CN104163858B - Pasteurella multocida acellular antigen, preparation method and applications thereof - Google Patents
Pasteurella multocida acellular antigen, preparation method and applications thereof Download PDFInfo
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Abstract
The invention relates to a pasteurella multocida acellular antigen, which is derived from deactivated supernate of pasteurella multocida culture medium, wherein the supernate contains polysaccharide and protein. The invention further provides a pasteurella multocida indirect hemagglutination test method, which is built on the basis of the antigen and has a very good specificity and sensitivity. The invention also provides a vaccine containing the antigen. The provided pasteurella multocida acellular antigen vaccine has the same immunity effect as that of whole cell vaccine, and has the advantages of no affection on animal growth, and little side effect on the vaccine injected part.
Description
Technical field
The present invention relates to veterinary biologics field, more particularly to a kind of pasteurella multocida acellular antigens.
Background technology
Pasteurella multocida（Pasteurell multoeida, Pm) Bacillus pasteurii disease of many animals can be caused, such as
The hueppe's disease of the animals such as fowl cholera, pig lung plague, cattle, the rabbit of birdss.Such disease is divided into acute, subacute and chronic three
Class, first two rapid onset, mortality rate are high, and the 3rd class is easily caused slow growth of animal, feed conversion rate reduction and secondary infection
Other antibacterials or virus, cause huge economic loss then.Effectively vaccination is to prevent and control having for such disease
Effect approach, the pasteurellosis bacilluss inactivated vaccine of various animals and attenuated vaccine are that the sick prevention and control are made that huge contribution.But mesh
Front used vaccine mostly is whole-bacterial-vaccine, although clinical effectiveness is pretty good, but because the Pseudomonas is in gram negative bacteria, high concentration
Bacterium solution in the not only antigenic substance containing high concentration, and the endotoxin containing high concentration, so that such vaccine is facing
Side reaction in bed application is larger, and in injection site scleroma is easily formed, and its intersecting protective is poor, and immune duration is not
It is long.The cross immunity effect of attenuated live vaccines is preferable, but stress is stronger after being inoculated with, and can not completely prevent sending out for the disease
It is raw.
Therefore, it is thought that removal thalline, retains effective antigenic component and be prepared into the more single vaccine of composition.Pod membrane
Polysaccharide is the important ingredient of pasteurella multocida, and research shows that capsular polysaccharide is not only relevant with the virulence of bacterial strain, and
Or the important protective antigen of pasteurella multocida.Then people extract pasteurella multocida using different methods
Capsular polysaccharide is prepared into subunit vaccine, just right in patent US005225194A early in the eighties in last century, Neylan A etc.
The preparation of pasteurella multocida polysaccharide vaccine has been described in detail, but polysaccharide antigen mostly is thymus independent antigen, lures
Lead humoral immunization and mainly produce IgM immunoglobulins, anamnestic response is faint or even disappearance, meanwhile, age immature animals table
Reveal the polysaccharide antigen unresponsiveness of age correlation.Therefore, research discovery later, capsular polysaccharide（Or polysaccharide）The immunity of vaccine
Effect is often barely satisfactory, and its immune effect is poorer than conventional whole-bacterial-vaccine.Sun Dejun et al. is in " rabbits pasteurellosis k antigen
Vaccine and conventional vaccine safety and the comparative experimental research of immune efficacy " [Chinese herding magazine, 2011,47（12）：70-72]
One is disclosed herein a kind of being inoculated in rabbits pasteurellosis strain in 0.1% cracking whole blood improvement martin's bouillon, then using physics side
Method is slightly carried, the method for preparing pod membrane vaccine, and has carried out the safety immune efficacy of pod membrane vaccine and conventional vaccine respectively
And immune period test, immune efficacy assay shows：The counteracting toxic substances protective rate of pod membrane vaccine is 60%, more conventional vaccine immunity effect
Power is low, but safety is better than conventional vaccine.
, used as another kind of main protective antigen of pasteurella multocida, many scholars are to its immunogenicity for outer membrane protein
Substantial amounts of research is carried out.Last century, YUE-SHOUNG LU etc. successively demonstrate the outer membrane protein of pasteurella multocida in rabbit
Physical ability produces good immunoreation, and can the counteracting toxic substances of homologous strain be provided with 100% protection.Into after 21 century, with right
What the composition of pasteurellosis bacilluss outer membrane protein, 26S Proteasome Structure and Function were studied gos deep into, and researchers are exempted to single outer membrane protein
The research of epidemic focus, as a result shows pasteurella multocida outer membrane protein H（OmpH）, lipoprotein E（PlpE）And lipoprotein B
（PlpB）There is good immunogenicity etc. single outer membrane protein, wherein PlpB also has different serotypes pasteurella multocida
The effect of cross protection.Pasteurella multocida 1 outer-membrane protein vaccine can carry out reality by the extracting directly of bacterial culturess
It is existing, such as described in the identification to five kinds of outer membrane protein of pasteurella multocida in 2004 such as Louisa B.Tabatabai
Sample, successively carries out thalline lysozyme, EDTA, Triton X-100 etc. and is processed, obtains through the link such as centrifugation and extraction
Outer membrane protein, what is so obtained mostly is outer membrane protein mixture；Pasteurella multocida 1 outer-membrane protein vaccine can also be by spy
Determine the gene cloning and expression of outer membrane protein and realize, such as Jeongmin Lee are being evaluated OmpH immune protective effects for 2007
Described in as, finally will after engineering bacterial cell disruption extract pasteurella multocida outer membrane protein method, be achieved in that
Outer membrane protein be the relatively single outer membrane protein of composition.The good outer membrane protein of both of which energy adaptive immune originality, but operate step
It is rapid loaded down with trivial details, it is difficult to accomplish scale production.
