CN104140940B - A kind of ablation method of tarda and its application - Google Patents
A kind of ablation method of tarda and its application Download PDFInfo
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- CN104140940B CN104140940B CN201410350754.XA CN201410350754A CN104140940B CN 104140940 B CN104140940 B CN 104140940B CN 201410350754 A CN201410350754 A CN 201410350754A CN 104140940 B CN104140940 B CN 104140940B
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Abstract
The present invention relates to the ablation method and its inactivated vaccine in aquiculture animal disease control field, more particularly to a kind of tarda, i.e., using H+Concentration range is 10‑7Mol/L -1mol/L acid solution, at 0 DEG C -100 DEG C, inactivates 10min -48h, and complete inactivation is carried out to tarda, and compared with prior art, inactivation tarda vaccine prepared by the present invention can effectively maintain the immunogenicity of tarda antigen.
Description
Technical field
The present invention relates to aquiculture animal disease control field, more particularly to a kind of ablation method of tarda and
It is applied.
Background technology
Tarda (Edwardsiella sp.) includes Edwardsiella tarda (E.tarda), catfish Ai Dehuashi
Bacterium (E.ictaluri) and guarantor section tarda (E.hoshinae), are one of the main pathogenic fungis in aquaculture, its place
Main scope is quite varied, and fish, amphibian animal, reptiles, birds and the mammality including the mankind have infection to report, and are in generation
Criticality be distributed, be prevalent in fresh water and briny environment, be mainly distributed on China, Australia, Japan, India, Israel,
The Perenniporia martius countries and regions such as Malay Archipelago, the U.S., Panama.From reporting first so far, the bacterium is in a variety of cultivation fishes
Trigger disease in class and cause massive losses.At present frequently with chemotherapeutant and antibiosis in fish disease preventing and treating
Element, but as their use, cause of disease drug resistance also strengthen therewith, and medicament residue and diffusion in the environment have caused
The problem of in terms of food security, environment and public health.Pollution will not be produced after fish vaccine use, will not be in immune fish body
It is interior to form residual, environmental pollution can be avoided and maintain the quality of fish body in itself, Reusability will not also produce drug resistance, application
Have a extensive future.
At present, fish vaccine in the application, include inactivated vaccine, attenuated live vaccine, DNA vaccination, subunit vaccine and
The forms such as ghost vaccine, wherein, the inactivated vaccine R＆D cycle is short, low manufacture cost, security are good, is guaranteed in Immune efficiency
In the case of be easiest to get the Green Light, therefore as current commercial vaccine main species.But research shows, Ai Dehuashi
Bacterium is different from many pathogens such as vibrios, and the tarda inactivated vaccine prepared using routine techniques can not produce to tested fish
Effective immune protective.Gutierrez and Miyazaki (1994, Dis Aquat Org.6:110117.) research in Fu Er
The relative protection ratio of Edwardsiella tarda injecting immune Cuscuta japonicoa of Malin's inactivation is only 12.5-25%, our previous experiments
The relative protection ratio (RPS) of the tarda inactivated vaccine made using not optimized routine techniques is also less than 40%；
(1995, the Fish Pathol.30 (4) such as Mekuchi:Intramuscular injection, immersion or oral inoculation side 251-256) is respectively adopted
Lefteye flounder is immunized in the Edwardsiella tarda of formula Formalin inactivation, but without the effective immune protective effect of acquirement；Sun etc.
(2011,Fish Shellfish Immunol.31(4):Although 595-599.) also reporting the inactivated vaccine of tarda,
The immune protective rate of the monovalent inactivated vaccine of tarda only has 33.3%, the only tarda when inactivation and Vibrio anguillarum
When being used in combination etc. another three kinds of bacterium, other three kinds of bacterium inactivated vaccines such as preferable immune protective rate, Vibrio anguillarum could be obtained in fact
The effect of immunologic adjuvant is served for tarda inactivated vaccine.On the whole, with reference to our own many experiments result
And most report, the immune effect of tarda inactivated vaccine is preferable not to the utmost, and this is also likely to be that people have to spend
Bigger cost carries out the reason for research of other kinds of vaccine.
