CN102988981A - Adjuvant for improving immunization effect of Edwardsiella vaccine and use method of adjuvant - Google Patents
Adjuvant for improving immunization effect of Edwardsiella vaccine and use method of adjuvant Download PDFInfo
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Abstract
The invention relates to an adjuvant for improving the immunization effect of Edwardsiella vaccine and a use method of the adjuvant. The adjuvant is characterized by being extracted from raw materials including grain, yeast and a part of fungi or algae. The effective component of the adjuvant is beta-1,3-glucan or the natural, artificially modified or synthetic product of the beta-1,3-glucan. The adjuvant can be used for increasing the specific immune protection ratio of any one or more inculcated Edwardsiella vaccine antigens including the inactivated bacteria, the bacteria disintegration component, the less-virulent strain, the attenuated strain, the protective antigen, the antigen subunit, the antigenic determinant clusters or the expression product of the antigen cell expression vector of the Edwardsiella, can be used together or not together with the vaccine antigen and can be prepared into a single preparation which is used together with the vaccine antigen.
Description
Technical field
The present invention relates to tarda vaccine immunity Prevention Technique, specifically a kind of adjuvant and application process thereof that can strengthen tarda vaccine virus immunization effect.
Background technology
Tarda (Edwardsiella sp.), it is one of the main pathogenic fungi in the current aquaculture, comprise at present Edwardsiella tarda (E. tarda) Channel-catfish fish tarda (E. ictaluri) and three kinds of guarantor section tarda (E. hoshinae), its host range is very extensive, Fish, amphibian, reptiles, birds and comprise that human mammals has the report of infection, and be worldwide distribution, be prevalent in fresh water and the briny environment, mainly be distributed in China, Australia, Japan, India, Israel, Malay Archipelago, the U.S., Panama waits the Perenniporia martius countries and regions.From reported first in 1962 so far, this cause of disease has already caused massive losses to multiple fish culture.The methods such as employing antibiotic and chemicals are prevented or are treated, and often bring the drawbacks such as pollution of drug resistance, drug residue and water environment such as cause of disease, have also caused the relevant issues such as food safety and environmental pollution.Current aspect the aquatic animal control and prevention of disease, the disease control technology so that the vaccine immunity technology is generally approved in the world, accepted and actively promote as the Comprehensive Preventing control techniques system on basis has good development prospect.At present, there has been comparatively widely research at domestic place to the tarda vaccine, and has obtained a series of achievement in research.Wherein traditional vaccine (comprising inactivated vaccine, cell extract, Adjuvanted vaccines) is made simply because having; the advantages such as cost is low, safety is high become the first-selection of various vaccine forms; but studies show that; tarda is different from other pathogen, and the immune protective effect that conventional inactivated vaccine produces is desirable not to the utmost.The relative protection ratio (RPS) of the tarda injecting immune Japan eel of formalin deactivation in Gutierrez and Miyazaki (1994) research only is 12.5-25%; Sun etc. (2011) report only has 33.3% with the RPS of the unit price inactivated vaccine immunity Paralichthys olivaceus (Paralichthys olivaceus) of tarda; The relative protection ratio (RPS) of the E. tarda inactivated vaccine that our previous experiments employing routine techniques is made is also less than 40%.In the recent period, (2011) such as Hossain and Kawai (2009) and Hossain have reported employing high pressure deactivation (600psi, the tarda inactivated vaccine that mode 5min) prepares has preferably immune effect, behind the lumbar injection Japan eel (Anguilla japonica), its RPS is in 80%-85% scope.But there is technical limitation in the method for high pressure, and for example the equipment cost is very high, and technical difficulty is large, is difficult to antibacterial in enormous quantities is carried out productive operation.In recent years, fast development along with biotechnology, the tarda new generation vaccine continues to bring out, and in the aquatic animal experiment, demonstrate good application prospect, such as dna vaccination (Jiao etc. (2009), subunit vaccine (Liu etc. (2005), Jiao etc. (2009), ghost vaccine (Kwon etc. (2007), attenuated live vaccine (Mo etc., 2007; More report Xiao etc., 2011) etc. is also arranged,, but consider that for factors such as application security, lead time and costs these new generation vaccines still can not get commercial application.On the whole, the immune effect of tarda vaccine is desirable not to the utmost, and in recent years, people have begun to pay attention to the application of adjuvant in its immunological technique, and have obtained certain achievement.Adjuvant is nonspecific immunity strengthening agent, mixes with antigen to use and can enhancing human body immunity replys or change type of immune response.Castro etc. (2008) result of study deactivation tarda vaccine and commercial adjuvant Montanide ISA 763 AVG(Seppic, France) unite use, 1 injection inoculation immunity turbot, its RPS still surpassed 90% after 6 months, and the RPS of single injecting immune deactivation group only has 20% at this moment; In the report of above-mentioned (2011) such as Sun, when the tarda of deactivation and Vibrio anguillarum (Vibrio anguillarum) etc. in addition three kinds of bacterium unite when using, its RPS can reach 70.9%, and other three kinds of bacterium inactivated vaccines such as Vibrio anguillarum have played the effect of tarda inactivated vaccine immunological adjuvant among this result.
