CN107224576A

CN107224576A - Vaccine of staphylococcus aureus mastitis in dairy cows subunit and its preparation method and application

Info

Publication number: CN107224576A
Application number: CN201710128915.4A
Authority: CN
Inventors: 钱泓; 吴有强; 张强; 贾宝琴; 查银河
Original assignee: Oceanic Rise Bio Tech Ltd Zhejiang
Current assignee: Oceanic Rise Bio Tech Ltd Zhejiang
Priority date: 2017-03-06
Filing date: 2017-03-06
Publication date: 2017-10-03

Abstract

The invention discloses a kind of vaccine of staphylococcus aureus mastitis in dairy cows subunit and its preparation method and application.The vaccine includes PVL S proteins and pharmaceutically acceptable adjuvant or PVL F proteins and pharmaceutically acceptable adjuvant；Its preparation method comprises the following steps：(1) clone of staphylococcus aureus PVL S proteins or PVL F proteins；(2) expression and purification of restructuring PVL S proteins or PVL F proteins；(3) restructuring PVL S proteins or PVL F proteins are obtained into recombinant subunit vaccine with the VG mixing and emulsifyings of ISA 201.The vaccine does not contain nucleic acid, and homologous recombination and itself virulence will not be caused to return the potential danger such as strong；Body can be induced and produce specific antibody, can promote cellular immunity, inducing immunological memory and cause extensive immune response, good cross protection reaction is produced, thus the vaccine more safety and stability, prepare it is simple, using convenient, cheap and time saving and energy saving.

Description

Vaccine of staphylococcus aureus mastitis in dairy cows subunit and preparation method thereof and Using

Technical field

The present invention relates to a kind of subunit vaccine of staphylococcus aureus mastitis in dairy cows and preparation method thereof and should With.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 Tissue is by a kind of inflammation caused by after microorganism infection, and multiple to be born in the postpartum breastfeeding phase, it is each that the disease is widely present in the world Ground, is milk cow common disease, frequently-occurring disease, is to cause one of Dairy Products Industry Implementing economic loss the most serious disease.Cause cow breast Scorching pathogenic microorganism kind about more than 150, mainly based on staphylococcus aureus, Escherichia coli, Streptococcusagalactiae, this Three kinds of bacterial mastitis for milk cows account for more than the 90% of total incidence, wherein especially using staphylococcus aureus as most.

The treatment of current mastitis for milk cows is mainly antibiotic therapy.Antibiotic is used to treat mastitis for milk cows existing 50 History for many years, its served in the preventing and treating of mastitis for milk cows it is certain, but due to long-term single, heavy dose of, not section Use antibiotic, causes sensitive bacteria elimination, and drug tolerant bacteria gradually account for main flow, especially Staphylococcus aureus The resistance problems of bacterium are increasingly serious, result in antibiotic therapy and produce little effect；On the other hand, with the improvement of living standards, Residue of Antibiotics in Milk is a very serious health problem.

There is good prospect with vaccine control mastitis for milk cows, first, vaccine can prevent milk cow infection pathogen and Cause mammitis；Secondly, vaccine helps to reduce the order of severity of intramammary infection, controls Subclinical mammitis；3rd, make Be not in antibiotic residue problem in milk with vaccine control mammitis；Be finally it is easy to operate, it is low-cost.Currently, grind Make successful vaccine seldom, and most of is weak malicious live vaccine or inactivated vaccine, in production practices, the preventing and treating to mammitis There is certain effect, however as the development of extensive intensive culture, the exquisite weak bacterial strain of people has homologous recombination, itself poison Power return it is strong etc. potentially possible, and inactivated vaccine there is also dosage it is big, the deficiency such as not thorough is inactivated, to improve traditional vaccine Security and immune protective efficiency, efficient, cheap new generation vaccine is developed particularly important.

Staphylococcus aureus (Staphylococcus aureus, abbreviation SA), is that a kind of leather is blue also referred to as " S. aureus L-forms " The positive coccus of Albert'stain Albert, 0.8 μm of diameter is arranged in thyrsiform under the microscope, it is possible to produce golden yellow pigment, Therefore gain the name.S. aureus L-forms are to cause one of the main pathogenic fungi of chronic/recessive mastitis for milk cows, can be with strong influence ox The yield and quality of milk, huge economic loss is brought to dairy industry.

