CN101862308A - Aeromonas hydrophila micro-capsular oral vaccine - Google Patents
Aeromonas hydrophila micro-capsular oral vaccine Download PDFInfo
- Publication number
- CN101862308A CN101862308A CN 201010125067 CN201010125067A CN101862308A CN 101862308 A CN101862308 A CN 101862308A CN 201010125067 CN201010125067 CN 201010125067 CN 201010125067 A CN201010125067 A CN 201010125067A CN 101862308 A CN101862308 A CN 101862308A
- Authority
- CN
- China
- Prior art keywords
- microcapsule
- aeromonas hydrophila
- vaccine
- micro
- oral vaccine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to an aeromonas hydrophila micro-capsular oral vaccine, which belongs to the technical field of biological pharmacy. The aeromonas hydrophila micro-capsular oral vaccine is prepared by using sodium alga acid and chitosan as wall materials, using the inactivated bacteria of aeromonas hydrophila as a core material and by using improved atomization and ion crosslinking technology. When the aeromonas hydrophila sodium alga acid-chitosan micro-capsular oral vaccine is used for immunizing a mouse and crucian, the phagocytosis activities of macrophages, the conversion efficiency of the splenic lymphocytes and the expression amount of cell factors such as IFN-gamma and Il-4 of the mouse and the crucian can be improved obviously. The relative protection rate of the vaccine for the crucian is 39.3 percent. A microcapsule is stable in a simulated gastrointestinal environment and shows high burst release and slow release; and the bacterial antigenicity of the microcapsule is well maintained and the vaccine has high safety and high storage stability.
Description
Technical field
The invention belongs to and relate to field of biological pharmacy.Particularly, the present invention relates to a kind of Aeromonas hydrophila oral vaccine.More particularly, the present invention relates to microcapsule antigen transmission system and based on the aeromonas hydrophila micro-capsular oral vaccine of this exploitation, this vaccine can be used for the hueppe's disease that prevents aeromonas hydrophila disease to cause.Also this microcapsule antigen transmission system can be applied to the exploitation of the oral vaccine of other cause of disease of Fish.
Background technology
Aeromonas hydrophila (Aeromonas hydrophila; Ah) can cause Fish, amphibian and reptile outburst hueppe's disease; cause fresh-water fishes skin hemorrhage (hueppe's disease) and mortality, already brought the tremendous economic loss for the scale freshwater fish culturing.Traditional remedies such as application antibiotic easily bring out bacterial resistance, a series of problems such as drug residue and environmental ecology destruction.
The Aeromonas hydrophila vaccine of being developed at present (whole-bacterial-vaccine, recombinant vaccine) is injecting immune or immersion immunity.Though compare with injecting immune, immersion immunity, the oral immunity effect is relatively poor, the immunity of lymphogranuloma inguinale clothes has good, the shoal of fish easy to use, safe stress be little, be suitable for scale and repeat characteristics such as immunity, is expected to become the most promising fish immunity approach.Because the destruction of Fish stomach acidity environment and enteral digestive enzyme, traditional vaccine is degraded before arriving intestinal mucosa in a large number, even there is a spot of antigen to arrive the immunostimulation that intestinal mucosa also can't form persistence, be difficult to bring out immunne response efficiently, improved immune cost greatly and improve immune effect by increase immunizing dose or immune time, immune effect is relatively poor.In recent years, domestic and international many scholars successively utilize various polymer microcapsule transmission systems to carry out oral vaccine research.
People such as A.P.Rodrigues utilized vegetable oil emulsifying method development Aeromonas hydrophila sodium alginate oral vaccine in 2006, but the particle diameter of prepared microsphere is bigger, mean diameter is about 50 μ m, the particle diameter that does not meet desirable microsphere requires (about 10 μ m), may influence the picked-up of antigen presenting cell to microsphere, the animal immune test effect does not appear in the newspapers.
