CN106635901A - Mycoplasma hyopneumoniae strain, vaccine and application thereof - Google Patents
Mycoplasma hyopneumoniae strain, vaccine and application thereof Download PDFInfo
- Publication number
- CN106635901A CN106635901A CN201611178118.9A CN201611178118A CN106635901A CN 106635901 A CN106635901 A CN 106635901A CN 201611178118 A CN201611178118 A CN 201611178118A CN 106635901 A CN106635901 A CN 106635901A
- Authority
- CN
- China
- Prior art keywords
- mycoplasma hyopneumoniae
- strain
- vaccine
- bacterial strains
- mycoplasma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/35—Mycoplasma
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Pulmonology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a mycoplasma hyopneumoniae strain. The strain is a mycoplasma hyopneumoniae HP-G strain; the preservation number of the strain is CCTCC NO: V2001661. The mycoplasma hyopneumoniae HP-G strain is obtained from peripheral tissues of the lung of an infected pig by culturing, separating and purifying. The invention also discloses application of the mycoplasma hyopneumoniae HP-G strain in preparation of a vaccine and a detection kit. Through sequencing and homology analysis, the homology of the mycoplasma hyopneumoniae HP-G strain disclosed by the invention and an unknown vaccine strain is up to 98.4 percent; compared with the existing strain, the mycoplasma hyopneumoniae HP-G strain has more remarkable immunogenicity; the pneumonia reduction rate can be remarkably improved; the immunocompetence of a pig can be enhanced; the incidence can be reduced.
Description
Technical field
The present invention relates to microbial technology field, and in particular to a kind of new mycoplasma hyopneumoniae bacterial strain and utilize this
Plant bacterial strain and make the multiple uses such as vaccine.
Background technology
Porcine mycoplasmal pneumonia, also known as mycoplasma pneumonia of swine, is the contact chronic respiratory of the boar caused by mycoplasma hyopneumoniae
Tract disease, it is generally popular in the world.According to document announcement, all over the world detached mycoplasma hyopneumoniae belongs to same serotype,
But antigenicity has very big difference between separation strains.Frey confirms first antigenicity between mycoplasma hyopneumoniae separation strains within 1992
Difference；1996, Artiushin and Minion further demonstrate that the viewpoint of Frey；1999, Kokotovic was similarly
Confirm this conclusion.Therefore, isolating the preferable mycoplasma hyopneumoniae bacterial strain of one plant of antigenicity carries out vaccine and diagnostic reagent
Box application, it is most important to preventing and treating porcine mycoplasmal pneumonia, one plant of bacterial strain for being adapted to prepare inactivated vaccine is particularly isolated, will fill out
Mend the domestic blank without independent research mycoplasma pneumonia of swine inactivated vaccine.
Mycoplasma hyopneumoniae is the pathogen of porcine mycoplasmal pneumonia.Porcine mycoplasmal pneumonia is that a kind of chronic debilitating of pig exhales
Road disease is inhaled, clinical infection rate is high.The disease cause persistently it is a few week persistent coughs, tarnished by hair, growth retardation and
Outward appearance is not sturdy.Swinery once infects mycoplasma hyopneumoniae, is just difficult to clean off, and not only has a strong impact on growing for swinery, uses
Dose increases, and easily scabies secondary infection PRRSV (PRRS virus), PCV (pig circular ring virus) etc., so as to cause various epidemic diseases
Seedling inoculation failure.Mycoplasma hyopneumoniae used as one of the important pathogen of porcine respiratory disease syndrome, estimate by annual economic loss
Between 200,000,000～2.5 hundred million dollars.Directly or indirectly huge economic loss is caused to pig industry.
At present, the effective means for preventing and controlling mycoplasma hyopneumoniae infection in the world are to carry out vaccine to swinery to exempt from
Epidemic disease.Most of vaccines are the whole cell inactivated vaccine containing adjuvant, and under normal circumstances these vaccines can provide preferably guarantor
Shield is acted on, and such as improves clinical symptoms, reduces injury of lungs, is reduced because of mycoplasma hyopneumoniae infection medication, improves growth efficiency etc..Mesh
Before, wide variety of vaccine is, using mycoplasma hyopneumoniae J strains or P strains as the inactivated vaccine of antigen, to be prepared using J strains or P strains
Vaccine is in global many country's sale and uses.However, due to the variation of the epidemic strain in swinery, clinically remaining to observation
Immune effect aspect has differences to after different swinery vaccine immunities, that is, there is the not good enough colony of some immune effects.It is clinical
Upper these reasons of discrepancies observed are due to having what antigenic difference was caused between the epidemic strain and vaccine strain in swinery
(Villarreal etc., BMC Veterinary Research 2012,8:2).There are some researches show, popular poison in current swinery
There is larger difference in the mycoplasma hyopneumoniae J strains or P strains that strain is used in terms of gene level, protein level and virulence with vaccine
Different (such as document Stakenborg, Vet Microbiol 2005,109:29-36；Calus etc., Vet Microbiol2007,
120:284-91.；The Vet Microbiol2003 such as Vicca, 97:177-190.；Meyns etc., Prev Vet Med2004,66:
The content reported in 265-275).Above-mentioned documents and materials show that the mycoplasma hyopneumoniae J strains that vaccine is used are to porcine mycoplasmal lung
Scorching clinical prevention and therapeutic effect be not good enough.
