CN104099286A - Inactivation method and inactivated vaccine for Edwardsiella sp. - Google Patents
Inactivation method and inactivated vaccine for Edwardsiella sp. Download PDFInfo
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- CN104099286A CN104099286A CN201410350881.XA CN201410350881A CN104099286A CN 104099286 A CN104099286 A CN 104099286A CN 201410350881 A CN201410350881 A CN 201410350881A CN 104099286 A CN104099286 A CN 104099286A
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Abstract
The invention discloses an inactivation method and inactivated vaccine for Edwardsiella sp., and belongs to the field of fish-used vaccine. Particularly, a sulfydryl reagent is taken as an inactivator; to the inactivation method and the prepared inactivated vaccine for Edwardsiella sp., the adopted inactivator is the sulfydryl reagent of which the volume fraction is less than or equal to 10%; the inactivation temperature range is 0-100 DEG C; the inactivation time range is 10 min-48 h. The inactivated vaccine for Edwardsiella sp. prepared by the method can effectively maintain immune protection force of Edwardsiella sp. antigen.
Description
Technical field
The present invention relates to fish vaccine field, be specifically sulfhydryl reagent as inactivator, the ablation method to tarda and the inactivated vaccine of preparation thereof.
Background technology
Tarda (Edwardsiella sp.) comprises Edwardsiella tarda (E.tarda), catfish tarda (E.ictaluri) and guarantor section tarda (E.hoshinae), it is one of the main pathogenic fungi in aquaculture, its host range is very extensive, fish, batrachians, reptiles, birds and the mammals that comprises the mankind have the report of infection, and be worldwide distribution, be prevalent in fresh water and briny environment, mainly be distributed in China, Australia, Japan, India, Israel, Malay Archipelago, the U.S., the Perenniporia martius countries and regions such as Panama.From reported first so far, this bacterium has caused disease and has caused massive losses in multiple cultured fishes.Normal chemotherapeutic and the microbiotic of adopting in fish disease control at present, but along with their use, cause of disease resistance also strengthens thereupon, and drug residue and the diffusion in environment have caused the problem aspect food safety, environment and public health.Fish vaccine can not produce pollution after using, and can in immune fish body, not form residually, can avoid environmental pollution and maintain the quality of fish body itself, and Reusability can not produce resistance yet, has a extensive future.
At present, at present, fish vaccine is in application, include the forms such as inactivated vaccine, attenuated live vaccine, DNA vaccination, subunit vaccine and ghost vaccine, wherein the inactivated vaccine R&D cycle is short, cost of manufacture is low, security is good, in the guaranteed situation of Immune efficiency, the most easily get the Green Light, therefore become the main kind of current commercialization vaccine.But research shows, many pathogenic bacterias such as tarda and vibrios are different, and tarda inactivated vaccine prepared by employing routine techniques can not produce effective immune protective to tested fish.Gutierrez and Miyazaki (1994, Dis Aquat Org.6:110 – 117.) relative protection ratio of Edwardsiella tarda injecting immune Japan eel of formalin deactivation is only 12.5-25% in research, our previous experiments adopts the relative protection ratio (RPS) of the tarda inactivated vaccine of making without the routine techniques of optimizing also less than 40%; Mekuchi etc. (1995, Fish Pathol.30 (4): 251-256) adopt respectively intramuscular injection, immersion or the oral vaccination ways Edwardsiella tarda immunity lefteye flounder of formalin deactivation, but all do not obtain effective immune protective effect; Sun etc. (2011; Fish Shellfish Immunol.31 (4): 595-599.) although also reported the inactivated vaccine of tarda; the immune protective rate of the unit price inactivated vaccine of tarda only has 33.3%; only have and combine while using when another three kinds of bacterium such as the tarda of deactivation and Vibrio anguillarums; could obtain good immune protective rate, other three kinds of bacterium inactivated vaccines such as Vibrio anguillarum have played the effect of immunological adjuvant in fact for tarda inactivated vaccine.On the whole, in conjunction with our many experiments result and most report, the present situation that the immune effect of tarda inactivated vaccine is desirable not to the utmost, this may be also people's reasons that the cost of Hua Geng great carries out the research of the vaccine of other kinds of having to.
