CN106620685A - Rabbit bordetella bronchiseptica subunit vaccine - Google Patents
Rabbit bordetella bronchiseptica subunit vaccine Download PDFInfo
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- CN106620685A CN106620685A CN201611251490.8A CN201611251490A CN106620685A CN 106620685 A CN106620685 A CN 106620685A CN 201611251490 A CN201611251490 A CN 201611251490A CN 106620685 A CN106620685 A CN 106620685A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- 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)
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- A—HUMAN NECESSITIES
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- 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
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- 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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Abstract
The invention provides a rabbit bordetella bronchiseptica subunit vaccine, particularly relates to rabbit bordetella bronchiseptica (QDBb01 strain with the preservation number of CCTCC M 2016607) identified through isolated culture, Bb gene specific primer PCR amplification and sequencing. Rabbit bordetella bronchiseptica OMP (osteoblast milk protein) is extracted, antigenic protein with the protein molecular weight larger than 40 kDa is purified, then the protein concentration is measured with a Bradford protein content measuring method and diluted to be 150 ug/ml, the subunit vaccine ( about 30 ug/ml for finished products) is prepared by emulsifying the protein and a aluminum hydroxide adjuvant in a volume ratio being 1:4, the vaccine is injected subcutaneously into a rabbit with the immunity weight being about 1.5 kg in the dosage of 1 ml for each rabbit, after 14 days, ear intravenous infusion challenge is performed with the rabbit bordetella bronchiseptica QDBb01 strain (LD50 being 4.8*105 CFU) according to 100 LD50, the rabbit is observed for 7 days after challenge, and the challenge protection rate of the rabbit is 10/10 through statistics.
Description
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to a kind of Rabbit Bordetella bronchiseptic stain subunit
Vaccine.
Background technology
Rabbit Bordetella bronchiseptic stain disease is multiple in autumn and winter season and early spring.Mainly by air borne, Jing respiratory tracts
And infect.Young rabbit invasion rate is high, and has death.Adult rabbit invasion is less.This bacterium is 64.4% in the pollution of group support rabbit, is dissipated
Foster rabbit is 20%.Under field conditions (factors), there is this bacterium parasitic in various mammal upper respiratory tracts, often cause chronic respiratory tract disease
Mutual infection.Especially in change in weather, the resistance of rabbit declines, and rabbit home health is bad, when air dirt, can draw
Play this disease to occur.This sick 7～10d of incubation period.Adult rabbits show rhinitis and bronchitis.There are serosity, the mucus of volume
Nose liquid flows out.Rhinitis does not heal for a long time, and nasal cavity flows out mucus or purulent secretion, sneezes, and has difficulty in breathing, and does not eat, and becomes thin.
Being in acute process young rabbit, the initial stage has just been seen after rhinitis symptom, i.e. performance is had difficulty in breathing more, rapid dead, 2～3d of the course of disease.This disease
Major lesions be rhinitis, suppurative rhinotracheitis, purulent bronchopneumonia, it is individual it is other occur septicaemia change.Nasal cavity, tracheae
Mucous membrane is congested, oedema.There are serosity, mucus or mucus purulent secretion in nasal cavity.On the side of hilus pulumonis peribronchial to lung
Edge is shown in Bronchopneumonia focus.Pathology is more common in lobus cardiacus, sharp leaf, and serious case involves the full lobe of the lung.Lesion protuberance, heavily fortified point
Firmly, in kermesinus, brown, and then for lark.There is the running sore for differing in size on some case lungs.Serious accounts for the 90% of lung
More than.Some liver surfaces have soya bean to the big running sore of broad bean.Running sore breaks the milky pus of rear visible sticky butyrous.Also
See there is pericarditis, pleurisy, thoracic cavity empyema and muscle abscess etc..