The content of the invention
An object of the present invention is to provide a kind of new mycoplasma hyopneumoniae bacterial strain, with good immunogenicity.
It is, up to above-mentioned purpose, a kind of mycoplasma hyopneumoniae bacterial strain to be provided in a scheme of the present invention, bacterial strain is pig lung
Scorching mycoplasma HP-G bacterial strains Mycoplasma hyopneumoniae HP-G strain, the deposit number of the bacterial strain is CCTCC
NO：V2001661.
The morphology of above-mentioned mycoplasma hyopneumoniae HP-G bacterial strains is described as follows：
Inoculation agar plate, forms tiny circle, neat in edge, slightly swells like dew drop-wise, middle densification on flat board
Petite；Culture smear, can be observed, with distinctive polymorphic thalline, there is point-like, ring-type, ball through Wright's staining
Shape and the two poles of the earth shape.
The gene order of above-mentioned mycoplasma hyopneumoniae HP-G bacterial strains is as shown in sequence table SEQ ID NO.1.
Based on the mycoplasma hyopneumoniae HP-G bacterial strains of the present invention, the invention also discloses the multiple use of the bacterial strain.
Application of the mycoplasma hyopneumoniae HP-G bacterial strains of the present invention in i (mycoplasma hyopneumoniae) vaccine is prepared.
Application of the mycoplasma hyopneumoniae HP-G bacterial strains in immunodiagnosis kit is prepared.
Application of the mycoplasma hyopneumoniae HP-G bacterial strains in i (mycoplasma hyopneumoniae) vaccine efficacy test.The application of efficacy test
Including the step that the culture of mycoplasma hyopneumoniae HP-G bacterial strains is carried out attacking poison.
Mycoplasma hyopneumoniae HP-G bacterial strains are preparing mycoplasma hyopneumoniae antibody or the application in hyper-immune serum.
Another object of the present invention is to be prepared into vaccine using mycoplasma hyopneumoniae HP-G bacterial strains.
In this regard, another scheme of the present invention is to disclose a kind of i (mycoplasma hyopneumoniae) vaccine, including by mycoplasma hyopneumoniae
The antigen vaccine that the process of HP-G bacterial strains is prepared into, and in the scheme of optimization, adjuvant is also included in vaccine；Adjuvant can be selected
The composition of any one or plurality of reagents in prior art, the function of adjuvant mainly includes：Strengthen the immunity of vaccine antigen
Originality；Promote cellular immunity and humoral immunity, optimize immune response, promote the immune response in the weaker crowd of immunocompetence；Increase
The transmission and immunity entered between antigen and mucous membrane is contacted；Reduce vaccine composition in antigen demand and in implementation process
Immunity inoculation number of times；Optimization antigenic structure, maintains antigen conformation etc..The abundant species of adjuvant, according to its function, can be divided into fortune
Carry transmission system and immunostimulating adjuvant.
Adjuvant has the material of immunogene property including the class that carrier can be combined with haptens, also includes to whole
Vaccine system plays transport transmission and sets off a class material of effect.According to the source of adjuvant, adjuvant can be bacillary, non-thin
Bacterium property, microbes, cytokine class and compound body class etc..According to physicochemical property, adjuvant can for graininess, gelation,
Latex adjuvant etc..With according to its performance, adjuvant can be therapeutic adjuvant, immunostimulating adjuvant, mucosal adjuvant and gene assistant
Agent etc..
Additionally, the invention also discloses a kind of method for preparing i (mycoplasma hyopneumoniae) vaccine, including by mycoplasma hyopneumoniae
The step of HP-G bacterial strain inactivation treatments.
In sum, the present invention has advantages below：
The mycoplasma hyopneumoniae HP-G bacterial strains of the present invention are homologous with known vaccine strains through sequencing and homology analysis
Property be up to more than 98.4%, compare existing bacterial strain with more significant immunogenicity, pneumonia pathology slip can be made notable
Improve, strengthen the immunocompetence of pig itself, reduce the incidence of disease.