Sodium alginate (C
6H
7O
8Na)
nBe to rely on 1 by α-L-mannuronic acid (M unit) and β-D-guluronic acid (G unit), the 4-glycosidic bond connects and the linear copolymer of composition.Sodium alginate powder is met water and is become wet, aquation and dissolving fully slowly.Alginate can be by the sodium ion and the bivalent cation (Ca of its golonic acid
2+, Ba
2+) exchange and instantaneous gelation balling-up.Chitosan is the natural polymer straight-chain polysaccharide by the de-acetyl chitin preparation.Owing on the chitosan molecule chain a large amount of primary amino radicals is arranged, a large amount of carboxyls is arranged on the strand of sodium alginate, chitosan and sodium alginate can form polyelectrolyte film by positive and negative charge attraction.Microcapsule antigen transmission system should have the protection immunogenicity of antigens and promote the effect of antigen presenting cell to antigenic picked-up.Calcium alginate microsphere has repellence to sour environment, and chitosan has repellence to alkaline environment.The microcapsule that both form is better to antigenic protectiveness, but how to adjust the particle diameter of microcapsule, it is easilier caught by antigen presenting cell, and how to control microcapsule becomes this research to problems such as antigenic slow release focus and difficult point.
Summary of the invention
Technical problem
The objective of the invention is to develop a kind of relatively stable in the Fish digestive system, particle diameter is moderate, is easily caught by macrophage, has slow-releasing and antigen protectiveness, and the microcapsule antigen transmission system of good biocompatibility.And this microcapsule antigen transmission system is applied to Aeromonas hydrophila, develop aeromonas hydrophila micro-capsular oral vaccine, to overcome the defective of the direct oral immunity weak effect of inactivated vaccine.With the Carassius auratus is animal pattern, has verified the immunological effect of this oral vaccine.This vaccine can be used for the immunoprophylaxis of the caused hueppe's disease of Fish Aeromonas hydrophila.
Technical scheme
A kind of aeromonas hydrophila micro-capsular oral vaccine is comprising calcium alginate-chitosan controlled-releasing microcapsule and be encapsulated in deactivation Aeromonas hydrophila in the described microcapsule.Comprise:
1.1 the screening of vaccine strain
The Aeromonas hydrophila J-1 strain that employed vaccine strain separates and preserves for this laboratory among the present invention.The J-1 strain belongs to Aeromonas hydrophila (Aeromonas hydrophila), and this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 6th, 2009, and its preserving number is CGMCC NO.3220.
1.2 the preparation of the full bacterium bacterium of deactivation liquid
With after the Aeromonas hydrophila J-1 strain rejuvenation in 28 ℃ of following amplification culture, after 0.4% formalin deactivation, use pH
7.2 PBS liquid washing antibacterial, bacterial concentration is adjusted into 1.0 * 10
10Cfu/mL.
1.3 the preparation of Aeromonas hydrophila calcium alginate-chitosan microcapsules (Ah-ACMS) oral vaccine
Under the stirring of homogenizing refiner, with a certain amount of sodium alginate, above-mentioned inactivated bacterial liquid and a certain amount of normal saline mix, and are made into sodium alginate-antigen emulsion (sodium alginate concentration is 2.0%W/V).Directly spray into the 5%CaCl that contains after the spray-dried machine atomizing of this emulsion through the magnetic stirrer stirring
2PBS solution in, form sodium alginate microcapsule, through centrifugal, collect sodium alginate microcapsule.Under the stirring of homogenizing refiner,, be distributed in 0.2% the chitosan solution collected sodium alginate microcapsule.After centrifugal, collect calcium alginate-chitosan microcapsules.Again with the microcapsule of collecting, be distributed in 8% the mannitol solution, be mixed, after the lyophilization, the capping packing.Envelop rate (96.52 ± 0.17) % of wherein said calcium alginate-chitosan controlled-releasing microcapsule parcel deactivation Aeromonas hydrophila carries bacterium amount (2.41 ± 0.32) * 10
8Cfu/mg.The particle diameter of wherein said calcium alginate-chitosan controlled-releasing microcapsule is 11.01 μ m.
