CN106267180A - A kind of actinomyces pseudonecrophorus acellular antigens and preparation method thereof and the vaccine of preparation - Google Patents
A kind of actinomyces pseudonecrophorus acellular antigens and preparation method thereof and the vaccine of preparation Download PDFInfo
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Abstract
The present invention relates to actinomyces pseudonecrophorus vaccines arts, particularly to a kind of actinomyces pseudonecrophorus acellular antigens and preparation method thereof and the vaccine of preparation.A kind of actinomyces pseudonecrophorus acellular antigens, for the actinomyces pseudonecrophorus cellular lysate product of inactivation.The present invention is through test of many times, use the mode of cracking thalline, the pyrolysis product obtained separates, remove the endotoxin and other invalid components contained, retain its Effective Antigens composition, the vaccine that the actinomyces pseudonecrophorus acellular antigens that inactivation obtains is made easily forms scleroma in injection site after overcoming currently available vaccines, existing vaccines inoculation, the side reaction such as partial gall or heating occurs, and the vaccine immunity of preparation is stable and reliable for performance.
Description
Technical field
The present invention relates to actinomyces pseudonecrophorus vaccines arts, in particular to a kind of actinomyces pseudonecrophorus acellular antigens and
Its preparation method and the vaccine of preparation.
Background technology
Foot rot is the one subacute high degree in contact sexually transmitted disease that the artiodactyls such as cattle, sheep, deer are common, clinical symptoms bag
Include the difficulty that rises and retires, unreal, the appetite of bearing a heavy burden when standing decline or useless absolutely, milk yield drastically declines；Severe patient hoof tips matter separates, even
Cause death.Foot rot causes tremendous economic to lose the most all to China or even global cattle, sheep, the aquaculture of deer.
At present, foot rot clinical prevention can be used without commercialized vaccine, mainly based on clinical treatment and antibiotic therapy, and control
Poor effect processed causes antibiotic to be abused the unfavorable situation in animal farming industry on the contrary；Ruminant aquaculture needs high-quality
Commercialized vaccine.At the end of last century, Gao Yun goes through to study for many years successfully to develop and is applicable to
The actinomyces pseudonecrophorus inactivation whole-bacterial-vaccine of cattle and deer.But easily form scleroma after this vaccination in injection site, local occurs
Swell and ache or the side reaction such as heating, have a strong impact on animal welfare, cause vaccine the most accepted by the public.
In view of this, the special proposition present invention.
Summary of the invention
The first object of the present invention is to provide a kind of actinomyces pseudonecrophorus acellular antigens, by by actinomyces pseudonecrophorus thalline
The supernatant inactivation that cracking obtains prepares, and solves actinomyces pseudonecrophorus inactivation whole-bacterial-vaccine in prior art and contains endotoxic lacking
Fall into.
The second object of the present invention is to provide the preparation method of a kind of described actinomyces pseudonecrophorus acellular antigens, the party
Method, by being cracked by actinomyces pseudonecrophorus thalline, is then peeled off, and no longer contains endotoxin, and prepare in the supernatant obtained
Antigen be well positioned to meet the demand preparing vaccine.
The third object of the present invention is to provide the vaccine prepared by above-mentioned antigen.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
A kind of actinomyces pseudonecrophorus acellular antigens, for the actinomyces pseudonecrophorus cellular lysate product of inactivation.
The present inventor inactivates whole-bacterial-vaccine research to the actinomyces pseudonecrophorus of prior art and finds, in injection after this vaccination
Position easily forms scleroma, occurs that the side reaction such as partial gall or heating is owing to this whole-bacterial-vaccine does not contain only the anti-of high concentration
Original matter, and containing high concentration endotoxin.The present invention, through test of many times, uses the mode of cracking thalline, and the cracking obtained is produced
Thing inactivates, and vaccine prepared by the actinomyces pseudonecrophorus acellular antigens obtained overcomes after currently available vaccines, existing vaccines is inoculated in injection part
The shortcoming that position forms the side reactions such as scleroma, partial gall and heating.