Panton-valentine leukocidins (panton-valentine leukocidin, PVL) are by golden yellow One of extracellular toxin that color staphylococcus produces, is also one of its main virulence factor.PVL by Van deleld in Find within 1894, and separated it from hemolysin by Panton and Valentine first in 1932.PVL is by two Kind of protein composition, i.e. PVL-S albumen and PVL-F albumen (LukS-PVT, LukF-PV), its molecular weight be respectively 34kDa and Amino acid sequence homology between 33kDa, and F protein and S protein has 36%.PVL belongs to film drilling toxin family, can lure Lead PMNs necrosis or adjust and die, be that LukS-PV is combined with the acceptor of the specific high-affinity on PMNs cell membranes first, secondly LukF-PV formation dimers in combination, then LukS-PV and LukF-PV knots platform successively, eventually form cyclic structure Heteromers.This heteromers internal diameter is 3nm, and external diameter is 9nm, molecular weight about 200 kDa, LukS-PV and LukF- contained therein PV molecular proportion is 1:1, the heteromers of this cyclic structure are inserted on PMNs cell membranes, form diameter about 2nm film Perforation, other PVL molecules enter cell by the hole, and set up duct on mitochondrial outer membrane, so as to destroy mitochondria Interior environment, and activate caspase 9, caspase 3 and release leukocidin C, inducing cell adjust dies.Therefore, PVL is One of important candidate albumen of S. aureus L-forms subunit vaccine, but in the presence of working as PVL-S and PVL-F all, it is likely to result in cell Mouse safety experiment has been done in toxicity, this laboratory to it, as a result identical with expection, contains PVL-S albumen simultaneously immune During with the vaccine of PVL-F albumen, mouse after immune 3-4 days it is just dead, but the PVL-S albumen of only immune same dose or In PVL-F albumen, 14 days tracked after being immunized, mouse is all normal, does not occur any exception, so, in subunit vaccine During selection, the candidate albumen for selecting one of which albumen as subunit vaccine is only capable of.

The content of the invention

The technical problem to be solved in the present invention：One is to provide a kind of new staphylococcus aureus mastitis in dairy cows Asia Subunit vaccine and preparation method thereof；Two to be that homologous recombination, itself virulence for overcoming live vaccine to exist are returned strong etc. potentially possible；Three Be overcome inactivated vaccine dosage big and inactivation that may be present thoroughly caused by homologous recombination the problems such as.

The invention provides a kind of new staphylococcus aureus mastitis in dairy cows subunit vaccine, the vaccine includes PVL-S albumen (i.e. staphylococcus aureus PVL-S albumen) is (i.e. golden yellow with pharmaceutically acceptable adjuvant or PVL-F albumen Color staphylococcus PVL-F albumen) and pharmaceutically acceptable adjuvant.

In technical scheme, it is preferable that the encoding gene of the PVL-S albumen is as shown in SEQ ID NO.1.

In technical scheme, it is preferable that the PVL-S albumen is protein (a1) or protein (b1)：Albumen The protein that matter (a1) is made up of the amino acid shown in SEQ ID NO.3；Amino acid of the protein (b1) in protein (a1) Sequence is by substitution, missing or one amino acid of addition or several amino acid and with PVL-S protein antigenicities by protein (a1) protein derived from.

In technical scheme, it is preferable that PVL-F protein coding genes are as shown in SEQ ID NO.2.

In technical scheme, it is preferable that the PVL-F albumen is protein (a2) or protein (b2)：Albumen The protein that matter (a2) is made up of the amino acid shown in SEQ ID NO.4；Amino acid of the protein (b2) in protein (a2) Sequence is by substitution, missing or one amino acid of addition or several amino acid and with PVL-F protein antigenicities by protein (a2) protein derived from.

In technical scheme, it is preferable that every part albumen containing PVL-S is 100 μ g or epidemic disease in described vaccine Every part albumen containing PVL-F is 100 μ g in seedling.

In technical scheme, it is preferable that the pharmaceutically acceptable adjuvant is the VG adjuvants of ISA 201.

In technical scheme, it is preferable that also contain preservative in the vaccine, the preservative is thimerosal, The concentration of the thimerosal is 2 μ g/ml.

