CN107201326A - One plant of actinobacillus pleuropneumoniae and its application - Google Patents
One plant of actinobacillus pleuropneumoniae and its application Download PDFInfo
- Publication number
- CN107201326A CN107201326A CN201710329663.1A CN201710329663A CN107201326A CN 107201326 A CN107201326 A CN 107201326A CN 201710329663 A CN201710329663 A CN 201710329663A CN 107201326 A CN107201326 A CN 107201326A
- Authority
- CN
- China
- Prior art keywords
- actinobacillus pleuropneumoniae
- actinobacillus
- hnapp1
- culture
- pleuropneumoniae
- 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.)
- Granted
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
-
- 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
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Mycology (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)
Abstract
The invention discloses one plant of actinobacillus pleuropneumoniae and its application, described actinobacillus pleuropneumoniae is Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) HNAPP1, deposit number：CGMCC NO：13333, preservation date：On November 22nd, 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 HNAPP1 in the present invention is isolated from the heart for occurring the growing and fattening pigs of typical case's expiratory dyspnea acute death, without other bacterial growths when being separated on TSA solid mediums, only actinobacillus pleuropneumoniae grows, and viral titer is high in TSB fluid nutrient mediums, up to 109More than CFU/mL.Bacterial strain HNAPP1 in the present invention has stronger pathogenicity to child care pig, causes the morbidity of child care pig dead, with good immunogenicity.
Description
Technical field
The present invention relates to field of molecular biotechnology, more particularly to one plant actinobacillus pleuropneumoniae and its should
With.
Background technology
Actinobacillus pleuropneumoniae (Actinobacillus Pleuropneumoniae, APP) is to cause pig to pass
The cause of disease of metachromia pleuropneumonia (Porcine infection Pleuropneumonia), is that the high degree in contact of a boar is passed
Metachromia breathing problem, is mainly shown as Outbreak, is characterized with cellulosic, hemorrhagic, necrotizing pneumonia, the incidence of disease
Between 8.5%~100%, the death rate is current growing and fattening pigs and boar main causes of death between 0.4%~100%
One of.Therefore the preventing and treating of actinobacillus pleuropneumoniae is significant for the sound development of pig industry.
Up to 15 kinds of Actinobacillus pleuropneumoniae serotype, and the Clinical isolation that also can not much shape at present,
Lack effective Cross immunogenicity between each serotype, the popular predominant serotype in country variant, area and pig farm is again not to the utmost
Identical, therefore, APP separation identification and parting are particularly important in the sick preventing and treating.Domestic popular serotype is mainly 1,
2,3,5,7 types, APP exotoxins cause a disease be immunized in play an important role, APP mono- have 4 kinds of exotoxins, i.e. toxin Apx I,
Apx II, Apx III and Apx IV, and toxin Apx I and Apx II play key effect in terms of APP virulence.Different serotypes
The exotoxin of secretion is different.Serum 1 type can excrete poison Apx I, Apx II and Apx IV simultaneously, be all serotype strains
The middle virulence most best strain of strong, immunogenicity, while being also a kind of popular predominant serotype strain of China.At present, need badly
Vaccine prepared by a kind of new bacterial strain with preferable immunogenicity.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide one plant of actinobacillus pleuropneumoniae and its should
With the Strain Virulence is strong, and the vaccine immunity of preparation is good.
To achieve these goals, the technical solution adopted in the present invention is：
One plant of actinobacillus pleuropneumoniae, described actinobacillus pleuropneumoniae is pleuropneumonia unwrapping wire
Bacillus (Actinobacillus pleuropneumoniae) HNAPP1, deposit number：CGMCC NO：13333, preservation date：
On November 22nd, 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.
Described actinobacillus pleuropneumoniae is the type of actinobacillus pleuropneumoniae 1.
A kind of actinobacillus pleuropneumoniae is in terms of actinobacillus pleuropneumoniae inactivated vaccine is prepared
Using.