Therefore, it is necessary that exploitation is a kind of being suitable for industrial-scale production in fact, possesses exempt from identical with conventional whole-bacterial-vaccine
Epidemic disease effect, while and the safe pasteurella multocida vaccine having no side effect.
The content of the invention
The technical problem to be solved is for the deficiencies in the prior art, there is provided a kind of pasteurella multocida without
Cellular antigens, the antigen preparation procedure is simple, it is easy to industrial-scale production, and with good immunogenicity；By the present invention
Acellular antigens agarose gel after, can be used to detect pasteurella multocida specific antibody, the detection method is special
The opposite sex and stability are preferable.Additionally, vaccine will be prepared into after the acellular antigens addition adjuvant and preservative of the present invention, have
The equal immune efficacy of whole-bacterial-vaccine, and do not affect the little advantage of growth of animal, the side reaction of vaccine injection site local.
In order to realize foregoing invention purpose, the present invention provides a kind of pasteurella multocida acellular antigens, wherein described
From the supernatant of the pasteurella multocida culture of inactivation, the supernatant contains polysaccharide and albumen to antigen.It is described to kill more
Property pasteurellosis bacilluss culture supernatant be by by pasteurella multocida culture be centrifuged or precipitate after take liquid component.
In a specific embodiment, the pasteurella multocida culture by fluid medium, in preference temperature, example
As cultivated certain hour at 37 DEG C, such as obtain after 12-16h；Can also be by after solid medium culture by sterile buffer
Or fluid medium washes lower bacterium colony（Or lawn）Obtain.
In another specific embodiment, in the culture medium in addition to conventional nutrients, also containing a certain amount of blood
Clearly.The concentration of serum is 2-20%（V/V）, preferably 5-15%（V/V）, further preferably 8-10%（V/V）, most preferably 10%（V/V）.
In a specific embodiment, the inactivator that the inactivation of the culture is used is in formalin or β-the third
Ester, it is also possible to inactivated by physics mode, is such as heated.
Preferably, the virulent strain that the pasteurella multocida is well known to those skilled in the art, including killing property rabbit more
Pasteurellosis bacilluss C51-2 strains, C57-17 strains and chicken pasteurella multocida C48-2 strains and 1502 plants, are also included by common platform
Pig source property, rabbit source property and other animal derived, the killing property Pasteur bars of such as cattle or sheep obtained with clinical case isolation identification more
Bacterium virulent strain.
Preferably, polyoses content is 0.5-0.7mg/ml in the antigen, and protein content is 8.8- in the antigen
10.6mg/ml。
Preferably, the pasteurella multocida acellular antigens are animal pasteurella multocida acellular antigens.Institute
Animal is stated for pig, rabbit, chicken, cattle or sheep.
Another object of the present invention is to provide a kind of method for preparing pasteurella multocida acellular antigens, the method
Including：Pasteurella multocida is inoculated in the culture medium containing serum, at 30-40 DEG C, at preferably 37 DEG C 8-24h is cultivated,
Pasteurella multocida culture is obtained, the pasteurella multocida culture centrifugation or Direct precipitation Jing after inactivation are abandoned thalline, taken
Supernatant can obtain the pasteurella multocida acellular antigens containing polysaccharide and albumen simultaneously.
Preferably, the concentration of the serum is 2-20% based on the culture medium（V/V）, preferably 5-15%（V/V）, then it is excellent
Select 5-10%（V/V）, most preferably 10%（V/V）.
Preferably, the culture medium can be brain heart infusion broth, Tryptose soy culture medium, Martin's culture medium, improvement
The commercially available culture mediums such as Martin's culture medium, or the formula culture containing nutritional labelings such as peptone, yeast extract, protolysates
Base, in a specific embodiment of the present invention, preferably improves Martin's culture medium.
Another goal of the invention of the present invention is to provide the pasteurella multocida acellular antigens in killing property Pasteur more
Application in bacillus Serologic detection.The Serologic detection includes the side such as indirect hemagglutination test, agar gel diffusion test or ELISA
Method.
In a specific embodiment, the present invention uses indirect hemagglutination test method, using pasteurella multocida without thin
The specific antibody of pasteurella multocida in extracellular antigen detection serum.For example, fresh anticoagulant Sheep Blood is gathered, is prepared after washing
Protoerythrocyte suspension, then uses glutaraldehyde hydroformylation, then relation with tannic acid, obtains relation sheep red blood cell (SRBC) liquid.This is relation
Sheep red blood cell (SRBC) liquid mixes with pasteurella multocida acellular antigens liquid of the present invention, sensitization, is made into sensitized erythrocyte and hangs
Liquid, can be used to detect pasteurella multocida specific antibody.
The present invention also aims to provide a kind of pasteurella multocida acellular vaccine, it contains the killing property bar more
Family name's bacillus acellular antigens, adjuvant and preservative.
Preferably, final concentration of 70-95% of the pasteurella multocida acellular antigens in vaccine（V/V）.
The example of the adjuvant in the acellular vaccine includes but is not limited to aluminium hydroxide gel, mineral oil, propolis, Gel01
（French SCIPPIC）、ISA206（French SCIPPIC）.Preferably aluminium hydroxide gel, propolis and/or white oil, more preferably hydrogen-oxygen
Change aluminium glue.
Final concentration of 4-29% of the adjuvant in vaccine（V/V）.
The preservative be thimerosal, its final concentration of 0.001-0.01% in vaccine（V/V）.
