CN103800900A

CN103800900A - Staphylococcus aureus strains and milk cattle mastitis vaccine comprising inactivated Staphylococcus aureus strains

Info

Publication number: CN103800900A
Application number: CN201410039970.2A
Authority: CN
Inventors: 李敏; 王林叶; 孙治华; 王欢; 杨鹏辉; 段跃强; 孙科; 孙艺萍
Original assignee: INNER MONGOLIA HUAXI BIOTECHNOLOGY Co Ltd
Current assignee: INNER MONGOLIA HUAXI BIOTECHNOLOGY Co Ltd
Priority date: 2014-01-27
Filing date: 2014-01-27
Publication date: 2014-05-21
Anticipated expiration: 2034-01-27
Also published as: CN103800900B

Abstract

The invention discloses Staphylococcus aureus strains and a milk cattle mastitis vaccine comprising inactivated Staphylococcus aureus strains. The milk cattle mastitis vaccine comprises inactivated strain SACP5-9, inactivated SACP8-6 and inactivated SACP336-3 as active ingredients. The milk cattle mastitis vaccine comprises inactivated strains and capsular polysaccharides as active ingredients, wherein the inactivated strains include inactivated strain SACP5-9, inactivated SACP8-6 and inactivated SACP336-3; capsular polysaccharides include capsular polysaccharide of strain SACP5-9, capsular polysaccharide of SACP8-6 and capsular polysaccharide of SACP336-3. The vaccine provided by the invention has the advantages of high efficiency, general use, good safety and stability, a variety of dosage forms, and the like. The milk cattle mastitis vaccine provides strong guarantee for safely, effectively and comprehensively preventing Staphylococcus aureus injection type milk cattle mastitis, and is a new milestone of development of Staphylococcus aureus mastitis vaccines.

Description

Staphylococcus aureus and the mammitis of cow vaccine that its deactivation is obtained

Technical field

The mammitis of cow vaccine that the present invention relates to staphylococcus aureus and its deactivation is obtained.

Background technology

Mammitis of cow (Bovine Mastitis) is the serious infectious diseases of harm animal husbandry development, food safety, particularly in recent years, along with the change of environment, antibiotic abuse, produce multiple drug-resistant bacteria and even occurred superbacteria, infect by antibacterial the mammitis of cow M & M causing soaring year by year, serious harm animal husbandry development and food safety, and then endangered human health.Because of the economic loss that mammitis of cow causes, the U.S. reaches 400,000,000 dollars of 3,000,000,000 dollars, Britain every year, and China is more than 3,500,000,000 yuan.Mammitis of cow is divided into clinical type and recessive type.The symptom of clinical type is: newborn district generates heat, swells and ache, and lactation amount reduces, and milk character changes, and bacterial population increases, somatic cell increases, and severe one multiple organ failure is even dead.Recessive type does not have special clinical manifestation, just can detect by somatic cell, milk yield and quality testing, but milch cow attack rate is more much higher than clinical type, causes further sending out of pathogenic microorganism, can change into clinical type under certain condition, actual loss is more much higher than clinical type.

The pathogen that causes mammitis of cow all reaches more than 140 and plants.Show according to clinical epidemiology data, the pathogen of China's mammitis of cow is with escherichia coli, staphylococcus aureus and streptococcus are main, account for 91.4% of antibacterial infection, wherein staphylococcus aureus accounts for 46%, and with 5 type staphylococcus aureuses (claiming again staphylococcus aureus capsular polysaccharide serotype 5 types), 8 type staphylococcus aureuses (claim not only staphylococcus aureus capsular polysaccharide Serotype8 type) and 336 type staphylococcus aureuses (but also claiming staphylococcus aureus capsular polysaccharide serotype 336 types) are main (three accounts for the more than 95% of infection of staphylococcus aureus), with current international report infection bacteria species, serotype and fashion trend are consistent.

Summary of the invention

The mammitis of cow vaccine that the object of this invention is to provide staphylococcus aureus and its deactivation is obtained.

The present invention protects bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 in the application of preparing in mammitis of cow vaccine.Described mammitis of cow can be the mammitis of cow that staphylococcus aureus causes.Described staphylococcus aureus can be 5 type staphylococcus aureuses, 8 type staphylococcus aureuses or 336 type staphylococcus aureuses.Described staphylococcus aureus specifically can be bacterial strain SACP5-9, bacterial strain SACP8-6, bacterial strain SACP336-3,5 type staphylococcus aureus ATCC49521,8 type staphylococcus aureus ATCC49525,336 type staphylococcus aureus ATCC55804, the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, the 8 serotype gold popular dominant strain of Portugal's bacterium mammitis of cow or the popular dominant strain of 336 serotypes gold Portugal's bacterium mammitis of cow.

The present invention also protects a kind of mammitis of cow vaccine, and its active component is bacterial strain SACP8-6 after bacterial strain SACP5-9, the deactivation after deactivation and the bacterial strain SACP336-3 after deactivation.Specifically, in described vaccine, the concentration of the bacterial strain SACP8-6 after bacterial strain SACP5-9, described deactivation after described deactivation and the bacterial strain SACP336-3 after described deactivation is 1 × 10 ¹⁰individual antibacterial/mL.Described vaccine also can comprise adjuvant.Described adjuvant is SP01 adjuvant, aluminium adjuvant or white flower oil adjuvant.The dosage form of described vaccine is papillary duct injection type, subcutaneous injection dosage form or intramuscular injection dosage form.Described mammitis of cow can be the mammitis of cow that staphylococcus aureus causes.Described staphylococcus aureus can be 5 type staphylococcus aureuses, 8 type staphylococcus aureuses or 336 type staphylococcus aureuses.Described staphylococcus aureus specifically can be bacterial strain SACP5-9, bacterial strain SACP8-6, bacterial strain SACP336-3,5 type staphylococcus aureus ATCC49521,8 type staphylococcus aureus ATCC49525,336 type staphylococcus aureus ATCC55804, the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, the 8 serotype gold popular dominant strain of Portugal's bacterium mammitis of cow or the popular dominant strain of 336 serotypes gold Portugal's bacterium mammitis of cow.

The present invention also protects a kind of mammitis of cow vaccine, and its active component is bacterial strain and the capsular polysaccharide after deactivation; Bacterial strain after described deactivation is bacterial strain SACP8-6 after bacterial strain SACP5-9, the deactivation after deactivation and the bacterial strain SACP336-3 after deactivation; Described capsular polysaccharide is the capsular polysaccharide of the capsular polysaccharide of described bacterial strain SACP5-9, described bacterial strain SACP8-6 and the capsular polysaccharide of described bacterial strain SACP336-3.Specifically, in described vaccine, the concentration of the bacterial strain SACP8-6 after bacterial strain SACP5-9, described deactivation after described deactivation and the bacterial strain SACP336-3 after described deactivation is 1 × 10 ¹⁰individual antibacterial/mL, the concentration of the capsular polysaccharide of the capsular polysaccharide of described bacterial strain SACP5-9, the capsular polysaccharide of described bacterial strain SACP8-6 and described bacterial strain SACP336-3 is 40 μ g/ml.Described vaccine also can comprise adjuvant.Described adjuvant is SP01 adjuvant, aluminium adjuvant or white flower oil adjuvant.Described mammitis of cow can be the mammitis of cow that staphylococcus aureus causes.The dosage form of described vaccine is papillary duct injection type, subcutaneous injection dosage form or intramuscular injection dosage form.Described staphylococcus aureus can be 5 type staphylococcus aureuses, 8 type staphylococcus aureuses or 336 type staphylococcus aureuses.Described staphylococcus aureus specifically can be bacterial strain SACP5-9, bacterial strain SACP8-6, bacterial strain SACP336-3,5 type staphylococcus aureus ATCC49521,8 type staphylococcus aureus ATCC49525,336 type staphylococcus aureus ATCC55804, the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, the 8 serotype gold popular dominant strain of Portugal's bacterium mammitis of cow or the popular dominant strain of 336 serotypes gold Portugal's bacterium mammitis of cow.

Described " bacterial strain after deactivation " can obtain by described bacterial strain is carried out to formalin-inactivated.Described " bacterial strain after deactivation " specifically obtains by the following method: get bacterial strain, resuspended with PBS buffer, then adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), 37 ℃, 150rpm oscillation incubation 36-48h, centrifugal (parameter of noncentricity specifically can be: 5000r/min, 20min) collects thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing, obtain " bacterial strain after deactivation " (containing 1 × 10 ¹⁰individual antibacterial/mL).

The preparation method of described capsular polysaccharide is specific as follows:

(1) get bacterial strain, be inoculated in BHI culture medium, 37 ℃, 150rpm shaken cultivation 18h.