In the application report of the attenuated live vaccines of tarda, Klesius etc. (2000) invention (US
Patent6153202 invention (the US of (2006) such as a kind of Edwardsiella ictaluri attenuated live vaccine, Evans) is disclosed
Patent7067122 a kind of live vaccine of the Edwardsiella tarda of genetic modification) is disclosed, Wang Qi will wait the invention of (2010)
(201010541646.2) one plant of Edwardsiella tarda gene delection attenuated live vaccines is disclosed.These patent descriptions are attenuated epidemic disease
Seedling has obtained more attention in tarda vaccine research.Although attenuated vaccine can guarantee that exempting from for tarda vaccine
Epidemic disease protective rate, but because attenuated vaccine still has certain bio-safety risk, it is more careful to be needed on safety evaluatio,
So that this vaccine development and realizing commercialized cycle length, cost is high.Necessarily special all be present additionally, due to different vaccines
Property, once there is the threat of not of the same race or different serotypes tardas, this attenuated vaccine just may no longer play immune
Protective effect.Other class new generation vaccines of tarda, such as DNA vaccination, subunit vaccine and ghost vaccine, although also having
There is certain immune effect, but consider production technology, cost, the problems such as security, above-mentioned vaccine is also difficult to put at present
In practical application.
The content of the invention
It is an object of the invention to provide a kind of tarda ablation method and its application, can be prepared effectively using this method
The inactivated vaccine of tarda antigen immunogenicity is maintained, with overcome the deficiencies in the prior art.
Tarda inactivator of the present invention, i.e., inactivation treatment is carried out to tarda using acid reagent.
Preferably, inactivation treatment is carried out using minimum inactivation intensity to tarda, minimum inactivation intensity instigates love moral
Fahrenheit bacterium is able to the combination of the minimum inactivation agent concentration of complete inactivation, inactivation temperature and inactivation time these three conditions, and three
Between it is negatively correlated each other.
Minimum inactivation intensity is to make the minimum dose of tarda complete inactivation, and the purpose using minimum inactivation intensity is
The immunogenicity of antigen is maintained while inactivating tarda as far as possible.Through above-mentioned minimum inactivation agent concentration, inactivation temperature
With the tarda of the combination inactivation treatment of inactivation time these three conditions, through inactivator is removed, and 100% place can be made
Tarda after reason loses the ability of growth and breeding completely in TSB fluid nutrient mediums, and can not recover in vivo
The ability of growth and breeding, then be considered as complete inactivation.The syntagmatic of these three conditions is three's negative correlation each other, formed by
The curved surface of continuous nearly reciprocal relation in the three-dimensional system of coordinate of these three conditions composition, the specific determination on curved surface be
Measure makes the minimum inactivation agent concentration of tarda complete inactivation, or the inactivation of certain concentration under specified temp and special time
Agent determines the shortest time for making tarda complete inactivation at a certain temperature.The invention demonstrates that using the of the same race of various dose
The time that inactivator prepares complete inactivation vaccine is different, and both relations present negatively correlated.
Preferably, the acid inactivator is H at room temperature+Concentration be 10-7Mol/L -1mol/L acid solution；Go out
Temperature range living is at 0 DEG C -100 DEG C；Inactivation time scope is 10min -48h.
Preferably, the inactivation condition that the inactivator uses is H+Concentration be 10-2Mol/L acid solution, inactivation temperature
Spend for 4 DEG C, inactivation time 24h, or H+Concentration be 10-4Mol/L acid solution, inactivation temperature are 16 DEG C, inactivation time
For 36h, or H+Concentration be 10-1Mol/L acid solution, inactivation temperature are 50 DEG C, inactivation time 20min, above-mentioned inactivation
Vaccine prepared by method tested fish can be provided relative protection ratio (RPS) be respectively 62.8%, 63.4%-65.7% and
60.3%-61.2% immune protective effect.