2.1 contain of the preparation of the fish of aeromonas hydrophila micro-capsular oral vaccine with bait
With prepared aeromonas hydrophila micro-capsular oral vaccine and a certain amount of after the commercialization fish feed powder mixes that the immersion of anhydrating was softened, the compacting slivering is dried, and makes granule.
2.2 the usage of aeromonas hydrophila micro-capsular oral vaccine
Immunization route: oral immunity.Behind shoal of fish fasting 24h, a certain amount of fish feed pellets that contains aeromonas hydrophila micro-capsular oral vaccine is dropped in the water, for shoal of fish free choice feeding.Immunizing dose is: the fish of every gram body weight is thrown in microcapsule oral vaccine 0.2mg (that is: the fish of every gram body weight immunity antibacterial 5.0 * 10
7CFU).After head exempts from 10 days, carry out two and exempt from, usage and dosage are the same.
Beneficial effect
1. originally to study employed wall material be natural macromolecular material to wall material wide material sources, and its wide material sources are cheap and have immunological enhancement, have been used for the additive or the adjuvant of medicine by the FDA approval, safety, avirulence.
2. the improvement spraying-ionomer technology that adopts of simple this institute of processing technology, processing technology is simple, and cost is lower, the favourable suitability for industrialized production of using.Preparation technology's agents useful for same safety, low toxicity does not produce noxious substance in the preparation process.
3. the more satisfactory prepared microcapsule rounding of microcapsule character, mean diameter less (11.010 ± 0.145 μ m), particle size distribution is more even; Antigen coated rate is (96.52 ± 0.17) % approximately; Drug loading is (2.41 ± 0.32) * 10
8Cfu/mg; Microcapsule is stable under the simulation gastrointestinal conditions, shows prominent preferably property released and slow-releasing; Bacterial antigen keeps good in the microcapsule, and safety and storage stability are better.
4. store and this vaccine of convenient transportation at normal temperatures (25 ℃) preserve more than 9 months and keep good antigenicity.Saved the cost of freezed storage and transportation, be convenient to promote.
5. easy to operate, the better oral immunity of immune effect stress be little to animal, and easy to operate, human cost is low, is convenient to large-scale promotion.This vaccine free from extraneous odour, palatability is good, and the shoal of fish easily searches for food, and immune effect is better.
6. have a extensive future the microcapsule antigen transmission system developed to resisting the stimulation of acid or alkali environment in the gastrointestinal; better to antigenic protection; and has slow releasing function; can form pulsed to body stimulates; can be applied to the exploitation of the oral vaccine of other cause of disease of Fish, accelerate the flow of research of Fish oral vaccine.
Description of drawings
The form of Figure 1A h-ACMS (* 400 times)
The form of Fig. 2 Ah-ACMS (* 200 times)
The form of the barren ACMS of Fig. 3 (* 400 times)
Fig. 4 Ah-ACMS microcapsule oral vaccine grading curve
Microcapsule is released bacterium time and accumulation release percentage curves (12h) under the different pH of Fig. 5
Microcapsule is released bacterium time and accumulation release percentage curves (120h) under the different pH of Fig. 6
Fig. 7 respectively organizes the dynamic change in time of Carassius auratus serum agglutinating antibody
Fig. 8 head-kidney macrophage phagocytic specific activity
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.It will be understood by those skilled in the art that these embodiment only to be used to the present invention is described and never scope of the present invention is constituted any restriction.Unless otherwise indicated, all scientific and technical terminologies among the application all have and the identical implication of one skilled in the art's common sense of the present invention.Arbitrary patent, patent application and the publication quoted among the application are hereby incorporated by.The experimental technique of unreceipted actual conditions in the following example, usually adopt normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the method for advising according to manufacturer.
