CN106929452A - One plant of Mycoplasma bovis and its application - Google Patents

One plant of Mycoplasma bovis and its application Download PDF

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CN106929452A
CN106929452A CN201710231073.5A CN201710231073A CN106929452A CN 106929452 A CN106929452 A CN 106929452A CN 201710231073 A CN201710231073 A CN 201710231073A CN 106929452 A CN106929452 A CN 106929452A
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mycoplasma bovis
mycoplasma
hnmb1
culture
bovis
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CN106929452B (en
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徐引弟
李海利
张青娴
王治方
郎利敏
朱文豪
焦文强
王克领
宋振宇
索延乐
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Institute of Animal Husbandry and Veterinary Medicine of Henan Academy of Agricultural Sciences
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    • C12R2001/35—Mycoplasma
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants

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  • Animal Behavior & Ethology (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses one plant of Mycoplasma bovis and its application, described Mycoplasma bovis are Mycoplasma bovis (Mycoplasma bovis) HNMB1, deposit number：CGMCC NO：13295, preservation date：On November 28th, 2016, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Bacterial strain HNMB1 in the present invention is isolated from the dead dairy calf of typical respiratory symptom, without other bacterial growths when being separated on PPLO solid mediums, only mycoplasma growth, and viral titer is high in PPLO fluid nutrient mediums, up to 109More than CFU/mL.Bacterial strain HNMB1 in the present invention has stronger pathogenicity to calf, causes calf to fall ill dead, with good immunogenicity.

Description

One plant of Mycoplasma bovis and its application
Technical field
The present invention relates to field of molecular biotechnology, more particularly to one plant Mycoplasma bovis and its application.
Background technology
Mycoplasma bovis (Mycoplasma bovis) mainly cause the pneumonia of ox, arthritis, mammitis, keratoconjunctivitis, Genital inflammation and miscarriage and the disease such as infertile.1961, American Hale etc. was first from suffering from the milk of mammitis milk cow In isolate Mycoplasma bovis.Mycoplasma bovis in 1976 are described as causing the Etiological of breathing problem, afterwards, Mycoplasma bovis and Its relevant disease diffuses to rapidly the cows in world's every country and area, and the sound development to world's cattle-raising is caused greatly Threat and serious economic loss.Since 2008, many ground of China occur in succession and it is pandemic to generate heat, cough and It is cardinal symptom to have a running nose, and the bovine respiratory infectious disease with necrotizing pneumonia as major lesions is also confirmed as Mycoplasma bovis infection Caused Mycoplasma bovis pneumonia, due to the sick conventional medicine unsatisfactory curative effect, and is prevented without effective vaccine, therefore to China Cattle-raising causes serious financial consequences.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide one plant of Mycoplasma bovis and its application, the Strain Virulence is strong, The vaccine immunity of preparation is good.
To achieve these goals, the technical solution adopted in the present invention is：
One plant of Mycoplasma bovis, described Mycoplasma bovis are Mycoplasma bovis (Mycoplasma bovis) HNMB1, and preservation is compiled Number：CGMCC NO：13295, preservation date：On November 28th, 2016, depositary institution：Chinese microorganism strain preservation conservator Can common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
A kind of application of Mycoplasma bovis in terms of Mycoplasma bovis inactivated vaccine is prepared.
In described Mycoplasma bovis inactivated vaccine, Mycoplasma bovis bacterium amount is 1 × 109CFU/mL。
The preparation method of described Mycoplasma bovis inactivated vaccine is：Described Mycoplasma bovis are sequentially passed through into culture, is harvested After obtaining vaccinogen liquid with inactivation, adjuvant is added to obtain final product vaccine.
Described adjuvant is aluminum hydroxide adjuvant.