Preferably, to tarda after above-mentioned inactivation treatment, carrying out bacterium, whether complete inactivation detects.Due to bacterium
Inactivation in, the influence of some physical factors, such as there may be possibility that some can influence inactivator and action effect because
Son, such as different bacterial concentration, medium component etc. may consume inactivation effect of some inactivators etc. during inactivation, therefore
In practical application, particularly under conditions of commercialization large-scale production, the incomplete problem of inactivation is may result in so as to influence
The quality stability and security of vaccine product, also therefore, in practical application it is necessary by the detection of actual inactivating efficacy (such as
Security, stability and application immune effect of vaccine etc.), formulate the respective standard in inactivation technology for above-mentioned factor.
The invention demonstrates that inactivation condition has a major impact to the immunogenicity of tarda inactivated vaccine, to Ai Dehuashi
The nucleic acid and analysis of protein of bacterium show that different inactivation condition processing there are to the nucleic acid integrality and protein structure of inactivated vaccine
It is obvious to influence.The application test result of tarda inactivated vaccine is carried out using zebra fish as model animal, it was demonstrated that using most
Inactivated vaccine prepared by the different inactivators of low inactivation dosage can be horizontal in fish Immune inducing in vivo different antibodies, produces different be immunized
Effect.Therefore, the tarda inactivated vaccine prepared for changing specific inactivation condition, as long as following minimum inactivation intensity
Principle carries out the preparation of inactivated vaccine, and obtains the tarda inactivated vaccine of effective immunoprotection, just nevertheless suffers from this specially
The protection of profit.
The immunoprotection force estimation that above-mentioned ablation method prepares inactivated vaccine can in the following ways, i.e., 1 time immune note
After penetrating tested fish 4 weeks, then with 10 times of half lethal dose (LD50) work tarda to tested fish infectable infection, compared with without
The control fish of inactivated vaccine immunity inoculation, the relative protection ratio (RPS) of Computation immunity group.
Present invention additionally comprises application of the obtained tarda inactivated vaccine in fish disease preventing and treating.
Preferably, the inactivated vaccine that above-mentioned ablation method is obtained can also reach with adjuvant use in conjunction in use
More preferable immune protective effect.Wherein, the effect of adjuvant is by immunological regulation, participation antigen submission, induces immune response, anti-
The modes such as original storage improve the immunogenicity of antigen and the sustainability of immune response.Available conventional adjuvant mainly has insoluble
Property aluminium salt class colloid, oil-water emulsions, microbe composition, nucleic acid and the like, polysaccharose substance, cell factor, liposome, exempt from
Epidemic disease stimulates compound, propolis, medicinal herb componentses etc..
Compared with prior art, the beneficial effects of the invention are as follows：The Edwardsiella inactivated vaccine that the present invention makes is to use
Minimum inactivation intensity carries out complete inactivation to tarda, can effectively maintain the immune protective efficiency of tarda antigen.Just
The application test result of step shows that the immune effect of tarda inactivated vaccine prepared by this method is preferable, and its RPS can reach
To more than 60% effect, this result will be exempted from apparently higher than the tarda vaccine that gained is handled using conventional inactivator
Epidemic disease protective rate.Tarda inactivated vaccine of the present invention meet commercial vaccine safely, effectively, it is practical low with cost
Requirement, have carry out commercial distribution or directly to commercialization cultivation fish implement immunity inoculation Development volue.
Brief description of the drawings
Fig. 1 is the electrophoresis pattern that different inactivators handle the influence to E.tarda bacterial genomes DNA；
Wherein：Swimming lane 1- lysols；Swimming lane 2- copper sulphate；Swimming lane 3- acetone；Swimming lane 4- glutaraldehydes；Low pH (the pH of swimming lane 5-
It is worth for 2)；Swimming lane 6-PBS (control).
The electrophoresis pattern of influence of Fig. 2 differences inactivator processing to E.tarda bacterioproteins；
Wherein, Marker is molecular weight marker；Swimming lane 1- acetone；The low pH of swimming lane 2- (pH value 2)；Swimming lane 3- lysols；
Swimming lane 4- copper sulphate；Swimming lane 5-PBS (control)；Swimming lane 6- glutaraldehydes.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples.
