CN102139102A - Preparation method of microcapsule vaccines capable of resisting infection of aeromonas hydrophila - Google Patents
Preparation method of microcapsule vaccines capable of resisting infection of aeromonas hydrophila Download PDFInfo
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Abstract
The invention discloses a preparation method of microcapsule vaccines capable of resisting the infection of aeromonas hydrophila, relating to the field of the preparation of antibacterial vaccines and solving the problems of time and labor wasting and difficult in large-scale popularization during the practical operations of cultivating fish and the like with immunotherapy by means of vaccine injection. The preparation method of the microcapsule vaccines capable of resisting the infection of the aeromonas hydrophila comprises the following steps of: (1) the preparation of aeromonas hydrophila suspension: inoculating the aeromonas hydrophila to prepare the aeromonas hydrophila suspension with the effective viable bacteria content of 1010 per ml, inactivating the aeromonas hydrophila suspension with formalin, and storing the suspension; and (2) the preparation of the microcapsule vaccines of the aeromonas hydrophila: uniformly mixing the aeromonas hydrophila suspension prepared in the step (1) and a 5% sodium alginate solution in the proportion of 1:1 in a vortex oscillator, and performing spraying and drying treatment on the mixture to obtain microcapsule particles. The method can be used for preparing the microcapsule vaccines for preventing and treating bacterial septicemia of sturgeon, particularly acipenser baeri.
Description
Technical field
The present invention relates to a kind of preparation method of preventing and treating fish bacterial septicemia vaccine, more specifically, relate to the preparation method that a kind of little glue that is used to prevent and treat the Siberia sturgeon bacterial septicemia that is caused by Aeromonas hydrophila is assisted vaccine.
Background technology
By the fish bacterial septicemia that Aeromonas hydrophila (Aeromonas hydrophila) causes, all can bring enormous economic loss to fish culture every year.Studies show that at present, inject nonspecific immunity activity and anti-infection ability that an amount of aeromonas hydrophila inactivated vaccine can significantly strengthen Fish, but be that the immunotherapy of means wastes time and energy in culturing practice with the vaccine injection, be unfavorable for large-scale promotion.
The Siberia sturgeon is under the jurisdiction of sturgeon section, sturgeon and belongs to, and is a kind of in existing 26 kinds of Acipenser Sinensiss, mainly is distributed in the western Ob of Russia to each river, Siberia between the Kolyma of east.China has formed huge breed scale so far since 1992 begin artificial cultivation, cultured output is up to several kilotons.Yet China Siberia sturgeon is cultured puzzlement, the especially bacterial septicemia that is caused by Aeromonas hydrophila that always is subjected to disease deeply, cultures all can for every year the Siberia sturgeon and bring enormous economic loss.Studies show that at present, inject nonspecific immunity activity and anti-infection ability that an amount of aeromonas hydrophila inactivated vaccine can significantly strengthen the Siberia sturgeon, but the immunotherapy that with the vaccine injection is means wastes time and energy in culturing practice, is unfavorable for large-scale promotion.In recent years, the application of microcapsule technology in people and the research of poultry oral vaccine is increasingly extensive, and the research of relevant aquatic animal microcapsule oral vaccine also obtains certain progress.But also there is not capsule vaccine production at the control of sturgeon bacterial septicemia.
Summary of the invention
In order to overcome vaccine injection is that the immunotherapy of means wastes time and energy in the fish culture practice, is unfavorable for the problem of large-scale promotion, and the inventor provides a kind of preparation method that is used for the microcapsule vaccine of Siberia sturgeon bacterial septicemia control.Can be used for preparation control Acipenser Sinensis, the especially microcapsule vaccine of Siberia sturgeon bacterial septicemia.