The vaccine adjuvant kind is less in aquaculture at present, and effect each is variant, the adjuvant report that wherein is applied to the tarda vaccine is very few, it is significant for the immune prevention and control of this cause of disease therefore to develop its new and effective adjuvant.β-1, the 3-glucosan is a kind of polysaccharide, is mainly derived to comprise in corn, yeast, part fungus and the Sargassum.Studies confirm that glucosan self has special molecular structure and makes it easily by immune system recognition and acceptance, effectively activating macrophage, neutrophils etc., stimulate the generation of the release cells factor and specific antibody, and induce the humoral and cellular immune response of body, improve the immunologic function of body.Beta glucan also has the free radical of removing, radioprotective, antitumor, dissolving cholesterol, the effect of prevention hyperlipemia, the infection that filterable virus, fungus, antibacterial etc. are caused also has certain resistant function, has than extensive use in industries such as medicine, food, cosmetics at present.The application of glucosan in aquaculture mainly is to use as nonspecific immunity strengthening agent, the common non-specific premunition that is used for improving prawn of adding in the shrimp feedstuff for example, also have as the fish feed additive in order to non-specifically to improve the premunition of fish, the interpolation of glucosan has the enhancing aquatic animal to the resistance of cause of disease, promote growth, and can improve fertility performance and the efficiency of feed utilization of animal, but at present not with research report, patent or the application of glucosan as the adjuvant of tarda vaccine.
Summary of the invention
The purpose of this invention is to provide a kind of adjuvant and application process thereof that strengthens tarda vaccine virus immunization effect, to improve the postvaccinal immune effect of tarda vaccine antigen.
A kind of adjuvant that strengthens tarda vaccine virus immunization effect of the present invention is the glucosan extract that obtains or the goods that contain the glucosan composition from corn, yeast, fungus or Sargassum; And and have and strengthen the immunity inoculation effect, above-mentioned glucosan extract or the goods that contain the glucosan composition are carried out artificial reconstructed, synthetic product.
Above-mentioned glucosan extract or contain the β-1 of these goods of the goods of glucosan composition, the 3-glucosan, or have the β-1 that strengthens the vaccination effect, artificial reconstructed, the synthetic product of 3-glucosan.
The using method of the adjuvant of enhancing tarda vaccine virus immunization effect of the present invention comprises following three kinds of modes:
1) being mixed and made into bacterin preparation with vaccine antigen uses;
2) make the adjuvant formulation that does not comprise vaccine antigen, before immunity inoculation with after vaccine antigen mixes, use simultaneously;
3) do not use simultaneously with vaccine antigen.
For the 1st kind of mode, join in injection, dipping bath agent or the oral agents that comprises vaccine antigen, above-mentioned adjuvant effective ingredient and vaccine antigen weight ratio are 1/10-1000/1, and its immunity inoculation approach can be immune for the immunity of injecting immune, wound, dipping bath, oral immunity or other import to any immunity inoculation approach in the aquatic animal body with adjuvant effective ingredient and vaccine antigen.
For the 2nd kind of mode, the preparation that contains above-mentioned adjuvant effective ingredient adds in the preparation that contains vaccine antigen before vaccine antigen carries out immunity inoculation to aquatic animal and abundant mixing temporarily, addition is 1/10-1000/1 of vaccine antigen weight, and its immunity inoculation mode can be injection, wound, dipping bath or oral immunity.
For the 3rd kind of mode, joining the effective ingredient of the above-mentioned adjuvant in aquatic animal feed or the dipping bath water body and feedstuff or dipping bath water body weight ratio is 1/100000-1/100, the interpolation time is that bacterin preparation treats that immune aquatic animal is injected, front 3 days of wound, dipping bath or oral immunization be to inoculating in rear 30 days, implements 1-5 days throw something and feed or the immunostimulant operation such as dipping bath.
Above immune application mode makes adjuvant effective ingredient β-1, the 3-glucosan reaches certain concentration in tarda vaccine antigen and aquatic animal immune system as the time spent, thereby allow immunocyte in the process of submission, processing, identification vaccine antigen, be subject to the stimulation of glucosan, make the activity of immunocyte be able to further raising, thereby activated immune system effectively strengthens the immune effect of vaccine antigen to the replying of vaccine antigen better.The present invention finds that the dosage of adjuvant effective ingredient has material impact to immune effect of vaccine, and in adjuvant safe concentration scope, both have certain dependency.When the using dosage of adjuvant effective ingredient surpasses the safe dose scope, can lethal effect be arranged to aquatic animal, therefore be necessary for the maximum dose level that can not cause aquatic animal death in 2-4 weeks after under immune type of aquatic and concrete immunization ways condition, carrying out the administering mode test of adjuvant or find out administration, the safe dose when determining that with this above-mentioned adjuvant effective ingredient uses in aquatic animal.The present invention confirm the dosage of effective ingredient in the aquatic animal body of above-mentioned adjuvant reach safe dose 1/100-4/5 the time can strengthen the immune effect of vaccine.