Embodiment 1：The determination of the minimum inactivation dosage of inactivator
Edwardsiella tarda can be incubated at LB culture mediums, and (tryptone 10g, yeast extract 5g, NaCl10g, goes
Ionized water is settled to 1L, pH7.0) or pancreas peptone soybean broth (TSB, tryptone 17g, phytone 3g, glucose
2.5g, NaCl5.0g, K2HPO42.5g, deionized water are settled to 1L, pH7.3) or 2216E culture mediums in (according to medium component
The presence or absence of middle seawater, two kinds can be divided into, one：Yeast extract 1g, tryptone 5g, FePO40.1g, seawater are settled to
1L, pH7.6-7.8；Secondly：Yeast extract 1g, tryptone 5g, FePO40.1g, NaCl34g, deionized water are settled to
1L, pH7.6-7.8).By the Wdwardsiella tarda of exponential phase with 1：100 ratio is seeded in a kind of above-mentioned culture medium,
28 DEG C of constant incubators, 5h, 6000rpm, 4 DEG C of centrifugation 10min of 180rpm shaken cultivations collect thalline, with sterile phosphate buffer
Three times, and thalline is resuspended with PBS in liquid (PBS) washing thalline, is made 1 × 109CFU/mL bacteria suspensions.
In laboratory inactivation dose screening, 96 porocyte culture plates are taken, the slow love moral that 200 μ L are prepared is added in every hole
Fahrenheit bacteria suspension.The inactivator of selection be respectively lysol (10%), acetone (AR), glutaraldehyde (25%), copper sulphate (AR) and
Concentrated hydrochloric acid (36% -38%), the dose gradient of use is as shown in table 1, wherein, lysol and acetone press volume fraction, and penta 2
Aldehyde and copper sulphate press its final concentration respectively, and concentrated hydrochloric acid is based on pH value, and 4 DEG C stand (period is repeatedly resuspended), and after 24h, each hole takes
The μ L of bacterium solution 20 are forwarded in the respective aperture of another 96 porocyte culture plates (each hole is added with fresh 200 μ L TSB culture mediums), 28 DEG C
Shaken cultivation is stayed overnight, to check inactivation situation.Observe the actual growing state of thalline in each hole (PBS groups are as control), such as original most
Low inactivation agent concentration end can reach the effect of complete inactivation, then the inactivation agent concentration in former different gradients is accordingly added,
The determination experiment of minimum inactivation agent concentration is re-started, method is same as above；Conversely, then can be according to the result of this experiment, it is determined that going out
Minimum inactivation concentration of the agent living to Edwardsiella tarda.
The screening of the different inactivator concentrations of table 1
The screening of above-mentioned inactivator concentration gradient, verified by corresponding expand, finally draw each chemical ablation agent most
Low inactivation dosage, such as table 2.
The minimum inactivation agent concentration of 2 different inactivators of table
In the present embodiment, acid reagent by taking HCL as an example, for Edwardsiella tarda other acid reagents can be effective
The minimum inactivator method for determination of amount of antigen immune protection is maintained to can be found in the present embodiment.
The acid inactivator of catfish tarda and Bao Ke tardas for Ai Dehuashi category can be tieed up effectively
The minimum inactivator method for determination of amount for holding antigen immune protection can be found in the present embodiment.
Embodiment 2：Different inactivators prepare tarda inactivated vaccine immune effect and compared
Edwardsiella tarda (1 × 109CFU/mL) prepared by bacteria suspension to see embodiment 1, and each inactivator prepares slow love moral
The inactivation parameter (inactivation dosage, inactivation time and temperature) that Fahrenheit inactivated vaccine uses is the same as table 2.The another Fu Er for preparing 1% concentration
Malin (4 DEG C, overnight) slow moral Fahrenheit bacteria vaccine of inactivation.Bacterial strain centrifuges 10min, nothing after inactivation, with 6000r/min in 4 DEG C
Bacterium PBS (pH7.2) washings vaccine 3 times, and PBS is resuspended in, 4 DEG C save backup.
From healthy zebra fish (0.38 ± 0.16) g, it is placed in the cultivation flume (3L/ grooves) of separation, water temperature is (25 ± 1)
℃.Daily lighting 12h.By fish body 3% is weighed during experiment feed and feed mixed feed day 2 times, temporarily supported 2 weeks before experiment.Spot
MS222 (1 is used before the injection of horse fish:10000 dilutions) carry out immersion anaesthetic treatment.Zebra fish is grouped at random, each group sets 3
Parallel group, per the tail of parallel group 30, experiment sets control group (injection PBS).Be injected intraperitoneally inactivated vaccine, injection dosage be 2 ×
105CFU/ tails.