In the application report of the attenuated live vaccines of tarda, the invention (US Patent 6153202) of Klesius etc. (2000) discloses a kind of catfish tarda attenuated live vaccine, the invention (US Patent 7067122) of Evans etc. (2006) discloses a kind of living vaccine of Edwardsiella tarda of genetic modification, and Wang Qi will wait the invention (201010541646.2) of (2010) to disclose a strain Edwardsiella tarda genetically deficient attenuated live vaccines.These patent description attenuated vaccines have obtained more attention in tarda vaccine research.Although attenuated vaccine can ensure the immune protective rate of tarda vaccine, because attenuated vaccine still exists certain Biosafety risk, it is more careful on safety evaluation, to need, and makes this vaccine development and realize the business-like cycle long, and cost is high.Because different vaccines all exist certain specificity, once there be the threat of the tarda of not of the same race or different serotypes, this attenuated vaccine may just no longer be brought into play immanoprotection action in addition.Other class new generation vaccines of tarda, as DNA vaccination, subunit vaccine and ghost vaccine etc., although also have certain immune effect, consider production technique, cost, and the problems such as security, above-mentioned vaccine is also difficult to put in practical application at present.
Summary of the invention
The object of this invention is to provide a kind of application of the ablation method and inactivated vaccine and this inactivated vaccine that effectively maintain tarda antigen immune protection, to overcome the deficiencies in the prior art.
For achieving the above object, the ablation method of tarda of the present invention, adopts sulfydryl inactivator to carry out inactivation treatment to tarda, can maintain the immunogenicity of tarda antigen.
Preferably, adopt minimum deactivation intensity to carry out inactivation treatment to tarda, described minimum deactivation intensity is between the sulfydryl inactivator of lowest dose level, minimum deactivation temperature and the shortest inactivation time and combines, and negative correlation each other between three.
When being deactivation tarda, the object that uses minimum deactivation intensity maintains as much as possible the immunogenicity of antigen.Through the tarda of the combination inactivation treatment of above-mentioned minimum inactivator dosage, deactivation temperature and these three kinds of conditions of inactivation time, through inactivator is removed, and can make 100% tarda after treatment in TSB liquid nutrient medium, lose the ability of growth and breeding completely, and cannot recover in vivo the ability of growth and breeding, be considered as complete inactivation.The syntagmatic of these three kinds of conditions is three's negative correlations each other, be formed on the curved surface of the continuous nearly reciprocal relation in the three-dimensional system of coordinate being formed by these three kinds of conditions, specific definite on curved surface is under specified temp and specified time, to measure the minimum inactivator dosage that makes tarda complete inactivation, or the inactivator of given dose is measured the shortest time that makes tarda complete inactivation under specified temp.The time that the present invention confirms to adopt the inactivator of the same race of various dose to prepare complete inactivation vaccine is different, and both relations present negative correlation.
Preferably, the dosage of described sulfydryl inactivator is to be less than or equal to 10% volume fraction; Deactivation temperature range is at 0 DEG C-100 DEG C; Inactivation time scope is 10min-48h.
Preferably, described sulfydryl inactivator is beta-mercaptoethanol.
Preferably; described deactivation condition is; volume fraction is that 6% beta-mercaptoethanol, deactivation temperature are that 4 DEG C, inactivation time are 24h; or volume fraction be 2% beta-mercaptoethanol, deactivation temperature is that 16 DEG C, inactivation time are 36h; or volume fraction be 8% beta-mercaptoethanol, deactivation temperature be that 50 DEG C, inactivation time are 40min, the prepared vaccine of above-mentioned ablation method to tested fish can provide that relative protection ratio (RPS) is respectively 50.7%, the immune protective effect of 56.2%-61.3% and 50.1%-54.6%.
The present invention also comprises tarda inactivated vaccine, and tarda after deactivation, is removed inactivator after testing, obtains the inactivated vaccine of tarda.
