CN107050451A - A kind of preparation method and application of Mycoplasma bovis inactivated vaccine - Google Patents
A kind of preparation method and application of Mycoplasma bovis inactivated vaccine Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of Mycoplasma bovis inactivated vaccine.Formalin-inactivated condition of the present invention from the preparation of Mycoplasma bovis inactivated vaccine, Mycoplasma bovis inactivated vaccine immunizing dose, the immunization wayses of Mycoplasma bovis inactivated vaccine, adjuvant that Mycoplasma bovis inactivated vaccine is used and probed into clinical practice, optimal Mycoplasma bovis formalin-inactivated condition is moused out, the immunizing dose of Mycoplasma bovis inactivated vaccine, the immunization wayses and immunologic adjuvant of Mycoplasma bovis inactivated vaccine, then animal body experiment is clinically carried out, as a result show that prevention of the Mycoplasma bovis inactivated vaccine of the present invention to ox M. bovis disease in clinical practice has good effect, it can be used as a kind of clinical expansion vaccine, the basis of science has been established for the prevention of M. bovis disease.
Description
Technical field
The present invention relates to a kind of preparation method of Mycoplasma bovis inactivated vaccine.
Background technology
Mycoplasma bovis (Mycoplasma bovis) is to cause the multisystems such as bovine respiratory inflammation, mastitis and arthritis scorching
One of important pathogenic pathogen of disease.Hale in 1961 etc. the U.S. is isolated from the milk for suffering from mastitis ox first should
Cause of disease；Hereafter it is separated to Mycoplasma bovis in the area such as Ireland, South America and Europe.2008, Hubei Province of China broke out
" infectiousness Mycoplasma bovis pneumonia " epidemic situation by principal character of necrotizing pneumonia, later in succession in Hubei, Guizhou, Ningxia, interior
The area such as Mongolia, Guangxi, Chongqing is introduced and the disease is found in calf, and the incidence of disease is 50% ~ 100%, and case fatality rate is up to 10% ~ 50%, gives
China's cattle-raising causes huge economic loss.The immunologic mechanism of Mycoplasma bovis is extremely complex and special, not yet complete so far
It is clear.Research shows that Mycoplasma bovis can produce two kinds of phenomenons with host immune cell interaction：Immune-enhancing effect and immunosupress.
Because the DNA high-frequencies of M. bovis genes are recombinated, surface lipoprotein has the specificity of height, and this just makes Mycoplasma bovis phenotype
It is changeable, also make it that the domestic and international flow of research about vaccine is slow, only the mycoplasma bovis vaccine of commercialization is delivered city by the U.S.
, only unique Respiratory Syncytial Virus(RSV), the type of parainfluenza 2 and 3 types, special mycoplasma and Mycoplasma bovis tetrad inactivated vaccine at present
For Mycoplasma bovis infection, of short duration protective effect can be provided and side effect is big.Someone is with Formalin inactivation ox branch in recent years
The full cell inactivation seedling of Mycoplasma bovis is have developed after substance, but it does not have effect to the mammitis triggered by Mycoplasma bovis.According to
The mycoplasma bovis vaccine protectiveness experiment of 1981 and 1987 finds that the vaccine existed at that time has to the mycoplasmal pneumonia of ox
There is certain effect, but the vaccine having can aggravate the infection of Mycoplasma bovis on the contrary.Barite in 1987 etc. is with hamster to Mycoplasma bovis
Vaccine and Formalin inactivation seedling detect, two kinds of vaccines can prevent Mycoplasma bovis pneumonia, but both reciprocations
It can not distinguish.Though there are two kinds of commercialized vaccines in the U.S., someone is tested the protectiveness of both vaccines within 2011, is passed through
27d calf immunity tests show that one kind can significantly improve antibody level in two kinds of vaccines, but to the protective rate about 44% of ox;
The immune effect of another vaccine is not notable.
The content of the invention
The invention discloses a kind of preparation method of Mycoplasma bovis inactivated vaccine.The present invention is from Mycoplasma bovis inactivated vaccine
The formalin-inactivated condition of preparation, Mycoplasma bovis inactivated vaccine immunizing dose, the immunization wayses of Mycoplasma bovis inactivated vaccine, Niu Zhiyuan
Adjuvant that body inactivated vaccine is used and probed into clinical practice, moused out optimal Mycoplasma bovis formalin-inactivated condition,
The immunizing dose of Mycoplasma bovis inactivated vaccine, the immunization wayses and immunologic adjuvant of Mycoplasma bovis inactivated vaccine.