In described actinobacillus pleuropneumoniae inactivated vaccine, actinobacillus pleuropneumoniae bacterium amount is 1
×109CFU/mL。
The preparation method of described actinobacillus pleuropneumoniae inactivated vaccine is：By described infectiousness pleura lung
Scorching Actinobacillus sequentially passes through culture, harvest and inactivation and obtained after vaccinogen liquid, adds adjuvant and produces vaccine.
Described adjuvant is aluminium hydroxide.
The preparation method of described actinobacillus pleuropneumoniae inactivated vaccine is：By contagious pleuropneumonia unwrapping wire
Bacillus strain HNAPP1 is inoculated into TSB fluid nutrient mediums, is placed in 37 DEG C of shaking tables and cultivates, culture is collected after 18h, determines concentration,
It is 2 × 10 to adjust the bacterium number in culture9CFU/mL, adds formaldehyde to final concentration of 0.2%, 12h is inactivated in 37 DEG C；After inactivation
Culture by volume 1:1 adds aluminum hydroxide adjuvant, is mixed to prepare actinobacillus pleuropneumoniae inactivated vaccine.
Beneficial effects of the present invention：
1st, the bacterial strain HNAPP1 in the present invention is isolated from the heart for occurring the growing and fattening pigs of typical case's expiratory dyspnea acute death,
Without other bacterial growths when being separated on TSA solid mediums, only actinobacillus pleuropneumoniae grows, in TSB liquid
Viral titer is high in culture medium, up to 109More than CFU/mL.
2nd, the bacterial strain HNAPP1 in the present invention has stronger pathogenicity to child care pig, causes the morbidity of child care pig dead, tool
There is good immunogenicity.
3rd, vaccine prepared by the bacterial strain HNAPP1 in the present invention has to the actinobacillus pleuropneumoniae disease of pig
There is preferable protecting effect.
Brief description of the drawings
Fig. 1 is bacterial strain HNAPP1 colonial morphologies.
Fig. 2 is form under bacterial strain HNAPP1 thalline 100 × microscopes of Gram's staining.
Fig. 3 is that the Marker in bacterial strain HNAPP1 PCR qualification results, figure is DL 2000bp, is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；Isosorbide-5-Nitrae is negative control；2 be that primer APP3, APP4 are infected to 1 type
Property Actinobacillus pleuropneumoniae type strain amplification, 3 be primer APP3, amplifications of the APP4 to bacterial strain HNAPP1；5 are
The amplification of primer APP1, APP2 to 1 type actinobacillus pleuropneumoniae type strain；6 be primer APP1, and APP2 is to bacterium
Strain HNAPP1 amplification.It can be seen that 5 and 6 be the amplification of actinobacillus pleuropneumoniae universal primer, piece
Section is 377bp, and 2,3 be the type primer amplified fragments of actinobacillus pleuropneumoniae 1, and size is 959bp, it was demonstrated that amplified fragments
For purpose fragment, meet desired design size.
Embodiment
The embodiment to the present invention is described in further detail with reference to embodiments.
The separation and identification of embodiment 1, actinobacillus pleuropneumoniae
1.1 pathological material of diseases are gathered
Pathological material of disease comes from occurs severe pneumonia expiratory dyspnea suddenly in November, 2016 on the large-scale pig farm in Ruzhou City of Henan Province
Property dead 5 monthly ages growing and fattening pigs.
The preparation of 1.2 culture mediums
TSB fluid nutrient mediums：By TSB gravy powders (Tryptic Soy broth, pancreas peptone soybean broth) 30g, it is dissolved in
In 1000mL ultra-pure waters, 115 DEG C of sterilizing 15min, plus hyclone 50mL, sterile 1%NAD (Nicotinamide adenine
Dinucleotide, NADH, cozymase) 1mL.
TSA solid mediums：By TSA agar powders (Tryptic Soy Agar, tryptose soya agar) 40g, it is dissolved in
In 1000mL ultra-pure waters, 115 DEG C of sterilizing 15min, plus hyclone 50mL, sterile 1%NAD (are added as needed on) 1mL；In 4
DEG C save backup.