Realize that technical scheme of the present invention is as follows:
The microcapsule vaccine preparation method that provides a kind of anti-Aeromonas hydrophila to infect may further comprise the steps:
1) preparation of Aeromonas hydrophila bacteria suspension
Aeromonas hydrophila is inoculated in the common nutrient broth of pH7.2, in 30 ℃, centrifuge washing behind the 150 commentaries on classics/min shaking table shaken cultivation 24-72 h, making effective viable bacteria content is 10
10The bacteria suspension of individual/ml, and use the formalin deactivation, preserve;
2) preparation of Aeromonas hydrophila microcapsule vaccine
With the bacteria suspension and the ratio of 5% sodium alginate soln with 1:1 of above-mentioned steps preparation, mix homogeneously on the vortex oscillation device carries out spray drying treatment, must microcapsule granule.
In the preparation of described step 1) Aeromonas hydrophila bacteria suspension, optimum condition is: Aeromonas hydrophila is inoculated in the common nutrient broth of pH7.2, and in 30 ℃, centrifuge washing behind 150 commentaries on classics/min shaking table shaken cultivation, 48 h, making effective viable bacteria content is 10
10The bacteria suspension of individual/ml, and use the formalin deactivation, preserve.
Described centrifugal be in centrifugal 20 min of 3000 commentaries on classics/min, described washing is with physiological saline solution washing 3 times, described saving as in 4 ℃ of refrigerators preserved.
Described step 2) in the preparation of middle Aeromonas hydrophila microcapsule vaccine, with the bacteria suspension of above-mentioned steps preparation and the 5% sodium alginate soln ratio with 1:1, mix homogeneously on the vortex oscillation device obtains material solution, it is carried out spray drying treatment, get microcapsule granule.
Described spray drying parameter is: air quantity 100%, 100 ℃-120 ℃ of inlet temperature, 45 ℃-55 ℃ of leaving air temps, inlet amount 30-40ml/h, cleansing pin frequency 5 s; Keeping throughput during the spray drying is 550-650 m
3/ h; Gas is filtered and is heated to after by aerator and is provided with temperature 110-120 ℃,
Described spray drying parametric optimization is: air quantity 100%, 115 ℃ of inlet temperature, 48 ℃ of leaving air temps, inlet amount 35 ml/h, cleansing pin frequency 5 s; Keeping throughput during the spray drying is 600 m
3/ h; Gas is filtered and is heated to after by aerator 115 ℃ of temperature are set.
Described material solution is sent to aerosol apparatus by peristaltic pump with material solution after carrying out spray drying treatment, collects microcapsule granule by cyclone separator.
At serious day by day bacterial disease, increasing aquaculture researcher tendency adopts the vaccination method to prevent and treat.Compare with other methods of vaccination, the oral immunity method is more suitable for not being subjected to size of fish and the restriction of time in the large-scale breed shoal of fish, and fish is not produced stress yet, especially is easy to repeatedly repeat immunity, thereby has solved the Fish technical barrier of immunity once more.
The present invention is an object of study with the microcapsule vaccine of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis, observed its effect to Siberia sturgeon nonspecific immunity and anti-infection ability, provide a kind of microcapsule vaccine preparation method that is exclusively used in the control of sturgeon bacterial septicemia, and the assay method that passes through to be provided has been verified the effectiveness of this preparation method products obtained therefrom.
Description of drawings
Fig. 1 BABL/C white mice antiserum antibody agglutination titer is measured;
Fig. 2 Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is to the influence of Siberia sturgeon peroxidase content;
Fig. 3 Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is to the influence of Siberia sturgeon serum alkaline phosphatase content;
Fig. 4 Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is molten to Siberia sturgeon serum
The influence of bacterium enzyme content.
The specific embodiment
Be to be understood that, unaccounted normal condition and method among the following embodiment, usually according to the conventional employing method of affiliated field experimenter: as " molecular cloning experiment guide " third edition of Sa nurse Brooker and Russell chief editor, or the step and the condition of advising according to manufacturer.