(2) get the bacterium liquid that step (1) obtains, be seeded to BHI culture medium, in fermentation tank, (fermentation initial time, the bacteria concentration of bacterial strain is 2 × 10 in 37 ℃ of fermentations ⁸individual/ml, fermentation jar temperature is 37 ℃, the initial speed of fermentation tank is 198r/min, tank pressure remains on 0.05MPa, regulating the pH of fermentation system with 8%NaOH aqueous solution or 5% aqueous hydrochloric acid solution is 7.0-7.2, be 25-35% by increasing fermentation tank rotating speed and/or increasing the dissolved oxygen amount that ventilation maintains fermentation system, the dissolved oxygen amount of fermentation system stops fermentation higher than 35% time);

(3) get the fermentation system that step (2) obtains, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), 25 ℃, 150rpm oscillation incubation 24h, then 4 ℃, the centrifugal 15min of 3000rpm, collect supernatant;

(4) get the supernatant that step (3) obtains, adding cetyl trimethyl ammonium bromide and making its concentration is 1g/100ml, room temperature, 120rpm oscillation incubation 1 hour, and then 4 ℃ leave standstill 16 hours, then the centrifugal 30min of 10000rpm, collecting precipitation (complex polysaccharide);

(5) get the precipitation that step (4) obtains, be dissolved in 2M calcium chloride water (precipitation is 0.2-0.5g:ml with the proportioning of calcium chloride water), grind, then add and the isopyknic water of 2M calcium chloride water, room temperature, 120rpm oscillation incubation 1h, then adding ethanol and making its concentration is 25%(volume ratio), 4 ℃ of standing 3h, then 4 ℃, the centrifugal 30min of 5000rpm, collect supernatant;

(6) get the supernatant that step (5) obtains, adding ethanol and making its concentration is 80% (volume ratio), 4 ℃ of standing 3h, then 4 ℃, the centrifugal 15min of 10000rpm, collecting precipitation, uses ethanol and washing with acetone successively, dry, obtain dry (raw sugar);

(7) get the dry that step (6) obtains, be dissolved in 10g/100mL aqueous sodium acetate solution (proportioning of dry and aqueous sodium acetate solution is 10-20mg:ml), add 2 times of phenol solutions to aqueous sodium acetate solution volume (100g crystalline phenol is dissolved in 40ml10g/100mL aqueous sodium acetate solution), 4 ℃, the 120rpm 20-30min that vibrates, then 4 ℃, 10000rpm, centrifugal 20min, get the superiors' liquid phase;

(8) get the liquid phase that step (7) obtains, add the phenol solution (100g crystalline phenol is dissolved in 40ml10g/100mL aqueous sodium acetate solution) of 2 times of volumes, 4 ℃, the 120rpm 20-30min that vibrates, then 4 ℃, 10000rpm, centrifugal 20min, get the superiors' liquid phase;

(9) get the liquid phase that step (8) obtains, in 0.1M calcium chloride water, dialyse;

(10) get the solution that step (9) obtains, adding ethanol and making its concentration is 75%(volume ratio), 4 ℃ of standing 3h, then 4 ℃, the centrifugal 20min of 10000rpm, collecting precipitation, uses ethanol and washing with acetone successively, dry, obtains dry (capsular polysaccharide).

Described SP01 adjuvant is specific as follows: solvent is water, containing 2g/100mL Squalene, 0.1g/100mL polyoxyethylene castor oil and 0.1g/100mL polyether polyol.

Described aluminium adjuvant specifically can be aluminium hydroxide.

Described vaccine specifically can be vaccine-I, vaccine-II, vaccine-III, vaccine-IV, vaccine-V, vaccine-VI, vaccine VII, vaccine VIII, vaccine IX, vaccine X, vaccine XI or the vaccine XII of preparation in embodiment 3.

Arbitrary described milch cow specifically can be china holstein cows above.

The present invention also protects bacterial strain SACP5-9 and/or bacterial strain SACP8-6 and/or bacterial strain SACP336-3.When between above three kinds of bacterial strains being " with " be related to time, the quantity of three bacterial strains is than specifically can be 1:1:1.

Staphylococcus aureus provided by the invention (Staphylococcus aureus) SACP5-9, be called for short bacterial strain SACP5-9, within 09th, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in December in 2013 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.8529.

Staphylococcus aureus provided by the invention (Staphylococcus aureus) SACP8-6, be called for short bacterial strain SACP8-6, within 09th, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in December in 2013 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.8530.

Staphylococcus aureus provided by the invention (Staphylococcus aureus) SACP336-3, be called for short bacterial strain SACP336-3, within 09th, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in December in 2013 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.8531.

For mammitis of cow, optimal preventions is for by vaccination, its object is exactly enhancing human body immunity responsing reaction, particularly the immune response of mammary gland tissue stops, neutralizes, kills the pathogenic microorganism that the external world enters, but because the physiological characteristics of breast itself brings a series of special challenges of knowing clearly to the generation of effective immunity.The first, the milk in mammary gland can dilute the concentration of anti-infective neutrophil cell, and some compositions in milk, can reduce the sterilizing ability of infection neutrophil cell as fat and casein.The second, raising dairy cattle has in the environment of the microorganism that can cause in a large number mastitis, and milk itself provides hotbed to pathogen growth.Therefore, make cow mammary gland produce effective immunity, reduce the incidence rate of mastitis, the difficult problem that need to capture is more, proposes the requirement higher than other infectious disease vaccine to the research and development of mammitis of cow vaccine.

Thereby vaccine is to make body obtain natural resistance against diseases by the function of enhancing human body immunity system, and vaccine has the features such as safe, stable, effective, and that therefore develops that safe and effective mammitis of cow vaccine becomes prevention and control mastitis must be by selecting.The vaccine of researching and developing the popular representative strain of suitable this country and environment is imperative.

Vaccine provided by the invention has the advantages such as efficient, general, safe, stable, dosage form is various, the present invention for safety, efficient, effectively prevention infection of staphylococcus aureus mammitis of cow provides strong guarantee comprehensively, by the new milestone that is staphylococcus aureus mastitis in dairy cows Vaccine Development.

The specific embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.PBS buffer in embodiment, be if no special instructions pH7.2,0.01M without thermal source PBS buffer.

The popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, the popular dominant strain of 8 serotype gold Portugal's bacterium mammitis of cow, the popular dominant strain of 336 serotype gold Portugal's bacterium mammitis of cow are all recorded in as Publication about Document: staphylococcus aureus mastitis in dairy cows inactivated vaccine and preparation method thereof (number of patent application: 201010543949.8; Application publication number CN101979089A).

Squalene: be purchased from SIGMA company, catalog number (Cat.No.) is 442785.Polyoxyethylene castor oil (density 1.05g/ml at25 ℃, viscosity 850cP): be purchased from SIGMA company, catalog number (Cat.No.) is C5135.Polyether polyol (density 1.05g/mlat25 ℃, viscosity 850cP): be purchased from SIGMA company, catalog number (Cat.No.) is 435449.SP01 adjuvant: solvent is water, containing 2g/100mL Squalene, 0.1g/100mL polyoxyethylene castor oil and 0.1g/100mL polyether polyol.White flower oil adjuvant: purchased from French Esso company, lot number 122735.Balb/C mice: purchased from Military Medical Science Institute's Experimental Animal Center.Milch cow (china holstein cows): be purchased from Inner Mongol Chifeng dairy cow farm in plenty.

Embodiment 1, bacterial strain acquisition and preservation

One, the screening of bacterial strain

From the Inner Mongol, 90 medium-and-large-sized cattle farms on 30 provinces and cities ground such as Chongqing, Guangzhou, Heilungkiang, Lanzhou, Hebei, Shandong gather 30,000 parts of milk Lac Bovis seu Bubali samples.The acquisition method of milk Lac Bovis seu Bubali sample: choose the milch cow that occurs mastitis clinical symptoms (or suspecting for Subclinical mastitis), first clean breast with warm water, clean breast by 0.2% bromo geramine again, finally use 70% alcohol wipe nipple, sampler is simultaneously with 70% alcohol wipe finger sterilization, each newborn chamber first squeeze decaptitates 2-3 milk, with the miscellaneous bacteria decontaminating, every cow head get newborn sample at least 5mL in sterile milk sample cup.

Separation and purification staphylococcus aureus strains from newborn sample.The colony morphology characteristic (37 ℃, cultivate 24-72h) of staphylococcus aureus: (1) TSA culture medium flat plate: can see the golden yellow bacterium colony of circle moistening, smooth, protuberance, picking colonies typical smear, dyeing microscopy, be botryoidalis and arrange; (2) nutrient broth: be muddy growth, there is a small amount of pale precipitation at the pipe end; (3) Mai Kangkai culture medium: do not grow; (4) blood agar plate (fresh defiber Sheep Blood): bacterium colony is larger, is golden yellow, circular, occurs obvious beta hemolysis around.The physiological and biochemical property of staphylococcus aureus: (1) catalase test: the positive; (2) clotting of plasma enzyme reaction: the positive; (3) sugar fermentating test: energy decomposition glucose, lactose, mannitol, maltose, sucrose fermentation, can not utilize arabinose, malonate; (4) oxidase test: feminine gender; (5) V-P test: the positive; (6) DNA enzyme test: the positive; (7) nitrate reduction reaction: the positive; (8) indole test: the positive; (9) gelatin liquefaction reaction: feminine gender; (10) Gram-positive.The conservative fragments of the encoding gene of the 16Sr RNA of staphylococcus aureus is as shown in the sequence 1 of sequence table.The staphylococcus aureus that separation is obtained carries out respectively genome sequencing, and the bacterial strain that genome sequencing comes to the same thing is same bacterial strain.Three strain staphylococcus aureuses are present in respectively in 25786,24653 and 26312 parts of newborn samples, show that this staphylococcus aureus is the popular dominant strain of China, will distinguish called after bacterial strain 1, bacterial strain 2 and bacterial strain 3.