1.3 bacteriums are separately cultured
The painstaking effort of aseptic collection sick pig are inoculated on the TSA solid mediums containing NAD, are cultivated in 37 DEG C of constant incubators
24h, grows consistent circular protrusions, translucent, smooth, moistening, and diameter is 1~1.5 millimeter of bacterium colony, 45 ° of refractions
Observation has obvious golden red band blue-fluorescence under light (see Fig. 1).Purify and be inoculated on the TSA solid mediums without NAD.
It can not be grown on the TSA culture mediums without NAD.Gram's staining (see Fig. 2), the bacterium is Gram-negative bacteria, coccobacillus, small
Bacillus, thread thalline is partly formed, be named as bacterial strain HNAPP1.
The identification of 1.4 bacterial strains
1.4.1 primer is designed
According to a pair of universal primers of sequences Design of actinobacillus pleuropneumoniae ApxIV genes, for infectiousness
The amplification of Actinobacillus pleuropneumoniae, primer sequence is as follows：
APP1：5’-GCTCACCAACGTTTGCTCAT-3’(SEQ ID NO.1)
APP2：5’-GGGGACGTAACTCGGTGATT-3’(SEQ ID NO.2)
Amplified fragments size is 377bp.
Simultaneously according to a pair of infectiousness pleura lungs of sequences Design of actinobacillus pleuropneumoniae pod membrane CPS1B genes
The specific primer of the scorching type of Actinobacillus 1, for the amplification of the type of actinobacillus pleuropneumoniae 1, primer sequence is as follows：
APP3：5’-CTGGAGTAATTACGGCGACTATTCC-3’(SEQ ID NO.3)
APP4：5’-AGGAGAAGCTAGTAGTACTTGCATTTTC-3’(SEQ ID NO.4)
Amplified fragments size is 959bp.
1.4.2PCR identification
Operating procedure according to DNA extraction kit extracts bacterial strain HNAPP1 DNA, and spectrophotometric determination concentration is
100 μ g/mL, enter performing PCR identification.Meanwhile, 1 type actinobacillus pleuropneumoniae type strain of setting is positive control, is set
The type type strain of haemophilus parasuis 4 is negative control.
Pcr amplification reaction system is 25 μ L：10 × buffer solution, 2.5 μ L, 2.5mM (each) dNTPs Mix 0.5 μ L, 10 μ
M/L universal primers APP1, APP2 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 pre-degeneration 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, is loaded per hole under 10 μ L, 1% agarose gel electrophoresis, ultraviolet light and observes result.
As a result 377bp fragments (see Fig. 3), sequencing and 8392 plants of ApxIVA genes of actinobacillus pleuropneumoniae are amplified
(GenBank NO:AF188867) 100% is homologous, and sequencing result is as follows, it was demonstrated that bacterial strain HNAPP1 is put for contagious pleuropneumonia
Line bar bacterium (Actinobacillus pleuropneumoniae).
GGGGACGTAACTCGGTGATTGATGCCGGTGCGGGTAATGATACGGTTAATGGCGGTAATGGCGATGACACCCTCATC
GGCGGCAAAGGTAATGATATTCTAAGAGGTGGCTACGGTGCGGACACCTATATCTTTAGCAAAGGACACGGACAGGA
TATCGTTTATGAAGATACCAATAATGATAACCGCGCAAGAGATATCGACACCTTAAAATTTACTGATATTAATTTAT
CCGAACTTTGGTTTAGCCGAGAAAATAACGATTTGATTATTAAATCATTATTAAGTGAGGATAAAGTCACGGTTCAA
AATTGGTATTCACACCAAGATCATAAAATAGAAAATATTCGTTTATCGAATGAGCAAACGTTGGTGAGC(SEQ ID
NO.5)
1.4.3 Serotype Identification
With reference to 1.4.2 methods, with specific primer APP3, the APP4 amplification of the type of actinobacillus pleuropneumoniae 1, knot
Fruit amplifies 959bp sizes fragment (see Fig. 3), sequencing and 1 type actinobacillus pleuropneumoniae, 4074 plants of (GenBank
NO：AF518558) 100% is homologous, it was demonstrated that bacterial strain HNAPP1 is 1 type actinobacillus pleuropneumoniae.