Among the embodiment, related part material source is as follows:
Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 purchases in Ministry of Agriculture fishery animals and plants cause of disease storehouse; Common nutrient broth, physiological saline solution, formalin, sodium alginate are purchased the chemical reagent company limited in traditional Chinese medicines group (Shanghai); The temperature control shaking table is purchased the Thermo Forma company in the U.S.; High speed centrifuge is purchased in SANYO GS company; The vortex oscillation instrument is purchased in the special Analytical Instrument Co., Ltd of Shanghai river in Henan Province; Small-sized spray drying instrument SY-6000 purchases in the effective company of Shanghai generation biological plant engineering far away; The Siberia sturgeon, healthy anosis wound, body weight 50～70 grams, long 13～16 centimetres of body is all purchased in sturgeon cultivation base, Thousand-Island Lake, Zhejiang Province; The BABL/C white mice is purchased in the Shanghai The 2nd Army Medical College; Peroxidase detection kit, lysozyme detection kit, protein quantification (biuret method) testing cassete are purchased and are built up bio-engineering research first branch in Nanjing; The common nutrient broth of pH7.2,5% sodium alginate soln are 1 * 10
5Standby behind Pa high pressure moist heat sterilization 20 min.
Embodiment one:
The preparation of Aeromonas hydrophila bacteria suspension
Under aseptic condition, the Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 that preserves on the test tube slant culture medium is inoculated in the common nutrient broth of pH7.2, in 30 ℃, behind 150 commentaries on classics/min shaking table shaken cultivation, 48 h in centrifugal 20 min of 3000 commentaries on classics/min, with physiological saline solution washing 3 times, making effective viable bacteria content is 10
10The Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 bacteria suspension of individual/ml, and use the formalin deactivation, preserve in 4 ℃ of refrigerators.
Embodiment two:
The preparation of Aeromonas hydrophila microcapsule vaccine
Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 bacteria suspension 200 ml and 5% sodium alginate soln, 200 ml mix homogeneously on the vortex oscillation device with 4 ℃ of refrigerator preservation deactivations, open aerator, and then open spray drying instrument, peristaltic pump and cleansing pin pump, parameter is set is: air quantity shows 100%, 115 ℃ of inlet temperature, 48 ℃ of leaving air temps, inlet amount 35 ml/h, cleansing pin frequency 5 s.Keeping throughput during the spray drying is 600 m
3/ h.Gas is filtered and is heated to after by aerator 115 ℃ of temperature is set, and peristaltic pump is sent to aerosol apparatus with material solution, collects microcapsule granule by cyclone separator, preserves in 4 ℃ of refrigerators.
Embodiment three:
The immunogenic mensuration of Aeromonas hydrophila microcapsule vaccine
1, picked at random healthy 4 ages in week 30 of female BABL/C white mice, be divided into 2 groups at random, promptly Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is mixed and is raised oral group, matched group, every group establish 3 parallel.Wherein, Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is mixed and is raised oral group of BABL/C white mice and throw something and feed every day and to contain the feedstuff of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, the additive capacity of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is 6 g/Kg feedstuffs, the matched group BABL/C white mice additive capacity that does not contain Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine of throwing something and feeding every day is 6 g/Kg feedstuffs, threw something and fed continuously 7 days, stopping to raise and immediately each group BABL/C white mice is carried out respectively eye socket afterwards gets blood and is collected in the centrifuge tube, room temperature leaves standstill behind 2 h 4 ℃ and spends the night, then in 4 ℃, get supernatant behind centrifugal 15 min under the 5000 commentaries on classics/min conditions, adopt indirect enzyme-linked immunosorbent assay to carry out the antibody agglutination titer and measure, mix with Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine that to raise the positive dilute serum of oral group of BABL/C white mice be antibody titer at the ratio of the absorbance at 405 nano wave length places greater than 2.1 o'clock positive serum antibody maximum dilution multiple at the absorbance at 405 nano wave length places and matched group BABL/C white mice negative serum.
2, the serum antibody agglutination titer of Aeromonas hydrophila microcapsule vaccine
The serum antibody agglutination titer measurement result of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is seen Fig. 1. experimental result shows, adopting indirect enzyme-linked immunosorbent assay to record Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, to mix the BABL/C white mice antiserum antibody agglutination titer of raising oral group be 6400.
Embodiment four:
The Aeromonas hydrophila microcapsule vaccine is to the mensuration of Siberia sturgeon nonspecific immunity effect
1, the healthy Siberia of picked at random sturgeon 600 tails are divided into 2 groups at random, and promptly Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is mixed and raised oral group, matched group, every group establish 3 parallel.Wherein, Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is mixed the Siberia sturgeon of raising oral group and is thrown something and fed every day and to contain the feedstuff of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, the additive capacity of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is 6 g/Kg feedstuffs, threw something and fed continuously 7 days, and changed the feedstuff that throwing does not contain Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine afterwards; Matched group Siberia sturgeon is thrown something and fed always and is not contained the feedstuff of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, and throw the amount of raising and be 3% of Siberia sturgeon TBW every day.After stopping raising the feedstuff that contains Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, in each group, got Siberia sturgeon blood sampling preparation serum respectively, measure the content of serum peroxidase, immunoglobulin, lysozyme according to the detection kit description every 7 days.Test period is 35 days, the continuous oxygenation of duration of test, and water temperature is controlled at 20～21 ℃.
2, result of the test
2.1 Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is to the influence of Siberia sturgeon serum peroxidase content
Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine the results are shown in Figure 2 to the influence of Siberia sturgeon serum peroxidase content.Experimental result shows that Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine can significantly improve the serum peroxidase content of Siberia sturgeon.Be in particular in: in continuous oral Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine 28 days after 7 days, Siberia sturgeon serum peroxidase content has remarkable rising than matched group, wherein Siberia sturgeon serum peroxidase content reaches maximum in continuous oral Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine the 21st day after 7 days, be 6.86 U/mgprot, improved 50.15% than matched group.
2.2 Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is to the influence of Siberia sturgeon serum immune globulin content
Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine the results are shown in Figure 3 to the influence of Siberia sturgeon serum immune globulin content.Experimental result shows that Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine can significantly improve the serum alkaline phosphatase content of Siberia sturgeon.Be in particular in: in continuous oral Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine 28 days after 7 days, Siberia sturgeon serum alkaline phosphatase content has remarkable rising than matched group, especially Siberia sturgeon serum alkaline phosphatase content reaches maximum in continuous oral Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine the 21st day after 7 days, be 67.37 U/L, improved 52.91% than matched group.
2.3 Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is to the influence of Siberia sturgeon serum lysozyme content
Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine the results are shown in Figure 4 to the influence of Siberia sturgeon serum lysozyme content.Experimental result shows that Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine can significantly improve the serum lysozyme content of Siberia sturgeon.Be in particular in: in continuous oral Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine 28 days after 7 days, Siberia sturgeon serum lysozyme content has remarkable rising than matched group, especially Siberia sturgeon serum lysozyme content reaches maximum in continuous oral Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine the 21st day after 7 days, be 191.43 U/ml, improved 92.51% than matched group.
Embodiment five:
The Aeromonas hydrophila microcapsule vaccine is to the mensuration of Siberia sturgeon relative immunity protective rate
1, the healthy Siberia of picked at random sturgeon 20 tails are divided into 2 groups at random, and promptly Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is mixed and raised oral group, matched group.Wherein, Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is mixed the Siberia sturgeon of raising oral group and is thrown something and fed every day and to contain the feedstuff of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, the additive capacity of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is 6 g/Kg feedstuffs, threw something and fed continuously 7 days, and changed the feedstuff that throwing does not contain Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine afterwards; Matched group Siberia sturgeon is thrown something and fed always and is not contained the feedstuff of Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine, and throw the amount of raising and be 3% of Siberia sturgeon TBW every day.Stop raising contain Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine feedstuff after 21 days be 1.0 * 10 with effective viable bacteria content
7Individual/ml Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 bacteria suspension carries out pectoral fin base portion infectable infection, and it is 0.2 ml that every endnote is penetrated dosage, the physiological saline solution of matched group injection equivalent.Observed 7 days continuously, mortality and the Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine of record matched group Siberia sturgeon are mixed the mortality of raising oral group of Siberia sturgeon, calculate the relative immunity protective rate.
2, Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine is to the mensuration of Siberia sturgeon relative immunity protective rate
Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine sees Table 1 to the measurement result of Siberia sturgeon relative immunity protective rate.Experimental result shows; Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine infects the anti-Siberia of Siberia sturgeon sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 and has the good immune protection effect, and its immune protective rate to the Siberia sturgeon is 50%.
Table 1 Aeromonas hydrophila bacterial strain S1 microcapsule vaccine is to the immune protective rate of Siberia sturgeon
Result treatment:
The Data Processing in Experiment method: all data are all carried out variance analysis, and carry out multiple comparisons.
This experiment is applied to the Aeromonas hydrophila microcapsule vaccine immunization experiment of Siberia sturgeon, obtained immune effect preferably, it can obviously improve the content of Siberia sturgeon serum peroxidase, immunoglobulin, lysozyme, reach maximum in continuous oral the 21st day after 7 days, and in 7 weeks still than still remaining on a higher level before the immunity.
Oxidation reaction between peroxidase catalyzing hydrogen peroxide and the hydrogen donor, thereby the part noxious substance hydrogen peroxide that produces in the decomposer.Therefore, peroxidase is considered to one of protective enzyme of organism.Peroxidase is being undertaken the critical function of body defence, and the serum peroxidase is weighed the index of Siberia sturgeon immune state as immune factor, does not appear in the newspapers so far.Therefore this experiment is studied the effect of oral microcapsule by this index, illustrates that the Aeromonas hydrophila microcapsule can strengthen the immunologic function of Siberia sturgeon as immune drug.
Immunoglobulin is as topmost immune mediator in the Fish specific humoral immune response, mainly secrete in blood and other body fluid with the antibody formation of solubility, situations such as its changes of contents and body health, nutrition and disease have confidential relation, can reflect the pathological state and the immunologic function of body.Immunoglobulin equally can be used as sturgeon humoral immunization evaluation index with serum antibody.About the influence of vaccine to Siberia sturgeon immunoglobulin, Kolman etc. studies show that the full vaccine of aeromonas salmonicida can make the content of the serum immune globulin of Siberia sturgeon bring up to 10.03 g/L by 6.04 g/L.This experimental result shows that the Aeromonas hydrophila microcapsule vaccine also can obviously improve the serum immune globulin content of Siberia sturgeon.
Lysozyme is as one of nonspecific immunity material of Fish, can when invading, microorganism play a role immediately, remove effectively and degrade pathogenic microorganism and other harmful substances, and this moment, antibody did not produce as yet, played an important role in the fish body is resisted the forefront defense mechanisms of infectious pathogenic bacterium.Therefore, the height of Fish serum peroxidase, immunoglobulin, antalzyme activity can be used as the index of weighing immune status.In recent years, the more research of domestic scholars Aeromonas hydrophila vaccine is to the influence of the nonspecific immunity of bony fishes such as Ctenopharyngodon idellus, crucian carp, mandarin fish.For example: Luo Xia etc. have studied and have had a liking for the single full vaccine of aqueous vapor to lysozyme content influence in the mandarin fish serum, and immunity back mandarin fish serum lysozyme content obviously improves, and reaches peak value on the 3rd day, but drops to original level after the 7th day.After Amway state etc. studies show that Cyprinus carpio injecting immune Aeromonas hydrophila whole-bacterial-vaccine, lysozyme content reached peak value on the 3rd day in the serum, dropped to original level after 1 week.This experiment finds that Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis bacterial strain S1 microcapsule vaccine can obviously improve Siberia sturgeon serum lysozyme content, lysozyme content to the reached peak value in 7 days in the serum of immunity back, though its content descends to some extent after 5 weeks, than still remaining on a higher level before the immunity.This shows that lysozyme content is in higher level in following period of time in the sturgeon serum of Siberia, immunity back, its nonspecific immunity acting duration long and this moment of serum antibody level also increases.These are different to the result of study of the full vaccine of Aeromonas hydrophila with Luo Xia, Amway state etc.
In sum, method of testing provided by the invention, confirmed that preparation method provided by the invention can prepare effective product, the Siberia sturgeon bacterial septicemia pathogenic hydrophila gingivalis microcapsule vaccine of the test dose of throwing something and feeding can strengthen the immune function of Siberia sturgeon.The present invention is ill for prevention and control China Siberia Acipenser Sinensis, improve aquatic product quality, alleviate and abuse the serious harm that brings to human health and ecological environment because of antibiotics a kind of feasible method is provided, and coming into operation of microcapsule vaccine has good economy, society and ecological significance.
Should be appreciated that the present invention with Aeromonas hydrophila bacterial strain S1 as the raw material of preparation method in order to the interpretation technique scheme, do not limit preparation method of the present invention and be applied to use of the use of other Aeromonas hydrophila bacterial strain as oral vaccine; As the vaccine effective object, do not represent effect of the present invention with the Siberia sturgeon, especially the limitation of Acipenser Sinensis effect to other Fish.
Claims (6)
1. microcapsule vaccine preparation method that anti-Aeromonas hydrophila infects may further comprise the steps:
1) preparation of Aeromonas hydrophila bacteria suspension
Aeromonas hydrophila is inoculated in the common nutrient broth of pH7.2, in 30 ℃, centrifuge washing behind the 150 commentaries on classics/min shaking table shaken cultivation 24-72 h, making effective viable bacteria content is 10
10The bacteria suspension of individual/ml, and use the formalin deactivation, preserve;
2) preparation of Aeromonas hydrophila microcapsule vaccine
With the bacteria suspension and the ratio of 5% sodium alginate soln with 1:1 of above-mentioned steps preparation, mix homogeneously on the vortex oscillation device obtains material solution, carries out spray drying treatment, must microcapsule granule.
2. preparation method as claimed in claim 1, it is characterized in that, in the preparation of described step 1) Aeromonas hydrophila bacteria suspension, Aeromonas hydrophila is inoculated in the common nutrient broth of pH7.2, in 30 ℃, centrifuge washing behind 150 commentaries on classics/min shaking table shaken cultivation, 48 h.
3. preparation method as claimed in claim 1 is characterized in that, centrifugal described in the step 1) is in centrifugal 20 min of 3000 commentaries on classics/min, and described washing is that described saving as in 4 ℃ of refrigerators preserved with physiological saline solution washing 3 times.
4. preparation method as claimed in claim 1 is characterized in that step 2) described in the spray drying parameter be: air quantity 100%, 100 ℃-120 ℃ of inlet temperature, 45 ℃-55 ℃ of leaving air temps, inlet amount 30-40ml/h, cleansing pin frequency 5 s; Keeping throughput during the spray drying is 550-650 m
3/ h; Gas is filtered and is heated to after by aerator and is provided with temperature 110-120 ℃.
5. preparation method as claimed in claim 1 is characterized in that, described spray drying parameter is: air quantity 100%, 115 ℃ of inlet temperature, 48 ℃ of leaving air temps, inlet amount 35 ml/h, cleansing pin frequency 5 s; Keeping throughput during the spray drying is 600 m
3/ h; Gas is filtered and is heated to after by aerator 115 ℃ of temperature are set.
6. preparation method as claimed in claim 1 is characterized in that step 2) described in material solution carry out spray drying treatment after, by peristaltic pump material solution is sent to aerosol apparatus, collect microcapsule granule by cyclone separator.
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| CN105031636A (en) * | 2014-09-15 | 2015-11-11 | 新乡医学院 | Bi-combined inactivate vaccine of aeromonas hydrophila and aeromonas veronii and preparation method thereof |
| CN104689310B (en) * | 2014-09-15 | 2017-06-09 | 新乡医学院 | A kind of Aeromonas hydrophila and Aeromonas veronii bigeminy oral slow-releasing microsphere vaccine and preparation method |
| CN105031636B (en) * | 2014-09-15 | 2018-06-19 | 新乡医学院 | A kind of Aeromonas hydrophila and Aeromonas veronii bivalent inactivated vaccine and preparation method |
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