After immune 4 weeks, 10 times of LD are injected intraperitoneally in zebra fish50The fresh Edwardsiella tarda (1.93 ± 0.30) of dosage ×
104CFU/g, carry out Experimental infection.28 days death rates for counting each test group after poison are attacked, calculate relative protection ratio (RPS),
RPS calculation formula are：RPS=[1 (the immune group death rate/control group death rate)] × 100%.
Result of calculation shows, above-mentioned 1 (lysol), 2 (copper sulphate), 3 (acetone), 4 (glutaraldehydes), 5 (pH value 2) and good fortune
The RPS values of your Malin's inactivation group are respectively 27.0%, 19.2%, 27.2%, 29.3%, 62.8% and 30.2%.This result
Show more other inactivators, tarda inactivated vaccine prepared by low pH of the present invention can more preferably maintain the cause of disease
Immune protective efficiency, a shot inoculation after, can to zebra fish produce more than 62% relative protection ratio.
The method of the present embodiment can also be used as hydrochloric acid to prepare catfish tarda or guarantor section tarda inactivated vaccine
Immune effect compare in application.The tarda inactivated vaccine prepared using the minimum inactivation dosage of other acid reagents
The comparison of immune effect, reference can be made to the method for the present embodiment.
Embodiment 3：The influence of Edwardsiella tarda inactivated vaccine prepared by different inactivators to zebra fish antibody titer
Each inactivator prepare Edwardsiella tarda inactivated vaccine use inactivation parameter (inactivation dosage, inactivation time and
Temperature) with table 2, experiment is with fish, aquaculture management, vaccine inoculation and artificial challenge with embodiment 2.After immune 28 days, every group takes 3
Tail zebra fish, takes 75mg to organize near tail fin respectively, adds 1mL homogenate buffers (100mM Tris-HCl, pH6.8；
1.0mM PMSF；6%SDS；2% beta -mercaptoethanol), it is placed in ice bath in homogenizer and grinds 20min.With 12,000g in 4 DEG C of centrifugations
10min, draw supernatant, -20 DEG C of storages.The antibody titer of above-mentioned tissue fluid is determined using micro-agglutination method.Draw 0.1mL tissues
In the 1st hole that 96 orifice plates that liquid adds U-shaped bottom are often gone, the sterile PBS of 0.05mL (1 ×) are added in other holes of the row, then
Take 0.05mL tissue fluid to add in the 2nd hole of colleague out of the 1st hole, take 0.05mL liquid to add colleague out of the 2nd hole again after mixing
Latter hole in, follow this, do 2 times of gradient dilutions, last hole suctions out 0.05mL liquid and discarded；Added again in each hole
0.05mL Edwardsiella tarda bacterium solutions, 37 DEG C of incubations, are observed, the maximum tissue liquid extension rate for occurring precipitating is after staying overnight
The potency of contained anti-Edwardsiella tarda antibody in tissue fluid.The antibody titer of each group the results are shown in Table 3.
Slow Edwardsiella vaccine prepared by the minimum inactivation dosage of 3. different inactivators of table is to zebra fish antibody titer
Influence
Result above show present invention determine that maintenance tarda antigen immune protection low pH inactivated vaccines compared with
Other 4 kinds of inactivators can preferably improve the antibody titer in immune fish tissues liquid.
Embodiment 4：Different inactivators handle the influence to Edwardsiella tarda genomic DNA
5 kinds of inactivators prepare Edwardsiella tarda inactivated vaccine use inactivation parameter (inactivation dosage, inactivation time and
Temperature) with embodiment 2.With bacterial genomes DNA extraction kit (Beijing Tiangeng biochemical technology Co., Ltd) to above-mentioned equivalent
Concentration be 108CFU/mL each inactivation thalline complete genome DNA is extracted, and the equivalent isoconcentration of same period culture is simultaneously resuspended
Control is used as in PBS Edwardsiella tarda.With the Ago-Gel of 2% concentration the DNA of extraction is carried out electrophoresis detection (see
Fig. 1).