Preferably, to tarda after above-mentioned inactivation treatment, carry out whether complete inactivation detection of bacterium.In deactivation due to bacterium, the impact of some physical factors, the possible factor that can affect inactivator and action effect etc. as existed some, for example different bacterial concentration when deactivation, medium components etc. may consume the deactivation effect of some inactivators etc., therefore in actual applications, particularly under the condition of commercialization scale operation, thereby may cause the incomplete problem of deactivation to affect quality stability and the security of vaccine product, also therefore, in practical application, be necessary that detection by actual inactivating efficacy is (as the security of vaccine, stability and application immune effect etc.), formulate for above-mentioned factor the respective standard at inactivation technology.
The present invention confirms that deactivation condition has material impact to the immunogenicity of tarda inactivated vaccine, and nucleic acid and analysis of protein to tarda show, nucleic acid integrity and protein structure that different deactivation conditions are processed inactivated vaccine have obvious impact.Carry out the application test result of tarda inactivated vaccine taking zebra fish as model animal, the inactivated vaccine that confirmation adopts the different inactivators of minimum deactivation dosage to prepare can be induced different antibodies level in fish body, produces different immune effects.Therefore, the tarda inactivated vaccine of preparing for the concrete deactivation condition of change, carries out the preparation of inactivated vaccine as long as follow the principle of minimum deactivation intensity, and obtains the tarda inactivated vaccine of effective immunoprotection, is just still subject to the protection of this patent.
Above-mentioned ablation method is prepared the immunoprotection force estimation of inactivated vaccine can be in the following ways, i.e. 1 immunization tested fish after 4 weeks, then use 10 times of medial lethal dose LD
50tarda to tested fish infectable infection, without the contrast fish of inactivated vaccine immunization, calculate the relative protection ratio (RPS) of immune group.
Preferably, the inactivated vaccine that above-mentioned ablation method obtains in use also can be with adjuvant combined utilization to reach better immune protective effect.Wherein, the effect of adjuvant is immunogenicity and the immunoreactive sustainability that mainly improves antigen by modes such as immunomodulatory, participation antigen presentation, induce immune response, antigen storages.Available conventional adjuvant mainly contains insoluble aluminium salt colloid, profit emulsion, microbe composition, nucleic acid and analogue thereof, polysaccharose substance, cytokine, liposome, immunostimulating complex, propolis, medicinal herb components etc.
The present invention also comprises the application that tarda inactivated vaccine is used as to preparation treatment tarda infection medicine.
Compared with prior art, the invention has the beneficial effects as follows: the Edwardsiella inactivated vaccine that the present invention makes is to adopt minimum deactivation intensity to carry out complete inactivation to tarda, can effectively maintain the immune protective efficiency of tarda antigen.Preliminary application test result shows that the immune effect of tarda inactivated vaccine prepared by the method is better; its RPS can reach 60% effect, and this result will be apparently higher than using conventional inactivator to process the immune protective rate of the tarda vaccine of gained.Tarda inactivated vaccine of the present invention meets safe, effective, practicality and the low requirement of cost of commercialization vaccine, has and carries out commercial distribution or directly the fish of commercialization cultivation are implemented to immunization exploitation being worth.
Brief description of the drawings
Fig. 1 is that different inactivators are processed the impact on Edwardsiella tarda genomic dna;
Wherein: swimming lane 1, lysol; Swimming lane 2, copper sulfate; Swimming lane 3, acetone; Swimming lane 4, glutaraldehyde; Swimming lane 5, beta-mercaptoethanol; Swimming lane 6, PBS (contrast);
Fig. 2 is that different inactivators are processed the impact on Edwardsiella tarda bacterioprotein.
Wherein: Marker is molecular weight marker; Swimming lane 1, acetone; Swimming lane 2, beta-mercaptoethanol; Swimming lane 3, lysol; Swimming lane 4, copper sulfate; Swimming lane 5, PBS (contrast); Swimming lane 6, glutaraldehyde.
Embodiment
The present invention is further described by the following examples.