Two, the amplification culture of bacterial strain

1, by bacterial strain 1, bacterial strain 2 or the primary bacterial strain of bacterial strain 3() distinguish streak inoculation to BHI culture medium flat plate, 37 ℃ leave standstill cultivation 20-24 hour, then rinse and collect the bacterium colony on flat board by the skim milk powder aqueous solution of 5g/100ml, be sub-packed in cryopreservation tube (being defined as P1 generation) ,-70 ℃ of following preservations.

2, get the cryopreservation tube that step 1 obtains, room temperature is thawed, and is then seeded to liquid B HI culture medium, 37 ℃, 150rpm shaken cultivation 20-24 hour, and every 100 milliliters add 5g defatted milk powder, are sub-packed in cryopreservation tube (being defined as P2 generation) ,-70 ℃ of following preservations.

3, get the cryopreservation tube that step 2 obtains, room temperature is thawed, and is then seeded to liquid B HI culture medium, and 37 ℃, 150rpm shaken cultivation 20-24 hour, add equal-volume glycerol, is sub-packed in cryopreservation tube (being defined as P3 generation) ,-70 ℃ of following preservations.

Three, the Serotype Identification of bacterial strain and pathogenic evaluation

P3 is respectively carried out to slide agglutination test (5 type staphylococcus aureus ATCC49521s for bacterial strain 2 and P3 for bacterial strain 3 for bacterial strain 1, P3; 8 type staphylococcus aureus ATCC49525; 336 type staphylococcus aureus ATCC55804; The method of preparing serum is: by immune BALB/c mouse after staphylococcus aureus deactivation, then gather in the crops serum).Result shows that bacterial strain 1 is 5 type staphylococcus aureuses (therefore called after bacterial strain SACP5-9), bacterial strain 2 is 8 type staphylococcus aureuses (therefore called after bacterial strain SACP8-6), and bacterial strain 3 is 336 type staphylococcus aureuses (therefore called after bacterial strain SACP336-3).

P3 is administered to respectively to 6-8 BALB/c mouse in age in week for bacterial strain SACP5-9, and (every by lumbar injection 3 × 10 ⁸cFU), 1.5-2 year rabbit (every by lumbar injection 1 × 10 ⁹and 3-4 year milch cow (injecting 800CFU by papillary duct for every) CFU).Observe to characterize and find that bacterial strain SACP5-9 can make BALB/c mouse and family's rabbit invasion, some animal is even dead in 1 week; Bacterial strain SACP5-9 can cause milch cow generation mastitis.Get respectively the liver of morbidity mice, the liver of morbidity rabbit, the breast of morbidity milch cow, carry out pathogen and heavily separate, carry out genome sequencing by heavily separating the bacterial strain obtaining, all consistent with the sequencing result of bacterial strain SACP5-9.

P3 is administered to respectively to 6-8 BALB/c mouse in age in week for bacterial strain SACP8-6, and (every by lumbar injection 3 × 10 ⁸cFU), 1.5-2 year rabbit (every by lumbar injection 1 × 10 ⁹and 3-4 year milch cow (injecting 800CFU by papillary duct for every) CFU).Observe to characterize and find that bacterial strain SACP8-6 can make BALB/c mouse and family's rabbit invasion, some animal is even dead in 1 week; Bacterial strain SACP8-6 can cause milch cow generation mastitis.Get respectively the liver of morbidity mice, the liver of morbidity rabbit, the breast of morbidity milch cow, carry out pathogen and heavily separate, carry out genome sequencing by heavily separating the bacterial strain obtaining, all consistent with the sequencing result of bacterial strain SACP8-6.

P3 is administered to respectively to 6-8 BALB/c mouse in age in week for bacterial strain SACP336-3, and (every by lumbar injection 3 × 10 ⁸cFU), 1.5-2 year rabbit (every by lumbar injection 1 × 10 ⁹and 3-4 year milch cow (injecting 800CFU by papillary duct for every) CFU).Observe to characterize and find that bacterial strain SACP336-3 can make BALB/c mouse and family's rabbit invasion, some animal is even dead in 1 week; Bacterial strain SACP336-3 can cause milch cow generation mastitis.Get respectively the liver of morbidity mice, the liver of morbidity rabbit, the breast of morbidity milch cow, carry out pathogen and heavily separate, carry out genome sequencing by heavily separating the bacterial strain obtaining, all consistent with the sequencing result of bacterial strain SACP336-3.

Four, the preservation of bacterial strain

Bacterial strain SACP5-9 full name is staphylococcus aureus (Staphylococcus aureus) SACP5-9.P3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on the 09th in December in 2013 for bacterial strain SACP5-9, and preserving number is CGMCC No.8529.

Bacterial strain SACP8-6 full name is staphylococcus aureus (Staphylococcus aureus) SACP8-6.P3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on the 09th in December in 2013 for bacterial strain SACP8-6, and preserving number is CGMCC No.8530.

Bacterial strain SACP336-3 full name is staphylococcus aureus (Staphylococcus aureus) SACP336-3.P3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on the 09th in December in 2013 for bacterial strain SACP336-3, and preserving number is CGMCC No.8531.

The characteristic of embodiment 2, bacterial strain

3 of the step 2 of the embodiment 1 cryopreservation tube room temperatures that obtain are thawed, P3 are carried out to following steps for bacterial strain:

One, the immunogenicity of bacterial strain

By P3, for bacterial strain SACP5-9 immunizing rabbit, (single immunization, every by subcutaneous injection 5 × 10 ⁷cFU/ml, injects 1 milliliter for every), heart extracting blood after 21 days, separation of serum.The IgG antibody titer that detects serum by ELISA (with PBST buffer gradient dilution, obtains serum dilution; Using P3 for bacterial strain SACP5-9 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt goat anti-rabbit igg ELIAS secondary antibody; Negative control is set, replaces serum dilution with PBST buffer; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above), be 1:12800.

By P3, for bacterial strain SACP8-6 immunizing rabbit, (single immunization, every by subcutaneous injection 5 × 10 ⁷cFU/ml, injects 1 milliliter for every), heart extracting blood after 21 days, separation of serum.The IgG antibody titer that detects serum by ELISA (with PBST buffer gradient dilution, obtains serum dilution; Using P3 for bacterial strain SACP8-6 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt goat anti-rabbit igg ELIAS secondary antibody; Negative control is set, replaces serum dilution with PBST buffer; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above), be 1:12800.

By P3, for bacterial strain SACP336-3 immunizing rabbit, (single immunization, every by subcutaneous injection 5 × 10 ⁷cFU/ml, injects 1 milliliter for every), heart extracting blood after 21 days, separation of serum.The IgG antibody titer that detects serum by ELISA (with PBST buffer gradient dilution, obtains serum dilution; Using P3 for bacterial strain SACP336-3 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt goat anti-rabbit igg ELIAS secondary antibody; Negative control is set, replaces serum dilution with PBST buffer; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above), be 1:12800.

Two, the immunogenicity of other contrast bacterial strain

Replace P3 for bacterial strain SACP5-9 the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, method according to step 1 detects that (which bacterial strain is the immunogenicity that detects which bacterial strain adopt as coating antigen, other same step 1), the tiring as 1:1600 of serum.

Replace P3 for bacterial strain SACP8-6 the popular dominant strain of 8 serotype gold Portugal's bacterium mammitis of cow, method according to step 1 detects that (which bacterial strain is the immunogenicity that detects which bacterial strain adopt as coating antigen, other same step 1), the tiring as 1:1600 of serum.

Replace P3 for bacterial strain SACP336-3 the popular dominant strain of 336 serotype gold Portugal's bacterium mammitis of cow, method according to step 1 detects that (which bacterial strain is the immunogenicity that detects which bacterial strain adopt as coating antigen, other same step 1), the tiring as 1:1600 of serum.

Replace P3 for bacterial strain SACP5-9 5 type staphylococcus aureus ATCC49521, detect (which bacterial strain is the immunogenicity that detects which bacterial strain adopt as coating antigen, other same step 1) according to the method for step 1, the tiring as 1:1600 of serum.

Replace P3 for bacterial strain SACP8-6 8 type staphylococcus aureus ATCC49525, detect (which bacterial strain is the immunogenicity that detects which bacterial strain adopt as coating antigen, other same step 1) according to the method for step 1, the tiring as 1:1600 of serum.

Replace P3 for bacterial strain SACP336-3 336 type staphylococcus aureus ATCC55804, method according to step 1 detects that (which bacterial strain is the immunogenicity that detects which bacterial strain adopt as coating antigen, other same step 1), the tiring as 1:1600 of serum.

Three, the universality of the serum obtaining after bacterial strain immunity to staphylococcus aureus

By P3 for bacterial strain SACP5-9 immunizing rabbit (the same step 1 of mode), heart extracting blood after 21 days, separation of serum.By ELISA detect serum IgG antibody titer (adopt bacterium to be measured replace P3 for bacterial strain SACP5-9 as coating antigen, other same step 1).Bacterium to be measured is 5 type staphylococcus aureus ATCC49521 or the popular dominant strain of 5 serotypes gold Portugal's bacterium mammitis of cow.Serum is 1:6400 to tiring of bacterium to be measured.

By P3 for bacterial strain SACP8-6 immunizing rabbit (the same step 1 of mode), heart extracting blood after 21 days, separation of serum.By ELISA detect serum IgG antibody titer (adopt bacterium to be measured replace P3 for bacterial strain SACP8-6 as coating antigen, other same step 1).Bacterium to be measured is 8 type staphylococcus aureus ATCC49525 or the popular dominant strain of 8 serotypes gold Portugal's bacterium mammitis of cow.Serum is 1:6400 to tiring of bacterium to be measured.