Further, any one or more during described actinomyces pseudonecrophorus derives from cattle, deer and sheep.
Present invention also offers the preparation method of actinomyces pseudonecrophorus acellular antigens, actinomyces pseudonecrophorus is cracked, from
Gains in depth of comprehension to supernatant after inactivation treatment, be described actinomyces pseudonecrophorus acellular antigens.
The preparation method of the actinomyces pseudonecrophorus acellular antigens that the present invention provides, cracks thalline so that it is content
Discharge, by separating, remove the endotoxin and other invalid compositions contained, retain its Effective Antigens composition, then
Inactivated and obtained actinomyces pseudonecrophorus acellular antigens.Preparing simple and easy to do, the actinomyces pseudonecrophorus acellular antigens obtained is made
Vaccine overcome the postvaccinal side reaction of currently available vaccines, existing vaccines.
Further, described cracking uses subtilin to carry out enzymolysis.Through multiple authentication, use subtilin to bad
Dead Fusobacterium carries out enzymolysis, and preparation method is convenient, and the antigen of preparation is effective.
Preferably, the pH value of the suspension of described enzymolysis is 7.7-7.8, and described suspension is the actinomyces pseudonecrophorus that will collect
Thalline suspends with aquesterilisa and prepares, and the pH aqueous slkali of described suspension is adjusted.By the pH of regulation thallus suspension liquid it is
Subtilin provides good enzymatic hydrolysis condition.
Preferably, described thalline is 1:3-5 with the weight ratio of described water.
Preferably, described aqueous slkali is strong alkali solution, and the mass concentration of described strong alkali solution is 8%-15%.
Further, the temperature of the resuspended thalline of described aqueous slkali is 37-40 DEG C.So that thalline resuspended uniformly, and be
Enzymolysis provides good condition.
Further, any one or two kinds of during described highly basic includes sodium hydroxide and potassium hydroxide.
Through test, under certain temperature conditions, enzymolysis is more complete, enzymatic hydrolysate be easier to remove containing endotoxin,
Preferably, the addition of described subtilin is the 4%-5% of thalline weight；The temperature of enzymolysis is 48-50 DEG C, enzymolysis time
For 12-17min.
In order to reach more preferable hydrolysis result, and follow-up separation, it is preferable that the mistake of subtilin enzymolysis thalline
Cheng Zhong, the pH keeping thallus suspension liquid is 7.2-7.6.
Further, described inactivation uses formalin to carry out.
Further, described inactivation is interpolation formalin in described supernatant, then processes 4.5-in 36-38 DEG C
5.5 days, period shook frequently.
Preferably, the protein concentration of described supernatant is 8-12mg/mL, and the formalin of interpolation accounts for the body of described supernatant
Long-pending percent is 0.45%-0.55%.
Further, the actinomyces pseudonecrophorus of cracking is: actinomyces pseudonecrophorus is inoculated in anaerobic culture medium, cultivates to logarithm raw
The thalline obtained for a long time.The thalli growth of exponential phase is vigorous, is also easy to cracking, and the endotoxin wherein accumulated is few.
Preferably, described anaerobic culture medium is brain heart leachate broth bouillon or sulphur glycollate culture medium.Both
Culture medium is existing, by commercially available purchase, or can configure according to commercially available formula.
Preferably, the rotating speed of described centrifugal employing is 12000-13000rpm.The thalline of cracking is centrifuged by this rotating speed,
To supernatant in eliminate endotoxin and other invalid compositions, remain Effective Antigens composition.
Further, described actinomyces pseudonecrophorus inoculum concentration is 5%-10%, and the temperature of cultivation is 35-39 DEG C, cultivation time
Between be 8-24h.By this inoculum concentration and cultivation temperature and time, the thalline obtained is in exponential phase.
Present invention also offers the actinomyces pseudonecrophorus vaccine prepared by above-mentioned actinomyces pseudonecrophorus acellular antigens.This vaccine
Prepared by the method that can use routine.
Further, arbitrary during described vaccine is white-oil adjuvant vaccine, aluminium glue Adjuvanted vaccines, Freund's complete adjuvant vaccine
Kind.