The preparation method of described Mycoplasma bovis inactivated vaccine is：Mycoplasma bovis are inoculated into PPLO fluid nutrient mediums, are put In 37 DEG C, 5%CO2Cultivated in incubator, collect culture after 3 days, determine concentration, the bacterium number in adjustment culture for 2 × 109CFU/mL, adds formaldehyde to final concentration of 0.2%, and 12h is inactivated in 37 DEG C；Culture after inactivation by volume 1:1 adds Aluminum hydroxide adjuvant, is mixed to prepare Mycoplasma bovis inactivated vaccine.
Beneficial effects of the present invention：
1st, the bacterial strain HNMB1 in the present invention is isolated from the dead dairy calf of typical respiratory symptom, in PPLO solids Without other bacterial growths when being separated on culture medium, only mycoplasma growth, viral titer is high in PPLO fluid nutrient mediums, reaches 109More than CFU/mL.
2nd, the bacterial strain HNMB1 in the present invention has stronger pathogenicity to calf, causes calf to fall ill dead, with good Immunogenicity.
3rd, vaccine prepared by the bacterial strain HNMB1 in the present invention has preferably protection effect to the Eaton agent pneumonia of ox Really.
Brief description of the drawings
Fig. 1 is form under bacterial strain HNMB1 4 × microscopes of bacterium colony.
Fig. 2 is form under bacterial strain HNMB1 thalline 100 × microscopes of Giemsa staining.
Fig. 3 is the PCR qualification results of bacterial strain HNMB1, and the Marker in figure is DL 2000bp, is followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；1 is the amplification knot of primer MB1, MB2 to Mycoplasma bovis type strain Really；2 is the amplification of primer MB1, MB2 to bacterial strain HNMB1；3 is the amplification of primer MB3, MB4 to bacterial strain HNMB1；From As can be seen that template PCR amplifications fragment is about 1390bp in figure, it was demonstrated that amplified fragments are purpose fragment, meet desired design big It is small.
Specific embodiment
Specific embodiment of the invention is described in further detail with reference to embodiments.
The separation and identification of embodiment 1, Mycoplasma bovis
1.1 pathological material of diseases are gathered
It is dead in the large-scale cattle farm generation severe pneumonia expiratory dyspnea in Henan Province Jiyuan City that pathological material of disease comes from November, 2016 The 2 monthly age Fresians died.
The preparation of 1.2 culture mediums
PPLO fluid nutrient mediums：PPLO gravy powder 10.5g, glucose 2.5g, dusty yeast 2.5g, are dissolved in 440mL ultra-pure waters In, 115 DEG C of sterilizing 15min, plus MEM culture mediums 5mL, horse serum 50mL, the unit of penicillin 80,000, aseptic 10% arginine 10mL 500 μ L phenol red with 1% (w/v).After culture is based on 115 DEG C of sterilizing 15min, saved backup in 4 DEG C.
PPLO solid mediums：The PPLO fluid nutrient mediums of 1.5% (w/v) agar powder.Culture is based on 115 DEG C of sterilizings After 15min, saved backup in 4 DEG C.
1.3 mycoplasmas are separately cultured
The sick lung tissue for gathering is cut into small pieces under aseptic condition.The tangent plane of fritter tissues is applied to PPLO solid mediums Surface, is placed in 37 DEG C, 5%CO2Cultivated in incubator.After 3~5d, without obvious other bacterial growths, minimum pin is visually seen Pointed bacterium colony, observes the form of bacterium colony on solid medium under low-powered microscope, form is that typical mycoplasma " decocts poached egg Sample " (see Fig. 1).There is the bacterium colony of " decocting poached egg sample " characteristic feature, purifying on picking PPLO solid mediums.Giemsa staining (see Fig. 2), oily Microscopic observation mycoplasma thalli morphology is polymorphic, in coccoid, thread, the helical form of bending, is named as bacterium Strain HNMB1.
The identification of 1.4 bacterial strains
1.4.1 primer is designed
A pair of universal primers of sequences Design according to Mycoplasma bovis 16S rRNA, for the amplification of Mycoplasma bovis, primer sequence Row are as follows：
MB1：5’-CCTTAGGCAGTTTCTTTATAA-3’(SEQ ID NO.1)
MB2：5’-CATGCATGTCGAGCGATGAT-3’(SEQ ID NO.2)
Amplified fragments size is 1390bp.