Embodiment 1: the minimum deactivation dosage of different inactivators determine
Edwardsiella tarda can be incubated at LB substratum (tryptone 10g, yeast extract 5g, NaCl10g, deionized water is settled to 1L, or pancreas peptone soybean broth (TSB, Tryptones 17g, phytone 3g pH7.0), glucose 2.5g, NaCl5.0g, K
2hPO
42.5g, deionized water is settled to 1L, pH7.3) or 2216E substratum in (according to having or not of seawater in medium component, can be divided into two kinds, one: yeast extract 1g, tryptone 5g, FePO
40.1g, seawater is settled to 1L, pH7.6-7.8; Its two: yeast extract 1g, tryptone 5g, FePO
40.1g, NaCl34g, deionized water is settled to 1L, pH7.6-7.8).The Wdwardsiella tarda of logarithmic phase is seeded in above-mentioned a kind of substratum with the ratio of 1:100,28 DEG C of constant incubators, 180rpm shaking culture 5h, 6000rpm, 4 DEG C of centrifugal 10min collect thalline, wash thalline three times with sterile phosphate damping fluid (PBS), and with the resuspended thalline of PBS, make 1 × 10
9cFU/mL bacteria suspension.
96 porocyte culture plates, in the deactivation dose screening of inactivator, are got in laboratory, the Edwardsiella tarda suspension that adds 200 μ L to prepare in every hole.The inactivator of selecting is respectively lysol (10%), acetone (AR), beta-mercaptoethanol (AR), glutaraldehyde (25%) and copper sulfate (AR), the dose gradient adopting is as shown in table 1, wherein, all mark meters by volume of the dosage of first 3 kinds of above-mentioned inactivator, rear 2 kinds by final concentration, 4 DEG C leave standstill (repeatedly resuspended during this time), after 24h, each hole is got bacterium liquid 20 μ L and is forwarded in the respective aperture of another 96 porocyte culture plate (each hole is added with fresh 200 μ L TSB substratum), 28 DEG C of shaking culture are spent the night, to check deactivation situation.Observe the actual growing state of thalline in each hole (PBS organizes in contrast), as former minimum inactivator dosage end can reach the effect of complete inactivation, the inactivator dosage in former different gradients is carried out to corresponding interpolation, re-start definite test of minimum inactivator dosage, method is the same; Otherwise, can be according to the result of this test, determine the minimum deactivation dosage of inactivator to Edwardsiella tarda.
The deactivation dose screening of the different inactivators of table 1
The screening of above-mentioned inactivator deactivation dose gradient, verifies through corresponding expansion, finally draws the minimum deactivation dosage of each chemical ablation agent, as table 2.
The minimum deactivation dosage of the different inactivators of table 2
In the present embodiment, sulfhydryl reagent is taking beta-mercaptoethanol as example, can be referring to the present embodiment for the minimum inactivator method for determination of amount that can effectively maintain antigen immune protection of other sulfhydryl reagent of Edwardsiella tarda.
The minimum inactivator method for determination of amount that can effectively maintain antigen immune protection of the catfish tarda belonging to for Ai Dehuashi and the different sulfhydryl reagent of Bao Ke tarda also can be referring to the present embodiment.
Embodiment 2: different inactivators are prepared the comparison of tarda inactivated vaccine immune effect
Edwardsiella tarda (1 × 10
9cFU/mL) collecting cells is shown in embodiment 1, and each inactivator is prepared the deactivation parameter (deactivation dosage, inactivation time and temperature) of slow Ai Dehuashi inactivated vaccine employing with table 2.The slow moral Fahrenheit of formalin (4 DEG C, spend the night) the deactivation bacteria vaccine of another preparation 1% concentration.Bacterial strain is after deactivation, and with 6000r/min, in 4 DEG C of centrifugal 10min, aseptic PBS (pH7.2) washs vaccine 3 times, and is resuspended in PBS, and 4 DEG C save backup.
Select healthy zebra fish (0.38 ± 0.16) g as laboratory animal, be placed in the cultivation flume (3L/ groove) of separation, water temperature is (25 ± 1) DEG C.Lighting every day 12h.Duration of test is thrown something and fed and is thrown something and fed mixed feed day 2 times by fish body weight 3%, before test, supports 2 weeks temporarily.Before zebra fish injection, soak anaesthetic treatment with MS222 (1:10000 dilution).Zebra fish is carried out to random packet, establish 3 parallel group for each group, every parallel group of 30 tails, control group (injection PBS) is established in test.Abdominal injection inactivated vaccine, injected dose is 2 × 10
5cFU/ tail.