The present invention mainly prepares a kind of mycoplasma bovis vaccine.It mainly includes the first of the preparation of Mycoplasma bovis inactivated vaccine
Aldehyde inactivates the screening of condition, and Mycoplasma bovis inactivated vaccine immunizing dose screening, the immunization wayses of Mycoplasma bovis inactivated vaccine are screened,
The adjuvant screening that Mycoplasma bovis inactivated vaccine is used.
The formalin-inactivated condition research of the preparation of Mycoplasma bovis inactivated vaccine
After the M.bovis bacterial strains frozen are recovered, M.bovis fluid nutrient mediums are inoculated into, are cultivated under certain condition, 3d
After pass on, measure CCU contents in forth generation M.bovis cultures, by a certain percentage concentrate after obtain 1.0 × 1010Ccu/ml's
M.bovis cultures are standby, and formaldehyde is added in culture（Analysis is pure）It is respectively 0.3%, 0.4%, 0.5% to make its final concentration, is filled
Cultivated under identical condition after point mixing, determine M.bovisCCU contents every 12h, each concentration mensuration 3 times is averaged
Value.Culture is centrifuged into 30min with 16000r/min when CCU contents are 0, obtained precipitation is washed with 0.1mol/L PBS
Wash 3 times, then with 1:In the generation of blind passage 3, is inoculated with solid medium observation inactivating efficacy simultaneously in 10 liquid medium withins.
Mycoplasma bovis inactivated vaccine immunizing dose research
12 experimental animals are divided into 4 groups of A, B, C, D, every group 3（Male and female are arranged in pairs or groups, body weight collocation）, remembered with immune the previous day
For 0d, do not add adjuvant 1.0 × 10 with what is preserved in experiment three in experiment 1d10CCU/ml vaccine by way of intramuscular injection to A,
B, C group experimental animal are immunized, and only, only, C groups 3ml/ is only by B groups 2ml/ by A groups 1ml/；During 15d in the same way and dosage
Booster immunization is carried out, D groups without any processing are negative control.Every 10d jugular vein blood collections from 0d, continuous 7 times, adopt every time
Blood 1ml is with 3000r/min rotating speed centrifugation 20min separation serum, and the refrigerator that serum is placed in into -80 DEG C is saved backup.
The immunization wayses research of Mycoplasma bovis inactivated vaccine
Experimental animal isolated rearing is adapted into experimental situation in one week, 9 experimental animals are divided into 3 groups of A, B, C, every group 3（It is female
Hero collocation, body weight collocation）, using immune the previous day as 0d, experiment 1d with preserved in experiment three do not add adjuvant 1.0 ×
1010Experimental animal is immunized CCU/ml vaccine, and A groups with the method injection 1ml/ of collunarium only, in the method for intramuscular injection note by B groups
Penetrate 1ml/ only, C groups carry out 1ml/ only in hypodermic method；During 15d in the same way with dosage booster immunization, from 0d
Rise every 10d jugular vein blood collections, continuous 7 times, the 1-2ml that takes a blood sample every time separates serum with 3000r/min rotating speed centrifugation 20min,
Refrigerator by serum in -80 DEG C is saved backup.
The adjuvant research that Mycoplasma bovis inactivated vaccine is used
By one week observation clinical symptoms of experimental animal isolated rearing, animal to be tested starts experiment after shaking down, by 12
Experimental animal is divided into 4 groups of A, B, C, D, every group 3（Male and female are arranged in pairs or groups, body weight collocation）, using immune the previous day as 0d, in experiment the
Experimental animal is immunized with the vaccine preserved in experiment three by 1d, A groups intramuscular injection 5 × 109CCU/ml aluminum hydroxide adjuvant goes out
Live seedling 1ml/, B groups intramuscular injection 5 × 109CCU/ml not formula Freund's complete adjuvant inactivated vaccine 1ml/, C groups intramuscular injection 5 × 109CCU/ml's
White-oil adjuvant inactivated vaccine 1ml/, D groups intramuscular injection × 109CCU/ml's does not add adjuvant inactivated vaccine 1ml/ only, with same during 15d
Mode and dosage injection same vaccine carry out booster immunization, continuous to take a blood sample 7 times from 0d every 10d jugular vein blood collections once, every time
1ml take a blood sample with 3000r/min rotating speed centrifugation 20min separation serum, the refrigerator by serum in -80 DEG C is saved backup.