Tarda vaccine adjuvant of the present invention can be applicable to Fish, the vaccine virus immunization of amphibian and reptiles comprises that marine fish is (such as Paralichthys olivaceus, turbot, Cynoglossus semilaevis, cabrilla, Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis), snapper, Rachycentron canadum, Atlantic salmon, puffer etc.), the brackish water aquaculture Fish are (such as tilapia, rainbow trout, Mugil cephalus etc.), straddling fish stocks are (such as anguilla japonica, Carnis Pseudosciaenae etc.), cultured freshwater fish is (such as Mylopharyngodon piceus, Hypophthalmichthys molitrix, Aristichthys nobilis, Cyprinus carpio, Carassius auratus, Silurus asotus fish, Siniperca chuatsi, Acipenser Sinensis, Australia gem perch etc.), pet fish is (such as Carassius auratus, Cyprinus carpio L. etc.), amphibian is (such as bull frog, Andrias davidianus Blanchard etc.) and reptiles (such as Trionyx sinensis Wiegmann, Testudinis, Serpentis etc.) vaccination.The safe dose of different aquatic animals is different with effective dose, for the concrete application dose of specific aquatic animal, should test separately.
Good effect of the present invention is: related adjuvant can improve specificity and the nonspecific immunity protection after the tarda vaccine antigen immunity inoculation.For this intracellular infection of tarda or vaccine with cause of disease of immune evasion ability; adjuvant of the present invention can effectively strengthen immanoprotection action; using method safety to operator and environmentally friendly, thereby has a good application prospect and market popularization value.
The specific embodiment
In view of the dosage of adjuvant effective ingredient to vaccine antigen immune effect important, the using dosage of adjuvant effective ingredient is crossed and is not lowly often reached the effect that strengthens immunity, and too high using dosage tends to aquatic animal is produced unfavorable or lethal effect.Therefore above-mentioned adjuvant in use should at first be determined safe dose scope and the best working concentration of above-mentioned adjuvant effective ingredient in concrete vaccine product for concrete type of aquatic, age and inoculation method etc. by the vaccine Protection.The concentration of adjuvant effective ingredient is 1mg/g-20mg/g(adjuvant effective ingredient weight/bacterin preparation weight in the tarda bacterin preparation), the concentration of adjuvant effective ingredient is 0.1g/kg-5g/kg(adjuvant effective ingredient weight/feedstuff weight in the aquatic animal feed), the concentration of adjuvant effective ingredient reaches 1.0 * 10 in the aquatic animal body after administration
-3Mg/g-5.0mg/g(adjuvant effective ingredient weight/aquatic animal body weight).
Antigenic component in the tarda bacterin preparation can stem from the Edwardsiellas such as Edwardsiella tarda, Channel-catfish tarda or guarantor section tarda antibacterial one or more cause of disease antigen or can in the aquatic animal body, produce corresponding antigen, also can be to comprise the united vaccine formulation that the antigen of the antigen that is derived from tarda and other pathogen or virus is made.The type of vaccine antigen can be any of the expression vector of inactivated bacteria body, ghost composition, low virulent strain, attenuated strain, protective antigen, Antigen Subunit, antigenic determinant or antigen gene or more than one; singly one or more exist with associating, integration or multivalence mode above-mentioned antigen in bacterin preparation; the tarda bacterin preparation of making also can comprise other the Multiple components such as adjuvant, protective agent, antiseptic, diluent, solvent except antigen maybe can produce the expression vector of antigen.Adjuvant of the present invention can mix with vaccine antigen use in application, use simultaneously after also can making unitary agent and vaccine antigen mixes, and can also not use simultaneously with vaccine antigen.Immunity inoculation mode in the application can be injecting immune, wound immunity, immersion immunity or oral immunity.
The present invention proves the β-1 in the yeast extract, and the 3-glucosan can effectively play the effect of tarda vaccine adjuvant, when using with the tarda inactivated vaccine is collaborative in the application, can significantly improve the immune protective rate of tarda inactivated vaccine.Described β-1, the 3-glucosan extracts from corn, yeast, fungus or Sargassum, the present invention has confirmed β-1, the 3-glucosan has the effect that strengthens tarda vaccine virus immunization effect, therefore the effect of effective ingredient of the present invention comprises β-1, the natural product of 3-glucosan, artificial reconstructed product or synthetic product.Natural β-1; the 3-glucosan can extract from corn, yeast, part fungus and Sargassum etc.; for not changing β-1; the artificial reconstructed product of 3-glucosan nuclear molecular structure or synthetic and imitated product may strengthen or weaken effect involved in the present invention; as long as this effect still keeps, just still belong within the protection domain of this patent.