By P3 for bacterial strain SACP336-3 immunizing rabbit (the same step 1 of mode), heart extracting blood after 21 days, separation of serum.By ELISA detect serum IgG antibody titer (adopt bacterium to be measured replace P3 for bacterial strain SACP336-3 as coating antigen, other same step 1).Bacterium to be measured is 336 type staphylococcus aureus ATCC55804 or the popular dominant strain of 336 serotypes gold Portugal's bacterium mammitis of cow.Serum is 1:6400 to tiring of bacterium to be measured.

Four, the stability of recombinant bacterium

Get P3 for bacterial strain SACP5-9, be seeded to liquid B HI culture medium, 37 ℃, 150rpm shaken cultivation 20-24 hour, obtain P4 for bacterial strain.Adopt said method to carry out continuous passage, reach P30 generation.Respectively P3 generation, P5 generation, P10 generation, P15 generation, P20 generation, P25 generation and P30 are carried out to genome sequencing for bacterial strain, sequencing result is all consistent, shows that bacterial strain SACP5-9 provided by the invention is highly stable.

Get P3 for bacterial strain SACP8-6, be seeded to liquid B HI culture medium, 37 ℃, 150rpm shaken cultivation 20-24 hour, obtain P4 for bacterial strain.Adopt said method to carry out continuous passage, reach P30 generation.Respectively P3 generation, P5 generation, P10 generation, P15 generation, P20 generation, P25 generation and P30 are carried out to genome sequencing for bacterial strain, sequencing result is all consistent, shows that bacterial strain SACP8-6 provided by the invention is highly stable.

Get P3 for bacterial strain SACP336-3, be seeded to liquid B HI culture medium, 37 ℃, 150rpm shaken cultivation 20-24 hour, obtain P4 for bacterial strain.Adopt said method to carry out continuous passage, reach P30 generation.Respectively P3 generation, P5 generation, P10 generation, P15 generation, P20 generation, P25 generation and P30 are carried out to genome sequencing for bacterial strain, sequencing result is all consistent, shows that bacterial strain SACP336-3 provided by the invention is highly stable.

The preparation of embodiment 3, vaccine

One, prepare capsular polysaccharide

1, get the colonies typical of 3-5 bacterial strain SACP5-9, be inoculated in BHI culture medium, 37 ℃, 150rpm shaken cultivation 18h.

2, get the bacterium liquid that 2 parts by volume steps 1 obtain, be seeded to 100 parts by volume BHI culture medium, in fermentation tank, (fermentation initial time, the bacteria concentration of bacterial strain SACP5-9 is 2 × 10 in 37 ℃ of fermentations ⁸individual/ml, fermentation jar temperature is 37 ℃, the initial speed of fermentation tank is 198r/min, tank pressure remains on 0.05MPa, regulating the pH of fermentation system with 8%NaOH aqueous solution or 5% aqueous hydrochloric acid solution is 7.0-7.2, be 25-35% by increasing fermentation tank rotating speed and/or increasing the dissolved oxygen amount that ventilation maintains fermentation system, the dissolved oxygen amount of fermentation system stops fermentation higher than 35% time).

3, get the fermentation system that step 2 obtains, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), 25 ℃, 150rpm oscillation incubation 24h, then 4 ℃, the centrifugal 15min of 3000rpm, collect supernatant.

4, get the supernatant that step 3 obtains, adding cetyl trimethyl ammonium bromide and making its concentration is 1g/100ml, room temperature, 120rpm oscillation incubation 1 hour, and then 4 ℃ leave standstill 16 hours, the then centrifugal 30min of 10000rpm, collecting precipitation (complex polysaccharide).

5, get the precipitation that step 4 obtains, be dissolved in 2M calcium chloride water (precipitation is 0.2-0.5g:ml with the proportioning of calcium chloride water), fully grind, then add and the isopyknic water of 2M calcium chloride water, room temperature, 120rpm oscillation incubation 1h, then adding ethanol and making its concentration is 25%(volume ratio), 4 ℃ of standing 3h, then 4 ℃, the centrifugal 30min of 5000rpm, collect supernatant.

6, get the supernatant that step 5 obtains, adding ethanol and making its concentration is 80% (volume ratio), the fully rear 4 ℃ of standing 3h of vibration, then 4 ℃, the centrifugal 15min of 10000rpm, collecting precipitation, use again washing with acetone twice twice by washing with alcohol, fully dry, obtain dry (raw sugar).

7, get the dry that step 6 obtains, be dissolved in 10g/100mL aqueous sodium acetate solution (proportioning of dry and aqueous sodium acetate solution is 10-20mg:ml), add 2 times of phenol solutions to aqueous sodium acetate solution volume (100g crystalline phenol is dissolved in 40ml10g/100mL aqueous sodium acetate solution), 4 ℃, the 120rpm 20-30min that vibrates, then 4 ℃, 10000rpm, centrifugal 20min are divided into three layers and (are followed successively by from top to bottom supernatant layer, albumin layer and phenol layer under upper; Wherein supernatant layer contains object polysaccharide), get supernatant layer.

8, get the supernatant layer that step 7 obtains, add the phenol solution (100g crystalline phenol is dissolved in 40ml10g/100mL aqueous sodium acetate solution) of 2 times of volumes, 4 ℃, the 120rpm 20-30min that vibrates, then 4 ℃, 10000rpm, centrifugal 20min are divided into three layers and (are followed successively by from top to bottom containing supernatant layer, albumin layer and phenol layer under upper; Wherein supernatant layer contains object polysaccharide), get supernatant layer.

9, get the supernatant layer that step 8 obtains, pack bag filter into, in 0.1M calcium chloride water, dialyse 12 hours for 4 ℃.

10, after completing steps 9, take out the solution in bag filter, adding ethanol and making its concentration is 75%(volume ratio), after fully vibrating, 4 ℃ of standing 3h, then 4 ℃, the centrifugal 20min of 10000rpm, collecting precipitation, use again washing with acetone twice twice by washing with alcohol, fully dry, obtain dry (bacterial strain SACP5-9 capsular polysaccharide).

Replace bacterial strain SACP5-9 to carry out above-mentioned steps bacterial strain SACP8-6, obtain bacterial strain SACP8-6 capsular polysaccharide.

Replace bacterial strain SACP5-9 to carry out above-mentioned steps bacterial strain SACP336-3, obtain bacterial strain SACP336-3 capsular polysaccharide.

With pyrogen-free 0.1M calcium chloride water dissolving bacterial strain SACP5-9 capsular polysaccharide, SACP8-6 capsular polysaccharide or bacterial strain SACP336-3 capsular polysaccharide, after filtration sterilization, carry out sterility test, serological test and each biochemical measurement.Identify respectively the quality (comprise solid amount, protein content, nucleic acid content, O-acetyl content, phosphorus content, polysaccharide molecular weight size, aseptic etc.) of capsular polysaccharide according to the method for pharmacopeia (Pharmacopoeia of People's Republic of China, two 〇 mono-〇 versions).Result shows, each capsular polysaccharide all meets the requirement of indices, and purity all reaches more than 95%, and be single each serotype capsular polysaccharide antigenic component, without exogenous factor.

Two, the preparation of vaccine

1, get P3 for bacterial strain SACP5-9, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

2, get P3 for bacterial strain SACP8-6, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

3, get P3 for bacterial strain SACP336-3, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

4, the PBS buffer after bacteria suspension step 1 being obtained, the bacteria suspension that step 2 obtains, bacteria suspension and sterilizing that step 3 obtains mixes, (in vaccine-I, the concentration of bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 is 1 × 10 to obtain vaccine-I ¹⁰individual antibacterial/mL).

5, the PBS buffer after bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide and sterilizing step 1 being obtained mixes, obtain vaccine-II (in vaccine-II, the concentration of bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide is 40 μ g/ml).

6, the PBS buffer after the bacteria suspension obtaining by step 1, the bacteria suspension that step 2 obtains, bacteria suspension that step 3 obtains, bacterial strain SACP5-9 capsular polysaccharide that step 1 obtains, bacterial strain SACP8-6 capsular polysaccharide that step 1 obtains, bacterial strain SACP336-3 capsular polysaccharide and sterilizing that step 1 obtains mixes, (in vaccine-III, the concentration of bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 is 1 × 10 to obtain vaccine-III ¹⁰individual antibacterial/mL, the concentration of bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide is 40 μ g/ml).

7, get vaccine-I, adding aluminium hydroxide and making its concentration is 0.5mg/ml, obtains vaccine-IV.

8, get vaccine-II, adding aluminium hydroxide and making its concentration is 0.5mg/ml, obtains vaccine-V.

9, get vaccine-III, adding aluminium hydroxide and making its concentration is 0.5mg/ml, obtains vaccine-VI.

10, the PBS buffer after bacteria suspension step 1 being obtained, the bacteria suspension that step 2 obtains, bacteria suspension and sterilizing that step 3 obtains mixes, obtain mixed liquor, then add and the isopyknic SP01 adjuvant of mixed liquor, (in vaccine VII, the concentration of bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 is 1 × 10 to obtain vaccine VII ¹⁰individual antibacterial/mL).