Description of the drawings
Fig. 1 is that one embodiment of the invention HP-G strain PCR identifies amplification；
Wherein, 1 is Maker III；2 represent subculture culture；The culture being separately cultured again after poison is attacked in 3 representatives；4
Represent J strains.
Fig. 2 is one embodiment of the invention homology analysis result figure.
Specific embodiment
1 material
1.1st, morbidity swine disease material：It is similar to mycoplasma pneumonia of swine that clinical symptoms are gathered throughout the country, it is doubtful for porcine mycoplasmal lung
The sick pig of inflammation infection, it is aseptic to take 15, the lungs with typical porcine mycoplasmal pneumonia pathology.
1.2nd, bacterium source
Standard control bacterial strain：Mycoplasma hyopneumoniae Jinan system velogen strain CVCC354, is reflected by China Veterinery Drug Inspection Office
Fixed, preservation and supply.
Experiment strain：Mycoplasma hyopneumoniae HP-G bacterial strains, carry out preservation, preservation day in China typical culture collection center
Phase:On December 6th, 2016, preserving number is CCTCC NO：V2001661.
1.3rd, culture medium：Improvement A26Culture medium is prepared by growth pharmaceutical Co. Ltd of China of Sichuan Province, the prescription of culture medium
And collocation method is as follows：
Culture medium preparation method：Above-mentioned composition is mixed, in 121 DEG C, 20 minutes autoclavings；Treat that culture medium temperature declines
During to 37 DEG C, 20% is added without specific antibody Swine serum (56 DEG C inactivate 30 minutes) 160ml, penicillin (250 units/ml) 20
Ten thousand, with NaOH pH value is adjusted to 7.5～7.6.
1.4th, main agents：
2 × Taq PCR MasterMix, Maker III, purchased from TIANGEN biotechnologies company；
Mycoplasma hyopneumoniae indirect hamagglutination detection reagent, by the manufacture of growth pharmaceutical Co. Ltd of China of Sichuan Province；
Emulsifying agent 28VG, purchased from seppic companies.
Mycoplasma hyopneumoniae indirect hamagglutination detection reagent：10% lyophilized antigen sensibilization red blood cell, with 1/ when using
15mol/L PBS (pH value 7.2) are diluted to 2% antigen sensibilization red blood cell, are made for IHA inspections and use.
1.5th, test pig：The susceptible pig of 14～21 ages in days health, by China of Sichuan Province growth pharmaceutical Co. Ltd fixed point pig farm
There is provided, Jing mycoplasma hyopneumoniaes IH detection mycoplasma hyopneumoniae antibody is negative (≤1:4).
The material of 1.6 IHs
10% lyophilized antigen sensibilization red blood cell, 2% antigen is diluted to when using with 1/15mol/L PBS (pH value 7.2)
Sensitized erythrocyte, is made for IHA inspections and uses.
Positive, negative control sera, 2～8 DEG C or Cryopreservation.
Micro-reaction plate：For 96 hole V-type poly (methyl methacrylate) plates or plastic plate.
Diluent：1/15mol/L PBS (pH value 7.2) containing 1% healthy rabbit anteserum (56 DEG C, inactivate for 30 minutes).
1.7 methods of operating
The process of 1.71 serum to be checked：56 DEG C of serum Jing to be checked is inactivated 30 minutes.
1.72 add PBS diluents：Respectively add the μ l of diluent 25 in each hole of reaction plate.
1.73 dilute serums：Add each 25 μ l of serum to be checked, the positive, negative serum respectively in first row hole, then multiple proportions is dilute
Release to the 6th hole, finally discard 25 μ l.
1.74 add antigen sensibilization red blood cell：Add the μ l of 2% antigen sensibilization red blood cell 25 per hole；The μ l diluents of antigen control 25+
25 μ l antigen sensibilization red blood cells.
1.75 reactions：Sample-adding is finished, and is put and vibrated 15 seconds or so on microoscillator, is stood 1～2 hour under room temperature and is judged knot
Really.
1.76 judge：When positive serum potency >=1:64, negative serum potency≤1:4, antigen sensibilization red blood cell control wells
Set up without test during self-solidifying.So that the serum highest dilution of (++) hemagglutination is presented as serum titer terminal.When blood to be checked
Clear potency >=1:When 16, the positive is judged to；Serum titer≤1:When 8, feminine gender is judged to.
1.77 criterion：So that the serum highest dilution of (++) hemagglutination is presented as serum titer terminal.
The separation of 2 experiment strains：
2.1st, the process of pathological material of disease
Using the edge tissues of aseptic operation clip test pig site of pathological change, edge tissues are shredded into sesame seed size,
Then 15 pipes improvement A is soaked into respectively26In culture medium, 37 DEG C of cultures are passed on during culture medium its colour changed into yellow, then carry out pathological material of disease separation
Identification.