1.5 virulence test
Experimental animal is healthy child care pig 8 of weaning at 2 monthly ages.Experimental animal is divided into two groups, control group 4, test group 4
Only.HNAPP1 is inoculated with after TSB fluid nutrient mediums, 37 DEG C of shaking table culture 18h, cell concentration, continuous 10 times of dilution spreads is determined
Count plate under solid plate, low power lens, calculates stoste cell concentration, is 2.8 × 109CFU/mL, then adjust to 108CFU/mL,
Test group animal infects 3mL actinobacillus pleuropneumoniae liquid cultures by collunarium, and control animals pass through tracheae
The TSB fluid nutrient mediums of interior inoculation 3mL sterilizings.Continuous Observation 2 weeks after injection, observe and record its feelings such as number and death toll of falling ill
Condition.
Test group animal shows the symptoms, body temperature such as expiratory dyspnea after inoculative infection Actinobacillus pleuropneumoniae 12h
Rise, ear, belly and buttocks cyanosis, the ground that finally crouches does not rise, jerk, death by suffocation, dead preceding mouth and nose elution band foam
Blood.2nd day dead 1, dead 3 after 2 days.Control animals appetite, spirit, body temperature during testing is normal.Experiment
Cutd open after end and kill test group animal, 4 control animals are killed while cuing open.Pathological material of disease is gathered, contagious pleuropneumonia unwrapping wire bar is carried out
Bacterium separates.Test group cut open inspection cardiopulmonary fibre disposition is oozed out, pericardiosymphysis, lung aubergine, tangent plane succulence, the outflow of color liquid, tracheae,
Bronchus is full of foam-like, courageous and upright mucus.Obvious pathological change does not occur for control group cut open inspection, and 4 parts of pathological material of diseases of control group are equal
It is not separated in actinobacillus pleuropneumoniae, 4 parts of pathological material of diseases of test group and is separated to contagious pleuropneumonia unwrapping wire bar
Bacterium, PCR qualification results are consistent with HNAPP1.
Embodiment 2, the preparation of vaccine, security and potency test
The preparation of 2.1 vaccines
Actinobacillus pleuropneumoniae strain HNAPP1 is inoculated into TSB fluid nutrient mediums, is placed in 37 DEG C of shaking tables and trains
Support, culture is collected after 18h, the bacterium number determined in concentration, adjustment culture is 2 × 109CFU/mL, adds formaldehyde to final concentration
For 0.2%, 12h are inactivated in 37 DEG C.Culture after inactivation by volume 1:1 adds aluminum hydroxide adjuvant, is mixed to prepare infection
Property Actinobacillus pleuropneumoniae inactivated vaccine.
The safety testing of 2.2 vaccines：
2 monthly ages healthy child care pig 10 is chosen, is divided into 2 groups, every group 5.Vaccine group：This vaccine of musculi colli injection 4mL；
Control group：Musculi colli injects 4mL sterile salines.Animal heat is determined, clinical manifestation is observed, observed 2 weeks altogether.Observation period
Between, the child care pig of vaccine group and control group breathing, appetite, the state of mind during whole observation is all normal, and injection site is without inflammation
Disease, illustrates that the inactivated vaccine security of the present invention is good.
The potency test of 2.3 vaccines
2 monthly age sodium selenite 30 is chosen, is divided into 6 groups, every group 5.Specially：Vaccine 1-4 groups：Every group is injected this respectively
Vaccine 0.5ml, 1ml, 1.5ml and 2ml, the 5th group is non-immune group, and the 6th group is healthy control group, injects 2mL sterile physiological salt
Water.Vaccine group and healthy control group after 3 weeks two exempt from inject Isodose vaccine or sterile saline.Two exempt from rear 14 days each groups
With 109CFU HNAPP1 bacterium solutions take collunarium to attack poison.The clinical manifestation of piglet is observed, and determines body temperature daily, is observed 2 weeks altogether.