Specifically, white-oil adjuvant vaccine is prepared by the following method: add Tween80 in the supernatant of the inactivation of preparation,
Then prepare white-oil adjuvant vaccine with adjuvant colloid mill emulsifying, determine lot number 1；
Wherein, the mass concentration of Tween80 is 3.5%-4.5%；Adjuvant is: No. 10 white oils, Span 80 and aluminium stearate
Mixture with mass ratio as 90:8:2.
Aluminium glue Adjuvanted vaccines is prepared by the following method: the supernatant of the inactivation of preparation adds for 3:6-8 by weight proportion
Aluminium adjuvant, being subsequently adding mass concentration is that 0.01% thimerosal fully vibrates, and places 24h at 2～10 DEG C of dark cold places, and period is frequently
Shake, makes fully to adsorb, prepares aluminium glue Adjuvanted vaccines, determine lot number 2.
Freund's complete adjuvant vaccine is prepared by the following method: the supernatant of the inactivation of preparation is 1% to add by mass concentration
Enter Tween80, then add Freund's complete adjuvant by weight for 5:6-4:5, use colloid mill emulsifying, prepare vaccine, determine lot number
3。
Three kinds of vaccines all move into 2～10 DEG C of dark cold places after aseptic, safety verification is qualified and preserve.Final guarantee formaldehyde is the denseest
Degree is less than 1%.
In addition, it is necessary to explanation, the present inventor also carries out alkaline lysis, E.C. 3.4.21.64 cracking, ultrasound wave to actinomyces pseudonecrophorus
Cracking and freezing-thawing and cracking etc., all remain more endotoxin in the supernatant of isolated after cracking, the vaccine made
Side reaction is basically identical with existing whole-bacterial-vaccine effect, all can not solve currently available vaccines, existing vaccines inoculation after in injection site easy shape
The problem become scleroma, the toxicity of partial gall or heating etc. occurring.
Compared with prior art, the invention have the benefit that
(1) present invention is by the way of cracking thalline, and pyrolysis product inactivates after separating, and the actinomyces pseudonecrophorus obtained is acellular
The vaccine that antigen is made easily forms scleroma after overcoming currently available vaccines, existing vaccines inoculation in injection site, it may appear that partial gall or send out
The toxicity of heat etc..
(2) present invention also offers the step of concrete cracking actinomyces pseudonecrophorus, by separating, it is interior that removal may contain
Toxin and other invalid antigenic components, retain its Effective Antigens composition, and the actinomyces pseudonecrophorus acellular antigens obtained is made
Vaccine overcome the postvaccinal side reaction of currently available vaccines, existing vaccines, and immune performance is stable.
(3) present invention also offers different types of vaccine, immune effect is reliable and stable.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but those skilled in the art will
Understanding, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.In embodiment unreceipted specifically
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument unreceipted production firm person, be
Can be by the commercially available conventional products bought and obtain.
Embodiment 1
A kind of actinomyces pseudonecrophorus acellular antigens, is prepared by the following method:
Cattle source actinomyces pseudonecrophorus is inoculated in anaerobic culture medium brain heart leachate broth bouillon, actinomyces pseudonecrophorus inoculum concentration
Being 5%, the temperature of cultivation is 39 DEG C, and the time of cultivation is 24h, cultivates and obtains being in the actinomyces pseudonecrophorus of exponential phase；
Actinomyces pseudonecrophorus bacterium solution cultivation obtained is centrifuged, the centrifugal continuous flow centrifuge that uses, use continuous flow centrifuge
During centrifugal actinomyces pseudonecrophorus culture, centrifuge rotor, tube for transfusion need boiling sterilization or autoclaving, install under aseptic condition
Rotor and tube for transfusion；
Bacterium solution enters the flow velocity of continuous flow centrifuge and is about 100mL/min, and centrifugal rotating speed is 12000rpm, centrifugal
5min, obtains bacterial sediment；
Bacterial sediment sterile purified water is resuspended, and bacterial sediment is 1:3 with the part by weight of distilled water, obtains thalline and suspends
Liquid；
Under the conditions of 37-40 DEG C, it is 7.7 that thallus suspension liquid 10%NaOH solution adjusts its pH value, rises high-temperature to 48
DEG C, adding the aqueous solution of subtilin, subtilin mass concentration in its aqueous solution is 50%, subtilin
Addition is the 4% of bacterial sediment thing weight；
The pH value of thallus suspension liquid is checked so that it is being maintained at 7.2-7.6, enzymolysis time is 12min every 15min；
After enzymolysis completes, being cooled to room temperature, 12000rpm is centrifuged 5min, collects supernatant；
Sample and measure tropina content according to Lowry method, utilize sterile purified water to be diluted to protein concentration for 8mg/mL,
Standby；
In the hydrolyzate of preparation, add formalin according to final concentration 0.45%, be placed in 36 DEG C of incubators inactivation 5.5
My god, period frequently shakes and guarantees that inactivation uniformly, obtains actinomyces pseudonecrophorus acellular antigens.