A pair of specific primers of contagious bovine pleuropneumonia mycoplasma are designed simultaneously, for contagious bovine pleuropneumonia branch The amplification of substance, primer sequence is as follows：
MB3：5’-ATATACTTCTGTTCTAGTAA TATG-3’(SEQ ID NO.3)
MB4：5’-CTGAT TATGATGACAGTGGTCA-3’(SEQ ID NO.4)
Amplified fragments size is 277bp.
1.4.2PCR identify
Bacterial strain HNMB1 is inoculated with PPLO fluid nutrient mediums, 37 DEG C of 5%CO are placed in2In incubator cultivate 3d after, culture medium by Red is changed into yellow, and collects thalline extracts the DNA of bacterial strain HNMB1, light splitting light according to the operating procedure of DNA extraction kit It is 100 μ g/mL that degree meter determines concentration, and respectively with MB1, MB2 and MB3, MB4 is expanded into performing PCR identification.Meanwhile, it is former that ox branch is set Body type strain is positive control.
Pcr amplification reaction system is 25 μ L：10 × buffer solution 2.5 μ L, 2.5mM (each) dNTPs Mix0.5 μ L, 10 μM/ L universal primers MB1, MB2 (or MB3, MB4) each the μ L of 1 μ L, 5U/ μ L rTaq 1, the μ L of 100 μ g/mL DNA profilings 1, add ddH2O To 25 μ L.
Reaction condition：95 DEG C of predegeneration 5min, into circulation：95 DEG C of 1min, 57 DEG C of 1min, 72 DEG C of 30s, totally 35 are followed Ring, last 72 DEG C of extensions 10min, 4 DEG C of preservations of PCR primer.
Amplified production carries out electrophoresis respectively, and 10 μ L are loaded per hole, and 1% agarose gel electrophoresis observes result under ultraviolet light (see Fig. 3).Fig. 3 finds out that MB1, MB2 is amplified and met expected size fragment, through sequencing and Mycoplasma bovis HB0801-P180 plants (Genbank No.CP007591) homology 100%, sequencing result is as follows.MB3, MB4 do not amplify fragment, it was demonstrated that separated Strain HNMB1 be Mycoplasma bovis (Mycoplasma bovis), be not contagious bovine pleuropneumonia mycoplasma.
CCTTAGGCAGTTTCTTTATAAACCGACTTCGGGCATTACCAGCTCCCATGGTTTGACGGGCGGTGTGTACAAGACCC GAGAACGTATTCACCGTAGCGTAGCTGATCTACGATTACTAGCGATTCCGACTTCATGAAGTCGAGTTGCAGACTTC AATCCGAACTGAGAACGGTTTTTTGAGGTTTGCTCCATGTCACCACTTCGCTTCTCTTTGTACCGTCCATTGTAGCA CGTGTGTAGCCCCACTCGTAAGAGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCCGATTACTCGGGCAGTCTC CTTAGAGTGCTCAACTAAATGGTAGTAACTAAGGATAAGGGTTGCGCTCGTTGCAGGACTTAACCGAACATCTCACG ACACGAGCTGACGACAACCATGCACCATCTGTCATTCTGTTAACCTCCACTATGTCTCCATAGCTTTGCAGAAGATG TCAAGAGTGGGTAAGGTTCTACGCGTAACATCAAATTAAACCACATGCTCCACCGCTTGTGCGGATCCCCGTCAATT CCTTTAAGTTTTATTCTTGCGAACGTACTACTCAGGCGGATCATTTAATGCGTTAGCTGCGTCGATGAGTTCCCCAT CAACTAATGATCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTCTCTCAG TGTCAGTGTATGCCCAGTTAGCTGCCTTCGCCATATTGATGTTCTTCCTTATATCTACGCATTTCACCGCTTCACAA GGAATTCCGCTAACCTCTACATAACTCTAGTCTGCCAGTATCCAACGCGTTTTGGGGTTGAGCCCCAAAATTTAACG CCAGACTTAACAAACAACCTACAGACGCTTTACGCCCAATAATTTCGGATAACGCTTGCAACCTATGTATTACCGCG GCTGCTGGCACATAGTTAGCCGTTGCTTTCTAA TCAGGTACCGTCAAGGTAGCATCATTTCCTATGCTACTTTTTCTTCCCTAACCACAGCAGTTTACAACCCATAGGGC CTTCATCCTGCACGCTGTGTCGCTCCATCAGACTTTCGTCCATTGTGGAATATTCCCTACTGCTGCCTCCCGTAGGA GTCTGGGCCGTATCTCAGTCCCAGTGTGGCGGATCAATCTCTCAACCCCGCTAAACATCATCGCCTTGGTGGGCCGT TACCTCACCAACTAGCTAATGTTGCGCACTCCGATCTTTTAGCGAAGCAAACGCTTCCTTTTATATTACTTTCATGC AAAAATAATAAGTATTCGGTATTATCGGATGTTTCCATCCGCTATCCCAATCTAAAAGGTACGTTGAGTACGTGTTA CTCACCCATTCGCCGCTATGATATTGCTATCATCGCTCGACATGCATG(SEQ ID NO.5)