From electrophoresis pattern, different inactivator processing have Different Effects to bacterial genomes DNA integrality.With it is right
Compare according to the DNA of group (swimming lane 6), inactivator copper sulphate (swimming lane 2) used is less to DNA damage in the present embodiment；Acetone (swimming
Road 3), glutaraldehyde (swimming lane 4) and lysol (swimming lane 1) have the degradation of dna effect reduced successively, wherein acetone treatment to DNA
DNA extracting may be made by difficulty, the band for showing as no purpose nucleic acid size in electrophoresis pattern occurs；(pH value is by low pH
2) processing (swimming lane 5) can cause DNA of bacteria to be broken, and show as nucleic acid fragment and substantially diminish compared with control group, and have and significantly " drag
Band " phenomenon, it was demonstrated that the processing generates irreversible lethal effect to bacterium.This result shows to include inactivator of the same race not
With the different inactivation conditions processing including dosage, obvious shadow there are to the nucleic acid integrality of tarda inactivated vaccine
Ring.
Embodiment 5：Different inactivators handle the influence to Edwardsiella tarda bacterioprotein
5 kinds of inactivators prepare Edwardsiella tarda inactivated vaccine use inactivation parameter (inactivation dosage, inactivation time and
Temperature) with embodiment 2.The concentration prepared to different inactivators is 108CFU/mL inactivated vaccine carries out whole bacterial protein SDS-
PAGE, the Edwardsiella tarda of the same concentrations for being resuspended in PBS of same period culture is as control.From Figure 2 it can be seen that compared with control group
The whole bacterial protein of (swimming lane 5), acetone (swimming lane 1) and pH value are that the influence that 2 (swimming lanes 2) are handled to whole bacterial protein is less, are obtained
Protein electrophoresis collection of illustrative plates is close with control group；And bacterium after lysol (swimming lane 3), copper sulphate (swimming lane 4) and glutaraldehyde (swimming lane 6) processing
Body, there is different degrees of reduction compared with control group, wherein the mycoprotein concentration after being handled especially with glutaraldehyde is minimum.Result above
Show the different inactivation conditions processing including the various dose of inactivator of the same race, to the albumen of tarda inactivated vaccine
Structure, which exists, to have a significant effect.
Embodiment 6：Immune application of the tarda inactivated vaccine to zebra fish
Preparation formalin and the slow Edwardsiella vaccine of acid reagent inactivation, acid reagent therein is with nitric acid
(HNO3) exemplified by.
Referring to the method for embodiment 1, it is determined that the minimum inactivation condition under the conditions of two groups of different inactivations, respectively organize 1：H+
Concentration is 10-4Mol/L HNO3, 16 DEG C, inactivate 36h；Group 2：H+Concentration is 10-1Mol/L HNO3, 50 DEG C, inactivate 20min.
Formalin group is separately set in experiment, i.e., with 0.5% formalin, 4 DEG C, inactivates 18h.Referring to method in embodiment 1, go to eliminate
Agent living, is made 4.0 × 109CFU/mL bacteria suspensions, each inactivated vaccine are coated with through solid plate and detected, and confirm that cause of disease is gone out completely
It is living.
From healthy zebra fish (0.32 ± 0.12) g as experimental animal, by the way of intraperitoneal injection.Control group is injected
Sterile PBS, immune group dosage of inoculation are 2.0 × 107CFU/ tails.After immune 4 weeks, with 10 times of LD50The E.tarda of dosage is carried out
Artificial challenge, the death rate is counted after 14 days.Twice test result indicates that, compared with control group, the RPS value models of immune group 1,2 and 3
Enclose respectively 63.4%-65.7%, 60.3%-61.2% and 28.2%-33.7%.
The method of the present embodiment can also be exempted from as nitric acid to catfish tarda and Bao Ke tarda inactivated vaccines
Epidemic disease effect compares in application.
The ratio of the immune effect of the tarda inactivated vaccine prepared using the minimum inactivation dosage of other acid reagents
Compared with the method that also can be found in the present embodiment.
