preparation of samples：By known information and dosage is needed, serum is subjected to appropriate dilution with confining liquid；
(5) it washs：With (2)；
(6) it is loaded：Dilute serum is added, while negative control, 37 DEG C of incubation 1h are with confining liquid；
(7) it washs：With (2)；
(8) add secondary antibody：Secondary antibody 100 the μ l, 37 DEG C of incubation 0.5h of the diluted HRP labels of appropriateness are added per hole；
(9) it washs：With (2)；
(10) it develops the color：The TMB developing solutions of 100 μ l, 37 DEG C of incubation 10min are added under the conditions of being protected from light per hole；
(11) it terminates：The 50 μ l terminate liquids (H of 2M are added per hole2SO4), terminate reaction；
(12) it detects：In 450nm wavelength determination sample OD values, data are analyzed；
(13) interpretation of result：Judge the standard of antibody positive：P/N >=2.1, OD450 >=0.1.
The present invention is illustrated by above embodiment, it is understood, however, that the present invention is not limited to institutes here
The particular example and embodiment of description.Purpose herein comprising these particular examples and embodiment is to help this field
In technical staff put into practice the present invention.Any those of skill in the art are easy to do not departing from spirit and scope of the invention
In the case of be further improved and perfect, therefore the present invention is only by the content of the claims in the present invention and limiting for range
System, intention, which covers, all to be included the alternative in the spirit and scope of the invention defined by appendix claim and waits
Same scheme.
Sequence table
<110>Zhejiang oceanic rise bio tech ltd
<120>Staphylococcus aureus mastitis in dairy cows subunit vaccine and its preparation method and application
<160> 4
<170> PatentIn version 3.3
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<211> 879
<212> DNA
<213>Recombination staphylococcus aureus PVL-S protein coding gene sequences
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aattcattta tcacatcatt aggtaaaatg tctggacatg atccaaattt atttgttgga 540
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agaagaacaa cacactatgg caatagttat ttagaaggat ctagaataca caacgcattt 780
gtaaacagaa attacacagt taaatatgaa gtgaactgga aaactcatga aattaaagtg 840
aaaggacata atctcgagca ccaccaccac caccactga 879
<210> 4
<211> 921
<212> DNA
<213>Recombination staphylococcus aureus PVL-F protein coding gene sequences
<400> 2
atgggagctc aacatatcac acctgtcagc gagaaaaaag tggatgacaa aatcactttg 60
tacaaaacga ctgctacatc agattctgac aaattaaaaa tttctcaaat tctaactttt 120
aattttatta aagacaaaag ttatgataaa gacacattaa tactaaaagc tgccggaaac 180
atttactcag gctataccca acccacttct gatagtagta taaattcaca attttattgg 240
ggagctaagt ataatgtttt tgttagctcg gagtccaaag attctgtaaa tattgttgac 300
tacgcgccta aaaatcaaaa tgaagaattt caagttcaac aaacattagg ttattcatat 360
ggcggagata ttaatataat aaatggatta actggtggat tgaacgggtc aaaatcattt 420
tcagaaacga ttaattataa gcaagaaagc tacagaacta cgattgatag gaaaataaat 480
cacaaattaa tcggctgggg tgtcgaggca cataaaatca tgaataatgg ttggggacca 540
tatggcagag atagtagtga ttcattatat ggaaacgaac tatttttagg tggcagacag 600
agtagctcga atgctaatca aaatttctta ccaacacatc aaatgcccat attagcacgt 660
ggtaatttca atccagaatt tataagcgta ctttctcaca aacaaaagga tgttaaaaaa 720
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ttgcactgga taggttataa tattaagaat caaaagagag caacacacac atcaatttat 840
gaaattgatt gggaaaaaca cacggttaaa ttagtagctt cgcaatctag cgaactcgag 900
caccaccacc accaccactg a 921
<210> 1
<211> 292
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<213>Recombination staphylococcus aureus PVL-S protein sequences
<400> 3
Met Gly Asn Ile Glu Asn Ile Gly Asp Gly Ala Glu Val Val Lys Arg
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Asn Thr Asp His Ile Lys Ala Met Arg Trp Pro Phe Gln Tyr Asn Ile
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Lys Asn Lys Ile Asp Ser Val Asn Val Ser Gln Thr Leu Gly Tyr Asn
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Ile Gly Gly Asn Phe Asn Ser Gly Pro Ser Thr Gly Gly Asn Gly Ser
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Phe Asn Tyr Ser Lys Thr Ile Ser Tyr Asn Gln Gln Asn Tyr Ile Ser
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Glu Val Glu Arg Gln Asn Ser Lys Ser Val Gln Trp Gly Ile Lys Ala
145 150 155 160
Asn Ser Phe Ile Thr Ser Leu Gly Lys Met Ser Gly His Asp Pro Asn
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Leu Phe Val Gly Tyr Lys Pro Tyr Ser Gln Asn Pro Arg Asp Tyr Phe
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Val Pro Asp Asn Glu Leu Pro Pro Leu Val His Ser Gly Phe Asn Pro
195 200 205
Ser Phe Ile Ala Thr Val Ser His Glu Lys Gly Ser Gly Asp Thr Ser
210 215 220
Glu Phe Glu Ile Thr Tyr Gly Arg Asn Met Asp Val Thr His Ala Thr
225 230 235 240
Arg Arg Thr Thr His Tyr Gly Asn Ser Tyr Leu Glu Gly Ser Arg Ile
245 250 255