1.5 virulence tests
Test ox is the Fresian of 2 monthly ages health, each 8 of bull, cow.Experimental animal is divided into two groups, control group 8 Head, test group 8.Two groups of experimental animals respectively include bull 2, the cow 2 of random packet.HNMB1 is inoculated with PPLO liquid Culture medium, 37 DEG C of 5%CO2After cultivating 3d in incubator, cell concentration, continuous 10 times of dilution spread solid plates, low power are determined Count plate under mirror, calculates stoste cell concentration, is 3.1 × 109CFU/mL, then adjust to 108CFU/mL, test group animal is led to Larynx tracheae inoculation 3mL mycoplasma liquid cultures are crossed, control animals are inoculated with the PPLO Liquid Cultures that 3mL sterilizes by larynx tracheae Base.Experimental period is 15d, the clinical manifestation of daily viewing test animal, measures body temperature, is cutd open immediately if experimental animal death Kill, observation respiratory disease disease collection pathological material of disease.When 15d off-tests, cut open and kill all experimental animals.
Test group animal shows respiratory secretions after inoculation mycoplasma 24h and the symptom, body temperature liter such as increases, coughs Height, does not eat, and ruminates stopping, dead 1 after 7 days, dead 2 after 10 days.Control animals body temperature between experimental period is normal and nothing Obvious respiratory symptom.Cutd open after off-test and kill test group animal, while cut open killing 4 control animals.Collection pathological material of disease, is carried out Mycoplasma is separated.Test group cut open inspection pleura has a large amount of fine element samples to ooze out, and lung separates existing meat sample consolidation, pleural effusion every leaf portion.It is right There is no obvious pathological change according to group cut open inspection, 4 parts of pathological material of diseases of control group are not separated to mycoplasma, 4 parts of diseases of test group Mycoplasma bovis are separated in material, colonial morphology shows as " decocting poached egg sample ", and PCR qualification results are consistent with HNMB1.
The preparation of embodiment 2, vaccine, security and potency test
The preparation of 2.1 vaccines
M. bovis strain HNMB1 is inoculated into PPLO fluid nutrient mediums, 37 DEG C, 5%CO is placed in2Cultivated in incubator, 3 Culture is collected after it, concentration is determined, the bacterium number in adjustment culture is 2 × 109CFU/mL, adds formaldehyde to final concentration of 0.2%, inactivate 12h in 37 DEG C.Culture after inactivation by volume 1:1 adds aluminum hydroxide adjuvant, is mixed to prepare ox branch former Body inactivated vaccine.