Present invention also offers a kind of method for preparing milk cow staphylococcus aureus subunit vaccine, the preparation side Method comprises the following steps：(1) clone of staphylococcus aureus PVL-S albumen or PVL-F albumen；(2) PVL-S albumen is recombinated Or the expression and purification of PVL-F albumen；(3) restructuring PVL-S albumen or PVL-F albumen are obtained with the VG mixing and emulsifyings of ISA 201 To recombinant subunit vaccine, wherein, restructuring PVL-S albumen or PVL-F albumen and the VG volume ratios of adjuvant ISA 201 are 46: 55。

The present invention provides a kind of restructuring PVL-S albumen, PVL-F albumen and is preparing milk cow staphylococcus aureus weight again Application in group subunit vaccine and dependent diagnostic reagent.Prepared invention further provides a kind of subunit vaccine for pre- Application in anti-or treatment staphylococcus aureus mastitis in dairy cows medicine.

Compared with prior art, the invention provides a kind of new staphylococcus aureus mastitis in dairy cows subunit Vaccine and its preparation method and application.The subunit vaccine does not contain nucleic acid, and homologous recombination and itself virulence will not be caused to return by force Etc. potential danger；Body can be induced and produce specific antibody, can promote cellular immunity, inducing immunological memory and cause extensive Immune response, produces the reaction of good cross protection, thus the vaccine more safety and stability, prepare it is simple, using convenient, valency Lattice are cheap, and time saving and energy saving.

Compared with prior art, mouse immune challenge viral dosage result of the invention is shown, either PVL-S albumen is still Subunit vaccine prepared by PVL-F albumen is suitable in terms of immune effect, and in the absence of safety issue, relative to others Toxin (such as alpha hemolysin and beta hemolysin) is when preparing subunit vaccine, it is not necessary in advance to albumen carry out inactivation treatment or Mutation processing is carried out to encoding gene just can directly prepare subunit vaccine, time saving, laborsaving, cost-effective.

Brief description of the drawings

Fig. 1 represents that agarose gel electrophoresis PCR expands PVL-F, PVL-S results.PVL-F gene PCR results, size is about For 906bp；PVL-S gene PCR results, size is about 849bp；M：DNA molecular amount standard DL2,000.

Fig. 2 represents 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 is 5,369bp and 904bp；pET28a-PVL-S：Plasmid PET28a-PVL-F XhoI and Nde I digestion qualification results, endonuclease bamhi size is 5,369bp and 849bp；M：DNA molecular Amount standard DL5,000.

Fig. 3 represents PVL-F albumen and PVL-S protein purification results.

Fig. 4 represents immune rear antibody titer testing result.

Fig. 5 represents immune rear challenge viral dosage result.

Embodiment

Below with reference to drawings and examples, the present invention will be further described, and embodiments of the invention are merely to illustrate Technical scheme, and the non-limiting present invention.

The source list of reagent and medicine of the present invention is as follows：

Chemical reagent and all commercially available prod of biological reagent；

Preservative thimerosal is purchased from Life Sciences；

The VG of ISA 201 are purchased from match BIC Corp of France.

Embodiment 1：Expression vector pET28a-PVL-S and pET28a-PVL-F- structure (two kinds of carrier construction method phases Together)

Milk cow staphylococcus aureus gene group to be clinically separated enters performing PCR as template, and primer is as shown in the table, As a result it is as shown in Figure 1：PVL-F gene PCR results, size is about 906 bp；PVL-S gene PCR results, size is about 849bp；It is all in the same size with expection.

1.1 sample-adding systems are (50 μ l):

1.2PCR amplification program:

1.3 glue reclaim DNA fragmentations:

(1) reaction solution of step 1.2 is subjected to 0.8% agarose gel electrophoresis (min of 110V 30)；

(2) under uviol lamp, gel extraction DNA fragmentation is in 1.5ml EP pipes；

(3) 500 μ l PC buffer, 50 DEG C, water-bath 10min are added in the 1.5ml EP pipes into step (2)；

(4) solution in step (3) is moved into adsorption column center, stands 2min, centrifugation, 12,000 rpm, 30s；

(5) waste liquid is abandoned, 600 μ l PW buffer are added to adsorption column center, 3min, 12,000rpm, 30s is stood；

(6) repeat step (5)；

(7) suction attached column is centrifuged, 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.

1.4 double digestions react (50 μ l systems)：

It is loaded, mixed according to above-mentioned system in 1.5ml EP pipes, the two 50 μ l reaction solutions is then placed in 37 In DEG C thermostat water bath, water-bath 3h.