Using method of the present invention is as follows:
(1) determining of adjuvant safe dose: preparation comprises 0.1%-10% PBS(pH7.2 of beta glucan) solution, membrane filtration degerming through 0.22 μ m, treat that with the above-mentioned adjuvant inoculation of variable concentrations immune aquatic animal, vaccination ways can be injecting immune, wound immunity, immersion immunity or oral immunity again.The immunity aquatic animal observed for 2 weeks, determined this adjuvant safe dose according to experimental result.
(2) tarda bacterin preparation preparation and using: in the inactivated vaccine preparation, add in the tarda suspension formaldehyde (36.5-38%, w/v), regulating its final concentration is 0.3%--1%, 4 ℃ are spent the night, use PBS(pH7.2) clean 3 times after, prepare certain density inactivated vaccine.With the adjuvant in the safe dose scope with treat immune aquatic animal after inactivated vaccine mixes according to a certain percentage and carry out immunity inoculation, again aquatic animal is carried out normal aquaculture management and gets final product.When using attenuated live vaccine, then need not to carry out inactivation treatment.
(3) the determining of adjuvant optimal dose in the vaccine product:
The using method of bacterin preparation can have following three kinds of modes: 1) be mixed and made into bacterin preparation with vaccine antigen and use; Perhaps 2) use simultaneously after making unitary agent and vaccine antigen mixing; Perhaps 3) do not use simultaneously with vaccine antigen.
For the 1st kind of mode, join that above-mentioned adjuvant effective ingredient and vaccine antigen weight ratio are 1/10-1000/1 in injection, dipping bath agent or the oral agents that comprises the tarda vaccine antigen, its immunity inoculation mode can be injection, wound, dipping bath or oral immunity.
For the 2nd kind of mode, above-mentioned adjuvant effective ingredient is interim the interpolation and abundant mixing before the tarda bacterin preparation carries out immunity inoculation to aquatic animal, addition is 1/10-1000/1 of vaccine antigen weight, and its immunity inoculation mode can be injection, wound, dipping bath or oral immunity.
For the 3rd kind of mode, joining the effective ingredient of the above-mentioned adjuvant in aquatic animal feed or the dipping bath water body and feedstuff or dipping bath water body weight ratio is 1/100000-1/100, the interpolation time is that the tarda bacterin preparation treats that immune aquatic animal is injected, front 3 days of wound, dipping bath or oral immunization be to inoculating in rear 15 days, implements 1-5 days throw something and feed or the immunostimulant operation such as dipping bath.
Until immune aquatic animal after the inoculation of above-mentioned bacterin preparation; 30-60 days laggard pedestrian worker's infection experiments; by the analysis of the immune indexes such as the relevant physiological biochemical indicator (such as rate of body weight gain, antibody titer etc.) after 14-60 days and relative protection ratio (RPS), to determine the best using dosage of adjuvant.
Embodiment 1: β-1, the 3-glucosan is determined turbot (Scophthatmus maximus) injection safety dosage
Select healthy turbot (11.4 ± 1.8) g, in cement pit, after temporarily foster 2 weeks, place the circular glass steel drum (to be filled with water 0.7 meter before the test
3) in, flowing water culture, water temperature is 18 ± 2 ℃, salinity 28 ‰ ± 2 ‰, continuous charge in 24 hours.Duration of test is by fish body weight 3% mixed feed of throwing something and feeding, and day throws something and feeds 3 times, changes water every day and removes contamination 2 times.Before the injection to turbot MS222(1:9000) soak anaesthetic treatment.
Be respectively the β-1 of 2,5,8,10 and 20 mg/mL with concentration, 3-glucosan lumbar injection 20 tail turbot, injected dose are 100 μ L/ tails, observe 14 days.
Death condition sees Table 1 in each experimental group turbot 14 days, determines β-1 by the result, and the 3-glucosan is 1000 μ g/ tails to the maximum safe lumbar injection dosage of turbot, i.e. 87.7 μ g/g(glucosan/fish body weight).
Table 1. β-1, the 3-glucosan is to turbot injection safety dosetest
Embodiment 2: β-1,3-glucosan make an addition in the formalin deactivation E. tarda vaccine, immunity inoculation turbot (S. maximus).
Test with fish and aquaculture management with embodiment 1.
E. tarda 1101 is inoculated in the pancreas peptone soybean broth (TSB), and 28 ℃ of shaken cultivation are spent the night, and are forwarded among the fresh TSB with 1:100 again, 28 ℃ of shaken cultivation 5h, collect bacterium liquid, 6000g, 4 ℃, 10min, aseptic PBS(pH7.2) cleans 3 times, be resuspended in PBS, and dilution spread is in tryptose soya agar (TSA), colony counting method is adopted in count of bacteria, makes 1 * 10
9The CFU/mL bacteria suspension adds formalin and (contains formaldehyde 36.5-38%, w/v), regulate its final concentration and be 1%, 4 ℃ and spend the night that make inactivated vaccine, PBS washs thalline 3 times (method is the same), 4 ℃ of preservations.The bacterium liquid 0.2mL that other gets after the deactivation is coated with TSA, cultivates 48h for 28 ℃, determines without colony growth.During vaccine safety detects, get vaccine and regulate concentration to 10
8CFU/mL, lumbar injection 20 tail turbot, injected dose are 100 μ L/ tails, observe 14 days, to determine the safety of bacterin preparation.