Actinobacillus pleuropneumoniae is carried out to organs such as dead pig lungs to be separately cultured.
Observe result：Vaccine group has obvious difference with non-immune group, attacks after poison second day, the piglet of non-immune group starts
There are clinical symptoms, body temperature is raised, and is had difficulty in breathing, start within the 3rd day death occur, dead preceding mouth and nose are bled, common dead 5 in four days
Head.There is hydrops in the dead pig thoracic cavity of the non-immune group of cut open inspection and abdominal cavity, and have fibrinous exudate, pericardiosymphysis.Lungs enlargement goes out
Blood, tracheae is full of courageous and upright foam thing.The piglet of healthy control group does not fall ill.And in vaccine group, 5 sons of 0.5ml vaccine groups
There are 2 clinical symptoms and case change occur in pig；There is 1 clinical symptoms occur in 5 piglets of 1ml vaccine groups and case becomes
Change；5 piglets of 1.5ml and 2ml vaccine groups do not fall ill.Immunization protection situation 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 those skilled in the art, the present invention can have
Various modifications and variations.Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., all should
Within protection scope of the present invention.
SEQUENCE LISTING
<110>Animal Busbandry ＆. Veterinary Medicine Inst., Henan Prov. Academy of Agriculture
<120>One plant of actinobacillus pleuropneumoniae and its application
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gctcaccaac gtttgctcat 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggggacgtaa ctcggtgatt 20
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
ctggagtaat tacggcgact attcc 25
<210> 4
<211> 28
<212> DNA
<213>Artificial sequence
<400> 4
aggagaagct agtagtactt gcattttc 28
<210> 5
<211> 377
<212> DNA
<213>Actinobacillus pleuropneumoniae（Actinobacillus pleuropneumoniae）
<400> 5
ggggacgtaa ctcggtgatt gatgccggtg cgggtaatga tacggttaat ggcggtaatg 60
gcgatgacac cctcatcggc ggcaaaggta atgatattct aagaggtggc tacggtgcgg 120
acacctatat ctttagcaaa ggacacggac aggatatcgt ttatgaagat accaataatg 180
ataaccgcgc aagagatatc gacaccttaa aatttactga tattaattta tccgaacttt 240
ggtttagccg agaaaataac gatttgatta ttaaatcatt attaagtgag gataaagtca 300
cggttcaaaa ttggtattca caccaagatc ataaaataga aaatattcgt ttatcgaatg 360
agcaaacgtt ggtgagc 377
Claims (7)
1. one plant of actinobacillus pleuropneumoniae, it is characterised in that：Described actinobacillus pleuropneumoniae is chest
Film Actinobacillus (Actinobacillus pleuropneumoniae) HNAPP1, deposit number：CGMCC NO：
13333, preservation date：On November 22nd, 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.
2. actinobacillus pleuropneumoniae according to claim 1, it is characterised in that：Described infectiousness pleura lung
Scorching Actinobacillus is the type of actinobacillus pleuropneumoniae 1.
3. a kind of actinobacillus pleuropneumoniae answering in terms of actinobacillus pleuropneumoniae inactivated vaccine is prepared
With.
4. application according to claim 3, it is characterised in that described actinobacillus pleuropneumoniae inactivated vaccine
In, actinobacillus pleuropneumoniae bacterium amount is 1 × 109CFU/mL。
5. application according to claim 3, it is characterised in that described actinobacillus pleuropneumoniae inactivated vaccine
Preparation method be：Described actinobacillus pleuropneumoniae is sequentially passed through into culture, harvest and inactivation and obtains vaccinogen
After liquid, add adjuvant and produce vaccine.
6. application according to claim 5, it is characterised in that described adjuvant is aluminium hydroxide.