Rabbit Bordetella bronchiseptic stain disease is mainly using bio-security and vaccinoprophylaxis.Wherein bio-safety
Measure mainly includes：Strengthen feeding and management, improve feeding environment, carry out epidemic preventing working；Preferably adhere to home-bred and autophytic in warren；To new
The rabbit of introduction, it is necessary to quarantine more than 1 month, Jing bacteriology can enter group with Serological testing for negative patient.This disease often with
Pasteurella, rabbit group once falls ill, it is necessary to ascertain the reason, and eliminates environmental stimuli factor, isolates infected rabbits, to control
Transmission of pathogen.The sick more refractory immunity healed, need to strengthen Intensive livestock farm of Rabbit Bordetella bronchiseptic stain, mainly uses divide at present
From to bordetella branchiseptica make inactivated vaccine, carry out immunization campaign, every rabbit hypodermic injection 1mL, annual 2 times；
Also useful rabbits pasteurellosis-bordetella bacilli Combined vaccine or Pasteurella-bordetella bacilli-rabbit hemorrhagic disease triple vaccine are pre-
Anti- inoculation (Du Yanan etc., 2006；Huang Yanyan etc., 2006).But Rabbit Bordetella bronchiseptic stain inactivated vaccine easily occurs thin
Verticillium toxin residue problem, often results in immune rabbit invasion, therefore, at present in the urgent need to it is a kind of being capable of the prevention and control sick securities, effect
The good vaccine of power, so as to make up the deficiencies in the prior art.
The content of the invention
It is an object of the invention to provide a kind of Rabbit Bordetella bronchiseptic stain subunit vaccine, so as to make up prior art
Deficiency.
Present invention firstly provides a kind of Rabbit Bordetella bronchiseptic stain, is Rabbit Bordetella bronchiseptic stain QDBb01 strains
(Rabbit Bordetella bronchiseptica QDBb01), was deposited in Wuhan, China on November 01st, 2016
State's Type Tissue Collection, deposit number is CCTCC NO:M 2016607.
The Rabbit Bordetella bronchiseptic stain that the present invention is screened is used to prepare vaccine；
The present invention another aspect provides a kind of Rabbit Bordetella bronchiseptic stain subunit vaccine, and wherein antigen is from upper
Extract in the Rabbit Bordetella bronchiseptic stain stated, with immunogenic albumen；
The extracting method of wherein albumen is as follows：After by the bacterial cell disruption of Rabbit Bordetella bronchiseptic stain QDBb01 strains, from
The heart remove it is unbroken fall microorganism and heavier bacterial cell inner cell organ；To be precipitated after supernatant high speed centrifugation；
After with buffer solution precipitation, after incubation at room temperature；High speed centrifugation is carried out again；After precipitation dissolving, use can retain protein molecular
Amount falls small molecular protein in the pellicle dialysis of more than 40kDa, and the albumen in dislysate is destination protein.
A kind of Rabbit Bordetella bronchiseptic stain subunit vaccine provided in the embodiment of the present invention, wherein aluminium hydroxide gel
Adjuvant；
In order to make up the deficiency of Rabbit Bordetella bronchiseptic stain vaccine now, the present invention is by by by the rabbit being separated to
Bordetella branchiseptica is cultivated, there is provided a kind of extracting method of Rabbit Bordetella bronchiseptic stain antigen protein, and
The antigen protein for extracting is mixed with aluminium hydroxide gel adjuvant emulsion, having prepared one kind can prevent rabbit bronchus sepsis ripple
The effective vaccine of family name's bacillosis.
Description of the drawings
Fig. 1：The separation of the Rabbit Bordetella bronchiseptic stain of the present invention and culture figure；
Fig. 2：Slide agglutination test figure；Wherein left (positive bacteria), in (sample bacterium) and the right side (physiological saline)
The feminine gender water control of the PCR amplification figures of Fig. 3 single bacterium colony 16S genes, Pm genes and Bb genes, wherein "-"；“+”
Positive bacteria is compareed.