2.2nd, the separation identification of pathological material of disease
2.2.1 the observation of cultural character：The 15 pipe bacterium solutions that will be cultivated in 2.1, are passaged to respectively A26Fluid nutrient medium and A26
Agar plate, observes cultural character of each separation bacterium under two kinds of cultivation conditions.
2.2.2 dyeing microscopic examination：Taking culture carries out smear, and with gram staining liquid and Wright's staining liquid dyeing sight is carried out
Examine.
The bacterial strain of the present invention can form tiny circle, neat in edge on flat board, drip like dew after seed agar flat board
The petite that shape, middle densification are slightly swelled；Culture smear, can be observed with distinctive polymorphic bacterium through Wright's staining
Body, there is point-like, ring-type, spherical and the two poles of the earth shape.Therefore, the bacterium colony with above-mentioned form is the pig of the present invention in incubation
Mycoplasma pneumoniae HP-G bacterial strains.
2.3 mycoplasma hyopneumoniaes purify the identification of bacterial strain
By popular mycoplasma hyopneumoniae bacterial strain selected in 2.2, further identification checking, method is as follows：
2.3.1PCR identify
A pair of specific primers are designed according to the mycoplasma hyopneumoniae P 36 genome sequence announced in Genbank, and is submitted to
Synthesize to Invitrogen, primer sequence is：P1：5-TTACAGCGGGAAGACC-3 ', P1：5’-CGGCGAGAAACTGGATA-
3’.The culture of the test of Jing subcultures and dyeing microscopic examination test for identification for mycoplasma hyopneumoniae will be selected to extract by literature method
Genomic DNA, enters performing PCR amplification, and amplification takes above-mentioned product 5ul and electrophoresis is being carried out on 1% agarose gel electrophoresis after terminating
Detection, testing result is as shown in Figure 1.
PCR qualification results：
Preliminary proof is entered into performing PCR amplification for the bacterial strain of mycoplasma pneumoniae, as a result with the expansion of mycoplasma hyopneumoniae J strain PCR
Increase result sizes consistent, and be mycoplasma pneumoniae with expected fragment 427bp bacterial strain of the same size.
2.3.2 serological Identification
2.3.2.1 metabolic inhibition test takes the A without serum26Culture medium, in 20% ratio rabbit-anti pig pneumonia original is added
Body J strain hyper-immune serums, by 1:5 culture medium capacity are inoculated with isolated strains culture, dispense 5 test tubes, and 2ml/ is propped up, while set up adding
Enter the A of 20% healthy rabbit anteserum and porcine mycoplasmal pneumonia negative healthy Swine serum26Culture medium dispenses 5 test tubes as control, and 37
DEG C culture 13d, carries out metabolic inhibition test, observes culture medium color change situation, identifies whether isolated strains are pig pneumonia
Substance.
Metabolic inhibition test result：
The culture of the mycoplasma pneumoniae identified through PCR is added into the training of rabbit-anti mycoplasma hyopneumoniae J strain hyper-immune serums
Nutrient solution, 37 DEG C of culture 13d, it may be observed that suspension fine particle, oil mirror inspection has a small amount of thalline with mycoplasma feature, culture
Liquid color and pH value are unchanged.And the separation bacterium culture through the mycoplasma pneumoniae of PCR identifications adds 20% Healthy Rabbits blood
The A26 culture mediums of cleer and peaceful porcine mycoplasmal pneumonia negative healthy Swine serum, it was observed that nutrient solution color is changed into yellow, pH value from redness
Drop to less than 7.0 by 7.6～7.5, microscopy is with the presence of a large amount of mycoplasma thalline.
2.3.2.2 growth inhibition test is no less than the A of 4mm with freshly prepd thickness263 pieces of agar plate so as to which volatilization is gone
Excessive moisture.The mycoplasma broth culture of logarithmic phase is diluted into 100～1000 times, its concentration is about 1.0 × 106CCU/
ML, takes 100 μ L and is inoculated on agar plate, and coating is uniform, places a moment.Preprepared antiserum is filtered with sterilizing tweezers
The scraps of paper (diameter 6mm, aseptic filter paper piece has adsorbed the anti-mycoplasma hyopneumoniae J strain serum of 1 drop, is dried) are attached at postvaccinal flat board
On, the scraps of paper 1.5～2.0cm of spacing, every piece of flat board places 3 sero-fast test scraps of paper, separately sets 1 serum-free virgin paper sheet pair
According to 7～10d of culture.Under low power lens, the antibacterial circle diameter of antiserum paper piece is measured.Criterion is：With diameter greater than 1.0mm
For the positive；It is feminine gender less than 0.5mm；When therebetween, it is possible to decrease serum or yeast extract concentration repeat 1 in culture medium
It is secondary.