7. application according to claim 6, it is characterised in that described actinobacillus pleuropneumoniae inactivated vaccine
Preparation method be：Actinobacillus pleuropneumoniae strain HNAPP1 is inoculated into TSB fluid nutrient mediums, 37 DEG C of shaking tables are placed in
Culture is collected after middle culture, 18h, the bacterium number determined in concentration, adjustment culture is 2 × 109CFU/mL, adds formaldehyde to end
Concentration is 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
Actinobacillus pleuropneumoniae inactivated vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710329663.1A CN107201326B (en) | 2017-04-11 | 2017-04-11 | Infectious actinobacillus pleuropneumoniae and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710329663.1A CN107201326B (en) | 2017-04-11 | 2017-04-11 | Infectious actinobacillus pleuropneumoniae and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107201326A true CN107201326A (en) | 2017-09-26 |
| CN107201326B CN107201326B (en) | 2020-06-12 |
Family
ID=59905796
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710329663.1A Active CN107201326B (en) | 2017-04-11 | 2017-04-11 | Infectious actinobacillus pleuropneumoniae and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107201326B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676900A (en) * | 2018-04-28 | 2018-10-19 | 湖北省农业科学院畜牧兽医研究所 | A kind of composite PCR parting kit and its application for distinguishing eight kinds of Serotyping of Actinobacilus pleuropneumoniae |
| CN110075289A (en) * | 2019-04-19 | 2019-08-02 | 河南省农业科学院畜牧兽医研究所 | A kind of haemophilus parasuis, Streptococcus suis and Actinobacillus pleuropneumoniae triple inactivated vaccine and its application |
| CN110540953A (en) * | 2019-09-29 | 2019-12-06 | 河南科技大学 | Porcine infectious pleuropneumonia actinobacillus culture medium, separation and identification method and culture method |
| RU2827226C1 (en) * | 2023-12-19 | 2024-09-23 | Общество с ограниченной ответственностью "Центр биотехнологической обработки продуктов питания при институте микроэкологии" (ООО "ЦБО Микроэкологии") | Actinobacillus pleuropneumoniae serotype 2 bacterial strain, intended for production of mono- and polyvalent immunogenic compositions aimed at specific prevention of porcine actinobacillus pleuropneumoniae |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102058880A (en) * | 2010-10-13 | 2011-05-18 | 青岛易邦生物工程有限公司 | Method for producing trivalent inactivated vaccines for porcine infectious pleuropneumonia |
| CN102391975A (en) * | 2011-11-24 | 2012-03-28 | 武汉科前动物生物制品有限责任公司 | Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method |
| CN103566364A (en) * | 2012-07-23 | 2014-02-12 | 普莱柯生物工程股份有限公司 | Porcine contagious pleuropneumonia and streptococcus suis disease combined inactivated vaccine and preparation method thereof |
-
2017
- 2017-04-11 CN CN201710329663.1A patent/CN107201326B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102058880A (en) * | 2010-10-13 | 2011-05-18 | 青岛易邦生物工程有限公司 | Method for producing trivalent inactivated vaccines for porcine infectious pleuropneumonia |
| CN102391975A (en) * | 2011-11-24 | 2012-03-28 | 武汉科前动物生物制品有限责任公司 | Actinobacillus pleuropneumoniae serotype 2 bacterial strain and its preparation method |
| CN103566364A (en) * | 2012-07-23 | 2014-02-12 | 普莱柯生物工程股份有限公司 | Porcine contagious pleuropneumonia and streptococcus suis disease combined inactivated vaccine and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 叶润全 等: "猪胸膜肺炎调查及地方适用型灭活疫苗的研制", 《畜牧与兽医》 * |
| 徐引弟 等: "1型传染性胸膜肺炎放线杆菌的分离鉴定及生物学特性", 《山西农业科学》 * |
| 朱士盛 等: "猪传染性胸膜肺炎HT株的分离、鉴定与定型试验", 《中国动物检疫》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676900A (en) * | 2018-04-28 | 2018-10-19 | 湖北省农业科学院畜牧兽医研究所 | A kind of composite PCR parting kit and its application for distinguishing eight kinds of Serotyping of Actinobacilus pleuropneumoniae |