Specific embodiment
Embodiment 1：The separation and culture of Rabbit Bordetella bronchiseptic stain
A collection of 1.5～3.0kg rabbits are bought from Shandong Province Weifang warren and do the pouring seedling test of swine fever spleen, occur falling ill
Anxious, dead fast, mouth and nose bleeding, the disease that Pulmonary hemorrhage, extravasated blood are characterized, the clinical manifestation, cut open inspection disease according to the dead rabbit of morbidity
Become, preliminary to suspect rabbits pasteurellosis or Rabbit Bordetella bronchiseptic stain infection, then taking lung tissue has carried out dividing for bacterium
From and culture, carry out slide agglutination test and verified；16s and Pasteurella (Pm) gene or Podbielniak are carried out to cultivating single bacterium colony
The PCR amplifications of bacillus (Bb) gene, and clone, be sequenced, making a definite diagnosis this batch of rabbit is caused by Rabbit Bordetella bronchiseptic stain infection
Death.
1.1 incidences buy a collection of 1.5～3.0kg rabbits and do the pouring seedling thermometric test of swine fever spleen, this batch from Shandong Weifang
Only rabbit pestilence seedling is crossed in immunity to rabbit, and other vaccines not immunity, integrality is good.
1.2 clinics and the general 2kg of one body weight of cut open inspection symptom or so test rabbit, die by visitation of God, mouth and nose spray blood is dead previous
Its state of mind, feeding and drinking-water are good, without any disease symptomses, without wound of fighting, and rabbit mouth and nose bleeding of falling ill, Pulmonary hemorrhage,
Extravasated blood, liver has a leaf into yellow-white.
1.3 laboratory diagnosis
1.3.1 bacteria distribution and culture
It is preliminary to suspect rabbits pasteurellosis or Rabbit Bordetella bronchiseptic stain infection, it is then aseptic to take morbidity rabbit lung tissue
Pathological material of disease is uniformly inoculated on chocolate agar plate culture medium, and culture forms medium bacterium colony in 24 hours, and in canescence, surface is wet
Profit, smooth, neat in edge.Refer to Fig. 1.
1.3.2 Bacteria Identification
1.3.2.1 gram, methylene blue dyeing identifies Jing Gram's staining and examines under a microscope determination, Gram's staining
For red globules bacillus, show that the bacterium is Gram-negative bacteria；With methylene blue, microscopy, it is seen that polymorphic, bipolar staining little
Bacillus.
1.3.2.2 slide agglutination test identification identifies the separation sample using Rabbit Bordetella bronchiseptic stain positive serum
Bacterium, and the positive and negative control are done respectively with Rabbit Bordetella bronchiseptic stain positive bacteria and physiological saline, concrete outcome is shown in figure
2。
1.3.3.3PCR prokaryotes are with 16S universal primers and Pasteurella (Pm) specific primer and set for identification and utilization detection
Bordetella bacilli (Bb) specific primer (upstream and downstream primer sequence refers to table 1) of meter enters performing PCR amplification to single bacterium colony, and in 1%
Electrophoresis in Ago-Gel, refers to Fig. 3.
The primer sequence table of table 1
1.3.3.4 objective gene sequence is determined and for 16S and Bb genes of interest to serve the raw work sequencing in sea, sequencing result point respectively
Analysis shows that the bacterium is Rabbit Bordetella bronchiseptic stain, is named as QDBb01 strains.
1.3.3.5 the preparation of Rabbit Bordetella bronchiseptic stain (QDBb01 strains) inactivated vaccine
Bacterial classification is inoculated in into Bao Jiang Shi culture mediums, culture 48h in 37 DEG C of shaking tables.With sterile saline centrifuge washing 2 times,
Again formaldehyde is added by cumulative volume 0.4% to being 20,000,000,000/ml containing bacterium number with normal saline dilution, mixed, 37 DEG C of inactivations 48 are little
When, mix once every 6h.Jing inspection inactivation eligibles are used as antigen for vaccine.With aluminium hydroxide gel adjuvant by volume=1：
4 carry out, with seedling, being prepared into Rabbit Bordetella bronchiseptic stain inactivated vaccine, and equivalent to finished product 4,000,000,000 inactivated bacteria/ml are contained.Immunity
As a result show, vaccine prepared by the QDBb01 strains of present invention screening has good immunity effect to Rabbit Bordetella bronchiseptic stain
Really.Also, carry out immunity with the Rabbit Bordetella bronchiseptic stain vaccine sold in the market, then with present invention screening
QDBb01 strains carry out challenge viral dosage；As a result the immune effect for showing the vaccine sold in the market is far below QDBb01 of the present invention
The effect of inactivated vaccine made by strain.It is presumably due to QDBb01 pnca genes and morphs cause the immune effect of current vaccine not
It is good.