11, the PBS buffer after bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide and sterilizing step 1 being obtained mixes, obtain mixed liquor, then add and the isopyknic SP01 adjuvant of mixed liquor, obtain vaccine VIII (in vaccine VIII, the concentration of bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide is 40 μ g/ml).

12, the PBS buffer after bacteria suspension step 1 being obtained, the bacteria suspension that step 2 obtains, bacteria suspension that step 3 obtains, bacterial strain SACP5-9 capsular polysaccharide that step 1 obtains, bacterial strain SACP8-6 capsular polysaccharide that step 1 obtains, bacterial strain SACP336-3 capsular polysaccharide and sterilizing that step 1 obtains mixes, obtain mixed liquor, then add and the isopyknic SP01 adjuvant of mixed liquor, (in vaccine-IX, the concentration of bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 is 1 × 10 to obtain vaccine-IX ¹⁰individual antibacterial/mL, the concentration of bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide is 40 μ g/ml).

13, the PBS buffer after bacteria suspension step 1 being obtained, the bacteria suspension that step 2 obtains, bacteria suspension and sterilizing that step 3 obtains mixes, obtain mixed liquor, then add and the isopyknic white flower oil adjuvant of mixed liquor, (in vaccine X, the concentration of bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 is 1 × 10 to obtain vaccine X ¹⁰individual antibacterial/mL).

14, the PBS buffer after bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide and sterilizing step 1 being obtained mixes, obtain mixed liquor, then add and the isopyknic white flower oil adjuvant of mixed liquor, obtain vaccine XI (in vaccine XI, the concentration of bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide is 40 μ g/ml).

15, the PBS buffer after bacteria suspension step 1 being obtained, the bacteria suspension that step 2 obtains, bacteria suspension that step 3 obtains, bacterial strain SACP5-9 capsular polysaccharide that step 1 obtains, bacterial strain SACP8-6 capsular polysaccharide that step 1 obtains, bacterial strain SACP336-3 capsular polysaccharide and sterilizing that step 1 obtains mixes, obtain mixed liquor, then add and the isopyknic white flower oil adjuvant of mixed liquor, (in vaccine-XII, the concentration of bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 is 1 × 10 to obtain vaccine-XII ¹⁰individual antibacterial/mL, the concentration of bacterial strain SACP5-9 capsular polysaccharide, bacterial strain SACP8-6 capsular polysaccharide and bacterial strain SACP336-3 capsular polysaccharide is 40 μ g/ml).

Each vaccine is placed in to 2-8 ℃ to be saved backup.

The effect of embodiment 4, vaccine

One, mouse experiment

6-8 week Balb/C mice, is divided into 7 groups, and 10 every group, grouping immunity following (subcutaneous injection):

First group: test the 1st day and the 15th day each immune vaccine-I of experiment 0.25 milliliter of each immunity;

Second group: test the 1st day and the 15th day each immune vaccine-II of experiment 0.25 milliliter of each immunity;

The 3rd group: test the 1st day and the 15th day each immune vaccine-III of experiment 0.25 milliliter of each immunity;

The 4th group: test the 1st day and the 15th day each immune vaccine-IV of experiment 0.25 milliliter of each immunity;

The 5th group: test the 1st day and the 15th day each immune vaccine-V of experiment 0.25 milliliter of each immunity;

The 6th group: test the 1st day and the 15th day each immune vaccine-VI of experiment 0.25 milliliter of each immunity;

The 7th group: test the 1st day and the 15th day each immune vaccine-VII of experiment 0.25 milliliter of each immunity;

The 8th group: test the 1st day and the 15th day each immune vaccine-VIII of experiment 0.25 milliliter of each immunity;

The 9th group: test the 1st day and the 15th day each immune vaccine-IX of experiment 0.25 milliliter of each immunity;

The tenth group: test the 1st day and the 15th day each immune vaccine-X of experiment 0.25 milliliter of each immunity;

The 11 group: test the 1st day and the 15th day each immune vaccine-XI of experiment 0.25 milliliter of each immunity;

The 12 group: test the 1st day and the 15th day each immune vaccine-XII of experiment 0.25 milliliter of each immunity;

The 13 group (negative control): test the 1st day and the 15th day each immune PBS buffer of experiment 0.25 milliliter of each immunity.

Test tail venous blood sampling separation of serum the 29th day.The IgG antibody titer that detects serum by ELISA (with PBST buffer gradient dilution, obtains serum dilution; Respectively using P3 for bacterial strain SACP5-9, P3 for bacterial strain SACP8-6 and P3 for bacterial strain SACP336-3 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt rabbit anti-mouse igg ELIAS secondary antibody; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above).The tiring of serum that adopts above first group of laboratory animal of three kinds of coating antigens to obtain is 1:51200, the tiring of serum that adopts above second group of laboratory animal of three kinds of coating antigens to obtain is 1:25600, adopt above tiring of the 3rd group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 4th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 5th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, adopt above tiring of the 6th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 7th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 8th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 9th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the tenth group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 11 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, adopt above tiring of the 12 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800.Result shows: the effect that the effect of thalline+capsular polysaccharide is better than thalline is better than the effect of capsular polysaccharide, and the effect of adding adjuvant is better than not adding adjuvant, and the effect of adding SP01 adjuvant is better than adding Alum adjuvant (aluminium hydroxide) or white flower oil adjuvant.

Two, milch cow experiment

2-5 year be about to the to enter lactation period healthy cow of (being antenatal 25d, conceived 8 months 20d), is divided into 37 groups, and 100 every group, grouping immunity is as follows:

First group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-I of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

Second group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-II of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 3rd group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-III of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 4th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-IV of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 5th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-V of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 6th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VI of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 7th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VII of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 8th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VIII of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 9th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-IX of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The tenth group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-X of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 11 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-XI of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 12 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-XII of experiment, 5 milliliters (injection of papillary duct single-point) of each immunity;

The 13 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-I of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 14 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-II of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 15 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-III of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 16 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-IV of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 17 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-V of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 18 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VI of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 19 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VII of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 20 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VIII of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 21 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-IX of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 22 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-X of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 23 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-XI of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 24 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-XII of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 25 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-I of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 26 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-II of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 27 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-III of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 28 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-IV of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 29 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-V of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 30 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VI of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 31 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VII of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 32 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-VIII of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 33 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-IX of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 34 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-X of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 35 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-XI of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 36 group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune vaccine-XII of experiment, 5 milliliters (injection of musculi colli single-point) of each immunity;

The 37 group (negative control): test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune PBS buffer of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity.

Test the 71st day, Jugular vessel is got blood separation of serum.The IgG antibody titer that detects serum by ELISA (with PBST buffer gradient dilution, obtains serum dilution; Respectively using P3 for bacterial strain SACP5-9, P3 for bacterial strain SACP8-6 and P3 for bacterial strain SACP336-3 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt goat-anti cattle IgG ELIAS secondary antibody; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above).Test the 71st day, get milk surum.The sIgA antibody titer that detects milk surum by ELISA (with PBST buffer gradient dilution, obtains milk surum diluent; Respectively using P3 for bacterial strain SACP5-9, P3 for bacterial strain SACP8-6 and P3 for bacterial strain SACP336-3 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt goat-anti cattle sIgA ELIAS secondary antibody; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above).

The tiring of serum that adopts above first group of laboratory animal of three kinds of coating antigens to obtain is 1:102400, the tiring of serum that adopts above second group of laboratory animal of three kinds of coating antigens to obtain is 1:51200, adopt above tiring of the 3rd group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 4th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 5th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 6th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 7th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 8th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 9th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the tenth group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 11 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 12 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600.

Adopt above tiring of the 13 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 14 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 15 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the 16 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the 17 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 18 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:1638400, adopt above tiring of the 19 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:1638400, adopt above tiring of the 20 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the 21 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:3276800, adopt above tiring of the 22 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the 23 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 24 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:1638400.

Adopt above tiring of the 25 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 26 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 27 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 28 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 29 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 30 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the 31 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200, adopt above tiring of the 32 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 33 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:1638400, adopt above tiring of the 34 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 35 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 36 group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:819200.

The tiring of milk surum that adopts above first group of laboratory animal of three kinds of coating antigens to obtain is 1:102400, the tiring of milk surum that adopts above second group of laboratory animal of three kinds of coating antigens to obtain is 1:51200, adopt above tiring of the 3rd group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 4th group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 5th group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, the tiring of milk surum that adopts above three kinds of coating antigen the 6th treated animals to obtain is 1:409600, adopt above tiring of the 7th group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:409600, adopt above tiring of the 8th group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, the tiring of milk surum that adopts above three kinds of coating antigen the 9th treated animals to obtain is 1:819200, adopt above tiring of the tenth group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 11 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, the tiring of milk surum that adopts above three kinds of coating antigen the 12 treated animals to obtain is 1:409600.

Adopt above tiring of the 13 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, adopt above tiring of the 14 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:25600, adopt above tiring of the 15 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 16 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 17 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, the tiring of milk surum that adopts above three kinds of coating antigen the 18 treated animals to obtain is 1:204800, adopt above tiring of the 19 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:204800, adopt above tiring of the 20 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, the tiring of milk surum that adopts above three kinds of coating antigen the 21 treated animals to obtain is 1:409600, adopt above tiring of the 22 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 23 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, the tiring of milk surum that adopts above three kinds of coating antigen the 24 treated animals to obtain is 1:204800.