The preparation and clinical practice of mycoplasma bovis vaccine
By Mycoplasma bovis with 1:100 ratio is inoculated into Mycoplasma bovis Thiaucourt ' s fluid nutrient mediums, 5% C02, 37
3d is cultivated under the conditions of DEG C and obtains F1 generation bacterium solution, then by F1 bacterium solutions with 1:100 are inoculated into fluid nutrient medium, 5% C02, 37 DEG C of bars
3d is cultivated under part and obtains F2, by F2 cultures with 1:10 are inoculated into fluid nutrient medium, 5% C02, 3d is cultivated under the conditions of 37 DEG C
F3 is obtained for bacterium solution；Then F3 is determined for bacterial concentration（CCU/ml）And formalin is added in F3 is for bacterium solution, make in bacterium solution
Formaldehyde final concentration of 0.4%, is inactivated under the conditions of 37 DEG C, and inactivated bacterial liquid is extracted after 2d and is inoculated in Thiaucourt ' s solid mediums
Carry out inspection work.After inactivation and the F3 of viable bacteria will be not detected by for inactivated bacterial liquid 16000r/min and centrifuge 30min, by obtained precipitation
Then sediment is diluted to original concentration by thing through being washed 3 times with reason brine buffer solution with physiological saline.Inactivated bacteria after washing
Liquid is mixed in equal volume with white-oil adjuvant, 100W, is spaced 2s, 10min ultrasonic emulsifications.Then it is inoculated in ox and only carries out clinical sight
Examine, pathologic examination and antibody level are detected.
Brief description of the drawings
Fig. 1 is change curve of the mycoplasma content with different inactivation times.
Fig. 2 is Temperature changing trend.
Fig. 3 is PC group pathology cut open inspection pathological tissues.
Fig. 4 is PC group lungs：Bronchiole reduces, and pleat increases, have in official jargon the epithelial cell 100 that comes off on a small quantity ×.
Fig. 5 is PC group lungs：Bronchiole reduces, and pleat increases 400 ×.
Fig. 6 is PC group lungs：Alveolar collapse, alveolar septa are thickened, and hyperblastosis 100 ×.
Fig. 7 is PC group lungs：Alveolar collapse, alveolar septa is thickened, have in alveolar space a small amount of cell detachment 400 ×.
Embodiment 1：
1st, the formalin-inactivated condition research of the preparation of Mycoplasma bovis inactivated vaccine
After the M.bovis bacterial strains frozen are recovered, M.bovis fluid nutrient mediums are inoculated into, are cultivated under certain condition, 3d
After pass on, measure CCU contents in forth generation M.bovis cultures, by a certain percentage concentrate after obtain 1.0 × 1010Ccu/ml's
M.bovis cultures are standby, and formaldehyde is added in culture（Analysis is pure）It is respectively 0.3%, 0.4%, 0.5% to make its final concentration, is filled
Cultivated under identical condition after point mixing, determine M.bovisCCU contents every 12h, each concentration mensuration 3 times is averaged
Value.Culture is centrifuged into 30min with 16000r/min when CCU contents are 0, obtained precipitation is washed with 0.1mol/L PBS
Wash 3 times, then with 1:In the generation of blind passage 3, is inoculated with solid medium observation inactivating efficacy simultaneously in 10 liquid medium withins.
As shown in Figure 1：The formaldehyde of various concentrations can thoroughly inactivate M.bovis bacterial strains, and the bigger inactivation time of concentration is more
It is short.
As shown in Table 1：After the formaldehyde treated of three kinds of concentration, content of formaldehyde is minimum after 0.4% formaldehyde treated.
In summary, 0.4% formaldehyde carries out 48h inactivations, and effect is best.