Present invention also offers a kind of method for preparing pasteurella multocida acellular vaccine, it comprises the steps：
Pasteurella multocida is inoculated in the culture medium containing serum, at 30-40 DEG C 8-24h is cultivated, obtain pasteurella multocida
Culture, the pasteurella multocida culture centrifugation or Direct precipitation Jing after inactivation, abandons thalline, obtains same by taking supernatant
The pasteurella multocida acellular antigens of Shi Hanyou polysaccharide and albumen, the antigen Jing after steriling test is qualified, add adjuvant and
Thimerosal, is pasteurella multocida acellular vaccine after mixing.
Present inventors have unexpectedly found that, pasteurella multocida acellular vaccine has equal immune efficacy with whole-bacterial-vaccine,
And the growth to animal and the impact of pelage quality are substantially less than the impact of whole-bacterial-vaccine.
The extracting method of pasteurella multocida acellular antigens provided by the present invention is simple, it is easy to scale metaplasia
Produce；After by pasteurella multocida acellular antigens agarose gel, can be used to detect pasteurella multocida specificity
Antibody, and with good specificity and sensitivity；Using prepared by pasteurella multocida acellular antigens of the present invention
Acellular vaccine immune effect can reach the immune effect of whole-bacterial-vaccine, while having been surprisingly found that after whole-bacterial-vaccine immune animal
Stress is larger, and not only the inflammatory reaction of injection site is more serious, and the weightening of animal is substantially less than acellular vaccine immunity
Group and matched group, and acellular vaccine immune group and matched group in terms of animal weightening without significant difference.Therefore, institute of the present invention
The pasteurella multocida acellular vaccine of offer is fully able to substitute current whole-bacterial-vaccine, while reducing vaccine injection institute
The side reaction for causing.After this vaccine is widely applied, meat animals are applicable not only to, are also applied for fleece animal, in prevention
While disease, the quality of animal product is not reduced.
Description of the drawings
The organization chartss of vaccine injection site are exempted from the test of Fig. 1 acellular vaccines immune group.
The organization chartss of vaccine injection site are exempted from the test of Fig. 2 whole-bacterial-vaccines immune group.
Specific embodiment
The embodiment of following offer illustrates specific embodiments of the present invention, but the invention is not restricted to following embodiments
Scope.
To make the present invention easier to understand, below in conjunction with embodiment the present invention is described in detail, these embodiments are only
Play illustrative effect, it is not limited to the range of application of the present invention, NM specific experiment method in the following example, generally
Carry out according to normal experiment method.
Bacterial strain used in the present invention, such as rabbit pasteurella multocida C51-2 strains, C51-17 strains；Killing property Pasteur chicken more
Bacillus C48-2 strains, 1502 plants；Bordetella branchiseptica BS039 strains and bacillus coli CMCC44149 strains, be
The biomaterial that the public can buy from domestic and international commercial channel, for example, can buy from China Veterinery Drug Inspection Office.
Pasteurella multocida acellular antigens of the present invention are discarded from the pasteurella multocida culture of inactivation
Supernatant after thalline, its not mycetome.Polysaccharide and albumen contained by antigen of the present invention is respectively killing property Pasteur's bars more
The ingredient of bacterium, pasteurella multocida par-tial polysaccharide and protein ingredient Jing after inoculation, culture are discharged into culture fluid, will be trained
Thalline is abandoned after nutrient solution inactivation, centrifugation or natural sedimentation and obtains the acellular antigens containing polysaccharide and albumen, and the antigen tool
There is good immunogenicity, can be used to prepare serodiagnosiss antigen and vaccine.
Embodiment one：The preparation of pasteurella multocida acellular antigens
Take rabbit pasteurella multocida C51-2 strains, C51-17 strains（It is purchased from China Veterinery Drug Inspection Office, bacterial strain preservation
Numbering is respectively CVCC499, CVCC1753）With chicken pasteurella multocida C48-2 strains, 1502 plants（It is purchased from Chinese veterinary drug
Product supervise institute, and bacterial strain deposit number is respectively CVCC44802, CVCC2082）Freeze-drying lactobacillus, respectively with 1-2ml sterile physiological salt
2-20% is inoculated in after water dissolution（V/V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 2-20%（V/V）Serum
Improvement martin's bouillon, puts 37 DEG C, and 200rpm concussion and cultivates 12h, bacterium solution adds 0.15% formalin, and 37 DEG C inactivate 24 hours
Afterwards, 10000rpm centrifugations 20min, supernatant is pasteurella multocida acellular antigens.
Wherein：Containing 2-20%（V/V）Improvement Martin's flat board of serum is prepared as follows：Weigh improvement Martin's agar（Purchase
From Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd）42g, adds 800-980ml distilled water, fully shakes up post-heating to abundant
Dissolving, 115 DEG C of high pressure steam sterilization 30min, temperature is down to 60 DEG C or so, is separately added into 20-200ml new-born calf serum（The four seasons
Green grass or young crops, purchased from Zhejiang Tian Hang bio tech ltd）, ware is down flat after fully shaking up.
Containing 2-20%（V/V）Serum improvement martin's bouillon is prepared as follows：Weigh improvement Martin's culture medium（Purchased from green grass or young crops
Island GaoKeYuan Hai Bo Bioisystech Co., Ltd）40g, adds 800-980ml distilled water, fully shakes up post-heating to fully molten
Solution, 115 DEG C of high pressure steam sterilization 30min add 20-200ml new-born calf serum after cooling, after mixing.
Embodiment two：The detection of pasteurella multocida acellular antigens main component
The measure of 1 acellular antigens protein content
Using nucleic acid-protein analyzer（German Eppendorf, BioPhotometer plus）Directly determine each acellular
The protein content of antigen, the results are shown in Table 1.