Healthy turbot is carried out random packet, and each group is established 3 parallel group.Matched group (injection PBS), β-1 are established in test, the β-1 of 3-glucosan group, inactivated vaccine group, variable concentrations, and 3-glucosan and inactivated vaccine compatibility group, the immunity test grouping sees Table 2.
The grouping of table 2. turbot immunity test
Adopt lumbar injection immunity turbot, injected dose is 100 μ L/ tails.
After the immunity 28 days, the fresh E. tarda of turbot lumbar injection bacterium liquid (1.35 * 10
4The CFU/ tail), carry out Experimental infection.
Added up the mortality rate of each test group in 14 days behind the counteracting toxic substances, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculate RPS.The results are shown in Table 3.
Table 3. glucosan is that E. tarda inactivated vaccine adjuvant is to the immune protective effect (mean+SD) of turbot
Above result shows, uses merely E. tarda formalin-inactivated vaccine (10
7The cfu/ tail) or 43.9 μ g/g (adjuvant dosage/body weight) β-1, the immune protective effect of 3-glucosan injection turbot is lower, and its RPS value is respectively 18.5% and 14.7%.Inactivated vaccine adds 21.9,43.9 and 87.7 μ g/g(adjuvant dosage/body weight) β-1, during the 3-glucosan, raising RPS that can be in various degree.Wherein add 43.9 μ g/g(adjuvant dosage/body weight with inactivated vaccine) β-1, the 3-glucosan has preferably immune protective effect, and RPS brings up to 64%.Above result proves that adjuvant of the present invention can strengthen the immune protective effect of vaccine preferably.
Embodiment 3: β-1,3-glucosan make an addition in the formalin deactivation E. tarda vaccine, and immune turbot is on the impact of serum antibody titer
Test with fish and aquaculture management with embodiment 1.
Bacterin preparation preparation and EXPERIMENTAL DESIGN (experiment grouping and immunization method) are with embodiment 2, and immunity was got 5 tail fishes for every group and extracted the 0.1mL tail vein after 28 days, and room temperature leaves standstill 2h, places 4 ° of C again, and 12h collects serum, mixes on the same group fish serum ,-20 ℃ of storages.
The antibody titer of serum is measured mouse monoclonal antibody (Aquatic Diagnostic Ltd, the Britain) operation instruction with reference to the brill IgM of the Chinese People's Anti-Japanese Military and Political College, and every hole adds 100 μ L 1.0 * 10
8E. tarda 1101 suspensions that cfu/mL is resuspended in the coating buffer are added a cover rear 4 ℃ of coated 96 hole ELISA Plate of spending the night, and then add 0.05% (v/v) glutaraldehyde of 50 μ L PBS dilution to each hole, and 22 ℃ leave standstill 20 min, wash plate 3 times with the less salt washing liquid; Add 250 μ L, 1% BSA sealing, 22 ℃ leave standstill 2h, and low saline solution is washed plate 3 times; Add respectively again 100 μ L in every hole with the turbot serum of PBS serial dilution, each dilution factor do 3 parallel, 22 ℃ leave standstill 3 h after high salt washing liquid wash plate 5 times, hatch for the last time 5min; Every hole adds the brill IgM of the Chinese People's Anti-Japanese Military and Political College monoclonal antibody of 100 μ L regeneration, washes plate 5 times by aforementioned high level salt solution plate washing method after leaving standstill 60min for 22 ℃; Add the sheep anti mouse two anti-(day root biochemical technology company limited) of the HRP labelling of 100 μ L dilution in 1: 1000 in every hole, 22 ℃ leave standstill 60 min, wash plate 5 times by aforementioned high level salt solution again; Every hole adds 100 μ L TMB solution afterwards, 22 ℃ of colour developing 10 min; Every hole adds 50 μ L 2M H2SO4 cessation reactions, surveys the OD value at the 450nm place with microplate reader.The OD value of analytic sample (P) and blank (N) is calculated P/N ratio, and P/N 〉=3.0 o'clock sample is positive, and P/N＜3.0 o'clock sample is negative.The antibody titer of each group the results are shown in Table 4.
Table 4. glucosan is that E. tarda inactivated vaccine adjuvant is on the impact (mean+SD) of the serum antibody titer of turbot
Above result shows, matched group (PBS), E. tarda formalin-inactivated vaccine (10
7The cfu/ tail), 43.9 μ g/g (adjuvant dosage/body weight) β-1, the serum antibody titer value of 3-glucosan injection turbot is very low, be respectively 106.7 ± 37.0,170.7 ± 73.9 and 213.3 ± 73.9, inactivated vaccine adds 21.9,43.9 and 87.7 μ g/g(adjuvant dosage/body weight) β-1, during the 3-glucosan, raising antibody titer that can be in various degree.Wherein add 43.9 μ g/g(adjuvant dosage/body weight with inactivated vaccine) β-1, the 3-glucosan has preferably immune protective effect, and the antibody titer value reaches 682.7 ± 147.8.Above result proves that adjuvant of the present invention can increase the antibody titer in the serum preferably.