The purification of the albumen of the Rabbit Bordetella bronchiseptic stain of embodiment 2 (QDBb01 strains)
The Rabbit Bordetella bronchiseptic stain (QDBb01 strains) for separating identification preservation is carried out into 3%TSB cultures, and 4000r/
Min collects thallines, then with the resuspended thalline of PH7.2Tris-Hcl, by resuspended thalline ultrasonic disruption, work 1s, interval time 3s,
All times 12min, 180 times altogether.4000r/min low-speed centrifugals remove it is unbroken fall microorganism and heavier bacterium it is thin
Intracellular organelle, takes supernatant in 100000r/min, ultracentrifugation 20min, removes supernatant.By the precipitation containing outer membrane protein with containing
The Tris-Hcl buffer solutions of 2%Triton X-100, are incubated 1h and remove inner membrane protein under room temperature, then carry out 100000r/
Min ultracentrifugation 30min, precipitation is resuspended again with buffer solution, and employing can retain molecular weight of albumen in more than 40kDa (comprising master
Want protective antigens albumen) pellicle dialysis fall small molecular protein, packing, put -20 DEG C of preservations.
Embodiment 3：It is prepared by Rabbit Bordetella bronchiseptic stain (QDBb01 strains) subunit vaccine
It is definitely fixed that the Rabbit Bordetella bronchiseptic stain albumen AAS for having purified is followed the steps below
Amount：(1) add the PBS 0.15ml and protein quantification Coomassie brilliant blue dye liquor 2.85ml that survey albumen in cuvette as right
According to being returned to zero；(2) add and survey albumen PBS 0.14ml, add testing protein solution 0.01ml and protein quantification bright with coomassie
It is 0.204 that blue dye liquor 2.85ml measures absorbance y in cuvette, show that albumen is dense by formula y=0.152x+0.068
Degree x=0.83mg/ml, as 895 μ g/ml, survey albumen used above is purchased from TIANGEN companies with reagent.The egg that this is purified
White solution is diluted to 150ug/ml, with aluminium hydroxide gel adjuvant by volume=1：4 carry out, with seedling, being prepared into rabbit bronchus sepsis
Bordetella bacilli subunit vaccine, finished product equivalent in every milliliter of aluminium hydroxide gel seedling contain the μ g/ml of albumen 30.
Embodiment 4：Rabbit Bordetella bronchiseptic stain inactivated vaccine and subunit vaccine efficacy test method
4.1 preparations for attacking poison bacterium solution will separate Rabbit Bordetella bronchiseptic stain (QDBb01 strains) recovery that identification is preserved
Picking single bacterium colony is inoculated with the calf serum Bao Jiang Shi culture mediums of 150ml 3%, 37 DEG C of overnight shaking table culture rabbit bronchus sepsis Podbielniaks
Bacillus, morning next day takes out, and carries out colony counting：Bacterium solution is carried out into 10-7Dilute again, taking 0.1ml carries out coated plate, then calculates bacterium
The number that falls is 48.As a result, bacterium number is 4.8 × 109cfu/ml。
4.2 attack toadstool liquid minimal lethal dose (LD50) determination select 25 rabbits, be randomly divided into 5 groups, 5 per group, will
Above-mentioned bacterium solution is according to 10-14.8 × 10 are diluted to successively7cfu/ml、4.8×106cfu/ml、4.8×105cfu/ml、4.8×
104Tetra- gradients of cfu/ml, carry out attacking bacterium with 1ml/ dosage only, stay 1 group as blank.Continuous Observation, record rabbit
Condition of morbidity death, anatomic observation pathology situation finally determines 4.8 × 105CFU is minimal lethal dose.