Table 1：The measurement result of protein content in pasteurella multocida acellular antigens
| Strain number | Protein content in acellular antigens（mg/ml） |
| C51-2 | 10.2 |
| C51-17 | 10.6 |
| C48-2 | 8.8 |
| 1502 | 9.5 |
2 acellular antigens measurements of the polysaccharide content (using phenol-sulfuric acid and colorimetric method)
Use anhydrous glucose（sigma）As reference substance, regression equation y=1.3914x+0.0086 is obtained（R2=0.997）, will
Each acellular antigens sample physiological saline solution is made after 20 times of dilutions, it to be determined in 490nm ripples using phenol-sulfuric acid and colorimetric method
Strong point absorbance, substitutes into the content of polysaccharide in regression equation calculation acellular antigens sample.The results are shown in Table 2.
Table 2：Measurement of the polysaccharide content in acellular antigens
Embodiment three：The foundation and its application of pasteurella multocida indirect hemagglutination test method
The preparation of 1 agarose gel
1.1 the collection and washing of sheep red blood cell (SRBC)
By fresh anticoagulant Sheep Blood, 4 DEG C preserve stable rear 2000rpm centrifugations 10min, abandon supernatant and leukocyte.Use PBS
（0.15mol/L, pH7.2）Washing 3-5 time, is made into by volume 5% red cell suspension, and 4 DEG C save backup.
The hydroformylation of 1.2 erythrocyte and relation
Glutaraldehyde water is made into into by volume 2.5% glutaraldehyde solution, 4 DEG C save backup.
The 5% of 4 DEG C of pre-coolings red cell suspension is placed in into magnetic stirring apparatuss, stirring at low speed, the volume ratio of 5 ︰ 1 is added dropwise over 4
The glutaraldehyde solution of DEG C pre-cooling.Then by red cell suspension in 30 DEG C of constant temperature oscillator, hydroformylation 5h under the conditions of 150rpm.Use PBS
(0.15mol/L, pH7.2) is washed 3 times, is made into 5% Hydroformylated red blood cell suspension, 4 DEG C of preservations.
Tannic acid and water are pressed into 1 ︰ 20000（g/ml）Tan-liquor is made into, it is now with the current.
Take the tan-liquor (1 of 5% Hydroformylated red blood cell suspension and isopyknic new preparation:20000) mix, put 37 DEG C of water-baths
30min, after washing 3 times with PBS (0.15mol/L, pH6.4), returns to original volume, is made into 5% relation sheep red blood cell (SRBC) liquid, and 4
DEG C save backup.
The sensitization of 1.3 sheep red blood cell (SRBC)s
The pasteurella multocida acellular antigens liquid of 10 times of dilutions of 5% relation sheep red blood cell (SRBC) and equal-volume is mixed
Close, 37 DEG C of water-bath sensitization 30min constantly gently vibrate therebetween, 3000rpm centrifugation 3min abandon supernatant, with PBS (0.15mol/
L, pH7.2) wash 3 times after, then be made into 1% sensitized erythrocyte suspension with PBS (0.15mol/L, pH7.2).
2 pasteurella multocida positive serums, the preparation of negative serum
By rabbit pasteurella multocida C51-2 strains, C51-17 strains, chicken pasteurella multocida C48-2 strains, 1502 plants and
The bordetella bacilli BS039 strains of trachea deteriorated blood and bacillus coli CMCC44149 strains are inoculated in respectively TSA solid mediums, and 37
Take single bacterium colony and be inoculated in respectively in 5mlTSB fluid mediums after DEG C culture 18h, 37 DEG C, concussion and cultivate under the conditions of 180rpm
14h, takes 0.1ml and is inoculated in 100mlTSB fluid mediums and be enlarged culture, 37 DEG C, samples after 200rpm concussion 10h and does
Count plate, adds 0.15% formalin to inactivate 24h to bacterium solution, and inactivated bacterial liquid is concentrated into 100 according to count plate result
Hundred million CFU/ml, are separately added into 20% aluminium hydroxide gel, and pasteurella multocida Pm serum is respectively after mixing（C51-2 strains）With
Antigen, pasteurella multocida Pm serum（C51-17 strains）With antigen, pasteurella multocida Pm serum（C48-2 strains）With anti-
Former, pasteurella multocida Pm serum（1502 plants）With antigen, bordetella branchiseptica Bb serum（BS039 strains）With anti-
Former, bacillus coli E.coli serum（CMCC44149 strains）Use antigen.Wherein, front 4 kinds of antigen is pasteurella multocida
Positive serum antigen, latter two is pasteurella multocida negative serum antigen.
Test rabbit 14 is taken, 7 groups are randomly divided into, 2/group, 6 groups of pasteurella multocida for being inoculated with above-mentioned gained respectively are positive
Property, negative serum antigen, i.e. respectively C51-2 strains, C51-17 strains, C48-2 strains, 1502 plants, BS039 strains and CMCC44149
Strain antigen, another group of inoculation physiological saline solution, 2ml/, dorsal sc injection；After 14 days same dose booster immunization once,
Each immune group test rabbit anteserum is gathered within 21 days after booster immunization respectively, i.e. respectively Pm serum（C51-2 strains）, Pm serum（C51-
17 plants）, Pm serum（C48-2 strains）, Pm serum（1502 plants）, Bb serum（BS039 strains）, E.coli serum（CMCC44149 strains）
And negative serum.