2nd, Mycoplasma bovis inactivated vaccine immunizing dose research
12 new zealand white rabbits are divided into 4 groups of A, B, C, D, every group 3（Male and female are arranged in pairs or groups, body weight collocation）, with immune previous
It is designated as 0d, does not add adjuvant 1.0 × 10 with what is preserved in experiment three in experiment 1d10CCU/ml vaccine is by way of intramuscular injection
A, B, C group experimental animal are immunized, only, only, C groups 3ml/ is only by B groups 2ml/ by A groups 1ml/；During 15d in the same way and
Dosage carries out booster immunization, and D groups without any processing are negative control.Every 10d jugular vein blood collections from 0d, continuous 7 times, often
Secondary blood sampling 1ml is with 3000r/min rotating speed centrifugation 20min separation serum, and the refrigerator that serum is placed in into -80 DEG C is saved backup.
As shown in Table 2, the test group of 3ml/ dosage 10d Mycoplasma bovis antibody level highests, immune effect after immune
It is best.
3rd, the immunization wayses research of Mycoplasma bovis inactivated vaccine
New zealand white rabbit isolated rearing is adapted into experimental situation in one week, 9 experimental animals are divided into 3 groups of A, B, C, every group 3
（Male and female are arranged in pairs or groups, body weight collocation）, using immune the previous day as 0d, experiment 1d with preserved in experiment three do not add adjuvant 1.0 ×
1010Experimental animal is immunized CCU/ml vaccine, and A groups with the method injection 1ml/ of collunarium only, in the method for intramuscular injection note by B groups
Penetrate 1ml/ only, C groups carry out 1ml/ only in hypodermic method；During 15d in the same way with dosage booster immunization, from 0d
Rise every 10d jugular vein blood collections, continuous 7 times, the 1-2ml that takes a blood sample every time separates serum with 3000r/min rotating speed centrifugation 20min,
Refrigerator by serum in -80 DEG C is saved backup.
As shown in Table 3：Three kinds of immunization wayses are attained by certain immune effect, wherein it is worst by effect after Nasal immunization,
When higher antibody level can be reached in ten days after subcutaneous and intramuscular injection but being maintained by being subcutaneously injected compared with intramuscular injection
Between it is shorter, therefore intramuscular injection be optimal immunization wayses.
4th, the adjuvant research that Mycoplasma bovis inactivated vaccine is used
By one week observation clinical symptoms of new zealand white rabbit isolated rearing, animal to be tested starts experiment after shaking down, will
12 experimental animals are divided into 4 groups of A, B, C, D, every group 3（Male and female are arranged in pairs or groups, body weight collocation）, using immune the previous day as 0d, in reality
Test 1d experimental animal is immunized with the vaccine preserved in experiment three, A groups intramuscular injection 5 × 109CCU/ml aluminium hydroxide assistant
Agent inactivated vaccine 1ml/, B groups intramuscular injection 5 × 109CCU/ml not formula Freund's complete adjuvant inactivated vaccine 1ml/, C groups intramuscular injection 5 × 109CCU/
Ml white-oil adjuvant inactivated vaccine 1ml/, D groups intramuscular injection 5 × 109CCU/ml not plus adjuvant inactivated vaccine 1ml/ only, during 15d with
Same mode and dosage injection same vaccine carry out booster immunization, continuously take a blood sample 7 from 0d every 10d jugular vein blood collections once
Secondary, blood sampling 1ml is with 3000r/min rotating speed centrifugation 20min separation serum every time, and the refrigerator by serum in -80 DEG C is saved backup.
From table 4 and table 5：All there is positive, B, C group antibody water after 10d is just exempted from after 10d is just exempted from all test groups
It is flat to reach highest, higher antibody level is always maintained in whole experiment process, A, D group result are similar, although antibody level
Highest is attained by, but positive rotary speed is slow compared with B, C group, and antibody level is weaker, and High antibody level is held time relatively
It is short, it is seen that the immune effect of B, C group is strong compared with A, D group.B, C group rabbit two is taken to determine antibody after exempting from the serum doubling dilution after 15d
Titre, as a result shows B group rabbits serum-dilution 102Times when antibody level start straight line decline, be diluted to 106When show as the moon
Property, i.e., antibody titer is 105, C group rabbits antibody titer is 106, serum-dilution to 107When show as feminine gender, it is clear that C group antibody
Level is higher compared with B groups, and the immunity of C groups is strong compared with B groups.Therefore white-oil adjuvant immune effect is optimal.