Embodiment 7：Lefteye flounder (Paralichthy is immunized with inactivation slow Edwardsiella vaccine compatibility in adjuvant
Solivaceus application mode)
In this application mode, adjuvant can from insoluble aluminium salt class colloid, oil-water emulsions, microbe composition, nucleic acid and its
Chosen in analog, polysaccharose substance, cell factor, liposome, immunostimulating complex, propolis, medicinal herb componentses etc. a kind of.
2 × 10 are prepared with the acid reagent that pH value is 1-48E.tarda CFU/mL inactivated vaccines, inactivation parameter (inactivation dosage, inactivation
Time and temperature) referring to embodiment 2 or example 6, after vaccine and adjuvant are mixed and made into bacterin preparation by a certain percentage, by 100 μ L/
The dosage of tail, intraperitoneal inoculation MS222 (1:9000) postanesthetic lefteye flounder is soaked, moves into fresh seawater and normally cultivates.It is immune
30-60 days afterwards, 10 times of LD are injected intraperitoneally to lefteye flounder50The fresh E.tarda bacterium solutions of dosage, carry out Experimental infection.After infection
14-30 days, the death rate is counted, and according to formula RPS=[1 (the immune group death rate/control group death rate)] × 100%, calculate
RPS, data acquired is analyzed, inactivating the immune lefteye flounder application mode of E.tarda compatibilities with hydrochloric acid to adjuvant assesses,
For RPS values more than 70%, it is qualified to be considered as.
In above-described embodiment, the preparation method of tarda inactivated vaccine can also use the acid reagent outside hydrochloric acid to add
With inactivation, prepared tarda inactivated vaccine also can be found in this example to the immune application mode of other fish.
Embodiment 8：Inactivate the application side of E.ictaluri vaccine immunities channel catfish (Ietalurus punetaus)
Formula
Tarda (inactivation dosage, goes out by taking E.ictaluri as an example referring first to the middle inactivation parameter of embodiment 2 or 6
Live time and temperature) E.ictaluri inactivated vaccines are prepared, immersion immunity is used in, channel catfish is placed in inactivation epidemic disease
Seedling final concentration of 108—109In CFU/mL fresh fresh water, after 10-30min, move into fresh fresh water and normally cultivate.14—30
After it, as above booster immunization 1 time.Another to feed containing E.ictaluri inactivated vaccine feeds, inactivated vaccine addition is in feed
(0.01-0.1g)/kg (inactivated vaccine weight/feed relative), continuously feeds 3-7 days, then carry out normal feeding management.Strengthen
30-60 days after immune, with the E.ictaluri of fresh cultured, artificial immersion infection experiment is carried out.14-30 days after infection, system
Each group RPS is counted, assesses E.ictaluri inactivated vaccine immunodotting Cha Wei Channel-catfish application effect, RPS values are considered as conjunction more than 60%
Lattice.
In above-described embodiment, the preparation method of tarda inactivated vaccine can also use the acid reagent outside hydrochloric acid to add
With inactivation, prepared tarda inactivated vaccine also can be found in this example to the immune application mode of other fish.
Embodiment 9：Bullfrog (Rana is immunized comprising the bivalent inactivated vaccine including tarda inactivated vaccine
Catesbeiana application mode).
Tarda is by taking E.tarda as an example, another inactivation epidemic disease in bigeminy vaccine in addition to E.tarda inactivated vaccines
Seedling, by taking Streptococcusagalactiae (Streptococcus agalactiae) as an example, the vaccine prepares the method referring to embodiment 1, i.e.,
Streptococcusagalactiae (S.agalactiae) is inactivated with the Formalin inactivation of lowest dose level.
E.tarda inactivated vaccines are prepared (inactivation parameter is referring to embodiment 2 or example 6) with the acid reagent that pH value is 1-4.
Above-mentioned vaccine is pressed 1：1 ratio is configured to bigeminy vaccine, and the final concentration of two kinds of inactivation of bacterial is 10 in the vaccine8CFU/
ML, by the dosage of 100 μ L/ only, immunity inoculation is carried out by the way of intramuscular injection.After 14-30 days, feed within continuous 5-7 days
The feed of bigeminy vaccine (0.01-0.2g vaccines dry weight/1kg feeds) containing above-mentioned inactivation carries out booster immunization.After 60 days
In artificial liver support, respectively with 10 times of LD50The fresh S.agalactiae and E.tarda bacterium solutions of dosage are carried out to 10 bullfrogs
Intraperitoneal injection, counts the death rate respectively, calculates RPS, as a result in 60% is reached to E.tarda and S.agalactiae RPS values,
Then being considered as the vaccine has preferable immune effect.