The foundation of 3 pasteurella multocida indirect hemagglutination test methods
Pasteurella multocida C51-2 and C48-2 are inoculated in into respectively the culture medium containing serum, through 37 DEG C, 200rpm
Concussion and cultivate 12h, adds 0.15% formalin, 37 DEG C of inactivation 24h supernatant to be taken after centrifugation i.e. respectively many in bacterium solution
The acellular antigens of killing property pasteurellosis bacilluss C51-2 and C48-2, step 1.1,1.2,1.3 sensitization are pressed by antigen Jing after 10 times dilute
Sheep red blood cell (SRBC), take the μ L of agarose gel 25 respectively with 25 μ L PBS (0.15mol/L, pH7.2), 25 μ L Pm（C51-2
And C48-2）Serum, 25 μ L Bb serum（BS039）, 25 μ L E.coli serum（CMCC44149）With 25 μ L negative serums in 96
On the V-type blood-coagulation-board of hole mix, put 37 DEG C effect 30min, as a result find C51-2 acellular antigens sensitization sheep red blood cell (SRBC) only with
Pm（C51-2）Serum there occurs agglutination, and remaining Kong Jun is settled into a dot；The silk floss of C48-2 acellular antigens sensitization
Sheep red blood cell only with Pm（C48-2）Serum there occurs agglutination, and remaining Kong Jun is settled into a dot, show prepared
Agarose gel can be used to detect the specific antibody of pasteurella multocida in serum.
4 pasteurella multocida indirect hemagglutination test methods are used to detect pasteurella multocida antibody in serum
4.1 indirect hemagglutination tests (IHA) method
Take the PBS containing 1% bovine serum albumin（0.15mol/L、pH7.2）25 μ l take Pm in 96 hole V-type blood clotting plate holes
Serum（C51-2）, Pm serum（C51-17）, Pm serum（C48-2）, Pm serum（1502）, Bb serum（BS039）, E.coli blood
Clearly（CMCC44149）25 μ l each with negative serum is respectively at the 1st hole of the first to seven row of two Sptting plates, doubling dilution to
11 holes, the 12nd hole as blank, then in the 1% sensitized erythrocyte suspension of the μ l of each Kong Zhongjia 25.It is light on microoscillator
Shake 1min so as to is sufficiently mixed, and after 37 DEG C of incubation 30min or incubation at room temperature 60min result is observed.With "-", " ++ ", " +++ ",
" # " represents the intensity of agglutination of erythrocyte.
4.2 criterion
" # " represents that the erythrocyte of coagulation is uniformly paved with bottom hole in film like, and during strong coagulation, coagulation shrinkage is agglomerating." +++ " table
The erythrocyte for showing coagulation is paved with bottom hole, but there is a small amount of erythrocyte sedimentation in central authorities into dot." ++ " represents that erythrocyte is sunken to bottom hole
Central authorities, surrounding still has the coagulation erythrocyte being dispersed in."-" represents that erythrocyte all sinks to bottom hole central authorities, red without what is be dispersed in around
Cell.With the hemagglutinative titer that the serum highest extension rate for " # " occur is the serum.
4.3 result
The testing result of indirect hemagglutination test method：Using pasteurella multocida indirect hemagglutination test side of the present invention
Method, to each blood serum sample to be checked indirect hemagglutination test is carried out, and its result shows：37 DEG C of effect 30min or room temperature effect 60min
Afterwards, the erythrocyte of blank control wells is settled into completely a dot, and with 100% coagulation to judge terminal, C51-2 is acellular
It is 1: 2 that the sheep red blood cell (SRBC) of antigen sensibilization measures the IHA potency of each Pm serum1~1: 26, wherein Pm serum（C51-2）IHA effect
Valency is up to 1: 26, Bb serum（BS039）, E.coli serum（CMCC44149）Feminine gender is with negative serum；C48-2 is without thin
It is 1: 2 that the sheep red blood cell (SRBC) of extracellular antigen sensitization measures the IHA potency of each Pm serum2~1: 27, wherein Pm serum（C48-2）IHA
Potency is up to 1: 27, Bb serum（BS039）, E.coli serum（CMCC44149）Feminine gender is with negative serum（Refer to table
3）, the result after 37 DEG C of effect 30min and room temperature effect 60min is consistent, from the result of table 3, between pasteurella multocida
Connect hemagglutination test method to can be used to detect whether containing pasteurella multocida antibody in serum, and with good stability,
IHA potency between homologous strain antigen-antibody is higher, and the IHA potency between heterologous strain antigen-antibody is then relatively low, shows this
The antigen that invention is provided has certain intercrossing between different strains, but the sensitivity between homologous strain is higher.
Table 3：The testing result of indirect hemagglutination test method
Example IV：The preparation of pasteurella multocida acellular vaccine
The steriling test of 1 pasteurella multocida acellular antigens
Pasteurella multocida acellular antigens are taken under aseptic technique, by 25 μ l/ wares aforesaid improvement horse is inoculated in
Fourth agar plate, 3-5 plate of every batch of sample inoculation, while nonvaccinated improvement Martin's agar plate is set as negative control,
Put 37 DEG C simultaneously to cultivate 48 hours, the plate and negative control plate of antigen inoculation is without bacterial growth.
The preparation of 2 pasteurella multocida acellular vaccines
The qualified pasteurella multocida acellular antigens of steriling test of learning from else's experience add by volume 20% aluminium hydroxide gel
（ALHYDROGEL,Brenntag Biosector）, addition preservative thimerosal, final concentration of the 0.01% of thimerosal（V/V）,
Pasteurella multocida acellular vaccine, 4 DEG C of preservations are after mixing.