Adopt above tiring of the 25 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:25600, adopt above tiring of the 26 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:12800, adopt above tiring of the 27 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, adopt above tiring of the 28 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, adopt above tiring of the 29 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:25600, the tiring of milk surum that adopts above three kinds of coating antigen the 30 treated animals to obtain is 1:102400, adopt above tiring of the 31 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:102400, adopt above tiring of the 32 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, the tiring of milk surum that adopts above three kinds of coating antigen the 33 treated animals to obtain is 1:204800, adopt above tiring of the 34 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:51200, adopt above tiring of the 35 group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:25600, the tiring of milk surum that adopts above three kinds of coating antigen the 36 treated animals to obtain is 1:102400.

Test the 71st day, get after blood, carry out counteracting toxic substances (using newborn pin that 600CFU5 type staphylococcus aureus ATCC49521,600CFU8 type staphylococcus aureus ATCC49525 and 600CFU336 type staphylococcus aureus ATCC55804 are injected in breast) with 5 type staphylococcus aureus ATCC49521,8 type staphylococcus aureus ATCC49525 and 336 type staphylococcus aureus ATCC55804, test the 72nd day-experiment the 85th day is the result judge phase.

Result is passed judgment on interim, if meet following (a) and (b), (c) and (d) in four more than two or two, be judged as mastitis morbidity individual: (a) mean body temperature of relative negative control group, fever 1.5-2.5 ℃; (b) in milk surum, there are grumeleuse and/or blood and/or pus; (c) there is symptom red and/or swollen and/or heating in cow breast; (d) the average lactation amount of relative negative control group, lactation amount reduces.Protective rate=(this group milch cow total individual number amount-this group mammitis of cow morbidity individual amount/this group milch cow total individual number amount) × 100%.

The protective rate of first group of laboratory animal is 92%, the protective rate of second group of laboratory animal is 91%, the protective rate of the 3rd group of laboratory animal is 93%, the protective rate of the 4th group of laboratory animal is 93%, the protective rate of the 5th group of laboratory animal is 92%, the protective rate of the 6th group of laboratory animal is 94%, the protective rate of the 7th group of laboratory animal is 94%, the protective rate of the 8th group of laboratory animal is 93%, the protective rate of the 9th group of laboratory animal is 95%, the protective rate of the tenth group of laboratory animal is 93%, the protective rate of the 11 group of laboratory animal is 92%, the protective rate of the 12 group of laboratory animal is 94%.

The protective rate of the 13 group of laboratory animal is 94%, the protective rate of the 14 group of laboratory animal is 93%, the protective rate of the 15 group of laboratory animal is 95%, the protective rate of the 16 group of laboratory animal is 95%, the protective rate of the 17 group of laboratory animal is 94%, the protective rate of the 18 group of laboratory animal is 96%, the protective rate of the 19 group of laboratory animal is 96%, the protective rate of the 20 group of laboratory animal is 95%, the protective rate of the 21 group of laboratory animal is 97%, the protective rate of the 22 group of laboratory animal is 95%, the protective rate of the 23 group of laboratory animal is 94%, the protective rate of the 24 group of laboratory animal is 96%.

The protective rate of the 25 group of laboratory animal is 93%, the protective rate of the 26 group of laboratory animal is 92%, the protective rate of the 27 group of laboratory animal is 94%, the protective rate of the 28 group of laboratory animal is 94%, the protective rate of the 29 group of laboratory animal is 93%, the protective rate of the 30 group of laboratory animal is 95%, the protective rate of the 31 group of laboratory animal is 95%, the protective rate of the 32 group of laboratory animal is 94%, the protective rate of the 33 group of laboratory animal is 96%, the protective rate of the 34 group of laboratory animal is 94%, the protective rate of the 35 group of laboratory animal is 93%, the protective rate of the 36 group of laboratory animal is 95%.

The protective rate of the 37 group of laboratory animal is 0%.

Result shows: the effect of adding adjuvant is better than not adding adjuvant, and the effect of adding SP01 adjuvant is better than adding Alum adjuvant (aluminium hydroxide) or white flower oil adjuvant.

Three, long-acting immune protective effect (natural occurrence)

It (is antenatal 25d that 2-5 year is about to enter lactation period, conceived 8 months 20d) healthy cow, be divided into 37 groups, every group 100, grouping immunity is as follows: test the 1st day, experiment the 29th day, test the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) and test the 368th day each immune vaccine, all same step 2 of immune packet mode, single immunization dosage and immunization ways.Test the 700th day-experiment and passed judgment on for 1 phase for result on the 714th day, test the 1070th day-test and passed judgment on for 2 phases for result on the 1083rd day.The same step 2 of result evaluation method.

Result passed judgment on for 1 phase: the protective rate of first group of laboratory animal is 89%, the protective rate of second group of laboratory animal is 88%, the protective rate of the 3rd group of laboratory animal is 90%, the protective rate of the 4th group of laboratory animal is 90%, the protective rate of the 5th group of laboratory animal is 89%, the protective rate of the 6th group of laboratory animal is 91%, the protective rate of the 7th group of laboratory animal is 91%, the protective rate of the 8th group of laboratory animal is 90%, the protective rate of the 9th group of laboratory animal is 92%, the protective rate of the tenth group of laboratory animal is 90%, the protective rate of the 11 group of laboratory animal is 89%, the protective rate of the 12 group of laboratory animal is 91%, the protective rate of the 13 group of laboratory animal is 91%, the protective rate of the 14 group of laboratory animal is 90%, the protective rate of the 15 group of laboratory animal is 92%, the protective rate of the 16 group of laboratory animal is 92%, the protective rate of the 17 group of laboratory animal is 91%, the protective rate of the 18 group of laboratory animal is 93%, the protective rate of the 19 group of laboratory animal is 93%, the protective rate of the 20 group of laboratory animal is 92%, the protective rate of the 21 group of laboratory animal is 94%, the protective rate of the 22 group of laboratory animal is 92%, the protective rate of the 23 group of laboratory animal is 91%, the protective rate of the 24 group of laboratory animal is 93%, the protective rate of the 25 group of laboratory animal is 90%, the protective rate of the 26 group of laboratory animal is 89%, the protective rate of the 27 group of laboratory animal is 91%, the protective rate of the 28 group of laboratory animal is 91%, the protective rate of the 29 group of laboratory animal is 90%, the protective rate of the 30 group of laboratory animal is 92%, the protective rate of the 31 group of laboratory animal is 92%, the protective rate of the 32 group of laboratory animal is 91%, the protective rate of the 33 group of laboratory animal is 93%, the protective rate of the 34 group of laboratory animal is 91%, the protective rate of the 35 group of laboratory animal is 90%, the protective rate of the 36 group of laboratory animal is 92%, the protective rate of the 37 group of laboratory animal is 39%.

Result passed judgment on for 2 phases: the protective rate of first group of laboratory animal is 88%, the protective rate of second group of laboratory animal is 87%, the protective rate of the 3rd group of laboratory animal is 89%, the protective rate of the 4th group of laboratory animal is 89%, the protective rate of the 5th group of laboratory animal is 88%, the protective rate of the 6th group of laboratory animal is 90%, the protective rate of the 7th group of laboratory animal is 90%, the protective rate of the 8th group of laboratory animal is 89%, the protective rate of the 9th group of laboratory animal is 91%, the protective rate of the tenth group of laboratory animal is 89%, the protective rate of the 11 group of laboratory animal is 88%, the protective rate of the 12 group of laboratory animal is 90%, the protective rate of the 13 group of laboratory animal is 90%, the protective rate of the 14 group of laboratory animal is 89%, the protective rate of the 15 group of laboratory animal is 91%, the protective rate of the 16 group of laboratory animal is 91%, the protective rate of the 17 group of laboratory animal is 90%, the protective rate of the 18 group of laboratory animal is 92%, the protective rate of the 19 group of laboratory animal is 92%, the protective rate of the 20 group of laboratory animal is 91%, the protective rate of the 21 group of laboratory animal is 93%, the protective rate of the 22 group of laboratory animal is 91%, the protective rate of the 23 group of laboratory animal is 90%, the protective rate of the 24 group of laboratory animal is 92%, the protective rate of the 25 group of laboratory animal is 89%, the protective rate of the 26 group of laboratory animal is 88%, the protective rate of the 27 group of laboratory animal is 90%, the protective rate of the 28 group of laboratory animal is 90%, the protective rate of the 29 group of laboratory animal is 89%, the protective rate of the 30 group of laboratory animal is 91%, the protective rate of the 31 group of laboratory animal is 91%, the protective rate of the 32 group of laboratory animal is 90%, the protective rate of the 33 group of laboratory animal is 92%, the protective rate of the 34 group of laboratory animal is 90%, the protective rate of the 35 group of laboratory animal is 89%, the protective rate of the 36 group of laboratory animal is 91%, the protective rate of the 37 group of laboratory animal is 26%.

Result shows, vaccine provided by the invention has the effect of long-effective protection.

The performance of embodiment 5, vaccine

One, the safety of vaccine (aseptic, mycoplasma test)

1, each vaccine of being prepared by embodiment 3 is seeded to respectively sulphur glycollate culture medium (streaming), cultivates 3 for 37 ℃.

2, draw the culture that step 1 obtains, be seeded to respectively 2 TG tubules (37 ℃ of cultivations are put 25 ℃ of cultivations for), 2 GA inclined-planes (37 ℃ of cultivations are put 25 ℃ of cultivations for) and 1 GP tubule (25 ℃ of cultivations), Continuous Observation 7 days, does not all observe bacterial growth.