His Asn Ala Phe Val Asn Arg Asn Tyr Thr Val Lys Tyr Glu Val Asn
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Trp Lys Thr His Glu Ile Lys Val Lys Gly His Asn Leu Glu His His
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His His His His
290
<210> 1
<211> 306
<212> PRT
<213>Recombination staphylococcus aureus PVL-F protein sequences
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Lys Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp
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Lys Asp Thr Leu Ile Leu Lys Ala Ala Gly Asn Ile Tyr Ser Gly Tyr
50 55 60
Thr Gln Pro Thr Ser Asp Ser Ser Ile Asn Ser Gln Phe Tyr Trp Gly
65 70 75 80
Ala Lys Tyr Asn Val Phe Val Ser Ser Glu Ser Lys Asp Ser Val Asn
85 90 95
Ile Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln
100 105 110
Gln Thr Leu Gly Tyr Ser Tyr Gly Gly Asp Ile Asn Ile Ile Asn Gly
115 120 125
Leu Thr Gly Gly Leu Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn
130 135 140
Tyr Lys Gln Glu Ser Tyr Arg Thr Thr Ile Asp Arg Lys Ile Asn His
145 150 155 160
Lys Leu Ile Gly Trp Gly Val Glu Ala His Lys Ile Met Asn Asn Gly
165 170 175
Trp Gly Pro Tyr Gly Arg Asp Ser Ser Asp Ser Leu Tyr Gly Asn Glu
180 185 190
Leu Phe Leu Gly Gly Arg Gln Ser Ser Ser Asn Ala Asn Gln Asn Phe
195 200 205
Leu Pro Thr His Gln Met Pro Ile Leu Ala Arg Gly Asn Phe Asn Pro
210 215 220
Glu Phe Ile Ser Val Leu Ser His Lys Gln Lys Asp Val Lys Lys Ser
225 230 235 240
Lys Ile Lys Val Thr Tyr Gln Arg Gln Met Asp Arg Tyr Glu Asn Phe
245 250 255
Trp Asn Asn Leu His Trp Ile Gly Tyr Asn Ile Lys Asn Gln Lys Arg
260 265 270
Ala Thr His Thr Ser Ile Tyr Glu Ile Asp Trp Glu Lys His Thr Val Lys
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Claims (9)
1. a kind of subunit vaccine composition of staphylococcus aureus mastitis in dairy cows, it is characterised in that：Subunit's epidemic disease
Seedling composition includes alpha hemolysin albumen, beta hemolysin albumen, PVL-S albumen and pharmaceutically acceptable adjuvant or α-molten
Sanguinin albumen, beta hemolysin albumen, PVL-F albumen and pharmaceutically acceptable adjuvant；Wherein, the alpha hemolysin albumen is
By the albumen of gene mutation processing lost hemolytic activity but retain immunogenicity, the beta hemolysin albumen is by gene
The albumen of mutation processing lost hemolytic activity but retain immunogenicity；The alpha hemolysin albumen, beta hemolysin albumen, PVL-
S protein is mixed according to equal mass ratioes, the alpha hemolysin albumen, beta hemolysin albumen, PVL-F albumen according to etc. mass ratioes it is mixed
It closes；The alpha hemolysin albumen is that the patent of invention of Patent No. 201610068723.4 prepares the alpha hemolysin albumen of mutation,
The beta hemolysin albumen is that the patent of invention of Patent No. 201610068816.7 prepares the beta hemolysin albumen of mutation.
2. subunit vaccine composition according to claim 1, which is characterized in that the alpha hemolysin albumen, beta hemolysis
Fibroin, PVL-S albumen are g/ parts of 100 μ.
3. subunit vaccine composition according to claim 1, which is characterized in that the alpha hemolysin albumen, beta hemolysis
Fibroin, PVL-F albumen are g/ parts of 100 μ.
4. subunit vaccine composition according to claim 1, which is characterized in that the pharmaceutically acceptable adjuvant is
201 VG adjuvants of ISA.
5. the subunit vaccine composition according to Claims 1-4 any claim, which is characterized in that the combination
Object also contains preservative.
6. subunit vaccine composition according to claim 5, which is characterized in that the preservative is thimerosal.
7. subunit vaccine composition according to claim 6, which is characterized in that the content of the thimerosal is 2 μ g/ heads
Part.
8. a kind of method preparing the subunit vaccine composition as described in claim 1~7 is any, which is characterized in that described
Method includes the following steps：
1) alpha hemolysin albumen, beta hemolysin albumen, PVL-F albumen, PVL-S albumen are prepared respectively；
2) alpha hemolysin albumen, beta hemolysin albumen, the PVL-F albumen that will be mixed with according to equal mass ratioes in step 1), or
Alpha hemolysin albumen, beta hemolysin albumen, the PVL-S albumen being mixed with according to equal mass ratioes are prepared into composition antigen liquid；
3) according to the volume of the composition antigen liquid prepared in step 2), thimerosal is got out, and ready thimerosal is added
Enter and is prepared into water phase into step 2) antigen liquid；
4) by the water phase and 201 VG adjuvants of ISA according to volume ratio 46:55 mixing and emulsifyings.
9. a kind of subunit vaccine composition as described in claim 1-7 any claims is being prepared for preventing and treating
Application in the vaccine of staphylococcus aureus mastitis in dairy cows.
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