Growth inhibition test result：
The A that 3 groups of separation bacterium are cultivated respectively26Agar plate on, around each anti-mycoplasma hyopneumoniae J strains serum scraps of paper,
Inhibition zone can be substantially observed, between antibacterial circle diameter 2.3mm～2.8mm, and be compareed around the scraps of paper not it is observed that inhibition zone,
As a result see the table below 1.
Table 1：The each growth inhibition circle size of 3 groups of isolated strains
| Experimental strain | First group | Second group | 3rd group | The control scraps of paper |
| The scraps of paper 1 (mm) | 2.3 | 2.6 | 2.6 | 0 |
| The scraps of paper 2 (mm) | 2.2 | 2.5 | 2.6 | 0 |
| The scraps of paper 3 (mm) | 2.5 | 2.8 | 2.7 | 0 |
| Antibacterial circle diameter (mm) | 2.3 | 2.6 | 2.6 | 0 |
2.3.3 isolated strains virulence determination
Test group group number is determined by the mycoplasma hyopneumoniae strain number number that isolated strains are identified, A26Culture medium is control group.Often
5 pigs of group, test group intrathecal 3ml, tracheae injection 2ml, the bacterium solution dilution (1 × 10 of common 5ml7CCU/ml), control group
Thoracic cavity 3ml, tracheae 2ml in the same way, the A of common 5ml26Culture medium, attacks isolated rearing after poison, Continuous Observation 25～30 days
Afterwards, all test pigs are carried out cuing open killing, observation lung pathologies change, with reference to Goodwin55 point of lung of the point-score to each test pig
Portion's disease pathology of breathing is scored.
Select 3 plants of isolated strains to carry out virulence test respectively, by healthy susceptible pig 20, be divided into 4 groups, 5 per group, wherein right
According to a group injection A26Culture medium, 3 groups of challenge test group injects respectively the fresh of 3 groups of (first group, second group, the 3rd group) separation strains
Culture, challenge test pig is attacked after poison there is poor appetite, listless, occurs the clinical condition such as cough, breathe in 10～16 days
Shape, indivedual challenge test pigs have the phenomenon of heating, and control group pig does not show any bad reaction.Attack 25～30 days after poison
Cut open inspection attacks malicious pig, it is seen that the sharp leaf of lungs, lobus cardiacus, middle leaf or lobus diaphragmaticus leading edge have different degrees of distinctive Eaton agent pneumonia
Pathology, in " change of pancreas sample " or " carnification ", Goodwin55 point of point-score score, lesion score is pressed to each test pig pulmonary lesion
It is shown in Table 2.From table 2 it can be seen that 3 groups are attacked malicious pig and different degrees of pulmonary lesion occur, it is aobvious with control group pulmonary lesion difference
Write, illustrate that the Strain Virulence of the present invention is preferably and suitable.
The test pig pneumonia lesion score of table 2
2.3.4 attack malicious swine disease and become separating again for mycoplasma in lung
With 2.1 method separating again and identifying for mycoplasma hyopneumoniae is carried out from attacking malicious swine disease and become in lung.
As a result：Viral disease change pig lung will be attacked to separate again by 2.1 method.Show that pH value declines after Secondary Culture；Smear staining
Microscopy has distinctive polymorphic thalline, has point-like, ring-type, spherical and the two poles of the earth shape, without living contaminants；PCR expand, as a result with
The amplification of mycoplasma hyopneumoniae J strain PCR is in the same size and in the same size with fragment 427bp before poison is attacked.
2.3.5 each isolated strains immunogenicity determining
The bacterial strain pure culture that separation is identified is carried out into inactivation emulsification to prepare.Per plant takes respectively 14～21 ages in days health easily
Sense pig (mycoplasma hyopneumoniae serum antibody is negative), each isolated strains immunity 5, each musculi colli vaccinates 1.0ml, connects
Press within 14th same dose and mode booster shot 1 time after kind, in addition 5 are not inoculated with as control, the raising with the conditions of.Connect first
55～60 days after kind, together with control group 5, the strong poison CVCC354 jellies of mycoplasma hyopneumoniae Jinan system of each tracheae injection 100MID
Dry lung-tissue virus, observe 25～30, cut open and kill total Test pig.By Goodwin55 point of pneumonia disease of the point-score to each test pig
Change is scored, and calculates pulmonary lesion average mark and pulmonary lesion slip.