Embodiment 2
A kind of actinomyces pseudonecrophorus acellular antigens, is prepared by the following method:
Cattle source actinomyces pseudonecrophorus is inoculated in anaerobic culture medium sulphur glycollate culture medium, and actinomyces pseudonecrophorus inoculum concentration is
8%, the temperature of cultivation is 37 DEG C, and the time of cultivation is 12h, cultivates and obtains being in the actinomyces pseudonecrophorus of exponential phase；
Actinomyces pseudonecrophorus bacterium solution cultivation obtained is centrifuged, the centrifugal continuous flow centrifuge that uses, use continuous flow centrifuge
During centrifugal actinomyces pseudonecrophorus culture, centrifuge rotor, tube for transfusion need boiling sterilization or autoclaving, install under aseptic condition
Rotor and tube for transfusion；
Bacterium solution enters the flow velocity of continuous flow centrifuge and is about 110mL/min, and centrifugal rotating speed is 12000rpm, centrifugal
5min, obtains bacterial sediment；
Bacterial sediment sterile purified water is resuspended, and bacterial sediment is 1:4 with the part by weight of distilled water, obtains thalline and suspends
Liquid；
Under the conditions of 37-40 DEG C, it is 7.8 that thallus suspension liquid 10%NaOH solution adjusts its pH value, rises high-temperature to 50
DEG C, adding the aqueous solution of subtilin, subtilin mass concentration in its aqueous solution is 50%, subtilin
Addition is the 4.5% of bacterial sediment thing weight；
The pH value of thallus suspension liquid is checked so that it is being maintained at 7.2-7.6, enzymolysis time is 15min every 15min；
After enzymolysis completes, being cooled to room temperature, 12000rpm is centrifuged 5min, collects supernatant；
Sample and measure tropina content according to Lowry method, utilize sterile purified water to be diluted to protein concentration for 10mg/
ML, standby；
In the hydrolyzate of preparation, uniformly add formalin according to final concentration 0.5%, be placed in 37 DEG C of incubators inactivation 5
My god, period frequently shakes and guarantees that inactivation uniformly, obtains actinomyces pseudonecrophorus acellular antigens.