The preparation method of four kinds of pasteurella multocida acellular vaccines is respectively：
The preparation of C51-2 strain acellular vaccines：Rabbit pasteurella multocida C51-2 strain freeze-drying lactobacillus are taken, is sterilized with 1-2ml
10% is inoculated in after physiological saline solution（V/V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 10%（V/V）Blood
Clear improvement martin's bouillon, puts 37 DEG C, and 200rpm concussion and cultivates 12h, bacterium solution adds 0.15% formalin, and 37 DEG C of inactivations 24 are little
Shi Hou, 10000rpm are centrifuged 20min, take supernatant and are pasteurella multocida C51-2 strain acellular antigens.The C51-2 strains
Acellular antigens add 20% aluminium hydroxide gel Jing after steriling test is qualified, by volume, add thimerosal, and its is final concentration of
0.01%, C51-2 strain acellular vaccines, 4 DEG C of preservations are obtained final product after mixing.
The preparation of C51-17 strain acellular vaccines：Rabbit pasteurella multocida C51-17 strain freeze-drying lactobacillus are taken, is gone out with 1-2ml
10% is inoculated in after bacterium physiological saline solution（V/V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 10%（V/V）
Serum improves martin's bouillon, puts 37 DEG C, and 200rpm concussion and cultivates 12h, bacterium solution adds 0.15% formalin, 37 DEG C of inactivations 24
After hour, 10000rpm centrifugation 20min take supernatant and are pasteurella multocida C51-17 strain acellular antigens.The C51-
17 plants of acellular antigens add 20% aluminium hydroxide gel Jing after steriling test is qualified, by volume, add thimerosal, and it is dense eventually
Spend for 0.01%, C51-17 strain acellular vaccines, 4 DEG C of preservations are obtained final product after mixing.
The preparation of C48-2 strain acellular vaccines：Chicken pasteurella multocida C48-2 strain freeze-drying lactobacillus are taken, is sterilized with 1-2ml
10% is inoculated in after physiological saline solution（V/V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 10%（V/V）Blood
Clear improvement martin's bouillon, puts 37 DEG C, and 200rpm concussion and cultivates 12h, bacterium solution adds 0.15% formalin, and 37 DEG C of inactivations 24 are little
Shi Hou, 10000rpm are centrifuged 20min, take supernatant and are pasteurella multocida C48-2 strain acellular antigens.The C48-2 strains
Acellular antigens add 20% aluminium hydroxide gel Jing after steriling test is qualified, by volume, add thimerosal, and its is final concentration of
0.01%, C48-2 strain acellular vaccines, 4 DEG C of preservations are obtained final product after mixing.
The preparation of 1502 plants of acellular vaccines：1502 plants of freeze-drying lactobacillus of chicken pasteurella multocida are taken, is given birth to 1-2ml sterilizings
Reason salt water dissolution is followed by planting in 10%（V/V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 10%（V/V）Serum
Improvement martin's bouillon, puts 37 DEG C, and 200rpm concussion and cultivates 12h, bacterium solution adds 0.15% formalin, and 37 DEG C inactivate 24 hours
Afterwards, 10000rpm centrifugations 20min, takes supernatant and is 1502 plants of acellular antigens of pasteurella multocida.This 1502 plants without thin
Extracellular antigen adds 20% aluminium hydroxide gel Jing after steriling test is qualified, by volume, adds thimerosal, and its is final concentration of
0.01%, 1502 plants of acellular vaccines, 4 DEG C of preservations are obtained final product after mixing.
Embodiment five：The effect comparative test of rabbit pasteurella multocida acellular vaccine and whole-bacterial-vaccine
1st, the preparation of whole-bacterial-vaccine
Take rabbit pasteurella multocida C51-2 strains, C51-17 strains（It is purchased from China Veterinery Drug Inspection Office, bacterial strain preservation
Numbering is respectively CVCC499, CVCC1753）Freeze-drying lactobacillus, after being dissolved with 1-2ml sterile salines respectively 10% is inoculated in（V/
V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 10%（V/V）Serum improves martin's bouillon, puts 37 DEG C
200rpm concussion and cultivates 12h, are concentrated into respectively 6.25 × 109CFU/ml（The immunizing agent of rabbit pasteurella multocida inactivated vaccine
Measure as 5.0 × 109CFU/ml, bacterial concentration is 6.25 × 109CFU/ml, after adding 20% aluminium hydroxide gel, bacteria containing amount is 5.0
×109CFU/ml）, after adding 0.2% 37 DEG C of inactivation 24h of formalin, add 20% by volume Jing after steriling test is qualified
Aluminium hydroxide gel and final concentration of 0.01% thimerosal, after mixing i.e. be respectively the full bacterium epidemic disease of pasteurella multocida C51-2 strains
Seedling and C51-17 strain whole-bacterial-vaccines.
2nd, the effect comparative test of rabbit pasteurella multocida acellular vaccine and whole-bacterial-vaccine
2.1 it is immune：The healthy susceptible rabbit of screening body weight 1.5-2.0kg（Purchased from Henan Condar laboratory animal company limited）
60, it is randomly divided into 6 groups, respectively C51-2 strains acellular vaccine immune group, whole-bacterial-vaccine immune group, matched group and C51-17
Strain acellular vaccine immune group, whole-bacterial-vaccine immune group, matched group, 10/group.Acellular vaccine used by immunity is embodiment
C51-2 strains acellular vaccine and C51-17 strain acellular vaccines prepared by four, whole-bacterial-vaccine is above-mentioned C51-2 strains whole-bacterial-vaccine
With C51-17 strain whole-bacterial-vaccines, immunizing dose is 1ml/ only, and immunization route is nape part subcutaneous injection.