The safety testing of 2.2 vaccines：
60 ages in days health Fresian 10 is chosen, is divided into 2 groups, every group 5.Vaccine group：Musculi colli injects 10mL This vaccine；Control group：Musculi colli injects 10mL sterile salines.Observation 30 days.During observation, vaccine group and control group The equal bouncing of milk cow, morning and evening body temperature, feed intake difference is not notable, illustrates inactivated vaccine safety of the invention.
The potency test of 2.3 vaccines
30 ages in days health Fresian 30 is chosen, is divided into 6 groups, every group 5.Specially：Vaccine 1-4 groups：Per component This vaccine 0.5ml, 1ml, 1.5ml and 2ml are not injected, and the 5th group is non-immune group, and the 6th group is healthy control group, and injection 2mL goes out Bacterium physiological saline.Vaccine group and healthy control group after 60 days two exempt from inject Isodose vaccine or sterile saline.Two exempt from 1 month each group uses 10 afterwards9The HNMB1 of CFU take tracheae inject malicious mode of attacking carry out attacking poison.Start daily within the 2nd day after attacking poison Collection nose swab, starts to adopt within every 3 days a nose swab on the 10th day, after nose swab filtering, does solid medium culture.After poison is attacked 10 days, 40 days, with reference to clinical setting, cut open inspection experimental animal, gathering the organs such as throat swab, lungs carried out Mycoplasma bovis separation training Support.
Observation result：Vaccine group has obvious difference with non-immune group milk cow, and after attacking poison, non-immune group starts for second day Existing clinical symptoms, body temperature is raised, runny nose, and expiratory dyspnea subtracts the maximum phase of an eclipse to going on a hunger strike, and ruminates stopping, starting death, 2 weeks occur after 7 days It is common dead 4 afterwards.Cut open inspection result：Lungs stick together seriously with thoracic cavity, pleural effusion, pulmonary consolidation.The milk cow of healthy control group Do not fall ill.And in vaccine group, there are 2 clinical symptoms occur in 5 cow heads of 0.5ml vaccine groups and case changes；1ml vaccines There is 1 clinical symptoms occur in 5 cow heads of group and case changes；5 cow heads of 1.5ml and 2ml vaccine groups are not fallen ill.Exempt from Epidemic disease is attacked malicious protection situation and be see the table below.
Note："-" represents inapplicable.
As can be seen from the above table, the minimum immunoprotection dosage of this vaccine is that 0.5ml bacteria containing amounts are 1 × 109CFU/mL.Say Bright inactivated vaccine of the invention has good protecting effect.
The optimal embodiment of the present invention is the foregoing is only, for a person skilled in the art, the present invention can have Various modifications and variations.All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., all should It is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Animal Busbandry ＆. Veterinary Medicine Inst., Henan Prov. Academy of Agriculture
<120>One plant of Mycoplasma bovis and its application
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
ccttaggcag tttctttata a 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
catgcatgtc gagcgatgat 20
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
atatacttct gttctagtaa tatg 24
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
ctgattatga tgacagtggt ca 22
<210> 5
<211> 1390
<212> DNA
<213>Mycoplasma bovis（Mycoplasma bovis）
<400> 5
ccttaggcag tttctttata aaccgacttc gggcattacc agctcccatg gtttgacggg 60
cggtgtgtac aagacccgag aacgtattca ccgtagcgta gctgatctac gattactagc 120
gattccgact tcatgaagtc gagttgcaga cttcaatccg aactgagaac ggttttttga 180
ggtttgctcc atgtcaccac ttcgcttctc tttgtaccgt ccattgtagc acgtgtgtag 240