After immunity 4 weeks, 10 times of LD of zebra fish abdominal injection
50the fresh Edwardsiella tarda (1.93 ± 0.30) × 10 of dosage
4cFU/g, carries out Experimental infection.Attack the rear mortality ratio of adding up each test group for 28 days of poison, calculate relative protection ratio (RPS), RPS calculation formula is: RPS=[1 – (immune group mortality ratio/control group mortality ratio)] × 100%.
Calculation result shows, the RPS value of above-mentioned 1 (lysol), 2 (copper sulfate), 3 (acetone), 4 (glutaraldehyde), 5 (beta-mercaptoethanol) group and formalin deactivation group is respectively 27.0%, 19.2%, 27.2%, 29.3%, 50.7% and 30.2%.This result shows the inactivator of other including formalin; tarda inactivated vaccine prepared by beta-mercaptoethanol of the present invention can better maintain the immune protective efficiency of this cause of disease; after shot inoculation, can produce more than 50% relative protection ratio to zebra fish.
The method of the present embodiment also can be used as during beta-mercaptoethanol relatively applies catfish tarda and Bao Ke tarda inactivated vaccine immune effect.
Adopt the comparison of the immune effect of tarda inactivated vaccine prepared by the minimum deactivation dosage of other sulfhydryl reagent, can be referring to the method for the present embodiment.
Embodiment 3: Edwardsiella tarda inactivated vaccine prepared by the different inactivators impact on zebra fish antibody titer
Each inactivator is prepared the deactivation parameter (deactivation dosage, inactivation time and temperature) of Edwardsiella tarda inactivated vaccine employing with table 2, and test uses fish, aquaculture management, vaccine inoculation and artificial challenge with embodiment 2.After immunity 28 days, get 3 tail zebra fishs for every group, from getting 75mg tissue near tail fin, add 1mL homogenate buffer respectively, be placed in homogenizer ice bath and grind 20min.In 4 DEG C of centrifugal 10min, draw supernatant ,-20 DEG C of storages with 12,000g.Adopt micro-agglutination to measure the antibody titer of above-mentioned tissue juice.Absorption 0.1mL tissue juice adds in the 1st hole of the every row of 96 orifice plate at the bottom of U-shaped, in other holes of this row, add the aseptic PBS of 0.05mL (1 ×), then add in colleague the 2nd hole from getting 0.05mL tissue juice in the 1st hole, after mixing, in the 2nd hole, get again 0.05mL liquid add colleague in a hole, follow this, do 2 times of gradient dilutions, last hole sucking-off 0.05mL liquid discards; In each hole, add again 0.05mL Edwardsiella tarda bacterium liquid, hatch for 37 DEG C, after spending the night, observe, occur that the maximum tissue liquid extension rate of precipitation is tiring of contained anti-Edwardsiella tarda antibody in tissue juice.The antibody titer of each group the results are shown in Table 3.
Slow Edwardsiella vaccine prepared by the minimum deactivation dosage of the different inactivators of table 3. impact on zebra fish antibody titer
Above result shows in inactivated vaccine prepared by different inactivators; beta-mercaptoethanol is compared compared with other 4 kinds of inactivators; antibody titer in the fish tissue juice of its generation is low; but its inactivated vaccine of preparing can produce better immune protective effect, also this inactivator can maintain preferably tarda antigen immune protection in inactivation process.
Embodiment 4: different inactivators are processed the impact on Edwardsiella tarda genomic dna
5 kinds of inactivators are prepared deactivation parameter (deactivation dosage, inactivation time and temperature) that Edwardsiella tarda inactivated vaccine adopts with embodiment 2.The concentration of above-mentioned equivalent is to 10 with bacterial genomes DNA extraction test kit (Beijing Tian Gen biochemical technology company limited)
8each deactivation thalline complete genome DNA of CFU/mL extracts, and the equivalent isoconcentration that cultivate the same period the Edwardsiella tarda that is resuspended in PBS are in contrast.The DNA extracting is carried out to electrophoresis detection (seeing Fig. 1) with the sepharose of 2% concentration.