| CN108676900B (en) * | 2018-04-28 | 2020-04-21 | 湖北省农业科学院畜牧兽医研究所 | Composite PCR typing kit for distinguishing eight porcine actinobacillus pleuropneumoniae serotypes and application thereof |
| CN110075289A (en) * | 2019-04-19 | 2019-08-02 | 河南省农业科学院畜牧兽医研究所 | A kind of haemophilus parasuis, Streptococcus suis and Actinobacillus pleuropneumoniae triple inactivated vaccine and its application |
| CN110540953A (en) * | 2019-09-29 | 2019-12-06 | 河南科技大学 | Porcine infectious pleuropneumonia actinobacillus culture medium, separation and identification method and culture method |
| RU2827226C1 (en) * | 2023-12-19 | 2024-09-23 | Общество с ограниченной ответственностью "Центр биотехнологической обработки продуктов питания при институте микроэкологии" (ООО "ЦБО Микроэкологии") | Actinobacillus pleuropneumoniae serotype 2 bacterial strain, intended for production of mono- and polyvalent immunogenic compositions aimed at specific prevention of porcine actinobacillus pleuropneumoniae |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107201326B (en) | 2020-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107245459A (en) | One plant of haemophilus parasuis and its application | |
| CN105327344B (en) | Vaccine composition and the preparation method and application thereof containing porcine circovirus 2 type antigen Yu haemophilus parasuis antigen | |
| CN109806389B (en) | Haemophilus parasuis trivalent inactivated vaccine and application thereof | |
| CN102847146B (en) | Vaccine used for preventing tilapia streptococcal disease | |
| CN106929451A (en) | One plant of Streptococcus suis and its application | |
| CN112316128B (en) | Canine distemper virus and canine parvovirus infectious hepatitis virus triple live vaccine | |
| CN107201326A (en) | One plant of actinobacillus pleuropneumoniae and its application | |
| CN104450559A (en) | New mycoplasma hyopneumoniae strain and vaccine composite of new mycoplasma hyopneumoniae | |
| CN106929452B (en) | Mycoplasma bovis and application thereof | |
| CN110812473A (en) | Triple inactivated vaccine for haemophilus parasuis disease, streptococcus suis disease and pasteurella multocida disease and preparation method thereof | |
| CN108066758B (en) | Raccoon dog canine distemper-parvovirus enteritis bigeminal live vaccine and preparation method and application thereof | |
| CN112251416B (en) | Canine distemper virus vaccine strain, vaccine and preparation method thereof | |
| CN111514285B (en) | Mycoplasma ovipneumoniae, type-A pasteurella multocida and type-D pasteurella multocida triple inactivated vaccine | |
| CN110124022B (en) | Mycoplasma hyopneumoniae, haemophilus parasuis, streptococcus suis and actinobacillus pleuropneumoniae quadruple inactivated vaccine and application thereof | |
| CN103194412B (en) | Serotype 5 haemophilus parasuis (HPs) vaccine strain | |
| CN107446859A (en) | One plant of chicken virus mycoplasma and its application | |
| CN107384837A (en) | One plant of chicken synovia mycoplasma and its application | |
| CN112195161A (en) | Canine parvovirus vaccine strain, vaccine and preparation method thereof | |
| CN110075289A (en) | A kind of haemophilus parasuis, Streptococcus suis and Actinobacillus pleuropneumoniae triple inactivated vaccine and its application | |
| CN109609418B (en) | Erysipelothrix rhusiopathiae and application thereof | |
| CN109745555A (en) | A kind of mycoplasma hyopneumoniae and haemophilus parasuis bivalent inactivated vaccine and its application | |
| CN104288762B (en) | A kind of vaccine combination and its preparation method and application | |
| CN106929480A (en) | Porcine reproductive and respiratory syndrome virus strain and its application | |
| CN113046271B (en) | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine | |
| CN107354114A (en) | One plant of haemophilus paragallinarum and its application |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |