CN102988981B - 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 current aquaculture, comprise Edwardsiella tarda (E. tarda) at present, Channel-catfish fish tarda (E.ictaluri) and guarantor section tarda (E. hoshinae) three kinds, its host range is very extensive, Fish, amphibian, reptiles, birds and the mammals comprising the mankind have and infect report, and in 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 in 1962 so far, this cause of disease causes massive losses to multiple fish culture industry.Adopt the method such as antibiotic and chemicals to carry out preventing or treating, often bring the drawbacks such as the pollution of the drug resistance of such as cause of disease, drug residue and water environment, also result in the relevant issues such as food safety and environmental pollution.Current in aquatic animal control and prevention of disease, the Synthetical prevention technical system based on vaccine immunity technology is generally approved in the world, accept the Disease management technology of also actively popularization, has good development prospect.At present, domestic place has had tarda vaccine to be studied comparatively widely, and achieves a series of achievement in research.Wherein traditional vaccine (comprising inactivated vaccine, cell extract, Adjuvanted vaccines) makes simply because having; the first-selection that cost is low, safety advantages of higher becomes various vaccine form; but research shows; tarda is different from other pathogen, and the immune protective effect that conventional inactivated vaccine produces is desirable not to the utmost.In Gutierrez and Miyazaki (1994) research, the relative protection ratio (RPS) of the tarda injecting immune Cuscuta japonicoa of Formalin inactivation is only 12.5-25%; The RPS of unit price inactivated vaccine immunity Paralichthys olivaceus (Paralichthys olivaceus) of Sun etc. (2011) report tarda only has 33.3%; The relative protection ratio (RPS) of the E. tarda inactivated vaccine that our previous experiments adopts routine techniques to make is also less than 40%.In the recent period, Hossain and Kawai (2009) and Hossain etc. (2011) report and adopt high pressure deactivation (600psi, tarda inactivated vaccine prepared by mode 5min) has good immune effect, after lumbar injection Cuscuta japonicoa (Anguilla japonica), its RPS is in 80%-85% scope.But the method for high pressure exists technical limitation, such as equipment cost is very high, and technical difficulty is large, is difficult to carry out productive operation to antibacterial in enormous quantities.In recent years, along with the fast development of biotechnology, tarda new generation vaccine continues to bring out, and good application prospect is demonstrated in aquatic animal experiment, 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; Xiao etc., 2011) etc. more report is also had, but consider 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 started to pay attention to the application of adjuvant in its immunological technique, and achieve certain achievement.Adjuvant is nonspecific immunity strengthening agent, can reply or change type of immune response by enhancing human body immunity with antigen is used in combination.Castro etc. (2008) result of study deactivation tarda vaccine and business adjuvant Montanide ISA 763AVG(Seppic, France) conbined usage, 1 injection inoculation immunity turbot, its RPS is after six months still more than 90%, and now the RPS of single injecting immune deactivation group only has 20%; In the report of above-mentioned Sun etc. (2011), when another three kinds of bacterium conbined usage such as tarda and Vibrio anguillarum (Vibrio anguillarum) of deactivation, its RPS can reach 70.9%, and in this result, other three kinds of bacterium inactivated vaccines such as Vibrio anguillarum serve the effect of tarda inactivated vaccine immunological adjuvant.
In aquaculture, vaccine adjuvant kind is less at present, and effect is each variant, and the adjuvant report being wherein applied to tarda vaccine is very few, therefore develops its new and effective adjuvant significant for the immune prevention and control of this cause of disease.β-1,3-glucosan is a kind of polysaccharide, is mainly derived from and comprises in corn, yeast, some fungi and Sargassum.Research confirms that glucosan self has special molecular structure and makes it easily by immune system recognition and acceptance, can 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 removes free radical, radioprotective, antitumor, dissolving cholesterol, the effect of prevention hyperlipemia, to the infection that filterable virus, fungus, antibacterial etc. cause, also there is certain resistant function, had comparatively extensive use in industries such as medicine, food, cosmetics at present.The application of glucosan in aquaculture mainly uses as nonspecific immunity strengthening agent, such as commonly to add in shrimp feedstuff for improving the non-specific premunition of prawn, also have as fish feed additive in order to non-specifically to improve the premunition of fish, the interpolation of glucosan has enhancing aquatic animal to the resistance of cause of disease, growth promoting effects, and fertility performance and the efficiency of feed utilization of animal can be improved, but at present not using glucosan as the research report of the adjuvant of tarda vaccine, patent or application.
Summary of the invention
The object of this invention is to provide a kind of adjuvant and the application process thereof that strengthen tarda vaccine virus immunization effect, to improve the postvaccinal immune effect of tarda vaccine antigen.
A kind of adjuvant strengthening tarda vaccine virus immunization effect of the present invention is the glucan extraction that obtains from corn, yeast, fungus or Sargassum or the goods containing glucosan composition; And and have and strengthen immunity inoculation effect, carry out product that is artificial reconstructed, synthesis to above-mentioned glucan extraction or containing the goods of glucosan composition.
β-1, the 3-glucosan of above-mentioned glucan extraction or these goods containing the goods of glucosan composition, or artificial reconstructed, the synthetic product with β-1, the 3-glucosan strengthening vaccination effect.
The using method of the adjuvant of enhancing tarda vaccine virus immunization effect of the present invention, comprises following three kinds of modes:
1) be mixed and made into bacterin preparation with vaccine antigen to use;
2) make the adjuvant formulation not comprising vaccine antigen, use after mixing with vaccine antigen before immunity inoculation simultaneously;
3) use time different from vaccine antigen.
For the 1st kind of mode, join comprise vaccine antigen injection, in dipping bath agent or oral agents, above-mentioned adjuvant effective ingredient and vaccine antigen weight ratio are 1/10-1000/1, its immunisation route can for injecting immune, wound immunity, immune, the oral immunity of dipping bath or other adjuvant effective ingredient and vaccine antigen are imported to any immunisation route in aquatic animal body.
For the 2nd kind of mode, preparation containing above-mentioned adjuvant effective ingredient adds also fully mixing in the preparation containing vaccine antigen to before vaccine antigen carries out immunity inoculation to aquatic animal 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 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 carries out injecting, first 3 days of wound, dipping bath or oral immunization to inoculating within latter 30 days, implement 1-5 days throw something and feed or the immunostimulant operation such as dipping bath.
Above immune application mode makes adjuvant effective ingredient β-1,3-glucosan reaches certain concentration when tarda vaccine antigen and the effect of aquatic animal immune system, thus allow immunocyte in the process of submission, processing, identification vaccine antigen, be subject to the stimulation of glucosan, the activity of immunocyte is made to be able to further raising, thus activated immune system, to the response of vaccine antigen, effectively strengthens the immune effect of vaccine antigen better.The present invention finds that the dosage of adjuvant effective ingredient has material impact to immune effect of vaccine, and within adjuvant safe concentration scope, both have certain dependency.When the using dosage of adjuvant effective ingredient exceedes safe-dosaging limits, lethal effect can be had to aquatic animal, therefore be necessary for carrying out the administering mode test of adjuvant or the maximum dose level of aquatic animal death can not be caused in 2-4 weeks after finding out administration under immune type of aquatic and concrete immunization ways condition, safe dose when determining that above-mentioned adjuvant effective ingredient uses in aquatic animal with this.The present invention confirms that the dosage of the effective ingredient of above-mentioned adjuvant in aquatic animal body can strengthen the immune effect of vaccine when reaching 1/100-4/5 of safe dose.
Tarda vaccine adjuvant of the present invention can be applicable to Fish, the vaccine virus immunization of amphibian and reptiles, comprise marine fish (as Paralichthys olivaceus, turbot, Cynoglossus semilaevis, cabrilla, Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis), snapper, Rachycentron canadum, Atlantic salmon, puffer etc.), brackish water aquaculture Fish are (as tilapia, rainbow trout, Mugil cephalus etc.), straddling fish stocks are (as anguilla japonica, Carnis Pseudosciaenae etc.), cultured freshwater fish is (as Mylopharyngodon piceus, Hypophthalmichthys molitrix, Aristichthys nobilis, Cyprinus carpio, Carassius auratus, Silurus asotus fish, Siniperca chuatsi, Acipenser Sinensis, Australia Jade Perch etc.), pet fish is (as Carassius auratus, Cyprinus carpio L. etc.), amphibian is (as bull frog, Andrias davidianus Blanchard etc.) and reptiles (as Trionyx sinensis Wiegmann, Testudinis, Serpentis etc.) vaccination.The safe dose of different aquatic animals is different with effective dose, for the embody rule dosage of specific aquatic animal, should test separately.
Good effect of the present invention is: involved adjuvant can improve the specificity after tarda vaccine antigen immunity inoculation and nonspecific immunity protection.For this intracellular infection of tarda or the vaccine of cause of disease with immune evasion ability; adjuvant of the present invention effectively can strengthen immanoprotection action; using method safety, to operator and environmentally friendly, thus has a good application prospect and market popularization value.
Detailed description of the invention
In view of the dosage of adjuvant effective ingredient is to vaccine antigen immune effect important, the using dosage of adjuvant effective ingredient is too low does not often reach the effect strengthening immunity, and too high using dosage often produces unfavorable or lethal effect to aquatic animal.Therefore first above-mentioned adjuvant in use should determine the safe-dosaging limits of above-mentioned adjuvant effective ingredient in concrete vaccine product and best working concentration for concrete type of aquatic, age and inoculation method etc. by vaccine Protection.In tarda bacterin preparation, the concentration of adjuvant effective ingredient is 1mg/g-20mg/g(adjuvant effective ingredient weight/bacterin preparation weight), in aquatic animal feed, the concentration of adjuvant effective ingredient is 0.1g/kg-5g/kg(adjuvant effective ingredient weight/feed relative), in aquatic animal body, the concentration of adjuvant effective ingredient reaches 1.0 × 10 upon administration
-3mg/g-5.0mg/g(adjuvant effective ingredient weight/aquatic animal body weight).
Antigenic component in tarda bacterin preparation can stem from the antibacterial of the Edwardsiella such as Edwardsiella tarda, Channel-catfish tarda or guarantor section tarda one or more cause of disease antigen or corresponding antigen can be produced in aquatic animal body, also can be the united vaccine formulation that the antigen comprising the antigen that is derived from tarda and other pathogen or virus is made.The type of vaccine antigen can be inactivated bacteria body, ghost composition, low virulent strain, attenuated strain, protective antigen, Antigen Subunit, any one of expression vector of antigenic determinant or antigen gene or more than one; above-mentioned antigen single one or more in bacterin preparation exist with associating, integration or multivalence mode; the tarda bacterin preparation made, except the expression vector that antigen maybe can produce antigen, also can comprise other the Multiple components such as adjuvant, protective agent, antiseptic, diluent, solvent.Adjuvant of the present invention in the application can be used in combination with vaccine antigen, also can make after unitary agent mixes with vaccine antigen and use simultaneously, can also different from vaccine antigen time use.Immunity inoculation mode in application can be injecting immune, wound immunity, immersion immunity or oral immunity.
The present invention proves that β-1, the 3-glucosan in yeast extract effectively can play the effect of tarda vaccine adjuvant, collaborative when using with tarda inactivated vaccine in application, can significantly improve the immune protective rate of tarda inactivated vaccine.Described β-1,3-glucosan extracts from corn, yeast, fungus or Sargassum, present invention demonstrates β-1,3-glucosan has the effect strengthening tarda vaccine virus immunization effect, therefore effect of effective ingredient of the present invention comprises the natural product of β-1,3-glucosan, artificial reconstructed product or synthetic product.Natural β-1; 3-glucosan can extract from corn, yeast, some fungi 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 retains, within the protection domain just still belonging to this patent.
Using method of the present invention is as follows:
(1) determination of adjuvant safe dose: preparation comprises the PBS(pH7.2 of 0.1%-10% of beta glucan) solution, degerming through the membrane filtration of 0.22 μm, treat immune aquatic animal with the above-mentioned adjuvant inoculation of variable concentrations again, vaccination ways can be injecting immune, wound immunity, immersion immunity or oral immunity.Immunity aquatic animal is observed 2 weeks, and experimentally result determines this adjuvant safe dose.
(2) preparation of tarda bacterin preparation and use: in inactivated vaccine preparation, add formaldehyde (36.5-38%, w/v) in tarda suspension, its final concentration is regulated to be 0.3%--1%, 4 DEG C are spent the night, with PBS(pH7.2) clean 3 times after, prepare certain density inactivated vaccine.Treat immune aquatic animal after being mixed according to a certain percentage with inactivated vaccine by adjuvant in safe-dosaging limits and carry out immunity inoculation, then aquatic animal is carried out normal aquaculture management.When using attenuated live vaccine, then without the need to carrying out inactivation treatment.
(3) determination of adjuvant optimal dose in 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; Or 2) make after unitary agent mixes with vaccine antigen and use simultaneously; Or use time 3) different from vaccine antigen.
For the 1st kind of mode, join comprise tarda vaccine antigen injection, above-mentioned adjuvant effective ingredient and vaccine antigen weight ratio are 1/10-1000/1 in dipping bath agent or oral agents, 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 before tarda bacterin preparation carries out immunity inoculation to aquatic animal to be added and fully mixing, 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 dipping bath water body and feedstuff or dipping bath water body weight ratio is 1/100000-1/100, the interpolation time is that tarda bacterin preparation treats that immune aquatic animal carries out injecting, first 3 days of wound, dipping bath or oral immunization to inoculating within latter 15 days, implement 1-5 days throw something and feed or the immunostimulant operation such as dipping bath.
Until immune aquatic animal after above-mentioned bacterin preparation inoculation; 30-60 days laggard row artificial liver support; by the analysis of the immune indexes such as relevant physiological biochemical indicator (as rate of body weight gain, antibody titer etc.) and relative protection ratio (RPS) after 14-60 days, to determine the best using dosage of adjuvant.
Embodiment 1: β-1,3-glucosan is to the determination of turbot (Scophthatmus maximus) injection safety dosage
Select healthy turbot (11.4 ± 1.8) g, support after 2 weeks temporarily in cement pit before test, be placed in circular glass steel drum and (be filled with water 0.7 meter
3) in, flowing water culture, water temperature is 18 ± 2 DEG C, salinity 28 ‰ ± 2 ‰, continuous charge in 24 hours.Duration of test to be thrown something and fed mixed feed by fish body weight 3%, throws something and feeds day 3 times, changes water every day and remove contamination 2 times.To turbot MS222(1:9000 before injection) carry out immersion anaesthetic treatment.
Be β-1, the 3-glucosan lumbar injection 20 tail turbot of 2,5,8,10 and 20 mg/mL respectively by concentration, injected dose is 100 μ L/ tails, observes 14 days.
Each experimental group turbot in 14 days death condition in table 1, determine that the maximum safe lumbar injection dosage of β-1,3-glucosan to turbot is 1000 μ g/ tails by result, i.e. 87.7 μ g/g(glucosan/fish body weight).
Table 1. β-1,3-glucosan is to turbot injection safety dosetest
Embodiment 2: β-1,3-glucosan makes an addition in Formalin inactivation E. tarda vaccine, immunity inoculation turbot (S. maximus).
Test uses fish and aquaculture management with embodiment 1.
Be inoculated in by E. tarda 1101 in pancreas peptone soybean broth (TSB), 28 DEG C of shaken cultivation are spent the night, then are forwarded in fresh TSB with 1:100,28 DEG C of shaken cultivation 5h, collect bacterium liquid, 6000g, 4 DEG C, 10min, aseptic PBS(pH7.2) clean 3 times, be resuspended in PBS, and dilution spread is in tryptose soya agar (TSA), count of bacteria adopts colony counting method, makes 1 × 10
9cFU/mL bacteria suspension, adds formalin (containing formaldehyde 36.5-38%, w/v), regulates its final concentration to be 1%, 4 DEG C and spends the night, make inactivated vaccine, and PBS washs thalline 3 times (method is the same), 4 DEG C of preservations.Separately get the bacterium liquid 0.2mL after deactivation and be coated with TSA, cultivate 48h, determine without colony growth for 28 DEG C.During vaccine safety detects, get vaccine and regulate concentration to 10
8cFU/mL, lumbar injection 20 tail turbot, injected dose is 100 μ L/ tails, observes 14 days, to determine the safety of bacterin preparation.
Random packet is carried out to healthy turbot, establishes 3 parallel group for each group.β-1,3-glucosan and the inactivated vaccine compatibility group of matched group (injection PBS), β-1,3-glucosan group, inactivated vaccine group, variable concentrations are established in test, and immunity test divides into groups in table 2.
Table 2. turbot immunity test divides into groups
Adopt lumbar injection immunity turbot, injected dose is 100 μ L/ tails.
After immune 28 days, turbot lumbar injection fresh E. tarda bacterium liquid (1.35 × 10
4cFU/ tail), carry out Experimental infection.
Within after counteracting toxic substances 14 days, add up the mortality rate of each test group, 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 the immune protective effect (mean+SD) of E. tarda inactivated vaccine adjuvant to turbot
Above result shows, simple with E. tarda formalin-inactivated vaccine (10
7cfu/ tail) or the immune protective effect of 43.9 μ g/g (adjuvant doses/body weight) β-1,3-dextran injection turbot lower, its RPS value is respectively 18.5% and 14.7%.Inactivated vaccine adds 21.9,43.9 and 87.7 μ g/g(adjuvant doses/body weight) β-1,3-glucosan time, can raising RPS in various degree.Wherein adding 43.9 μ g/g(adjuvant doses/body weight with inactivated vaccine) β-1,3-glucosan has good 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 makes an addition in Formalin inactivation E. tarda vaccine, and immune turbot is on the impact of serum antibody titer
Test uses fish and aquaculture management with embodiment 1.
Bacterin preparation preparation and EXPERIMENTAL DESIGN (experiment grouping and immunization method) are with embodiment 2, and immunity is after 28 days, and often organize and get 5 tail fishes extraction 0.1mL tail veins, room temperature leaves standstill 2h, then is placed in 4 ° of C, 12h, collects serum, and mixing is with organizing fish serum ,-20 DEG C of storages.
The antibody titer of serum measures mouse monoclonal antibody (AquaticDiagnostic Ltd, the Britain) operation instruction with reference to Chinese People's Anti-Japanese Military and Political College brill IgM, and every hole adds 100 μ L 1.0 × 10
8cfu/mL E. tarda 1101 suspension be resuspended in coating buffer adds a cover rear 4 DEG C of bags that spend the night by 96 hole ELISA Plate, and then 0.05% (v/v) glutaraldehyde of 50 μ L PBS dilutions is added to each hole, 22 DEG C leave standstill 20 min, wash plate 3 times by less salt washing liquid; Add 250 μ L 1% BSA to close, 22 DEG C of standing 2h, low salt solutions washes plate 3 times; Again respectively to the turbot serum adding 100 μ L PBS serial dilutions in every hole, each dilution factor do 3 parallel, 22 DEG C leave standstill high salt washing liquid after 3 h and wash plate 5 times, hatch 5min for the last time; Every hole adds Chinese People's Anti-Japanese Military and Political College's brill IgM monoclonal antibody that 100 μ L regenerate, and washes plate 5 times after 22 DEG C of standing 60min by aforementioned high level salt solution plate washing method; In every hole, add the sheep anti mouse two anti-(Tian Gen biochemical technology company limited) of the HRP labelling that 100 μ L 1: 1000 dilute again, 22 DEG C leave standstill 60 min, wash plate 5 times by aforementioned high level salt solution; Every hole adds 100 μ L TMB solution afterwards, 22 DEG C of colour developing 10 min; Every hole adds 50 μ L 2M H2SO4 cessation reactions, surveys OD value by microplate reader at 450nm place.Analyze the OD value of sample (P) and blank (N), calculate P/N ratio, during P/N >=3.0, sample is positive, and during P/N < 3.0, sample is negative.The antibody titer of each group the results are shown in Table 4.
Table 4. glucosan is the impact (mean+SD) of E. tarda inactivated vaccine adjuvant on the serum antibody titer of turbot
Above result shows, matched group (PBS), E. tarda formalin-inactivated vaccine (10
7cfu/ tail), 43.9 μ g/g (adjuvant doses/body weight) β-1, the serum antibody titer value of 3-dextran 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 doses/body weight) β-1, during 3-glucosan, can raising antibody titer in various degree.Wherein adding 43.9 μ g/g(adjuvant doses/body weight with inactivated vaccine) β-1,3-glucosan has good immune protective effect, and antibody titer value reaches 682.7 ± 147.8.Above result proves that adjuvant of the present invention can increase the antibody titer in serum preferably.
Embodiment 4: β-1,3-glucosan makes an addition to formalin Mie Huo Channel-catfish fish tarda (Edwardsiella ictaluri)
In vaccine, the application mode of immune Pelteobagrus fulvidraco (Pelteobagrus fulvidraco).
Select healthy Pelteobagrus fulvidraco, first carry out the determination of β-1,3-glucosan and the best applications dosage of E. ictaluri inactivated vaccine compatibility in feedstuff with reference to the method in experimental example 1 and 2.Determine the addition of glucosan in feedstuff based on this, first adopt immersion immunity in application, Pelteobagrus fulvidraco being placed in final concentration is 10
8in the fresh fresh water of CFU/mL deactivation E. ictaluri vaccine, after 10-20min, move in fresh water and normally cultivate.After 14-30 days, booster immunization is 1 time (determined by the specification of fish by the booster immunization interval, body size is less, and the time can shorten, the body size comparatively large then time can proper extension), the immunization feedstuff of throwing something and feeding containing glucosan and E. ictaluri inactivated vaccine, in immunization feedstuff, inactivated vaccine addition is 0.1g/kg(inactivated vaccine weight/feed relative), throw something and feed continuously 5 days, then carry out normal feeding and management.After booster immunization 30-60 days, calculate the rate of body weight gain of duration of test, separately get part Experiment Pelteobagrus fulvidraco lumbar injection 10 times of LD
50the fresh E. ictaluri bacterium liquid of dosage, carries out Experimental infection.Infect latter 14-30 days, statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] × 100%.Calculate RPS, to obtained data analysis, the adjuvant immunity Pelteobagrus fulvidraco application mode of glucosan as E. ictaluri inactivated vaccine is assessed, there is good gaining effect and RPS value more than 70%, it is qualified to be considered as.
Embodiment 5: β-1,3-glucosan makes an addition in deactivation Edwardsiella tarda (E. tarda) vaccine, the application mode of immune tilapia (Oreochromis niloticus).
First preparation has immunogenic Edwardsiella tarda (E. tarda) vaccine, and antigen contained in this vaccine can be the deactivation thalline of tarda, ghost composition, less-virulent strain or attenuated strain.In application, with reference to the method in experimental example 1 and 2, healthy tilapia is carried out to the determination of β-1,3-glucosan and the best applications dosage of E. tarda vaccine compatibility in feedstuff.Determine the addition of glucosan in feedstuff based on this, adopt immersion immunity to tilapia in application, tilapia being placed in final concentration is 10
8in the water body of CFU/mL E. tarda vaccine, after 10-20min, move in fresh water body and normally cultivate.After 14-30 days, carry out booster immunization 1 time (booster immunization is determined by the specification of fish interval time, and body size is less, and the time can shorten, body size comparatively large then time can proper extension).The immunization feedstuff of throwing something and feeding containing glucosan and E. tarda vaccine compatibility, in immunization feedstuff, vaccine addition is 0.1g/kg(vaccine weight/feed relative), throw something and feed continuously 5 days, after 60 days, calculate the rate of body weight gain of duration of test, separately to tilapia lumbar injection 10 times of LD
50the fresh E. tarda bacterium liquid of dosage, carries out Experimental infection.Infect latter 14-30 days, statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] × 100%.Calculating RPS, to obtained data analysis, is that E. tarda vaccine adjuvant immunity tilapia application mode is assessed to glucosan, there is certain gaining effect and RPS value more than 70%, it is qualified to be considered as.
Embodiment 6: β-1,3-glucosan makes an addition in esrB, evpC dual-gene disappearance attenuated strain of E. tarda, the application mode of immune turbot (S. maximus).
Select healthy turbot, first carry out the determination of esrB, evpC dual-gene disappearance attenuated strain immersion turbot maximum safe concentration of β-1,3-glucosan optimum addition and E. tarda in feedstuff with reference to the method in experimental example 1 and 2.First immersion immunity 5-10min in the seawater in application, the concentration of soaking esrB, evpC dual-gene disappearance attenuated strain of E. tarda used in sea water is 1/10 of above-mentioned maximum safe concentration, then moves in fresh seawater and normally cultivate.After 14-30 days, within continuous 5 days, throw something and feed containing the feedstuff of best applications dosage β-1,3-glucosan, then carry out normal feeding and management.After booster immunization 30-60 days, calculate the rate of body weight gain of duration of test, separately to turbot lumbar injection 10 times of LD
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 obtained data analysis, assess as E. tarda attenuated live vaccine adjuvant immunity turbot application mode β-1,3-glucosan, RPS value is more than 70%, and it is qualified to be considered as.
The application mode of the streptococcus agalactiae (Streptococcus agalactiae) of embodiment 7: β-1,3-glucosan and deactivation and Edwardsiella tarda (E. tarda) bivalent inactivated vaccine compatibility immunity bull frog (Rana catesbeiana).
First traditional ablation method (as Formalin inactivation, hot deactivation, high pressure deactivation etc.) is used to prepare streptococcus agalactiae (S. agalactiae) and Edwardsiella tarda (E. tarda) vaccine of deactivation.Become bigeminy vaccine by the proportions of 1:1, in vaccine, the final concentration of two kinds of inactivation of bacterial is 10
8cFU/mL.Immunization feedstuff prepare, by 0.25,0.5,0.75 and 1g/kg(adjuvant effective ingredient weight/feed relative) proportioning β-1, the 3-glucosan of various dose is added in feedstuff.In application, take out after first healthy bull frog being soaked bigeminy vaccine 10-20min, after 14-30 days, within continuous 5 days, throw something and feed containing glucosan feedstuff.After 60 days, compare in feedstuff and add the impact of various dose glucosan on rate of body weight gain, be separately divided into two groups at random to the bull frog in each proportioning group, each group uses 10 times of LD respectively
50fresh S. agalactiae and the E. tarda bacterium liquid of dosage carry out lumbar injection, carry out Experimental infection.Infect latter 14-30 days, statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] × 100%.Calculate RPS, to obtained data analysis, glucosan is assessed as S. agalactiae and E. tarda bivalent inactivated vaccine adjuvant immunity tooth bull frog application mode, in infection experiment result to its RPS of any one of above-mentioned two kinds of cause of diseases more than 70%, and there is the glucosan addition of certain gaining effect corresponding to it be considered as applying addition, it is qualified that its adjuvant effect is also considered as.
Embodiment 8: β-1,3-glucosan makes an addition in multiple vaccines prepared by Formalin inactivation, the application mode of immune Paralichthys olivaceus (Paralichthys olivaceus).
First traditional ablation method (as Formalin inactivation, hot deactivation, high pressure deactivation etc.) is used to carry out deactivation to Vibrio anguillarum (Vibrio anguillarum), Aeromonas hydrophila (Aeromonas hydrophila) and Edwardsiella tarda (E. tarda), again three is become triple vaccine by the proportions of 1:1:1, in this vaccine, the final concentration of three kinds of inactivation of bacterial is 10
8cFU/mL.Immunization feedstuff prepare, by 0.25,0.5,0.75 and 1g/kg(adjuvant effective ingredient weight/feed relative) proportioning β-1, the 3-glucosan of various dose is added in feedstuff.In application, after first healthy Paralichthys olivaceus being soaked in triple vaccine 10-20min, then moving in fresh seawater and normally cultivate.After 14-30 days, within continuous 5 days, throw something and feed containing glucosan feedstuff.After 60 days, compare in feedstuff and add the impact of various dose glucosan on rate of body weight gain, be separately divided into three groups at random to the fish in each proportioning group, each group uses 10 times of LD respectively
50fresh V. anguillarum, the A. hydrophila of dosage and E. tarda bacterium liquid, carry out Experimental infection, infects and adopt lumbar injection mode.Infect latter 14-30 days, statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] × 100%.Calculate RPS, to obtained data analysis, glucosan is assessed as V. anguillarum, A. hydrophila and E. tarda triple inactivated vaccine adjuvant immunity Paralichthys olivaceus application mode, in infection experiment result to its RPS of any one of above three kinds of bacterium more than 70%, and there is the glucosan addition of certain gaining effect corresponding to it be considered as applying addition, it is qualified that its adjuvant effect is also considered as.
Embodiment 9: the confirmation of suitable proportion in β-1,3-glucosan and vaccine compatibility in oral vaccine delivery agent
Make an addition to β-1,3-glucosan in Edwardsiella tarda (E. tarda) vaccine of deactivation and oral immunity is implemented for example to Japanese eel (Anguilla japonica), the suitable proportion of adjuvant in vaccination and antigen concentration is confirmed.
First preparation has immunogenic slow Edwardsiella vaccine, antigen contained in this vaccine can be the deactivation thalline of antibacterial, ghost composition, less-virulent strain, attenuated strain, protective antigen albumen, Antigen Subunit, antigenic determinant or antigen gene expression carrier one or more of expression product with associating, integration or multivalence mode.In application, select healthy anguilla japonica, each test group all establishes 3 parallel group, 20 tails/parallel group, and matched group experimental session is thrown something and fed chow diet always, and in each immune group, adjuvant effective ingredient, vaccine and feedstuff ratio are in table 5.
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, then chow diet of throwing something and feeding.After 30 days, each group of corresponding feedstuff of throwing something and feeding again carries out booster immunization once, after 60 days, calculates the rate of body weight gain of duration of test, separately to anguilla japonica lumbar injection 10 times of LD
50the fresh Edwardsiella tarda bacterium liquid of dosage, carries out Experimental infection.Infect latter 14-30 days, statistics mortality rate, and according to formula RPS=[1 – (immune group mortality rate/matched group mortality rate)] × 100%.Calculate RPS, to obtained data analysis, in different glucosan and slow Edwardsiella vaccine proportioning, there is certain gaining effect in feedstuff and RPS value more than 70%, be considered as the suitable proportion of adjuvant and antigen in this oral vaccine delivery agent.
Claims (4)
1. β-3-glucosan strengthens the application in the adjuvant of tarda vaccine virus immunization effect in preparation.
2. apply as claimed in claim 1, it is characterized in that the antigen of described tarda vaccine comprises one or more the vaccine antigen stemming from Edwardsiella tarda, Channel-catfish tarda or guarantor section tarda; Vaccine antigen type comprise deactivation thalline, ghost composition, less-virulent strain, attenuated strain, protective antigen, Antigen Subunit, any one of expression product of antigenic determinant or antigen gene expression carrier or more than one.
3., as right wants the application as described in 1, it is characterized in that described adjuvant and vaccine antigen are mixed and made into bacterin preparation and use, or make not containing vaccine antigen adjuvant formulation but use with vaccine antigen simultaneously, or to use time different from vaccine antigen.
4. apply as claimed in claim 1, it is characterized in that three kinds of application modes of described adjuvant are:
(1) be mixed and made into bacterin preparation with vaccine antigen when using, join comprise tarda vaccine antigen injection, the effective ingredient of above-mentioned adjuvant and vaccine antigen weight ratio are 1/10-1000/1 in dipping bath agent or oral agents;
(2) make not containing vaccine antigen adjuvant formulation but use with vaccine antigen simultaneously time, the effective ingredient of above-mentioned adjuvant is interim before bacterin preparation carries out immunity inoculation to aquatic animal to be added and fully mixing, and addition is 1/10-1000/1 of vaccine antigen weight;
(3) when using time different from vaccine antigen, the effective ingredient joining the above-mentioned adjuvant in aquatic animal feed or 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 first 3 days of immunity inoculation to inoculating within latter 30 days, can administration 1 time or repeatedly.
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| CN104099286B (en) * | 2014-07-22 | 2017-12-08 | 中国水产科学研究院黄海水产研究所 | The ablation method and its inactivated vaccine of a kind of tarda |
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