From electrophoretogram, with the DNA comparison of control group (swimming lane 6), inactivator copper sulfate (swimming lane 2) and beta-mercaptoethanol (swimming lane 5) used in the present embodiment are less to DNA damage; Acetone (swimming lane 3), glutaraldehyde (swimming lane 4) and lysol (swimming lane 1) have the degradation of dna effect reducing successively to DNA, wherein acetone treatment may make the extracting of DNA suffer difficulty, shows as without the band of object nucleic acid size and occur in electrophoretogram.This result shows the different deactivation condition processing including the various dose of inactivator of the same race, and the nucleic acid integrity of tarda inactivated vaccine is had to obvious impact.
Embodiment 5: different inactivators are processed the impact on Edwardsiella tarda bacterioprotein
5 kinds of inactivators are prepared deactivation parameter (deactivation dosage, inactivation time and temperature) that Edwardsiella tarda inactivated vaccine adopts with embodiment 2.Concentration prepared by different inactivators is 10
8the inactivated vaccine of CFU/mL carries out whole bacterial protein SDS-PAGE, and the Edwardsiella tarda of the same concentrations that is resuspended in PBS of cultivating the same period in contrast.As seen from Figure 2, compared with the whole bacterial protein of control group (swimming lane 5), acetone (swimming lane 1) and beta-mercaptoethanol group (swimming lane 2) are processed less on the impact of whole bacterial protein, and the protein electrophoresis collection of illustrative plates and the control group that obtain are close; And lysol (swimming lane 3), copper sulfate (swimming lane 4) and glutaraldehyde (swimming lane 6) are processed rear thalline, there is reduction in various degree compared with control group, wherein especially minimum with glutaraldehyde tropina concentration after treatment.Above result shows the different deactivation condition processing including the various dose of inactivator of the same race, and the protein structure of tarda inactivated vaccine is existed and had a significant effect.
Embodiment 6: the immunity application of tarda inactivated vaccine to zebra fish
The slow Edwardsiella vaccine of formalin and sulfhydryl reagent deactivation for preparation, sulfhydryl reagent is wherein taking beta-mercaptoethanol as example.Referring to the method for embodiment 1, determine two groups of minimum deactivation conditions under different deactivation conditions, be respectively the beta-mercaptoethanol of group 1:2%, 16 DEG C, deactivation 36h; The beta-mercaptoethanol of group 2:8%, 50 DEG C, deactivation 40min.In experiment, separately establish formalin group, with 0.5% formalin, 4 DEG C, deactivation 18h.Referring to method in embodiment 1, remove inactivator, make 4.0 × 10
9cFU/mL bacteria suspension, each inactivated vaccine detects through solid plate coating, confirms cause of disease complete inactivation.
Select healthy zebra fish (0.32 ± 0.12) g as laboratory animal, adopt the mode of abdominal injection.Control group is injected aseptic PBS, and immune group dosage of inoculation is 2.0 × 10
7cFU/ tail.After immunity 4 weeks, with 10 times of LD
50the E.tarda of dosage carries out artificial challenge, after 14 days, adds up mortality ratio.The experimental result of twice shows, compared with control group, immune group 1,2 and 3 RPS value scope be respectively 56.2%-61.3%, 50.1%-54.6% and 28.2%-33.7%.
The method of the present embodiment also can be used as during beta-mercaptoethanol relatively applies catfish tarda and Bao Ke tarda inactivated vaccine immune effect.
Adopt the comparison of the immune effect of tarda inactivated vaccine prepared by the minimum deactivation dosage of other sulfhydryl reagent, also can be referring to the method for the present embodiment.
Embodiment 7: the application mode of adjuvant and deactivation slow Edwardsiella vaccine compatibility immunity turbot (Scophthalmus maximus).
In this application mode, sulfhydryl reagent is taking beta-mercaptoethanol as example, and adjuvant is taking Freund's incomplete adjuvant as example.