Experimentation and result：
Immune group test pig is divided into 3 groups, 5 per group, respectively immunity is by 3 plants of (J strains, AV747 strains, HP-G) isolated strains
The vaccine prepared respectively, by the immunity of 2.3.5 methods, after inoculation, do not it is found that test pig has any bad reaction, feeding and essence
Refreshing state is good, and head carries out attacking poison after exempting from 55～60 to all test pigs, and Continuous Observation 25～30 days finds control group examination
Test pig and attack after poison and poor appetite occur, it is listless, occur the clinical symptoms such as cough, breathe in 7～14 days, in immune group
AV747 strain test groups have 3 pigs the respiratory symptoms such as cough, only 2/5 protection occur, and J strain test groups have 2 pigs to occur coughing
The respiratory symptom such as cough, in 3/5 protection, all immune swines of HP-G test groups have no any bad reaction, feeding and the state of mind
It is good, 5/5 protection.Poison is attacked after 28 days, all pigs are carried out cuing open to kill, observe pulmonary lesion, by Goodwin55 point of point-score pair
Pulmonary lesion is scored, and calculates pneumonia pathology slip, the results are shown in Table 3.The test of immunity AV747 strains as can be seen from Table 3
In group, secondly test pig pulmonary lesion most serious are J strains, and HP-G strain test pig pulmonary lesions are hardly obvious, its pathology
Less rate is more up to 84.2%, with good immunogenicity.
The test pig pneumonia pathology of table 3 reduces situation
The test by more than learns that we are finally recovered identification from the pathological material of disease of the doubtful mycoplasma hyopneumoniae in 15 plants of whole nations
Go out HP-G strain mycoplasma hyopneumoniae bacterial strains, the immunogenicity of HP-G strains is all good compared with other two plants in prior art, it is possible to select
Determine the vaccine strain that HP-G is Eaton agent pneumonia vaccine development.
2.4 vaccine strain sequence analyses
By 2.3 tests, by HP-G strains as vaccine strain, the culture of the vaccine strain is entered into performing PCR amplification, product is carried out
Sequencing, and sequence corresponding with 4 world main representative vaccine strains in GenBank carries out sequence analysis and homology analysis.
Reference strains title and its number of logging in GenBank for comparing be：J(NCO07295)、168(NC017509)、232
(NC006360)、7448(NC007332)。
The basic HP-G for selecting Eaton agent pneumonia vaccine development vaccine strain is entered into performing PCR amplification, amplification is shown in Fig. 1, and
Invitrogen companies are sent to be sequenced, sequencing result Jing NCBI-Blast and DNAStar compare analysis, mycoplasma hyopneumoniae HP-G bacterium
The gene order of strain is as shown in sequence table SEQ ID NO.1.The sequence of HP-G respectively J strains, 168 plants, 232 plants, 7448 plants compare,
Individual base has difference, with J strain sequence (genes number：NC007295) compare and find to go out many G bases in the 8th base, the
134th, it is inconsistent at 224,341 bases；With 168 plants of sequence (genes number：NC017509) reverse complemental, and in the 8th base
Go out many G bases, it is inconsistent at the 134th, 224,341,382 bases；With 232 plants of sequence (genes number：NC006360), and
Go out many G bases in the 8th base, it is inconsistent at the 27th, 134,171,224,341,350 bases；With 7448 plants of sequences
Row (gene number：NC007332) compare, go out many G bases in the 8th base, at the 134th, 224,341,381 bases
It is inconsistent.
2.5 homology analysis
The HP-G and NCBI gene pool of the Eaton agent pneumonia vaccine development vaccine strain of the present invention is selected into 4 plants of references both at home and abroad
The gene of bacterial strain carries out sequence analysis, comparison result such as Fig. 2.Sequencing result Jing NCBI-Blast and DNAStar compare analysis,
Aim sequence and J strain sequence (genes number：NC007295) homology is 99.1%；With 168 plants of sequence (genes number：NC017509)
Homology be 98.8%；With 232 plants of sequence (genes number：NC006360 homology) is 98.4%；With 7448 plants of sequence (bases
Cause number：NC007332 homology) is 98.8%.
Sequence analysis and homology analysis result show that basic selected HP-G strains refer to epidemic disease with domestic and international Eaton agent pneumonia
Seedling strain homology more than 99%, therefore can using selected HP-G as Eaton agent pneumonia vaccine development vaccine strain.
The invention also discloses HP-G bacterial strains are used to be prepared into antigen vaccine and the preparation method of the vaccine, vaccine
The step of inactivating HP-G strains is included in preparation method, also including addition adjuvant the step of.Additionally, of the invention obtain vaccine inactivation
The preparation method of method and vaccine can select any one method in prior art, and any one additive method also may be selected.