In above-described embodiment, the preparation method of Edwardsiella inactivated vaccine can also use the acid reagent outside hydrochloric acid to be subject to
Inactivate, the immune application mode of the bigeminy vaccine including bag armful tarda inactivated vaccine, also can be found in this example.
Embodiment 10：Tilapia mossambica is immunized comprising the triple inactivated vaccine including Edwardsiella tarda inactivated vaccine
The application mode of (Oreochromis niloticus)
Other two inactivated vaccine in triple vaccine in addition to E.tarda inactivated vaccines, with Vibrio anguillarum (Vibrio
Anguillarum) and exemplified by Aeromonas hydrophila (Aeromonas hydrophila), the preparation of both inactivations can use often
Ablation method (such as Formalin inactivation, heat inactivation, high pressure inactivation) is advised to carry out.E.tarda inactivated vaccines use with pH value be 1-
4 acid solution is prepared (inactivation parameter is referring to embodiment 2 or example 6).Three is pressed 1 again：1：1 ratio is configured to three epidemic diseases
Seedling, the final concentration of three kinds of inactivation of bacterial is 10 in the vaccine8CFU/mL.It is immune to be prepared with feed, by 0.3g vaccines dry weight/
The ratio of 1kg feeds makes an addition to above-mentioned triple vaccine in feed.In, first by healthy Rofe fish soaking triple vaccine
Taken out after 10-20min, after 14-30 days, feed above-mentioned immunization feedstuff within continuous 5 days, after 60 days, respectively with 10 times of LD50 dosage
Fresh E.tarda, V.anguillarum and A.hydrophila 10 Tilapia mossambicas are injected intraperitoneally, statistics is dead respectively
Die rate, calculate RPS, as a result in E.tarda, V.anguillarum and A.hydrophila RPS is respectively reached 50% with
On, then being considered as the vaccine has preferable immune effect.
In above-described embodiment, the preparation method of Edwardsiella inactivated vaccine can also use the acid reagent outside hydrochloric acid to be subject to
Inactivation.The immune application mode of three (more) vaccines including tarda inactivated vaccine, it also can be found in this example.
Each embodiment described above is only the preferred embodiment of the present invention, it should be pointed out that：For the general of the art
For logical technical staff, under the premise without departing from the principles of the invention, some improvement can also be made, these improvement should be regarded as this
The protection domain of invention.
Claims (6)
1. a kind of ablation method for maintaining tarda antigen immunogenicity, it is characterised in that using acid reagent to love
Moral Fahrenheit bacterium carries out inactivation treatment, can maintain tarda antigen immunogenicity, and the acid inactivator is H at room temperature+It is dense
Spend for 10-4Mol/L -1mol/L acid solution.
2. ablation method according to claim 1, it is characterised in that carried out to tarda using minimum inactivation intensity
Inactivation treatment, the minimum inactivation intensity are acid inactivator, minimum inactivation temperature and the most short inactivation time of least concentration
Between combine, it is and negatively correlated each other between three.
3. ablation method as claimed in claim 2, it is characterised in that the inactivation temperature is 0~50 DEG C.
4. ablation method according to claim 2, it is characterised in that the inactivation time is 10min~36h.
5. ablation method according to claim 1, it is characterised in that the inactivator is H+Concentration is 10-2Mol/L acid
Property solution, inactivation temperature are 4 DEG C, inactivation time 24h, or H+Concentration is 10-4Mol/L acid solution, inactivation temperature 16
DEG C, inactivation time 36h, or H+Concentration is 10-1Mol/L acid solution, inactivation temperature are 50 DEG C, inactivation time 20min.
6. according to the ablation method described in claim any one of 1-5, it is characterised in that carry out inactivation treatment to tarda
Tarda whether complete inactivation is detected afterwards.
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