Embodiment 4: β-1,3-glucosan make an addition to formalin Mie Huo Channel-catfish fish tarda (Edwardsiella ictaluri)
In the vaccine, the application mode of immune Pelteobagrus fulvidraco (Pelteobagrus fulvidraco).
Select healthy Pelteobagrus fulvidraco, at first carry out β-1 with reference to the method in experimental example 1 and 2, the determining of 3-glucosan and the best applications dosage of E. ictaluri inactivated vaccine compatibility in feedstuff.Determine based on this addition of glucosan in the feedstuff, at first adopt immersion immunity in the application, it is 10 that Pelteobagrus fulvidraco has been placed final concentration
8In the fresh fresh water of CFU/mL deactivation E. ictaluri vaccine, behind 10-20min, move into normally cultivation in the fresh water.After 14-30 days, booster immunization is 1 time (decided by fish size by the booster immunization interval, the less then time of body size can shorten, but body size is the time proper extension more then), throw something and feed and contain the immunization feedstuff of glucosan and E. ictaluri inactivated vaccine, the inactivated vaccine addition is 0.1g/kg(inactivated vaccine weight/feedstuff weight in the immunization feedstuff), threw something and fed continuously 5 days, carry out again normal feeding and management.Behind the booster immunization 30-60 days, calculate the rate of body weight gain of duration of test, other gets the part experiment with 10 times of LD of Pelteobagrus fulvidraco lumbar injection
50The fresh E. ictaluri bacterium liquid of dosage carries out Experimental infection.Infected rear 14-30 days, the statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculate RPS, to obtaining data analysis, glucosan is assessed as the adjuvant immunity Pelteobagrus fulvidraco application mode of E. ictaluri inactivated vaccine, have preferably gaining effect and RPS value above 70%, it is qualified to be considered as.
Embodiment 5: β-1,3-glucosan make an addition in deactivation Edwardsiella tarda (E. tarda) vaccine, the application mode of immune tilapia (Oreochromis niloticus).
At first preparation has immunogenic Edwardsiella tarda (E. tarda) vaccine, and antigen contained in this vaccine can be deactivation thalline, ghost composition, less-virulent strain or the attenuated strain of tarda.In the application, with reference to the method in experimental example 1 and 2 healthy tilapia is carried out β-1, the determining of 3-glucosan and the best applications dosage of E. tarda vaccine compatibility in feedstuff.Determine based on this addition of glucosan in the feedstuff, in the application tilapia is adopted immersion immunity, it is 10 that tilapia is placed final concentration
8In the water body of CFU/mL E. tarda vaccine, behind 10-20min, move into normally cultivation in the fresh water body.After 14-30 days, carry out booster immunization 1 time (booster immunization is decided by fish size blanking time, and the less then time of body size can shorten, but body size time proper extension more then).Throw something and feed and contain the immunization feedstuff of glucosan and E. tarda vaccine compatibility, the vaccine addition is 0.1g/kg(vaccine weight/feedstuff weight in the immunization feedstuff), threw something and fed continuously 5 days, after 60 days, calculate the rate of body weight gain of duration of test, in addition to 10 times of LD of tilapia lumbar injection
50The fresh E. tarda bacterium liquid of dosage carries out Experimental infection.Infected rear 14-30 days, the statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculating RPS, to obtaining data analysis, is that E. tarda vaccine adjuvant immunity tilapia application mode is assessed to glucosan, has certain gaining effect and RPS value and surpasses 70%, and it is qualified to be considered as.
Embodiment 6: β-1,3-glucosan make an addition in the dual-gene disappearance of esrB, the evpC attenuated strain of E. tarda, the application mode of immune turbot (S. maximus).
Select healthy turbot, at first carry out β-1 with reference to the method in experimental example 1 and 2, the 3-glucosan dual-gene disappearance of esrB, the evpC of optimum addition and E. tarda attenuated strain in feedstuff is soaked determining of turbot maximum safe concentration.Immersion immunity 5-10min in sea water at first in the application, the concentration of soaking the dual-gene disappearance of esrB, the evpC attenuated strain of used E. tarda in the sea water is 1/10 of above-mentioned maximum safe concentration, moves into normally cultivation in the fresh seawater again.After 14-30 days, throwing something and feeding in continuous 5 days contains best applications dosage β-1, and the feedstuff of 3-glucosan carries out normal feeding and management again.Behind the booster immunization 30-60 days, calculate the rate of body weight gain of duration of test, in addition to 10 times of LD of turbot lumbar injection
50The fresh wild E. tarda bacterium liquid of dosage carries out Experimental infection.14-30 days statistics mortality rates after infecting, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculate RPS, to obtaining data analysis, to β-1, the 3-glucosan is assessed as E. tarda attenuated live vaccine adjuvant immunity turbot application mode, and the RPS value surpasses 70%, and it is qualified to be considered as.