Based on the mycoplasma hyopneumoniae HP-G bacterial strains of the present invention, the invention also discloses the multiple use of the bacterial strain.For example
Application of the mycoplasma hyopneumoniae HP-G bacterial strains of the present invention in i (mycoplasma hyopneumoniae) vaccine is prepared；Mycoplasma hyopneumoniae HP-G bacterium
Application of the strain in immunodiagnosis kit is prepared；Mycoplasma hyopneumoniae HP-G bacterial strains are examined in i (mycoplasma hyopneumoniae) vaccine effect
Application in testing；The application of efficacy test includes carrying out the culture of mycoplasma hyopneumoniae HP-G bacterial strains to attack the step of poison；Pig
Mycoplasma pneumoniae HP-G bacterial strains are preparing mycoplasma hyopneumoniae antibody or the application in hyper-immune serum.These applications are not limited to this
The concrete form of invention indication, these application specific embodiment can using it is existing any one use, inspection or
Identification, preparation method.
SEQUENCE LISTING
<110>Growth pharmaceutical Co. Ltd of China of Sichuan Province
<120>A kind of mycoplasma hyopneumoniae bacterial strain, vaccine and its application
<130>Nothing
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 428
<212> DNA
<213>Mycoplasma hyopneumoniae（M.hyopneumoniae）
<400> 1
ttacagcggg gaagaccaca aaaaccgggt gaaactcggc ttgaattagt agctgataac 60
atccgaatta tccgggaaat tgcactaaaa gtcaaagaaa gtggctttag tggaataagt 120
attattgttg ctagtcctgt tgatataatt acaagggctt accgggatgc atctggattt 180
tccgatcaaa aagttatcgg tagtggaact gttttagata cagtaaggct tcaatttgca 240
atcgcaaaaa gagcaaaagt atcgcctaat tcggttcagg cctacgtgat gggtgaacat 300
ggtgattcat cttttgttgc ttattcaaat attaaaattg gcggtgaatg tttctgtgct 360
tattctaaac taaccggaat tgatagctca aattacgaaa aagaacttga atatccagtt 420
tctcgccg 428
Claims (10)
1. a kind of mycoplasma hyopneumoniae bacterial strain, it is characterised in that：The bacterial strain is mycoplasma hyopneumoniae HP-G bacterial strains, the bacterial strain
Deposit number is CCTCC NO：V2001661.
2. mycoplasma hyopneumoniae bacterial strain as claimed in claim 1, it is characterised in that：The mycoplasma hyopneumoniae HP-G bacterial strains
Morphology is described as follows：
Inoculation agar plate, formed on flat board tiny circle, neat in edge, like dew drop-wise, middle densification slightly swell it is little
Bacterium colony；Culture smear, can be observed with distinctive polymorphic thalline through Wright's staining, have point-like, ring-type, it is spherical and
The two poles of the earth shape.
3. mycoplasma hyopneumoniae bacterial strain as claimed in claim 1, it is characterised in that：The mycoplasma hyopneumoniae HP-G bacterial strains
Gene order is as shown in sequence table SEQ ID NO.1.
4. application of the mycoplasma hyopneumoniae HP-G bacterial strains described in claim 1 in i (mycoplasma hyopneumoniae) vaccine is prepared.
5. a kind of i (mycoplasma hyopneumoniae) vaccine, it is characterised in that：Including what the process of mycoplasma hyopneumoniae HP-G bacterial strains was prepared into
Antigen vaccine.
6. a kind of method for preparing i (mycoplasma hyopneumoniae) vaccine, it is characterised in that：Including mycoplasma hyopneumoniae HP-G bacterial strains are gone out
The step of process living.
7. application of the mycoplasma hyopneumoniae HP-G bacterial strains described in claim 1 in immunodiagnosis kit is prepared.
8. application of the mycoplasma hyopneumoniae HP-G bacterial strains described in claim 1 in i (mycoplasma hyopneumoniae) vaccine efficacy test.
9. application as claimed in claim 8, it is characterised in that：The application of the efficacy test is included mycoplasma hyopneumoniae
The culture of HP-G bacterial strains carries out attacking the step of poison.