3, each vaccine of being prepared by embodiment 3 is seeded to respectively TSA culture medium (semisolid), 37 ℃ of cultivations, and initial culture 21 days, inferior culture 21 days, does not all observe mycoplasma growth.

Two, hemolytic test

1, get the Cavia porcellus that body weight is 350g left and right, gather fresh blood 1ml, washed corpuscles.

2, with PBS buffer washing hemocyte, then prepare cell suspension (volume ratio of hemocyte and PBS buffer is 2:98) with PBS buffer.

3, with 2 times of diluents of PBS buffer each vaccine that respectively prepared by Preparation Example 3,4 times of diluents and 8 times of diluents.

4, the vaccine diluent that the cell suspension 1 parts by volume step 2 being obtained and 1 parts by volume step 3 obtain is mixed, and room temperature leaves standstill 8 hours, then judges (complete hemolysis: solution is clear and bright, red, the pipe end is acellular residual; Part haemolysis: solution is clear and bright, red or brown, the pipe end has part erythrocyte residual; Without haemolysis: erythrocyte precipitates completely, and supernatant liquid color is clear and bright).Result shows: blood cell does not all occur and break, without haemolysis.

Three, acute toxicity test

Body weight is each vaccine prepared by 12～18g Balb/C mice: single intraperitoneal injection embodiment 3, and every mice 0.5ml observes active state, body weight change and the survival rate of mice for continuous 2 weeks, within 14 days, puts to death and dissects inspection afterwards; Mice all survives, the ill symptoms such as do not occur perpendicular hair, lethargy, be slow in action, and body weight presents increase, and having no internal organs has pathological change.

Body weight is the Beagle Canis familiaris L. of 8～10kg: each vaccine prepared by single intramuscular injection embodiment 3, and every Canis familiaris L. 15ml, continuous 2 weeks observed behaviors, body weight and survival rates, put to death and dissect inspection for 14 days afterwards; Canis familiaris L. has no toxic reaction, and behavior is normal, there is no death, and body weight increases to some extent, and having no internal organs has obvious pathological change.

Four, hypersensitive test

Body weight is 250～350g Hartley Cavia porcellus: test each vaccine (at every turn inoculating 0.5ml) prepared by the 1st day, experiment the 3rd day and experiment each subcutaneous vaccination in the 5th day embodiment 3, test the 26th day, each vaccine (inoculation 0.5ml) prepared by embodiment 3 of ear vein inoculation, continues to observe animal for three days on end; Cavia porcellus is without death, and without allergic symptoms such as rhinocnesmus, sneeze, dysphoria, dyspnea, shock, spasm.

Five, rabbit thermal source matter test

Body weight is 2～3kg rabbit: measure 2 times body temperature, 30 minutes, interval, requires 2 temperature difference to be not more than 0.2 ℃, 2 mean temperature 38.6-39.5 ℃ of rabbit.After the 2nd thermometric in 15 minutes, inject each vaccine prepared by embodiment 3 from vein in one's ear, after injection every 30 minutes take temperatures 1 time, tie-in 6 times, the individual intensification of rabbit do not exceed 0.2 ℃.

Six, immunopathogenesis damage test

Mice, rabbit and milch cow: each vaccine prepared by immune embodiment 3, the detections such as peripheral blood antibody subtype, the interestization factor, inflammatory factor, eosinophilic granulocyte, neutrophilic granulocyte, basophil and trachea, lungs, liver, spleen, kidney, Th2/Th1 immunne response tends to balance, and there is no the sign of immune organ damage.

Seven, the stability of vaccine

Get each vaccine prepared by embodiment 3, place 24 months, 37 ℃ placements for 2-8 ℃ and place 3 months for 1 month or 25 ℃.

Without phenomenons such as variable color layerings, pH value is between 7.0-7.2, and electron microscopic observation size is consistent.

The step 2 that each is placed to vaccine after treatment and carry out embodiment 4, result is all consistent with the result of the step 2 of embodiment 2.

Comparative example,

One, prepare capsular polysaccharide

Replace bacterial strain SACP5-9 to carry out the step 1 of embodiment 3 the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, obtain the popular dominant strain capsular polysaccharide of 5 serotype gold Portugal's bacterium mammitis of cow.

Replace bacterial strain SACP5-9 to carry out the step 1 of embodiment 3 the popular dominant strain of 8 serotype gold Portugal's bacterium mammitis of cow, obtain the popular dominant strain capsular polysaccharide of 8 serotype gold Portugal's bacterium mammitis of cow.

Replace bacterial strain SACP5-9 to carry out the step 1 of embodiment 3 the popular dominant strain of 336 serotype gold Portugal's bacterium mammitis of cow, obtain the popular dominant strain capsular polysaccharide of 336 serotype gold Portugal's bacterium mammitis of cow.

Replace bacterial strain SACP5-9 to carry out the step 1 of embodiment 35 type staphylococcus aureus ATCC49521, obtain 5 type staphylococcus aureus ATCC49521 capsular polysaccharides.

Replace bacterial strain SACP5-9 to carry out the step 1 of embodiment 38 type staphylococcus aureus ATCC49525, obtain 8 type staphylococcus aureus ATCC49525 capsular polysaccharides.

Replace bacterial strain SACP5-9 to carry out the step 1 of embodiment 3 336 type staphylococcus aureus ATCC55804, obtain 336 type staphylococcus aureus ATCC55804 capsular polysaccharides.

Two, the preparation of vaccine

1, get the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

2, get the popular dominant strain of 8 serotype gold Portugal's bacterium mammitis of cow, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

3, get the popular dominant strain of 336 serotype gold Portugal's bacterium mammitis of cow, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

4, get 5 type staphylococcus aureus ATCC49521, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

5, get 8 type staphylococcus aureus ATCC49525, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

6, get 336 type staphylococcus aureus ATCC55804, as solvent, obtain bacterium liquid with PBS buffer; Get bacterium liquid, adding 37-40% formalin and making concentration of formaldehyde is 0.4%(volume ratio), in 37 ℃, 150rpm oscillation incubation 36h(practical application 36h-48h all can), then 4 ℃, the centrifugal 15min collection of 6500rpm thalline, with the PBS buffer washing thalline after sterilizing, then with the PBS buffer suspension thalline after sterilizing.

7, the PBS buffer after bacteria suspension step 1 being obtained, the bacteria suspension that step 2 obtains, bacteria suspension and sterilizing that step 3 obtains mixes, (in control vaccine first, the concentration of the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, the 8 serotype gold popular dominant strain of Portugal's bacterium mammitis of cow and the popular dominant strain of 336 serotypes gold Portugal's bacterium mammitis of cow is 1 × 10 to obtain control vaccine first ¹⁰individual antibacterial/mL).

8, the bacteria suspension that step 1 is obtained, the bacteria suspension that step 2 obtains, the bacteria suspension that step 3 obtains, the popular dominant strain capsular polysaccharide of 5 serotype gold Portugal's bacterium mammitis of cow that step 1 obtains, the popular dominant strain capsular polysaccharide of 8 serotype gold Portugal's bacterium mammitis of cow that step 1 obtains, PBS buffer after the 336 serotype gold popular dominant strain capsular polysaccharides of Portugal's bacterium mammitis of cow and sterilizing that step 1 obtains mixes, obtain mixed liquor, then add and the isopyknic SP01 adjuvant of mixed liquor, obtain control vaccine second (in control vaccine second, the popular dominant strain of 5 serotype gold Portugal's bacterium mammitis of cow, the concentration of the 8 serotype gold popular dominant strain of Portugal's bacterium mammitis of cow and the popular dominant strain of 336 serotypes gold Portugal's bacterium mammitis of cow is 1 × 10 ¹⁰individual antibacterial/mL, the concentration of the popular dominant strain capsular polysaccharide of 5 serotype gold Portugal's bacterium mammitis of cow, the 8 serotype gold popular dominant strain capsular polysaccharides of Portugal's bacterium mammitis of cow and the popular dominant strain capsular polysaccharide of 336 serotypes gold Portugal's bacterium mammitis of cow is 40 μ g/ml).

9, the PBS buffer after bacteria suspension step 4 being obtained, the bacteria suspension that step 5 obtains, bacteria suspension and sterilizing that step 6 obtains mixes, (in control vaccine third, the concentration of 5 type staphylococcus aureus ATCC49521,8 type staphylococcus aureus ATCC49525 and 336 type staphylococcus aureus ATCC55804 is 1 × 10 to obtain control vaccine third ¹⁰individual antibacterial/mL).

8, the bacteria suspension that step 4 is obtained, the bacteria suspension that step 5 obtains, the bacteria suspension that step 6 obtains, the 5 type staphylococcus aureus ATCC49521 capsular polysaccharides that step 1 obtains, the 8 type staphylococcus aureus ATCC49525 capsular polysaccharides that step 1 obtains, PBS buffer after 336 type staphylococcus aureus ATCC55804 capsular polysaccharides and sterilizing that step 1 obtains mixes, obtain mixed liquor, then add and the isopyknic SP01 adjuvant of mixed liquor, obtain control vaccine fourth (in control vaccine fourth, 5 type staphylococcus aureus ATCC49521, the concentration of 8 type staphylococcus aureus ATCC49525 and 336 type staphylococcus aureus ATCC55804 is 1 × 10 ¹⁰individual antibacterial/mL, the concentration of 5 type staphylococcus aureus ATCC49521 capsular polysaccharides, 8 type staphylococcus aureus ATCC49525 capsular polysaccharides and 336 type staphylococcus aureus ATCC55804 capsular polysaccharides is 40 μ g/ml).