First prepare 2 × 10 with beta-mercaptoethanol
8e.tarda CFU/mL inactivated vaccine, deactivation parameter (deactivation dosage, inactivation time and temperature) is referring to embodiment 2, by the vaccine of equivalent and Freund's incomplete adjuvant (Freund ' s Adjuvant Incomplete, Sigma) suck respectively in two asepsis injectors, between two syringes, be connected with aseptic sebific duct, then alternately promote needle tubing, until form the emulsion of thickness.The emulsifying agent preparing splashes in cold water, does not disperse if emulsion droplet keeps complete, becomes to drip shape and bubbles through the water column, and it is qualified to be considered as.MS222 for abdominal injection (1:9000) soaks the turbot after anaesthetic treatment, and dosage of inoculation is 100 μ L/ tails, moves into normally cultivation in fresh seawater.Latter 30-60 days of immunity, to 10 times of LD of turbot abdominal injection
50the fresh E.tarda bacterium liquid of dosage, carries out Experimental infection.Infect latter 14-30 days, statistics mortality ratio, and according to formula RPS=[1 – (immune group mortality ratio/control group mortality ratio)] × 100%, calculate RPS, to obtained data analysis, adjuvant and hydrochloric acid deactivation E.tarda compatibility immunity turbot application mode are assessed, and RPS value exceedes 70%, and it is qualified to be considered as.
The immunity application of tarda inactivated vaccine prepared by other sulfhydryl reagent, also can be referring to this application mode.In application, selected adjuvant also can be the effective adjuvant except Freund's incomplete adjuvant, as insoluble aluminium salt colloid, profit emulsion, microbe composition, nucleic acid and analogue thereof, polysaccharose substance, cytokine, liposome, immunostimulating complex, propolis, medicinal herb components etc.
Embodiment 8: the application mode of deactivation E.ictaluri vaccine immunity Yellow catfish (Pelteobagrus fulvidraco).
Sulfhydryl reagent is taking beta-mercaptoethanol as example, first can effectively maintain the immunogenic deactivation E.ictaluri of cause of disease vaccine (referring to embodiment 1 and 2) with beta-mercaptoethanol preparation, in application, adopt immersion immunity, it is 10 that Yellow catfish is placed in to inactivated vaccine final concentration
8-10
9in the fresh fresh water of CFU/mL, after 10-30min, move into normally cultivation in fresh fresh water.After 14-30 days, carry out booster immunization 1 time (booster immunization interval time is determined by fish body weight size, and the less time of body weight can shorten, and more the time can proper extension for body weight).Throw something and feed and contain E.ictaluri inactivated vaccine feed, in feed, inactivated vaccine addition is (0.01-0.1) g/kg (inactivated vaccine weight/feed weight), throws something and feeds continuously 5 days, then carries out normal feeding and management.After booster immunization 30-60 days, calculate the rate of body weight gain of duration of test, separately to 10 times of LD of lefteye flounder abdominal injection
50the fresh E.ictaluri bacterium liquid of dosage, carries out Experimental infection.Infect latter 14-30 days, statistics mortality ratio, and according to formula RPS=[1 – (immune group mortality ratio/control group mortality ratio)] × 100%.Calculate RPS, to obtained data analysis, E.ictaluri inactivated vaccine immunity lefteye flounder application mode is assessed, RPS value exceedes 60%, and it is qualified to be considered as.
The immunity application of tarda inactivated vaccine prepared by other sulfhydryl reagent, also can be referring to this application mode.
Embodiment 9: comprise the application mode of Edwardsiella tarda inactivated vaccine interior bivalent inactivated vaccine immunity bullfrog (Rana catesbeiana).
In bigeminy vaccine respectively taking E.tarda inactivated vaccine and streptococcus agalactiae (Streptococcus agalactiae) inactivated vaccine as example.Referring to the method for embodiment 2, prepare the E.tarda vaccine of beta-mercaptoethanol deactivation.The preparation of streptococcus agalactiae vaccine is referring to the method for embodiment 1, use the deactivation of lowest dose level formalin to carry out deactivation to streptococcus agalactiae (S.agalactiae), the substratum of S.agalactiae growth is selected brain heart infusion (Brian Heart Infusion, BHI) substratum.
In application, above-mentioned vaccine is mixed with to bigeminy vaccine in the ratio of 1:1, in this vaccine, the final concentration of two kinds of deactivation bacteriums is 10
8cFU/mL.In immunization feedstuff, contain β-1, the bigeminy vaccine (0.2g vaccine dry weight/1kg feed) of 3-dextran (0.5g dextran/1kg feed) and above-mentioned deactivation.In application, first will after healthy bullfrog immersion bigeminy vaccine 10-20min, take out, after 14-30 days, the continuous 5 days above-mentioned immunization feedstuffs of throwing something and feeding, after 60 days, calculate rate of body weight gain.In artificial challenge's experiment, use respectively 10 times of LD
50the fresh S.agalactiae of dosage and E.tarda bacterium liquid carry out abdominal injection to 10 bullfrogs, add up respectively mortality ratio, and according to formula RPS=[1 – (immune group mortality ratio/control group mortality ratio)] × 100%.Calculate RPS, in result, the RPS of E.tarda and S.agalactiae is reached respectively to 60%, and have certain gaining effect, it is qualified to be considered as.