ccccactcgt aagaggcatg atgatttgac gtcgtcccca ccttcctccc gattactcgg 300
gcagtctcct tagagtgctc aactaaatgg tagtaactaa ggataagggt tgcgctcgtt 360
gcaggactta accgaacatc tcacgacacg agctgacgac aaccatgcac catctgtcat 420
tctgttaacc tccactatgt ctccatagct ttgcagaaga tgtcaagagt gggtaaggtt 480
ctacgcgtaa catcaaatta aaccacatgc tccaccgctt gtgcggatcc ccgtcaattc 540
ctttaagttt tattcttgcg aacgtactac tcaggcggat catttaatgc gttagctgcg 600
tcgatgagtt ccccatcaac taatgatcat cgtttagggc gtggactacc agggtatcta 660
atcctgtttg ctccccacgc tttcgtctct cagtgtcagt gtatgcccag ttagctgcct 720
tcgccatatt gatgttcttc cttatatcta cgcatttcac cgcttcacaa ggaattccgc 780
taacctctac ataactctag tctgccagta tccaacgcgt tttggggttg agccccaaaa 840
tttaacgcca gacttaacaa acaacctaca gacgctttac gcccaataat ttcggataac 900
gcttgcaacc tatgtattac cgcggctgct ggcacatagt tagccgttgc tttctaatca 960
ggtaccgtca aggtagcatc atttcctatg ctactttttc ttccctaacc acagcagttt 1020
acaacccata gggccttcat cctgcacgct gtgtcgctcc atcagacttt cgtccattgt 1080
ggaatattcc ctactgctgc ctcccgtagg agtctgggcc gtatctcagt cccagtgtgg 1140
cggatcaatc tctcaacccc gctaaacatc atcgccttgg tgggccgtta cctcaccaac 1200
tagctaatgt tgcgcactcc gatcttttag cgaagcaaac gcttcctttt atattacttt 1260
catgcaaaaa taataagtat tcggtattat cggatgtttc catccgctat cccaatctaa 1320
aaggtacgtt gagtacgtgt tactcaccca ttcgccgcta tgatattgct atcatcgctc 1380
gacatgcatg 1390

Claims (6)

1. one plant of Mycoplasma bovis, it is characterised in that：Described Mycoplasma bovis are Mycoplasma bovis (Mycoplasma bovis) HNMB1, deposit number：CGMCC NO：13295, preservation date：On November 28th, 2016, depositary institution：Chinese microorganism strain Preservation administration committee common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. application of a kind of Mycoplasma bovis in terms of Mycoplasma bovis inactivated vaccine is prepared.
3. application according to claim 2, it is characterised in that in described Mycoplasma bovis inactivated vaccine, Mycoplasma bovis bacterium Measure is 1 × 109CFU/mL。
4. application according to claim 2, it is characterised in that the preparation method of described Mycoplasma bovis inactivated vaccine is： Described Mycoplasma bovis are sequentially passed through into culture, is harvested and after inactivation obtains vaccinogen liquid, add adjuvant to obtain final product vaccine.
5. application according to claim 4, it is characterised in that described adjuvant is aluminum hydroxide adjuvant.
6. application according to claim 5, it is characterised in that the preparation method of described Mycoplasma bovis inactivated vaccine is： Mycoplasma bovis are inoculated into PPLO fluid nutrient mediums, 37 DEG C, 5%CO is placed in2Cultivated in incubator, culture is collected after 3 days, surveyed Determine concentration, the bacterium number in adjustment culture is 2 × 109CFU/mL, adds formaldehyde to final concentration of 0.2%, in 37 DEG C of inactivations 12h；Culture after inactivation by volume 1:1 adds aluminum hydroxide adjuvant, is mixed to prepare Mycoplasma bovis inactivated vaccine.
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CN107446858A (en) * 2017-09-02 2017-12-08 河南省农业科学院畜牧兽医研究所 One plant of Mycoplasma columbinum and its application
CN107488612A (en) * 2017-09-02 2017-12-19 河南省农业科学院畜牧兽医研究所 One plant of mycoplasma hyopneumoniae and its application
CN109652357A (en) * 2019-02-21 2019-04-19 华中农业大学 Cell co-cultures Mycoplasma bovis mutant strain and the application of lower growth defect
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CN109652357A (en) * 2019-02-21 2019-04-19 华中农业大学 Cell co-cultures Mycoplasma bovis mutant strain and the application of lower growth defect
CN110317749A (en) * 2019-06-19 2019-10-11 山东省农业科学院奶牛研究中心 One plant of Mycoplasma bovis velogen strain and its application

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