1.5 glue reclaim DNA fragmentations：

(1) reaction solution of step 1.4 is subjected to 0.8% agarose gel electrophoresis (min of 110V 30)；

(2) under uviol lamp, gel extraction DNA fragmentation is in 1.5ml EP pipes；

(6) repeat step (5)；

(7) suction attached column is centrifuged, 12,000rpm, 1min；

(9) collection step (8) DNA sample carries out electrophoresis.

1.6 coupled reactions (10 μ l systems)：

It is loaded, mixed according to above-mentioned system in 1.5ml EP pipes, above-mentioned reaction solution is then placed in 16 DEG C, water Take out, 65 DEG C, inactivated after water-bath 15min after bath 16h, by 4 DEG C of preservations of sample.

1.7 transformation experiment：

(1) the coupled reaction liquid of step 1.6 is taken out, 100 μ l E.coli DH5 α competent cells are added thereto, is mixed It is even；

(2) ice bath 30min；

(3) 42 DEG C, water-bath 100s；

(4) ice bath 2min；

(5) take out, 600 μ l LB liquid mediums, 37 DEG C, the h of water-bath 1 are added into EP pipes；

(6) sample cell is taken out, is centrifuged (8,000rpm, 2min), removes 600 μ l, remaining 100 μ l LB and thalline is resuspended；

(7) take bacterium solution to be plated in LK flat boards (Kan concentration is 50 μ g/ml), LK flat boards are placed in biochemical constant incubator In, 37 DEG C of culture 12h.

1.8 recombinant plasmids are extracted and digestion identification：

(1) picking monoclonal is into 3ml LK fluid nutrient mediums from conversion flat board, and 37 DEG C, 260rpm shakes bacterium and stayed overnight；

(2) 1ml bacterium solutions are taken into 1.5ml EP pipes, centrifuges (12,000rpm, 2min), abandons supernatant；

(3) 250 μ l P1 buffer are added in the EP pipes into step (2), thalline is resuspended；

(4) 250 μ l P2 buffer are added into step (3) solution, it is gentle to mix, stand 2 min；

(5) 350 μ l P3 buffer are added into step (4) solution, it is gentle to mix；

(6) by step (5) solution, centrifuge (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, is centrifuged (9,000 g, 30s)；

(9) repeat step (8)；

(10) suction attached column centrifugation (9,000g, 1min)；

(11) 30 μ l Elution buffer are added to adsorption column, stands 2min, centrifuged (12,000 rpm, 2min)；

(12) collection step (11) DNA sample carries out electrophoresis；

(13) as shown in step 1.4, digestion identification is carried out to the plasmid extracted, 0.8% Ago-Gel is then carried out Electrophoresis.

(14) recombinant plasmid digestion qualification result is as shown in Figure 2：：pET28a-PVL-F：Plasmid pET28a-PVL-F XhoI and 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 is 5,369bp and 849bp.

Embodiment 2：Convert e. coli bl21

Draw 1 μ l plasmids to add in 100 μ l BL21 competent cells, ice bath 30min；

42 DEG C of heat shock 90s；

Ice bath 2min；

The LB nutrient solutions of 900 μ l non-resistants are added in super-clean bench；

37 DEG C of 180rpm shake 1h；

Draw 100 μ l bacterium solutions card-coating that resistance LB flat boards, 37 DEG C of incubated overnights.

Embodiment 3：A large amount of induced expressions

Choose bacterium：Picking monoclonal is into 50ml cards that resistance LB nutrient solutions, 37 DEG C of incubated overnights；

Switching：By 1:100 ratios transfer bacterium solution to 500ml cards that resistance LB nutrient solutions, and 3.5L, 37 DEG C of 220rpm are shaken altogether Cultivate 2-2.5h to OD₆₀₀It is worth 0.6；

Induction：Bacterium solution OD₆₀₀After being worth 0.6,500 μ l IPTG (1M) are added to the final concentration of 1mmol/L of IPTG, 37 DEG C 220rpm Fiber differentiations 4h；

Microorganism collection：Bacterium solution 6,000rpm centrifugation 10min, collects thalline；Thalline, 6,000rpm are cleaned with 40ml PBS 10min is centrifuged, thalline is collected, is placed in -20 DEG C of preservations；

Embodiment 4：PVL-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 is added in obtained thalline into embodiment 3 NaH₂PO₄(pH 8.0), 500mM NaCl, 20mM imidazoles；Membrane filtration using 0.8 μm), blown and beaten with 50mL syringes uniform To without graininess fungus block；Thalline sample is poured into cell homogeneous instrument sample cell, outlet is prepared to collect sample with beaker； Using 90% outflow outlet of sample as a circulation, after the completion of a circulation, the sample that outlet beaker is collected is refunded Sample cell, continues to repeat 5 circulations.