Embodiment 3
A kind of actinomyces pseudonecrophorus acellular antigens, is prepared by the following method:
Cattle source actinomyces pseudonecrophorus is inoculated in anaerobic culture medium brain heart leachate broth bouillon, actinomyces pseudonecrophorus inoculum concentration
Being 10%, the temperature of cultivation is 35 DEG C, and the time of cultivation is 8h, cultivates and obtains being in the actinomyces pseudonecrophorus of exponential phase；
Actinomyces pseudonecrophorus bacterium solution cultivation obtained is centrifuged, the centrifugal continuous flow centrifuge that uses, use continuous flow centrifuge
During centrifugal actinomyces pseudonecrophorus culture, centrifuge rotor, tube for transfusion need boiling sterilization or autoclaving, install under aseptic condition
Rotor and tube for transfusion；
Bacterium solution enters the flow velocity of continuous flow centrifuge and is about 120mL/min, and centrifugal rotating speed is 13000rpm, centrifugal
3min, obtains bacterial sediment；
Bacterial sediment sterile purified water is resuspended, and bacterial sediment is 1:5 with the part by weight of distilled water, obtains thalline and suspends
Liquid；
Under the conditions of 37-40 DEG C, it is 7.8 that thallus suspension liquid 10%NaOH solution adjusts its pH value, rises high-temperature to 50
DEG C, adding the aqueous solution of subtilin, subtilin mass concentration in its aqueous solution is 50%, subtilin
Addition is the 5% of bacterial sediment thing weight；
Checking the pH value of thallus suspension liquid every 15min so that it is be maintained at 7.2-7.6, enzymolysis time is 17min；
After enzymolysis completes, being cooled to room temperature, 13000rpm is centrifuged 3min, collects supernatant；
Sample and measure tropina content according to Lowry method, utilize sterile purified water to be diluted to protein concentration for 12mg/
ML, standby；
In the hydrolyzate of preparation, add formalin according to final concentration 0.5%, be placed in 38 DEG C of incubators inactivation 4.5
My god, period frequently shakes and guarantees that inactivation uniformly, obtains actinomyces pseudonecrophorus acellular antigens.
Test example
The actinomyces pseudonecrophorus acellular antigens of embodiment 2 preparation is prepared as vaccine respectively, and the kind of vaccine is white-oil adjuvant
Vaccine, aluminium glue Adjuvanted vaccines, Freund's complete adjuvant vaccine；
White-oil adjuvant vaccine is prepared by the following method: add Tween80 in the supernatant of the inactivation of preparation, then with assistant
Agent colloid mill emulsifying prepares white-oil adjuvant vaccine, is No. 1 vaccine；
Wherein, the mass concentration of Tween80 is 4%；Adjuvant is: No. 10 white oils, Span 80 and aluminium stearate are with mass ratio
Mixture for 90:8:2；
Aluminium glue Adjuvanted vaccines is prepared by the following method: the supernatant of the inactivation of preparation adds aluminum for 3:7 by weight proportion
Adjuvant, being subsequently adding mass concentration is that 0.01% thimerosal fully vibrates, and places 24h at 2～10 DEG C of dark cold places, and period shakes frequently
Dynamic, make fully to adsorb, prepare aluminium glue Adjuvanted vaccines, be No. 2 vaccines；
Freund's complete adjuvant vaccine is prepared by the following method: the supernatant of the inactivation of preparation is 1% to add by mass concentration
Enter Tween80, then add Freund's complete adjuvant by weight for 1:1, use colloid mill emulsifying, be prepared into vaccine, be No. 3 epidemic diseases
Seedling；
Three kinds of vaccines all move into 2～10 DEG C of dark cold places after aseptic, safety verification is qualified and preserve.
Different types of vaccine of preparation carries out immunity test.
1, rabbit immunity test
Testing rabbit, be randomly divided into 4 groups for 20, often group 5, point cage routine is raised.No. 1 vaccine of first group of subcutaneous injection
0.5mL, No. 2 vaccine 0.5mL of second group of subcutaneous injection, No. 3 vaccine 0.5mL of the 3rd group of subcutaneous injection, the injection site basal part of the ear is subcutaneous,
4th group is blank group.Take a blood sample before vaccinating and after vaccinating, and 28d tests rabbit to each group after vaccinating
The actinomyces pseudonecrophorus (10 of 2 fatal dose of basal part of the ear subcutaneous injection8Cells/mL, 2mL), observe the changes such as spirit, appetite, in detail
Record.Result is as shown in Table 1 and Table 2.
Table 1 rabbit challenge test result
The antibody change in 3 kinds of vaccine rabbit anteserums inoculated by table 2
Note: have " X " only to detect antibody in " X/Y " expression " Y ", as " 3/4 " represents that four rabbits have three to detect antibody.
As it can be seen from table 1 the vaccine that the present invention provides only has slight untoward reaction, in injection site after vaccination
Scleroma will not be formed, also do not have the toxicity such as partial gall or heating.And immune effect is preferable.