Embodiment 7: β-1, the application mode of the streptococcus agalactiae of 3-glucosan and deactivation (Streptococcus agalactiae) and Edwardsiella tarda (E. tarda) bivalent inactivated vaccine compatibility immunity bull frog (Rana catesbeiana).
At first use streptococcus agalactiae (S. agalactiae) and Edwardsiella tarda (E. tarda) vaccine of traditional ablation method (such as formalin deactivation, hot deactivation, high pressure deactivation etc.) preparation deactivation.Ratio in 1:1 is mixed with bigeminy vaccine, and the final concentration of two kinds of deactivation antibacterials is 10 in the vaccine
8CFU/mL.Immunization feedstuff preparation, by 0.25,0.5,0.75 and 1g/kg(adjuvant effective ingredient weight/feedstuff weight) proportioning with the β-1 of various dose, the 3-glucosan is added in the feedstuff.In the application, at first will be healthy bull frog take out after soaking bigeminy vaccine 10-20min, after 14-30 days, threw something and fed in continuous 5 days and to contain the glucosan feedstuff.After 60 days, relatively add the various dose glucosan in the feedstuff to the impact of rate of body weight gain, in addition the bull frog in each proportioning group is divided into two groups at random, each group uses respectively 10 times of LD
50The fresh S. agalactiae of dosage and E. tarda bacterium liquid carry out lumbar injection, carry out Experimental infection.Infected rear 14-30 days, the statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculate RPS, to obtaining data analysis, glucosan is assessed as S. agalactiae and E. tarda bivalent inactivated vaccine adjuvant immunity tooth bull frog application mode, any its RPS to above-mentioned two kinds of cause of diseases among the infection experiment result surpasses 70%, and have its corresponding glucosan addition of certain gaining effect and be considered as using addition, it is qualified that its adjuvant effect also is considered as.
Embodiment 8: β-1,3-glucosan make an addition in the multiple vaccines of formalin deactivation preparation, the application mode of immune Paralichthys olivaceus (Paralichthys olivaceus).
At first use traditional ablation method (such as formalin deactivation, hot deactivation, high pressure deactivation etc.) that Vibrio anguillarum (Vibrio anguillarum), Aeromonas hydrophila (Aeromonas hydrophila) and Edwardsiella tarda (E. tarda) are carried out deactivation, again the three is mixed with triple vaccine in the ratio of 1:1:1, the final concentration of three kinds of deactivation antibacterials is 10 in this vaccine
8CFU/mL.Immunization feedstuff preparation, by 0.25,0.5,0.75 and 1g/kg(adjuvant effective ingredient weight/feedstuff weight) proportioning with the β-1 of various dose, the 3-glucosan is added in the feedstuff.In the application, at first healthy Paralichthys olivaceus is soaked in triple vaccine 10-20min after, move into again in the fresh seawater normally cultivation.After 14-30 days, threw something and fed in continuous 5 days and to contain the glucosan feedstuff.After 60 days, relatively add the various dose glucosan in the feedstuff to the impact of rate of body weight gain, in addition the fish in each proportioning group is divided into three groups at random, each group uses respectively 10 times of LD
50Fresh V. anguillarum, the A. hydrophila of dosage and E. tarda bacterium liquid carry out Experimental infection, infect the lumbar injection mode that adopts.Infected rear 14-30 days, the statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculate RPS, to obtaining data analysis, glucosan is assessed as V. anguillarum, A. hydrophila and E. tarda triple inactivated vaccine adjuvant immunity Paralichthys olivaceus application mode, any its RPS to above three kinds of bacterium among the infection experiment result surpasses 70%, and have its corresponding glucosan addition of certain gaining effect and be considered as using addition, it is qualified that its adjuvant effect also is considered as.
Embodiment 9: β in the vaccine oral agents-1, the affirmation of suitable proportion in 3-glucosan and the vaccine compatibility
With β-1, it is example that the 3-glucosan makes an addition in Edwardsiella tarda (E. tarda) vaccine of deactivation Japanese eel (Anguilla japonica) enforcement oral immunity, and the suitable proportion of adjuvant in the vaccination and antigen concentration is confirmed.
At first preparation has immunogenic slow Edwardsiella vaccine, in this vaccine contained antigen can for one or more of the expression product of deactivation thalline, ghost composition, less-virulent strain, attenuated strain, protective antigen albumen, Antigen Subunit, antigenic determinant or the antigen gene expression carrier of antibacterial with associating, integration or multivalence mode.In the application, select healthy anguilla japonica, each test group is all established 3 parallel group, 20 tails/parallel group, the matched group experimental session normal feedstuff of always throwing something and feeding, and adjuvant effective ingredient, vaccine and feedstuff ratio see Table 5 in each immune group.