(2) complete sample will be crushed in step (1) to be dispensed into 250mL Beckman centrifuge tubes, 12,000rpm, 4 DEG C centrifugation 30min, Supernatant samples are crossed after 0.8 μm of film as loading sample, and reserving 80 μ L samples is used for SDS-PAGE detections；

(3) column equilibration：With the ultrapure column volume of water balance 2~3 (CV), the ethanol of discharge 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), takes 80 μ L to be examined for SDS-PAGE Survey.

(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) post is washed, 5mL/min rinses 80 Individual column volume.

(6) 2 are rinsed：With (the elution buffer 1 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) post is washed, reduce Triton X-114 residual.

(7) elute：(the 50%Elution buffer 2 of 50% elution buffer component 2：50mM NaH₂PO₄(pH 7.4), 500mM NaCl, 250mM imidazoles, the membrane filtration using 0.8 μm) elution destination protein, wash flat to baseline, speed 5ml/ Min, is collected, and takes 80 μ L to be analyzed for SDS-PAGE after mixing；The 100% de- component of buffer solution 2 cleaning pillar (100% Elution buffer 2：50mM NaH₂PO₄(pH 7.4), 500mM NaCl, 500mM imidazoles, uses 0.8 μm of filter membrane mistake Filter), wash flat to baseline, 5mL/min is collected, and takes 80 μ L to be analyzed for SDS-PAGE after mixing.

(8) HiPrep Desalting desalting columns column equilibration：With the ultrapure column volume of water balance 2~3 (CV), discharge 20% Ethanol preserve liquid；Then 3-4 column volumes (CV), speed 10mL/min are balanced with elution buffer component 1.

(9) loading：Speed 10mL/min is injected (inject), and maximum injection (inject) amount is 13mL.

(10) collect：Stop after loading, into load patterns, 10mL/min, UV rises to 1mAU and starts collection, i.e. egg White sample starts appearance, and 10ml/ pipes are collected, and treats that UV is down to below 5mAU, stops collecting.

(11) balance：Flow velocity 10ml/min, balances 2-3CV.

(12) 7.10~7.12 are circulated, until completion of the sample.

(13) aseptic filtration：The protein solution of collection is at 4 DEG C, and 12,000rpm centrifugation 15min collect supernatant, move to life Thing safety cabinet, crosses the low syringe needle filter of 0.2 μm of protein binding rate, and -80 DEG C of refrigerator preservations are placed in after filtering.

(14) purification result is as shown in Figure 3：PVL-S albumen and the purity of PVL-F albumen after purification can reach 90% More than；By calculating, in the case of no optimization fermented and cultured, the expression quantity of PVL-S albumen and PVL-F albumen can reach To more than 500mg/L.

Embodiment 5：Vaccine is prepared (1ml/ parts)

(1) needed according to experiment, calculate the amount of each composition of vaccine solution, make to recombinate PVL-S albumen or PVL-F in vaccine Final concentration of protein is 100 μ g/ml, makes the final concentration of 2 μ g/ml of thimerosal in vaccine, then by load weighted recombinant protein with Load weighted thimerosal mixing, as aqueous phase, is 46 according to aqueous phase and the VG volume ratios of adjuvant ISA 201:55, measure adjuvant；

(2) measured aqueous phase and adjuvant are placed in thermostat water bath and are heated to 32 DEG C ± 1 DEG C, after after temperature stabilization Antigen is added in adjuvant pipe, oscillator concussion 10min 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 Emulsified on broken instrument；

(4) after emulsification terminates, emulsifying effectiveness is observed：Part vaccine is taken to be placed in centrifuge tube, 3,000 rpm centrifugation 15min, Vaccine is not stratified to be qualified；

(5) detect that qualified vaccine is dispensed into 15ml centrifuge tubes, mark that sealed membrane sealing is placed in 4 DEG C of preservations.