2.2 immune rabbit body weight change：Weigh to testing rabbit within 21 days before immunity, after immunity, respectively to full bacterium epidemic disease
The Gain weight of Seedling immune group, acellular vaccine immune group and matched group test rabbit carries out statistical analysiss.
The counteracting toxic substances of rabbit are tested after 2.3 immunity 21 days：The 21st day after immunity, together with matched group test rabbit using minimum lethal
Dosage carries out counteracting toxic substances by subcutaneous injection.Continuous Observation 7 days after counteracting toxic substances, the death condition of record test rabbit.
2.4 immune rabbit body weight statistical results
10 test rabbits of each group are weighed for 21 days before immunity, after immunity, and calculates its weightening, adopted with SPSS softwares
Analyzed with Duncan ' s multiple range test methods, as a result shown, 21 after the test rabbit immunity of whole-bacterial-vaccine immune group
It body weight and the weightening within 21 day time are below acellular vaccine immune group and matched group, and significant difference（P≤
0.05）, compared with matched group, its difference is not notable, refers to table 4 for the body weight change of acellular vaccine immune group test rabbit.
Table 4：The Gain weight of rabbit is tested during test
Remarks：Same row difference letter representation significant difference（P≤0.05, n=10）.
Result after 2.5 counteracting toxic substances：
21 days counteracting toxic substances after immunity, C51-2 strains and C51-17 strains matched group test rabbit occur 100%（10/10）Death,
C51-2 strain acellular vaccine immune group and C51-17 strain whole-bacterial-vaccine immune group are each dead 1, and remaining immune group does not occur extremely
The protective rate of phenomenon, i.e. whole-bacterial-vaccine and the acellular vaccine immune group of dying has reached more than 90%, refers to table 5.As a result show,
Acellular vaccine has equal immune efficacy with whole-bacterial-vaccine.
Table 5：The effect comparative test result of pasteurella multocida acellular vaccine and whole-bacterial-vaccine
Embodiment six：The effect comparative test of chicken pasteurella multocida acellular vaccine and whole-bacterial-vaccine
1st, the preparation of whole-bacterial-vaccine：
Chicken pasteurella multocida C48-2 strains and 1502 plants of freeze-drying lactobacillus are taken, is dissolved with 1-2ml sterile salines respectively
After be inoculated in 10%（V/V）After serum improvement Martin agar plate recovery, colony inoculation is taken in 10%（V/V）Serum improves Martin's meat
Soup, puts 37 DEG C, and 200rpm concussion and cultivates 12h are concentrated into respectively 6.25 × 109CFU/ml（Chicken pasteurella multocida inactivates epidemic disease
The immunizing dose of Seedling is 5.0 × 109CFU/ml, bacterial concentration is 6.25 × 109CFU/ml, after adding 20% aluminium hydroxide gel,
Bacteria containing amount is 5.0 × 109CFU/ml）, after adding 0.2% 37 DEG C of inactivation 24h of formalin, Jing after steriling test is qualified body is pressed
Product is respectively pasteurella multocida than the aluminium hydroxide gel for adding 20% and final concentration of by 0.01% thimerosal, after mixing
C48-2 strains whole-bacterial-vaccine and 1502 plants of whole-bacterial-vaccines.
2nd, the effect comparative test of chicken pasteurella multocida acellular vaccine and whole-bacterial-vaccine
2.1 it is immune：Select the healthy susceptible chicken of 10 ages in days of not inoculated fowl cholera vaccine（Using purchased from Beijing Cimmeria
Hatching egg oneself hatching of Wei Tong laboratory animals company is simultaneously raised in isolator）120,6 groups are randomly divided into, respectively C48-2
Strain acellular vaccine immune group, whole-bacterial-vaccine immune group, matched group and 1502 plants of acellular vaccine immune group, whole-bacterial-vaccine immunity
Group, matched group, 20/group.Acellular vaccine used by immunity be in example IV prepared C48-2 strains acellular vaccine and
1502 plants of acellular vaccines, whole-bacterial-vaccine is above-mentioned C48-2 strains whole-bacterial-vaccine and 1502 plants of whole-bacterial-vaccines, and immunizing dose is
Only, immunization route is nape part subcutaneous injection to 1ml/.
The change of 2.2 immune chicken body weight：Each test chicken was weighed in 21 days before immunity, after immunity, it is right respectively
The Gain weight of whole-bacterial-vaccine immune group, acellular vaccine immune group and matched group test rabbit carries out statistical analysiss.
The counteracting toxic substances of test chicken after 2.3 immunity 21 days：The 21st day after immunity, 10 tests are randomly selected per group together with matched group
Chicken, carries out counteracting toxic substances using minimal lethal dose by subcutaneous injection.Continuous Observation 14 days after counteracting toxic substances, record the death of test chicken
Situation.
2.4 immune chicken body weight statistical results
10 test chickens of each group are weighed for 21 days before immunity, after immunity, and calculates its weightening, adopted with SPSS softwares
Analyzed with Duncan ' s multiple range test methods, as a result shown, weight differences are notable before immunity, but full bacterium
Vaccine immunity group test chicken immune after 21 days body weight and the weightening within 21 day time be below acellular vaccine immune group and
Matched group, and significant difference（P≤0.05）, the body weight change of acellular vaccine immunity test chicken compared with matched group, its difference
Not significantly, table 6 is referred to.
Table 6：The Gain weight of test chicken during test
Remarks：Same row difference letter representation significant difference（P≤0.05, n=10）.