Include the immunity application of the bigeminy vaccine of tarda inactivated vaccine prepared by other sulfhydryl reagent, also can be referring to this application mode.
Embodiment 10: comprise the application mode of Edwardsiella tarda inactivated vaccine interior triple inactivated vaccine immunity tilapia (Oreochromis niloticus).
Another two kinds of inactivated vaccines in triple vaccine except E.tarda vaccine, taking Vibrio anguillarum (Vibrio anguillarum) and Aeromonas hydrophila (Aeromonas hydrophila) as example, the preparation of these two kinds of deactivations can adopt conventional ablation method (as formalin deactivation, hot deactivation, high pressure deactivation etc.) to carry out.
In inactivated vaccine prepared by sulfhydryl reagent, taking beta-mercaptoethanol as example, first referring to embodiment 2, preparation E.tarda inactivated vaccine.Again three is mixed with to triple vaccine in the ratio of 1:1:1, in this vaccine, the final concentration of three kinds of deactivation bacteriums is 10
8cFU/mL.Immunity, with in feed preparation, makes an addition to above-mentioned triple vaccine in feed in the ratio of 0.3g vaccine dry weight/1kg feed.In application, first after healthy tilapia being soaked to triple vaccine 10-20min, take out, after 14-30 days, the continuous 5 days above-mentioned immunization feedstuffs of throwing something and feeding, after 60 days, with fresh E.tarda, V.anguillarum and the A.hydrophila of 10 times of LD50 dosage, 10 tilapias are carried out to abdominal injection respectively, add up respectively mortality ratio, and according to formula RPS=[1 – (immune group mortality ratio/control group mortality ratio)] × 100%.Calculate RPS, in result, the RPS of E.tarda, V.anguillarum and A.hydrophila is reached respectively to 50%, it is qualified to be considered as.
Include the immunity application of the triple vaccine of tarda inactivated vaccine prepared by other sulfhydryl reagent, also can be referring to this application mode.
The above each embodiment is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvement, these improvement should be considered as protection scope of the present invention.
Claims (10)
1. a tarda ablation method, is characterized in that, adopts sulfydryl inactivator to carry out inactivation treatment to tarda, can maintain the immunogenicity of tarda antigen.
2. tarda ablation method according to claim 1, it is characterized in that, adopt minimum deactivation intensity to carry out inactivation treatment to tarda, described minimum deactivation intensity is between the sulfydryl inactivator of lowest dose level, minimum deactivation temperature and the shortest inactivation time and combines, and negative correlation each other between three.
3. tarda ablation method according to claim 2, is characterized in that, the dosage of described sulfydryl inactivator is to be less than or equal to 10% volume fraction.
4. tarda ablation method according to claim 3, is characterized in that, described deactivation temperature is 0～100 DEG C.
5. tarda ablation method according to claim 3, is characterized in that, described inactivation time is 10min～48h.
6. tarda ablation method according to claim 3, is characterized in that, described sulfydryl inactivator is beta-mercaptoethanol.
7. the tarda inactivated vaccine making according to method described in the arbitrary claim of claim 1～6, is characterized in that, tarda, after the whole deactivations of minimum deactivation intensity, after inactivator is removed, obtains described vaccine.
8. tarda inactivated vaccine according to claim 7, is characterized in that, also can contain adjuvant in described tarda inactivated vaccine.
9. tarda inactivated vaccine according to claim 8, it is characterized in that, described adjuvant is one or more in insoluble aluminium salt colloid, profit emulsion, microbe composition, nucleic acid and analogue, polysaccharose substance, cytokine, liposome, immunostimulating complex, propolis or medicinal herb components.
10. tarda inactivated vaccine according to claim 7, can be used as the application of preparation treatment tarda infection medicine.
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