Embodiment 6：Mouse immune challenge viral dosage

6.1 immunization experiment

(1) vaccine is taken into (25 DEG C) placement 2h of room temperature or so, vaccine temperature is returned to normal temperature；

(2) weigh, be grouped to mouse and mark, two groups are immunity test group (every group of 10 mouse), and PVL- is immunized respectively S subunit vaccines and PVL-F subunit vaccines；Another group is control group (n=10), PBS is immunized, and record numerical value；

(3) 1ml vaccines are drawn with 1ml syringes, notes draining in bubble；Then back leg flesh is given with 75% cotton ball soaked in alcohol Meat is sterilized, the inserting needle in the middle part of muscle masses, and left and right back leg respectively injects 50 μ l vaccines；

(4) 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 latter 14 days collection serum, detect antibody titer.

(5) bioactivity result is as shown in Figure 4：Antibody titer result after detection is incorporated into a figure, after two exempt from 2 groups of immune group relative antibody titers can reach 12, more than 800, and three, which exempt from rear 2 groups of immune group relative antibody titers, can reach More than 80,000；Either two exempt from after or three exempt from after, the antibody titers of PVL-S immune groups is above the anti-of PVL-F immune groups Body potency, this is also corresponding with attacking malicious result.

6.2 challenge viral dosages (three exempt to carry out within latter 14 days attacking poison)

(1) picking SA single bacterium colonies (bacterial strain is the CQ339 bacterial strains that this laboratory is preserved) are in 5 ml liquid broths In, 220rpm, 37 DEG C shake bacterium and stay overnight；

(2) microbionation overnight will be shaken in 100ml fresh liquid broth bouillons by centesimal volume (1ml) In, 220rpm, 37 DEG C shake bacterium and 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, finally all thalline is resuspended with 5ml PBS and mixed；

(4) counted after bacterium solution being done into 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) weighed to 2 groups of immune groups and control group mice, one group of immune PVL-S Protein assays group (n=10), another group Immune PVL-F Protein assays group (n=10)；Last group records numerical value for control group (n=10)；

(6) bacterium solution is drawn with 1ml syringes, tail vein injection, injection volume is carried out according to the corresponding mouse of each concentration bacterium For 200 μ l/20g mouse.

(7) survival state of Continuous Observation mouse, records the death time of each mouse：It is each in the morning, afternoon and evening to check once.

(8) mouse survival rate result after poison is attacked as shown in Figure 5：Within the 308h of observation, dead 8 of control group, PVL- Dead 1 of S immune groups, dead 2 of PVL-F immune groups, therefore, control group survival rate only have 20%, and 2 groups of immune groups are survived Rate is above 80%, and PVL-S immune groups are high by 10% compared with PVL-F immune group survival rates.

Embodiment 7：ELISA antibody titers are detected

(1) it is coated with：Detection albumen (the PVL-S of purifying is diluted with coating buffer (50mM carbonate buffer solutions, pH 9.5) 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 were placed after sealed membrane is sealed Night；

(2) wash：Taken out from refrigerator after ELISA Plate, be put into board-washing machine and wash, cleaning solution PBST；

(3) close：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 consumption is needed, serum is subjected to appropriateness dilution with confining liquid；

(5) wash：With (2)；

(6) it is loaded：Dilute serum is added, while negative control is made of confining liquid, 37 DEG C of incubation 1h；

(7) wash：With (2)；

(8) secondary antibody is added：Secondary antibody 100 the μ l, 37 DEG C of incubation 0.5h of the HRP marks of appropriateness dilution are added per hole；

(9) wash：With (2)；

(10) develop the color：100 μ l TMB nitrite ions, 37 DEG C of incubation 10min are added under the conditions of lucifuge per hole；

(11) terminate：50 μ l terminate liquids (2M H are added per hole₂SO₄), terminating reaction；

(12) detect：In 450nm wavelength determination sample OD values, analyze data；

(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 here Described particular example and embodiment.Purpose herein comprising these particular examples and embodiment is to help this The technical staff practice present invention in field.Any those of skill in the art be easy to do not depart from spirit of the invention and It is further improved in the case of scope and perfect, therefore the present invention is only by the content and model of the claims in the present invention The limitation enclosed, its intention covers alternative in all spirit and scope of the invention for being included in and being limited by appendix claim Scheme and equivalent.