5th, the preparation and clinical practice of mycoplasma bovis vaccine
By Mycoplasma bovis with 1:100 ratio is inoculated into Mycoplasma bovis Thiaucourt ' s fluid nutrient mediums, 5% C02, 37
3d is cultivated under the conditions of DEG C and obtains F1 generation bacterium solution, then by F1 bacterium solutions with 1:100 are inoculated into fluid nutrient medium, 5% C02, 37 DEG C of bars
3d is cultivated under part and obtains F2, by F2 cultures with 1:10 are inoculated into fluid nutrient medium, 5% C02, 3d is cultivated under the conditions of 37 DEG C
F3 is obtained for bacterium solution；Then F3 is determined for bacterial concentration（CCU/ml）And formalin is added in F3 is for bacterium solution, make in bacterium solution
Formaldehyde final concentration of 0.4%, is inactivated under the conditions of 37 DEG C, and inactivated bacterial liquid is extracted after 2d and is inoculated in Thiaucourt ' s solid mediums
Carry out inspection work.After inactivation and the F3 of viable bacteria will be not detected by for inactivated bacterial liquid 16000r/min and centrifuge 30min, by obtained precipitation
Then sediment is diluted to original concentration by thing through being washed 3 times with reason brine buffer solution with physiological saline.Inactivated bacteria after washing
Liquid is mixed in equal volume with white-oil adjuvant, 100W, is spaced 2s, 10min ultrasonic emulsifications.
Collection bull nose swab carries out the separation identification of mycoplasma simultaneously before 15 first 2 monthly age He Sitan calves of purchase, purchase
Blood sample separation serum is gathered, Mycoplasma bovis antibody test is carried out using Mycoplasma bovis antibody assay kit.Choose detection
To be negative and be not detected by the former calf of mycoplasmosis as subjects.15 qualified bulls are divided into 3 groups every group 5.
Respectively negative control group（NC）, positive controls（PC）, vaccine group.Every ox of vaccine group is spaced intramuscular injection in two weeks
5ml 1ˣ1010CCU/ml Mycoplasma bovis inactivated vaccine 1 time, totally 2 times；PC group intramuscular injection 5ml 1 1010CCU/ml dosage
Mycoplasma bovis strain is infected.The 15d after the 2nd vaccine immunity, vaccine group intramuscular injection 5ml 1 1010CCU/ml dosage
Mycoplasma bovis strain carry out infection experiment, Thiaucourt ' the s liquid of consecutive infection 3d, NC group intramuscular injection Isodose
Culture medium, continuous observation 28d.
In 0-15d, daily 7:00-8:00 measurement body temperature once, after 15d every other day surveys a body temperature, until infection
Off-test；0d（It is immune for the first time）After 0d, blood was gathered every 1 week by the last week collection serum 1 time respectively for 3 groups of test ox
And separate serum 1 time, until infection terminates for 3 weeks after terminating.28d after infection, only carries out pathology by PC, NC and test group ox and cuts open
Examine, and gather ox lungs, tracheae, larynx, heart, renal tissue and be fixed on 10% formalin solution, fix after 7 d, paraffin
Embedding, HE dyeing, optical microphotograph Microscopic observation.
Start after zoogenetic infection, nose swab is gathered 1 time to 3 groups of test ox weekly, and be diluted with PBS.By nose swab
It is seeded in Thiaucourt ' s fluid nutrient mediums and 5 % CO2 of solid medium CO2 incubators, 37 DEG C of cultures, after culture 2d
Colonial morphology on microscopy solid medium；, filter bacterium solution using 0.22 μm of biofilter, and be inoculated in fluid nutrient medium
Culture DNA is extracted after row culture, culture 2d and ox is carried out using the progress mycoplasma identification of 16S rRNA primers and uvrC primers
Mycoplasma and mycoplasma agalactiae identification.Ox pathology cut open inspection, collection ox joint fluid, thoracic cavity product are carried out after zoogenetic infection examination 28d
Liquid, pericardial fluid and lung tissue.By the lung tissue surface flame sterilization of collection, the fritter of clip lungs deep tissue one, so
It is inoculated in afterwards under aseptic technique in Mycoplasma bovis Thiaucourt ' s liquid screening mediums and Mycoplasma bovis
Thiaucourt ' s solid screening and culturing mediums, every part of samples are inoculated with 3 fluid nutrient mediums and 3 solid mediums.Gu
Body culture medium is placed in after 5 % CO2 of CO2 incubators, 37 DEG C of 2 d of culture, micro- Microscopic observation colonial morphology；Liquid Culture is based on
Culture medium color change situation is observed after 37 DEG C of d of constant-temperature table culture 2, by the nutrient solution of the flavescence degerming filter membranes of 0.22 um
Filtering, repeats to extract DNA progress Mycoplasma bovis molecular biology identifications after culture filtered fluid.