Table 2 result shows, inoculates three kinds of vaccine rabbits before 14d, is exempted from rabbit anteserum counter immunoelectrophoresis and be negative, after 3 weeks
Gradually producing antibody, trace detection to 22 week, tested rabbit anteserum counter immunoelectrophoresis is still positive.
In table 2, the actinomyces pseudonecrophorus bacterium solution starting to inoculate 2 fatal dose in 4 weeks after immunity due to test rabbit is attacked
Poison, investigates the counteracting toxic substances protection of vaccine；Occur so having the experimental rabbit phenomena of mortality；Even if there is no death, owing to choosing test rabbit
Carry out anatomy experiment, the situation of change of the substantial viscera of viewing test rabbit, compare analysis with matched group, so having test
Rabbit quantity reduces situation and occurs.
2, three kinds of vaccines immunity test to milch cow
544 milch cows of the packet of 2.1 milch cows are randomly divided into 3 groups, 1 group 167,2 groups 231,3 groups 146, often organize again with
Machine is divided into 2 groups, and for vaccinating group and matched group, concrete packet situation is as shown in table 3.
Table 3 immunity milch cow packet mode
| Grouping serial number | Sum | Vaccinate group | Matched group |
| 1 | 167 | 87 | 80 |
| 2 | 231 | 126 | 105 |
| 3 | 146 | 92 | 54 |
Group 1,2,3 vaccinates group 1,2, No. 3 vaccines of correspondence injection, every cow head basal part of the ear subcutaneous injection vaccine 4mL respectively；
Matched group does not vaccinates；Statistics morbidity number, concrete outcome is as shown in table 4.
Table 4 immunity test result
After No. 1 vaccine and No. 2 vaccines are to cattle immunity, there is indivedual cattle that spirit, inappetence, transference cure after 3d occur；No. 3
After vaccine is to cattle immunity, local non-bacterial suppurates to have more than 10 cattle to occur, ulceration after month, always protracted course of disease, in field
The wound of veterinary cut-away portions cattle, fully recovers after process, and untreated cattle is suppurated after half a year and fades away.Suspect No. 3 vaccines be due to
Additive in addition to antigen causes immune animal cattle more uncomfortable phenomenon occur.
It addition, change the interpolation of each composition in white-oil adjuvant vaccine, aluminium glue Adjuvanted vaccines and Freund's complete adjuvant vaccine
Amount, the most within the scope of the present invention to obtain more superior immune effect.
Application SPSS software carries out double factor analysis to table 4, three kinds of different vaccine F=1.344, P=0.273 > 0.05, no
With the immune effect zero difference between adjuvant；Application vaccine before and after F=323.151, P=0.0001 < 0.01, i.e. application vaccine before and after
Effect is notable.
The vaccine utilizing preparation has carried out field test, by the SPSS software field test to table 4 on this animal milch cow
Interpretation of result, application paired-samples T-test, T=-5.675, P=0.000 < 0.01, significant difference before and after vaccine immunity, vaccine is described
Respond well.
It addition, the actinomyces pseudonecrophorus acellular antigens that embodiment 1 and embodiment 3 are also prepared by the present invention uses and embodiment
It is complete that the identical method of actinomyces pseudonecrophorus acellular antigens of 2 preparations prepares white-oil adjuvant vaccine, aluminium glue Adjuvanted vaccines, Freund respectively
Full Adjuvanted vaccines, and it is carried out the checking of performance, the vaccine effect that result is all prepared with embodiment 2 is consistent.
The cattle source actinomyces pseudonecrophorus that the present invention provides has good immunogenicity, and the present invention prepares cattle the most at home
Necrobacillus disease vaccine, animal experiment proves, vaccine safety is effective.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that without departing substantially from the present invention's
May be made that in the case of spirit and scope many other change and amendment.It is, therefore, intended that in the following claims
Including all such changes and modifications belonged in the scope of the invention.