Sequence table

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<120>Staphylococcus aureus mastitis in dairy cows subunit vaccine and its preparation method and application

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<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

tctaaaatta aagtgactta tcaaagacaa atggatcggt atgaaaattt ttggaacaac 780

ttgcactgga taggttataa tattaagaat caaaagagag caacacacac atcaatttat 840

gaaattgatt gggaaaaaca cacggttaaa ttagtagctt cgcaatctag cgaactcgag 900

caccaccacc accaccactg a 921

<210> 1

<211> 292

<212> PRT

<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

1 5 10 15

Thr Glu Asp Thr Ser Ser Asp Lys Trp Gly Val Thr Gln Asn Ile Gln

20 25 30

Phe Asp Phe Val Lys Asp Lys Lys Tyr Asn Lys Asp Ala Leu Ile Leu

35 40 45

Lys Met Gln Gly Phe Ile Asn Ser Lys Thr Thr Tyr Tyr Asn Tyr Lys

50 55 60

Asn Thr Asp His Ile Lys Ala Met Arg Trp Pro Phe Gln Tyr Asn Ile

65 70 75 80

Gly Leu Lys Thr Asn Asp Pro Asn Val Asp Leu Ile Asn Tyr Leu Pro

85 90 95

Lys Asn Lys Ile Asp Ser Val Asn Val Ser Gln Thr Leu Gly Tyr Asn

100 105 110

Ile Gly Gly Asn Phe Asn Ser Gly Pro Ser Thr Gly Gly Asn Gly Ser

115 120 125

Phe Asn Tyr Ser Lys Thr Ile Ser Tyr Asn Gln Gln Asn Tyr Ile Ser

130 135 140

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

165 170 175

Leu Phe Val Gly Tyr Lys Pro Tyr Ser Gln Asn Pro Arg Asp Tyr Phe

180 185 190

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

260 265 270

Trp Lys Thr His Glu Ile Lys Val Lys Gly His Asn Leu Glu His His

275 280 285

His His His His

290

<210> 1

<211> 306

<212> PRT

<213>Recombination staphylococcus aureus PVL-F protein sequences

<400> 4

Met Gly Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp

1 5 10 15

Lys Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Ser Asp Lys Leu

20 25 30

Lys Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp

35 40 45

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

275 280 285

Leu Val Ala Ser Gln Ser Ser Glu Leu Glu His His His His His His

290 295 300 305

Claims

1. a kind of vaccine of staphylococcus aureus mastitis in dairy cows subunit, it is characterised in that：The vaccine includes PVL-S eggs In vain with pharmaceutically acceptable adjuvant or PVL-F albumen and pharmaceutically acceptable adjuvant.

2. vaccine according to claim 1, it is characterised in that the encoding gene of the PVL-S albumen such as SEQ ID NO.1 It is shown.

3. vaccine according to claim 1, it is characterised in that the encoding gene of the PVL-F albumen such as SEQ ID NO.2 It is shown.

4. vaccine according to claim 1, it is characterised in that in the vaccine every part albumen containing PVL-S be 100 μ g, Or every part albumen containing PVL-F is 100 μ g in the vaccine.

5. vaccine according to claim 1, it is characterised in that the pharmaceutically acceptable adjuvant is helped for the VG of ISA 201 Agent.

6. according to any described vaccine of Claims 1 to 5, it is characterised in that the vaccine also contains preservative.

7. vaccine according to claim 6, it is characterised in that the preservative is thimerosal, the concentration of the thimerosal For 2 μ g/ml.

8. a kind of method for preparing the vaccine as described in claim 1~7 is any, it is characterised in that methods described includes following step Suddenly：

(1) clone of staphylococcus aureus PVL-S albumen or PVL-F albumen；

(2) expression and purification of restructuring PVL-S albumen or PVL-F albumen；

(3) restructuring PVL-S albumen or PVL-F albumen are obtained into recombinant subunit vaccine with the VG mixing and emulsifyings of adjuvant ISA 201, Wherein, restructuring PVL-S albumen or PVL-F albumen and the VG volume ratios of adjuvant ISA 201 are 46:55.

9. a kind of restructuring PVL-S albumen, PVL-F albumen according to claim 1~7 any claim is preparing milk cow Application in staphylococcus aureus recombinant subunit vaccine and dependent diagnostic reagent.

10. a kind of subunit vaccine according to claim 1-7 any claims is being prepared for preventing or treating milk Application in the medicine of ox staphylococcus aureus mastitis.