During testing, NC groups body temperature is maintained between 39.10 DEG C~39.50 DEG C always, within ox normal body temperature；PC groups
39.50 DEG C are started above from 15d, and in elevated trend, 40.40 DEG C of highest body temperature, the largest body temperature difference with NC groups is
1.15 DEG C, significant difference（P<0.05）；The 1st vaccine inoculation Temperature changing of inactivated vaccine group be not obvious, after the 2nd vaccine inoculation
Body temperature slightly has rise, and 39.60 DEG C of highest body temperature is not notable with NC groups difference（P>0.05）, highest body temperature 39.6 after infection experiment
DEG C, difference is not notable compared with NC groups（P>0.05）（Fig. 2）.
Inactivated vaccine group cut open inspection is found：Lungs, larynx, heart, liver, tracheae, kidney and joint tissue can without naked eyes
See lesion.PC groups cut open inspection is found：Lung tissue is adhered, substantive meat change and size occur for Pulmonary hemorrhage extravasated blood, lung tissue not
Deng canescence is cheesy or suppurative necrosis region, lung tissue inside bleeding and have suppurative and caseous necrosis stove, other
Tissue is visible by naked eyes pathological change（Fig. 3）.
Inactivated vaccine group lungs and tracheal tissue are without obvious Histopathologic changes（Fig. 4-Fig. 7）, the visible ramuscule gas of PC groups
Pipe reduces, and pleat increases, and has the epithelial cell come off on a small quantity in official jargon, alveolar collapse, alveolar septa are thickened, hyperblastosis, alveolar
Intracavitary has a small amount of cell detachment.
Inactivated vaccine group 1 week antibody level after inoculation Mycoplasma bovis inactivated vaccine starts rise, not notable with 0 week difference
（P>0.05）, the 2nd week Mycoplasma bovis antibodies positive simultaneously continues rise, extremely notable with 0 week difference（P<0.01）, reach within the 3rd week
Maximum, Mycoplasma bovis antibody level is in relatively stable state within-the 6 week the 4th week；After 6th week Mycoplasma bovis pathogen infection,
Mycoplasma bovis antibody level reached maximum at the 7th week, and in stable state.NC group ox Mycoplasma bovis antibody level is more
Stable to be negative, difference is not notable（P>0.05）.NC groups are negative and anti-in whole experimental stage Mycoplasma bovis antibody test
Body level is relatively stablized, and difference is not notable（P>0.05）.PC groups ox Mycoplasma bovis antibody is constantly in higher after infecting one week
Level, be positive in whole experimental stage.
In summary：Mycoplasma bovis inactivated vaccine is in clinical practice compared with negative and positive control, and antibody can be maintained
In higher level；After Animal infection experiments, inactivated vaccine group calf is not detected by Mycoplasma bovis without Histopathologic changes
The presence of cause of disease.Illustrate, Mycoplasma bovis inactivated vaccine of the invention has good effect for the prevention of Mycoplasma bovis cause of disease.
Claims (6)
1. a kind of Mycoplasma bovis inactivated vaccine, it is characterised in that the preparation method of Mycoplasma bovis formalin-killed vaccine.
2. the preparation method of Mycoplasma bovis formalin-killed vaccine as claimed in claim 1, it is characterised in that Mycoplasma bovis is inactivated
The formalin-inactivated condition of the preparation of vaccine, Mycoplasma bovis inactivated vaccine immunizing dose, the immunization wayses of Mycoplasma bovis inactivated vaccine,
The adjuvant that Mycoplasma bovis inactivated vaccine is used.