Result after 2.5 counteracting toxic substances：
Counteracting toxic substances protective rate：21 days counteracting toxic substances after immunity, C48-2 strains and 1502 plants of matched group test chickens occur 100%（10/
10）Death, C48-2 strain acellular vaccine immune group, C48-2 strains and 1502 plants of whole-bacterial-vaccine immune group are each dead 1,1502
Strain acellular vaccine immune group it is dead 2, i.e., the protective rate of whole-bacterial-vaccine and acellular vaccine immune group reached 80% with
On, refer to table 7.As a result show, acellular vaccine has equal immune efficacy with whole-bacterial-vaccine.
Table 7：The effect comparative test result of pasteurella multocida acellular vaccine and whole-bacterial-vaccine
Embodiment seven：The detection of vaccine injection local side reaction
21 days immune group after rabbit pasteurella multocida acellular vaccine and whole-bacterial-vaccine are immune in random Example five
Test rabbit it is each 2, air tap inserting method put to death after cut open inspection vaccine injection site, check vaccine injection site local side reaction, by with
Lower standard is scored：Person remembers 4 points to touch tuberosity before dissection；There is suppuration phenomenon person to remember 3 points after dissection, have obvious redness person to remember
2 points, rarely seen injection vestige person remembers 1 point, and acellular vaccine immune group and whole-bacterial-vaccine immune group test rabbit injection part are counted respectively
Position side reaction score, adopts Duncan ' s multiple range test methods to analyze with SPSS softwares.
As a result find, the vaccine injection site of whole-bacterial-vaccine immune group test rabbit can touch hard tubercle before dissecting, and dissect
There is suppuration phenomenon afterwards, and the vaccine injection site of acellular vaccine immune group test rabbit can not touch hard tubercle before dissecting,
Also suppuration is had no after dissection, the red and swollen inflammatory reaction in rarely seen part, according to the scoring criteria of local side reaction, acellular vaccine is exempted from
2.5 points of epidemic disease group injection site local side reaction average, substantially less than whole-bacterial-vaccine immune group injection site local side reaction
7.5 points of average, refers to Fig. 1 and Fig. 2.
As can be seen here, pasteurella multocida acellular antigens provided by the present invention is added and made after adjuvant, preservative
It is standby that into acellular vaccine, the acellular vaccine has an equal immune efficacy of whole-bacterial-vaccine, and with not affecting growth of animal, epidemic disease
The little advantage of Seedling injection site local side reaction.
Embodiment disclosed above is only illustrated demonstration, the invention is not limited in this.Obtained using different culture media
Pasteurella multocida bacterium solution, or after solid medium culture with the liquid such as physiological saline solution wash it is lower after the bacterium solution that obtains,
The culture supernatant i.e. acellular antigens obtained through different separation methods belong to the protection domain of this patent；Using difference
Pasteurella multocida acellular vaccine containing polysaccharide and phage surface protein prepared by bacterial strain belongs to this patent protection domain；
In addition, except rabbit and chicken involved in embodiment, the pasteurella multocida acellular vaccine of other animal originality belongs to
In the protection domain of this patent.
Claims (9)
1. a kind of pasteurella multocida acellular antigens, it is characterised in that killing property Pasteur bars of the antigen from inactivation more
The supernatant of bacterium culture, the supernatant contains polysaccharide and albumen；Wherein, polyoses content is 0.5-0.7mg/ in the antigen
Ml, protein content is 8.8-10.6mg/ml in the antigen；
The preparation method of the antigen includes：Pasteurella multocida is inoculated in the culture medium containing serum, at 30-40 DEG C
Culture 8-24h, obtains pasteurella multocida culture, pasteurella multocida culture centrifugation or directly heavy Jing after inactivation
Form sediment, abandon thalline, by taking supernatant the pasteurella multocida acellular antigens containing polysaccharide and albumen simultaneously are obtained.
2. pasteurella multocida acellular antigens according to claim 1, it is characterised in that killing property Pasteur's bars more
Bacterium acellular antigens are animal pasteurella multocida acellular antigens.
3. pasteurella multocida acellular antigens according to claim 2, it is characterised in that the animal be pig, rabbit,
Chicken, cattle or sheep.
4. pasteurella multocida acellular antigens according to claim 1, it is characterised in that the concentration base of the serum
In the culture medium be 2-20% (V/V).
5. pasteurella multocida acellular antigens according to claim 1, it is characterised in that the culture medium is improvement
Martin's culture medium.
6. the antigen described in any one of claim 1-5 is in for the pasteurella multocida Serologic detection of non-diagnostic purpose
Application.
7. a kind of pasteurella multocida acellular vaccine, it is characterised in that containing many as described in any one of claim 1-5
Killing property pasteurellosis bacilluss acellular antigens, adjuvant and preservative.
8. vaccine according to claim 7, it is characterised in that the pasteurella multocida acellular antigens are in vaccine
Final concentration of 70-95% (V/V)；Final concentration of 4-29% (V/V) of the adjuvant in vaccine；The preservative is sulfur willow
Hydrargyrum, its final concentration of 0.001-0.01% (V/V) in vaccine.
9. a kind of method for preparing pasteurella multocida acellular vaccine, it is characterised in that the method includes：By killing property bar more
Family name bacillus is inoculated in the culture medium containing serum, and at 30-40 DEG C 8-24h is cultivated, and obtains pasteurella multocida culture, and this is more
Killing property pasteurellosis bacilluss culture centrifugation or Direct precipitation Jing after inactivation, abandon thalline, while being contained polysaccharide by taking supernatant
With the pasteurella multocida acellular antigens of albumen, the antigen adds adjuvant and thimerosal, mixes Jing after steriling test is qualified
Pasteurella multocida acellular vaccine is afterwards.
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