10. the mycoplasma hyopneumoniae HP-G bacterial strains described in claim 1 are in mycoplasma hyopneumoniae antibody or hyper-immune serum is prepared
Application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611178118.9A CN106635901A (en) | 2016-12-19 | 2016-12-19 | Mycoplasma hyopneumoniae strain, vaccine and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611178118.9A CN106635901A (en) | 2016-12-19 | 2016-12-19 | Mycoplasma hyopneumoniae strain, vaccine and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106635901A true CN106635901A (en) | 2017-05-10 |
Family
ID=58833336
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611178118.9A Pending CN106635901A (en) | 2016-12-19 | 2016-12-19 | Mycoplasma hyopneumoniae strain, vaccine and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106635901A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117305192A (en) * | 2023-12-01 | 2023-12-29 | 北京瑞阳瑞泰生物科技有限公司 | Mycoplasma hyopneumoniae RT02 strain, vaccine composition and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031258B (en) * | 2012-06-29 | 2014-12-17 | 普莱柯生物工程股份有限公司 | Novel mycoplasma hyopneumoniae bacterial strain and vaccine composition thereof |
| CN104450559A (en) * | 2012-06-29 | 2015-03-25 | 普莱柯生物工程股份有限公司 | New mycoplasma hyopneumoniae strain and vaccine composite of new mycoplasma hyopneumoniae |
| CN104894009A (en) * | 2015-05-15 | 2015-09-09 | 北京中海生物科技有限公司 | Mycoplasma hyopneumoniae strain and application thereof |
| CN103740625B (en) * | 2014-01-26 | 2016-06-22 | 江苏省农业科学院 | A kind of mycoplasmal pneumonia of swine attenuated live vaccine and application thereof |
-
2016
- 2016-12-19 CN CN201611178118.9A patent/CN106635901A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031258B (en) * | 2012-06-29 | 2014-12-17 | 普莱柯生物工程股份有限公司 | Novel mycoplasma hyopneumoniae bacterial strain and vaccine composition thereof |
| CN104450559A (en) * | 2012-06-29 | 2015-03-25 | 普莱柯生物工程股份有限公司 | New mycoplasma hyopneumoniae strain and vaccine composite of new mycoplasma hyopneumoniae |
| CN103740625B (en) * | 2014-01-26 | 2016-06-22 | 江苏省农业科学院 | A kind of mycoplasmal pneumonia of swine attenuated live vaccine and application thereof |
| CN104894009A (en) * | 2015-05-15 | 2015-09-09 | 北京中海生物科技有限公司 | Mycoplasma hyopneumoniae strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 白文彬等: "《动物传染病诊断学》", 30 September 2002, 中国农业出版社 * |
| 龚文波等: "猪支原体肺炎疫苗免疫效果评价", 《四川畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117305192A (en) * | 2023-12-01 | 2023-12-29 | 北京瑞阳瑞泰生物科技有限公司 | Mycoplasma hyopneumoniae RT02 strain, vaccine composition and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103031258B (en) | Novel mycoplasma hyopneumoniae bacterial strain and vaccine composition thereof | |
| CN102258776B (en) | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof | |
| CN104450559B (en) | New mycoplasma hyopneumoniae bacterial strain and its vaccine combination | |
| CN111789941B (en) | Bivalent inactivated vaccine for mycoplasma pneumonia and chlamydia psittaci disease of goats and preparation method thereof | |
| CN109806389B (en) | Haemophilus parasuis trivalent inactivated vaccine and application thereof | |
| CN112843228B (en) | Bivalent inactivated vaccine for rabbit pasteurellosis and preparation method thereof | |
| CN107513506A (en) | Mycoplasma hyopneumoniae, vaccine combination and its application | |
| CN112481154A (en) | Mycoplasma ovipneumoniae vaccine strain, vaccine composition prepared from vaccine strain and application of vaccine composition | |
| CN115287270B (en) | Subtype B avian metapneumovirus (APV) subculture attenuated strain and application thereof | |
| CN107338208B (en) | Porcine atrophic rhinitis D type virus-producing pasteurella multocida vaccine strain and application thereof | |
| CN100419073C (en) | Live vaccine for mycoplasma pneumonia of swine and production method thereof | |
| CN109609468A (en) | A kind of porcine pseudorabies virus of six gene delection, pseudorabies disease vaccine and preparation method | |
| CN106085936B (en) | A kind of 10 gene-deleted strain of streptococcus suis 2-type and application | |
| CN109745555B (en) | Mycoplasma hyopneumoniae and haemophilus parasuis combined inactivated vaccine and application thereof | |
| CN111789942B (en) | Bivalent inactivated vaccine for mycoplasma ovis pneumonia and chlamydia psittaci disease and preparation method thereof | |
| CN106635901A (en) | Mycoplasma hyopneumoniae strain, vaccine and application thereof | |
| CN105754905A (en) | Pseudomonas aeruginosa of minks and application of pseudomonas aeruginosa | |
| CN113046271B (en) | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine | |
| CN112442473B (en) | Avian Escherichia coli vaccine strain | |
| CN103623400B (en) | Vaccine composition for resisting pig mycoplasma pneumonia and infectious pleuropneumonia and preparation method and preparation method | |
| CN110819599B (en) | Vaccine strain for preventing taiwan infectious bronchitis | |
| CN104338128B (en) | A kind of vaccine combination and its preparation method and application | |
| CN102391975B (en) | Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method | |
| CN107446858B (en) | Pigeon mycoplasma and application thereof | |
| CN104645324A (en) | Application of swine enzootic hyopneumoniae vaccine strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |
|
| RJ01 | Rejection of invention patent application after publication |