Each vaccine is placed in to 2-8 ℃ to be saved backup.

Three, mouse experiment

6-8 week Balb/C mice, is divided into 5 groups, and 10 every group, grouping immunity following (subcutaneous injection):

First group: test the 1st day and the 28th day each immune control vaccine first of experiment 0.25 milliliter of each immunity;

Second group: test the 1st day and the 28th day each immune control vaccine second of experiment 0.25 milliliter of each immunity;

The 3rd group: test the 1st day and the 28th day each immune control vaccine the third of experiment 0.25 milliliter of each immunity;

The 4th group: test the 1st day and the 28th day each immune control vaccine fourth of experiment 0.25 milliliter of each immunity;

The 5th group (negative control): test the 1st day and the 28th day each immune PBS buffer of experiment 0.25 milliliter of each immunity.

Test tail venous blood sampling separation of serum the 42nd day.The IgG antibody titer that detects serum by ELISA (with PBST buffer gradient dilution, obtains serum dilution; Respectively using P3 for bacterial strain SACP5-9, P3 for bacterial strain SACP8-6 and P3 for bacterial strain SACP336-3 as coating antigen, coated concentration is 1 × 10 ⁹individual bacterium/ml; Adopt rabbit anti-mouse igg ELIAS secondary antibody; Detect the light absorption value of 450nm; 2.1 times of the negative contrast of light absorption value are judged as the positive above).The tiring of serum that adopts above first group of laboratory animal of three kinds of coating antigens to obtain is 1:6400, the tiring of serum that adopts above second group of laboratory animal of three kinds of coating antigens to obtain is 1:12800, adopt above tiring of the 3rd group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:3200, adopt above tiring of the 4th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:6400.

Four, milch cow experiment

2-5 year be about to the to enter lactation period healthy cow of (being antenatal 25d, conceived 8 months 20d), is divided into five groups, and 100 every group, grouping immunity is as follows:

First group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune control vaccine first of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

Second group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune control vaccine second of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 3rd group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune control vaccine the third of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 4th group: test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune control vaccine fourth of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity;

The 5th group (negative control): test the 1st day, experiment the 29th day and the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) each immune PBS buffer of experiment, 5 milliliters (cervical region bilateral subcutaneous injection) of each immunity.

The tiring of serum that adopts above first group of laboratory animal of three kinds of coating antigens to obtain is 1:12800, the tiring of serum that adopts above second group of laboratory animal of three kinds of coating antigens to obtain is 1:25600, adopt above tiring of the 3rd group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:6400, adopt above tiring of the 4th group of serum that laboratory animal obtains of three kinds of coating antigens to be 1:12800.

The tiring of milk surum that adopts above first group of laboratory animal of three kinds of coating antigens to obtain is 1:3200, the tiring of milk surum that adopts above second group of laboratory animal of three kinds of coating antigens to obtain is 1:6400, adopt above tiring of the 3rd group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:1600, adopt above tiring of the 4th group of milk surum that laboratory animal obtains of three kinds of coating antigens to be 1:3200.

Test the 71st day, get after blood, carry out counteracting toxic substances (using newborn pin that 600CFU5 type staphylococcus aureus ATCC49521,600CFU8 type staphylococcus aureus ATCC49525 and 600CFU336 type staphylococcus aureus ATCC55804 are injected in breast) with 5 type staphylococcus aureus ATCC49521,8 type staphylococcus aureus ATCC49525 and 336 type staphylococcus aureus ATCC55804, test the 72nd day-experiment the 85th day is the result judge phase.Result evaluation method is with the step 2 of embodiment 4.

The protective rate of first group of laboratory animal is that the protective rate of 82%, the second group of laboratory animal is that the protective rate that protective rate that the protective rate of 83%, the three group of laboratory animal is 72%, the four group of laboratory animal is 74%, the five group of laboratory animal is 0%.

Five, long-acting immune protective effect

It (is antenatal 25d that 2-5 year is about to enter lactation period, conceived 8 months 20d) healthy cow, be divided into 5 groups, every group 100, grouping immunity is as follows: test the 1st day, experiment the 29th day, test the 57th day (being antenatal 25 days, 3 days puerperal, 31 days puerperal) and test the 368th day each immune vaccine, all same step 3 of immune packet mode, single immunization dosage and immunization ways.Test the 700th day-experiment and passed judgment on for 1 phase for result on the 714th day, test the 1070th day-test and passed judgment on for 2 phases for result on the 1083rd day.Result evaluation method is with the step 2 of embodiment 4.

Result passed judgment on for 1 phase: the protective rate of first group of laboratory animal is that the protective rate of 81%, the second group of laboratory animal is that the protective rate of 82%, the three group of laboratory animal is that the protective rate that the protective rate of 71%, the four group of laboratory animal is 72%, the five group of laboratory animal is 36%.

Result passed judgment on for 2 phases: the protective rate of first group of laboratory animal is that the protective rate of 80%, the second group of laboratory animal is that the protective rate of 81%, the three group of laboratory animal is that the protective rate of 70%, the four group of laboratory animal is that the protective rate of 71%, the five group of laboratory animal is 32%.

Claims

1. bacterial strain SACP5-9, bacterial strain SACP8-6 and bacterial strain SACP336-3 are in the application of preparing in mammitis of cow vaccine; Described bacterial strain SACP5-9 is staphylococcus aureus (Staphylococcus aureus) SACP5-9CGMCCNo.8529; Described bacterial strain SACP8-6 is staphylococcus aureus (Staphylococcus aureus) SACP8-6CGMCC No.8530; Described bacterial strain SACP336-3 is staphylococcus aureus (Staphylococcus aureus) SACP336-3CGMCC No.8531.

2. application as claimed in claim 1, is characterized in that: described mammitis of cow is the mammitis of cow that staphylococcus aureus causes.

3. a mammitis of cow vaccine, its active component is the bacterial strain after deactivation; Bacterial strain after described deactivation is bacterial strain SACP8-6 after bacterial strain SACP5-9, the deactivation after deactivation and the bacterial strain SACP336-3 after deactivation; Described bacterial strain SACP5-9 is staphylococcus aureus (Staphylococcus aureus) SACP5-9CGMCC No.8529; Described bacterial strain SACP8-6 is staphylococcus aureus (Staphylococcus aureus) SACP8-6CGMCC No.8530; Described bacterial strain SACP336-3 is staphylococcus aureus (Staphylococcus aureus) SACP336-3CGMCC No.8531.

4. vaccine as claimed in claim 3, is characterized in that: in described vaccine, the concentration of the bacterial strain SACP8-6 after bacterial strain SACP5-9, described deactivation after described deactivation and the bacterial strain SACP336-3 after described deactivation is 1 × 10 ¹⁰individual antibacterial/mL.

5. a mammitis of cow vaccine, its active component is bacterial strain and the capsular polysaccharide after deactivation; Bacterial strain after described deactivation is bacterial strain SACP8-6 after bacterial strain SACP5-9, the deactivation after deactivation and the bacterial strain SACP336-3 after deactivation; Described capsular polysaccharide is the capsular polysaccharide of the capsular polysaccharide of described bacterial strain SACP5-9, described bacterial strain SACP8-6 and the capsular polysaccharide of described bacterial strain SACP336-3; Described bacterial strain SACP5-9 is staphylococcus aureus (Staphylococcus aureus) SACP5-9CGMCC No.8529; Described bacterial strain SACP8-6 is staphylococcus aureus (Staphylococcus aureus) SACP8-6CGMCC No.8530; Described bacterial strain SACP336-3 is staphylococcus aureus (Staphylococcus aureus) SACP336-3CGMCC No.8531.

6. vaccine as claimed in claim 5, is characterized in that: in described vaccine, the concentration of the bacterial strain SACP8-6 after bacterial strain SACP5-9, described deactivation after described deactivation and the bacterial strain SACP336-3 after described deactivation is 1 × 10 ¹⁰individual antibacterial/mL, the concentration of the capsular polysaccharide of the capsular polysaccharide of described bacterial strain SACP5-9, the capsular polysaccharide of described bacterial strain SACP8-6 and described bacterial strain SACP336-3 is 40 μ g/ml.

7. as the vaccine as described in arbitrary in claim 3 to 6, it is characterized in that: described vaccine also comprises adjuvant.

8. vaccine as claimed in claim 7, is characterized in that: described adjuvant is SP01 adjuvant, aluminium adjuvant or white flower oil adjuvant.

9. as the vaccine as described in arbitrary in claim 3 to 8, it is characterized in that: described mammitis of cow is the mammitis of cow that staphylococcus aureus causes.

10. bacterial strain SACP5-9 and/or bacterial strain SACP8-6 and/or bacterial strain SACP336-3; Described bacterial strain SACP5-9 is staphylococcus aureus (Staphylococcus aureus) SACP5-9CGMCC No.8529; Described bacterial strain SACP8-6 is staphylococcus aureus (Staphylococcus aureus) SACP8-6CGMCC No.8530; Described bacterial strain SACP336-3 is staphylococcus aureus (Staphylococcus aureus) SACP336-3CGMCC No.8531.