4.3 Rabbit Bordetella bronchiseptic stain inactivated vaccines, subunit vaccine protest test by prepare this
Inactivated vaccine and subunit vaccine finished product are respectively according to the rabbit each 10 of 1ml/ dose subcutaneous injecting immune body weight 1.5kg or so
Only, 10 are stayed to be only used as non-immunized controls group.14 days after exempting from, with Rabbit Bordetella bronchiseptic stain QDBb01 strain (LD50For 4.8 ×
105CFU) according to 100LD50Carry out attacking poison, attack after poison and observe 7, count rabbit attacks malicious protective rate, and the vaccine is judged with this
Protected effect.As a result, all morbidities of control group 10, dissect upper respiratory tract pathology and are bordetella branchiseptica typical case
Symptom；And subunit vaccine and inactivated vaccine immune group are attacked after poison to be good for and lived, morbidity is showed no, protective rate is 10/10.But dissect
The position of the injection of two kinds of vaccines finds that inactivated vaccine absorbs incomplete, and the subunit vaccine for preparing absorbs complete, therefore, system
Standby Rabbit Bordetella bronchiseptic stain (QDBb01 strains) subunit vaccine has more preferable security compared with inactivated vaccine.And, with
Inactivated vaccine prepared by QDBb01 strains is similar to, and subunit vaccine prepared by the present invention is to the immune effect of QDBb01 strains also in statistics
Learn the similar vaccine being better than in meaning on market.
Claims (7)
1. a kind of Rabbit Bordetella bronchiseptic stain, it is characterised in that the preservation of described Rabbit Bordetella bronchiseptic stain is compiled
Number be CCTCC M 2016607.
2. application of the Rabbit Bordetella bronchiseptic stain described in claim 1 in vaccine is prepared.
3. a kind of inactivated vaccine, it is characterised in that the antigen in described vaccine is the rabbit gas described in the claim 1 of inactivation
Pipe sepsis bordetella bacilli.
4. a kind of Rabbit Bordetella bronchiseptic stain subunit vaccine, it is characterised in that the antigen of described subunit vaccine is
Extract from the Rabbit Bordetella bronchiseptic stain described in claim 1, with immunogenic albumen.
5. subunit vaccine as claimed in claim 4, it is characterised in that described albumen, is that molecular weight is not less than 40kDa
Albumen.
6. the subunit vaccine as described in claim 4 or 5, it is characterised in that described albumen, its extracting method is as follows：Will
After the bacterial cell disruption of the Rabbit Bordetella bronchiseptic stain described in claim 1, centrifugation remove it is unbroken fall microorganism and
Heavier bacterial cell inner cell organ；To be precipitated after supernatant high speed centrifugation；After buffer solution precipitation, incubation at room temperature
Afterwards；High speed centrifugation is carried out again；After precipitation dissolving, use can retain molecular weight of albumen and fall in the pellicle dialysis of more than 40kDa
Small molecular protein, the albumen in dislysate is destination protein.
7. subunit vaccine as claimed in claim 4, it is characterised in that the vaccine adjuvant in described subunit vaccine is hydrogen
Oxidation aluminium glue adjuvant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108721614A (en) * | 2018-06-12 | 2018-11-02 | 福建省农业科学院畜牧兽医研究所 | Rabbit staphylococosis, Disease Caused By Bordetella Avium bigeminy Attenuated vaccine and preparation method |
| CN116983396A (en) * | 2023-08-09 | 2023-11-03 | 浙江省农业科学院 | Rabbit bronchogenic bordetella inactivated vaccine emulsion, and preparation and application thereof |
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Cited By (3)
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| CN108721614A (en) * | 2018-06-12 | 2018-11-02 | 福建省农业科学院畜牧兽医研究所 | Rabbit staphylococosis, Disease Caused By Bordetella Avium bigeminy Attenuated vaccine and preparation method |
| CN116983396A (en) * | 2023-08-09 | 2023-11-03 | 浙江省农业科学院 | Rabbit bronchogenic bordetella inactivated vaccine emulsion, and preparation and application thereof |
| CN116983396B (en) * | 2023-08-09 | 2024-05-07 | 浙江省农业科学院 | Rabbit bronchogenic bordetella inactivated vaccine emulsion, and preparation and application thereof |
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