3. the formalin-inactivated condition of the preparation of Mycoplasma bovis inactivated vaccine as claimed in claim 2, it is characterised in that Niu Zhiyuan
The incubation time of body bacterial strain, the dosage of formaldehyde, formalin-inactivated time：After the M.bovis bacterial strains frozen are recovered, it is inoculated into
M.bovis fluid nutrient mediums, are cultivated under certain condition, are passed on, are measured in forth generation M.bovis cultures after 3~4d
CCU contents, 1.0 × 10 are obtained after concentrating by a certain percentage10Ccu/ml M.bovis cultures are standby, added in culture
Formaldehyde（Analysis is pure）It is respectively 0.3%, 0.4%, 0.5% to make its final concentration, is cultivated under identical condition after being sufficiently mixed, every
12h determines M.bovisCCU contents, and each concentration mensuration 3 times is averaged；When CCU contents be 0 when by culture with
16000r/min centrifuges 30min, obtained precipitation is washed 3 times with 0.1mol/L PBS, then with 1:10 liquid medium withins
In the middle generation of blind passage 3, is inoculated with solid medium observation inactivating efficacy simultaneously.
4. Mycoplasma bovis inactivated vaccine immunizing dose as claimed in claim 2, it is characterised in that selection Mycoplasma bovis immunizing agent
Amount selection：12~16 experimental animals are divided into 4 groups of A, B, C, D, every group 3~4（Male and female are arranged in pairs or groups, body weight collocation）, in order to avoid
Epidemic disease the previous day is designated as 0d, does not add adjuvant 1.0 × 10 with what is preserved in experiment three in experiment 1d10CCU/ml vaccine passes through intramuscular injection
Mode A, B, C group experimental animal are immunized, A groups 1ml/ only, B groups 2ml/ only, C groups 3ml/；With same during 15d
Mode and dosage carry out booster immunization, and D groups without any processing are negative control；Every 10d jugular vein blood collections from 0d, continuously
7 times, blood sampling 1ml is with 3000r/min rotating speed centrifugation 20min separation serum every time, and the refrigerator that serum is placed in into -80 DEG C preserves standby
With.
5. the immunization wayses of the Mycoplasma bovis inactivated vaccine as described in right 2, it is characterised in that intramuscular inoculation, subcutaneous vaccination and drop
Rhinovaccination：Experimental animal isolated rearing is adapted into experimental situation in one week, 9~12 experimental animals are divided into 3 groups, every group of A, B, C
3~4（Male and female are arranged in pairs or groups, body weight collocation）, using immune the previous day as 0d, do not add adjuvant with what is preserved in experiment three in experiment 1d
1.0×1010Experimental animal is immunized CCU/ml vaccine, and with the method injection 1ml/ of collunarium only, B groups are with intramuscular injection for A groups
Only, C groups carry out 1ml/ only to method injection 1ml/ in hypodermic method；Exempt from the same way with dosage reinforcement during 15d
Epidemic disease, every 10d jugular vein blood collections from 0d, continuous 7 times, blood sampling 1-2ml is with 3000r/min 20min points of rotating speed centrifugation every time
From serum, the refrigerator by serum in -80 DEG C is saved backup.
6. the adjuvant that Mycoplasma bovis inactivated vaccine as claimed in claim 2 is used, it is characterised in that complete Freund's adjuvant and not
Complete Freund's adjuvant, aluminum hydroxide adjuvant, white-oil adjuvant：Clinical symptoms are observed into experimental animal isolated rearing for one week, it is to be tested
Animal starts experiment after shaking down, 12~16 experimental animals are divided into 4 groups of A, B, C, D, every group 3~4（Male and female are taken
Match somebody with somebody, body weight collocation）, using immune the previous day as 0d, experimental animal is exempted from the vaccine preserved in experiment three in experiment 1d
Epidemic disease, A groups intramuscular injection 5 × 109CCU/ml aluminum hydroxide adjuvant inactivated vaccine 1ml/, B groups intramuscular injection 5 × 109CCU/ml not formula is complete
Adjuvant inactivated vaccine 1ml/, C groups intramuscular injection 5 × 109CCU/ml white-oil adjuvant inactivated vaccine 1ml/, D groups intramuscular injection × 109CCU/ml
Not plus adjuvant inactivated vaccine 1ml/ only, carry out booster immunization with dosage injection same vaccine in the same way during 15d, from 0d
Every 10d jugular vein blood collections once, continuous to take a blood sample 7 times, blood sampling 1ml is with 3000r/min rotating speed centrifugation 20min separation blood every time
Clearly, the refrigerator by serum in -80 DEG C is saved backup.
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| CN111856005A (en) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | Application of mycoplasma bovis secretory protein MbovP280 |
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| CN111856005B (en) * | 2020-06-22 | 2021-07-30 | 华中农业大学 | Application of mycoplasma bovis secretory protein MbovP280 |
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