Feed ingredient in each immune group of table 5. anguilla japonica immunity test
After throwing something and feeding continuously 5 days by 3% of fish body body weight, the normal feedstuff of throwing something and feeding again.After 30 days, each is organized the corresponding feedstuff of throwing something and feeding again and carries out booster immunization once, after 60 days, calculates the rate of body weight gain of duration of test, in addition to 10 times of LD of anguilla japonica lumbar injection
50The fresh Edwardsiella tarda bacterium liquid of dosage carries out Experimental infection.Infected rear 14-30 days, the statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] * 100%.Calculate RPS, to obtaining data analysis, have certain gaining effect and RPS value in the feedstuff in different glucosans and the slow Edwardsiella vaccine proportioning above 70%, be considered as the suitable proportion of adjuvant and antigen in this vaccine oral agents.
Claims (10)
1. an adjuvant that strengthens tarda vaccine virus immunization effect is characterized in that above-mentioned adjuvant is the glucosan extract that obtains or the goods that contain the glucosan composition from corn, yeast, fungus or Sargassum; And and have and strengthen the immunity inoculation effect, above-mentioned glucosan extract or the goods that contain the glucosan composition are carried out artificial reconstructed, synthetic product.
2. the adjuvant of enhancing tarda vaccine virus immunization effect as claimed in claim 1, the effective ingredient that it is characterized in that described glucosan extract or contain the goods of glucosan composition is β-1, the 3-glucosan.
3. the adjuvant of enhancing tarda vaccine virus immunization effect as claimed in claim 1, the goods that it is characterized in that described glucosan extract or contain the glucosan composition are β-1, the 3-glucosan, or have the β-1 that strengthens the vaccination effect, artificial reconstructed, the synthetic product of 3-glucosan.
4. the application of the adjuvant of enhancing tarda vaccine virus immunization effect claimed in claim 1 is and tarda vaccine antigen coupling immunity inoculation aquatic animal.
5. application as claimed in claim 4, the vaccine antigen that it is characterized in that described adjuvant coupling immunity inoculation comprise one or more the vaccine antigen of the antibacterial of Edwardsiellas such as stemming from Edwardsiella tarda, Channel-catfish tarda or guarantor section tarda; The vaccine antigen type comprises expression product any of deactivation thalline, ghost composition, less-virulent strain, attenuated strain, protective antigen, Antigen Subunit, antigenic determinant or antigen gene expression carrier or more than one.
6. application as claimed in claim 4, the application that it is characterized in that described adjuvant and vaccine antigen coupling immunity inoculation aquatic animal comprises following three kinds of modes: above-mentioned adjuvant and vaccine antigen are mixed and made into bacterin preparation and use, perhaps make the adjuvant formulation that does not contain vaccine antigen but use simultaneously with vaccine antigen, perhaps do not use simultaneously with vaccine antigen.
7. application as claimed in claim 4 is characterized in that three kinds of application modes of described adjuvant are:
When (1) being mixed and made into the bacterin preparation use with vaccine antigen, the effective ingredient and the vaccine antigen weight ratio that join above-mentioned adjuvant in injection, dipping bath agent or the oral agents that comprises the tarda vaccine antigen are 1/10-1000/1;
(2) make the adjuvant formulation that do not contain vaccine antigen but when using simultaneously with vaccine antigen, the effective ingredient of above-mentioned adjuvant is interim the interpolation and abundant mixing before bacterin preparation carries out immunity inoculation to aquatic animal, and addition is 1/10-1000/1 of vaccine antigen weight;
When (3) not using simultaneously with vaccine antigen, the effective ingredient that joins the above-mentioned adjuvant in aquatic animal feed or the dipping bath water body adds by 1/100000-1/100 of feedstuff or dipping bath water body weight, the interpolation time is that bacterin preparation is treated immune aquatic animal and carried out front 3 days of immunity inoculation to inoculating in rear 30 days, can administration 1 time or repeatedly.
8. application as claimed in claim 6, when it is characterized in that described aquatic animal is carried out immunity inoculation, can adopt that the immunity of injecting immune, wound, dipping bath are immune, oral immunity or other import to any immunity inoculation approach in the aquatic animal body with adjuvant effective ingredient and vaccine antigen.
9. application as claimed in claim 4, it is characterized in that described adjuvant is before application, different application mode for above-mentioned adjuvant, find out under immune condition after the administration that effective ingredient is to the safe dose scope of aquatic animal body in 14 days, determine the actual amount of above-mentioned adjuvant, make that the content of effective ingredient in the aquatic animal body is 1/100-4/5 of above-mentioned safe dose after the administration.
10. application as claimed in claim 4, the aquatic animal object that it is characterized in that described adjuvant and the common vaccine virus immunization of using comprises amphibian and the reptiles of marine fish, brackish water aquaculture Fish, straddling fish stocks, cultured freshwater fish, fancy fishes and freshwater aquiculture.
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