Claims (10)
1. an actinomyces pseudonecrophorus acellular antigens, it is characterised in that for the actinomyces pseudonecrophorus cellular lysate product of inactivation.
Actinomyces pseudonecrophorus acellular antigens the most according to claim 1, it is characterised in that described actinomyces pseudonecrophorus derives from
Any one or more in cattle, deer and sheep.
3. the preparation method of the actinomyces pseudonecrophorus acellular antigens described in claim 1 or 2, it is characterised in that by necrosis shuttle bar
Bacterium cracks, and the centrifugal supernatant obtained is described actinomyces pseudonecrophorus acellular antigens after inactivation treatment.
Preparation method the most according to claim 3, it is characterised in that described in be cracked into: use subtilin carry out enzyme
Solve；
Preferably, the pH value of the suspension of described enzymolysis is 7.7-7.8, and described suspension is the actinomyces pseudonecrophorus thalline that will collect
Preparing with aqueous suspension, described thalline is preferably 1:3-5 with the weight ratio of described water, and the pH aqueous slkali of described suspension is adjusted
Joint；
Preferably, described aqueous slkali is strong alkali solution, and the mass concentration of described strong alkali solution is 8%-15%, and described alkali is molten
The temperature of the resuspended thalline of liquid is 37-40 DEG C.
Preparation method the most according to claim 4, it is characterised in that the addition of described subtilin is thalline weight
4%-5%, the temperature of enzymolysis is 48-50 DEG C, and enzymolysis time is 12-17min；
Preferably, during subtilin enzymolysis thalline, the pH keeping thallus suspension liquid is 7.2-7.6.
Preparation method the most according to claim 3, it is characterised in that described inactivation uses formalin to carry out；
Further, described inactivation is interpolation formalin in described supernatant, then processes 4.5-5.5 days in 36-38 DEG C,
Period shakes frequently；
Preferably, the protein concentration of described supernatant is 8-12mg/mL, and the formalin of interpolation accounts for the volume hundred of described supernatant
Mark is 0.45%-0.55%.
7. according to the preparation method described in any one of claim 3-6, it is characterised in that the actinomyces pseudonecrophorus of cracking is: by bad
Dead Fusobacterium is inoculated in anaerobic culture medium, cultivates the thalline obtained to exponential phase；
Described anaerobic culture medium is preferably brain heart leachate broth bouillon or sulphur glycollate culture medium；
Preferably, the rotating speed of described centrifugal employing is 12000-13000rpm.
8. according to the preparation method described in any one of claim 3-6, it is characterised in that described actinomyces pseudonecrophorus inoculum concentration is
5%-10%, the temperature of cultivation is 35-39 DEG C, and the time of cultivation is 8-24h.
9. the actinomyces pseudonecrophorus vaccine prepared by the actinomyces pseudonecrophorus acellular antigens described in claim 1 or 2.
Vaccine the most according to claim 9, it is characterised in that described vaccine is white-oil adjuvant vaccine, aluminium glue adjuvant epidemic disease
Any one in Seedling, Freund's complete adjuvant vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610813747.8A CN106267180B (en) | 2016-09-09 | 2016-09-09 | Fusobacterium necrophorum cell-free antigen, preparation method thereof and prepared vaccine |
Applications Claiming Priority (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2043770C1 (en) * | 1993-02-15 | 1995-09-20 | Лавченко Елена Григорьевна | Method of preparing vaccine against necrobacteriosis in animals |
| CN105106946A (en) * | 2015-09-08 | 2015-12-02 | 四川省畜牧科学研究院 | Oil emulsion vaccine for cattle and sheep foot rot and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2043770C1 (en) * | 1993-02-15 | 1995-09-20 | Лавченко Елена Григорьевна | Method of preparing vaccine against necrobacteriosis in animals |
| CN105106946A (en) * | 2015-09-08 | 2015-12-02 | 四川省畜牧科学研究院 | Oil emulsion vaccine for cattle and sheep foot rot and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 苗利光等: "坏死梭杆菌FN(A)型毒力株保护性抗原筛选及初步鉴定", 《畜牧兽医学报》 * |
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