(1) preparation of Aeromonas hydrophila sodium alginate-chitosan microcapsules (Ah-ACMS) oral vaccine
1.1 the selection of vaccine strain
Employed vaccine strain is Aeromonas hydrophila J-1 strain (Ah J among the present invention
1), be to separate and get from Jiangsu morbidity fish.Belong to Aeromonas hydrophila (Aeromonas hydrophila), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 6th, 2009, and its preserving number is CGMCC NO.3220.
This bacterium is the Grain-negative brevibacterium, also can be two spherical or thread sometimes.On ordinary nutrient agar, can grow, cultivate the 48h colony diameter for 25 ℃ and can reach 2～3mm, smooth surface.On blood plate, can produce β haemolysis circle.Well-grown on the Mai Kangkai culture medium is selected culture medium TCBS or is not grown in 6%NaCl in vibrio, and it is insensitive that vibrio is suppressed bacterium O/129, most azymic lactose, oxidase positive.Guan Jian biochemical indicator is in addition: the glucose aerogenesis, fermentation mannitol, sucrose utilize arabinose, hydrolysis Esculin/salicin, ODC Ornithine decarboxylase feminine gender.Meet as above-mentioned 6 indexs, then decidable is an Aeromonas hydrophila.Aeromonas has pathogenic widely, infection comprises multiple animal such as Fish, birds and the mammals of cold blooded animal, causing local infections such as septicemia or skin ulcer, is the especially modal pathogenic bacterium of Fish of aquatic animal, can cause outbreak of epidemic in the summer that water temperature is high.Human infection mobility Aeromonas causes acute gastroenteritis etc., this bacterium is included in the conventional sense scope of diarrhoea pathogenic bacteria at present abroad, is the object of inspection for food hygiene.
1.2 the preparation of inactivated vaccine
Will be through infecting the Ah J of Carassius auratus rejuvenation
1Be inoculated in the flask of the 500mL that 250mL LB meat soup is housed, in 28 ℃, 150rpm, shake is cultivated 24h.Behind the dyeing microscopic examination, the adding final concentration is 0.4% formalin, and behind effect 3h under 28 ℃, 4 ℃ act on 24h down.Take a morsel then bacterium liquid inoculation LB meat soup after the deactivation, whether in 28 ℃, shake is cultivated 24h, thorough with the check deactivation.With the bacterium liquid of complete inactivation behind the centrifugal 10min of 8000rpm, with the PBS liquid washing antibacterial of pH 7.2 three times.The antibacterial final concentration is adjusted to 1.0 * 10
10Cfu/mL, 4 ℃ of preservations are standby.
1.3 the preparation of Aeromonas hydrophila sodium alginate-chitosan microcapsules (Ah-ACMS) oral vaccine
According to table 1 orthogonal design scheme, set up 9 levels, the important technological parameters such as bacterial concentration, sodium alginate concentration, spray velocity and mixing speed that influence microcapsule oral vaccine characteristic are carried out the quadrature screening, characteristics such as slow-releasing according to particle diameter, year bacterium amount, production efficiency and microcapsule, established following optimal preparation technology: get a certain amount of above-mentioned bacterial suspension, behind the centrifugal 10min of 8000rpm, resuspended with the PBS liquid of pH7.2, adjusting bacterial concentration is 5.0 * 10
9Cfu/mL.Take by weighing a certain amount of sodium alginate (SA), join in the above-mentioned bacterium liquid, utilize homogenizing refiner 15000rpm, effect 5min dissolves sodium alginate fully and forms sodium alginate-antibacterial emulsion (SA concentration is 2.0%W/V).Be provided with the spray dryer parameter as follows: nozzle diameter: 0.5mm; Inlet temperature: 25 ℃; Leaving air temp: 25 ℃; Air-flow: 3bar; Spray velocity: 7mL/min; Cleansing pin: 20 times/min.Beaker with 500mL holds 10 times of 5%CaCl that contain to above-mentioned sodium alginate soln volume
2PBS solution, be placed on the magnetic stirrer, adjust rotating speed and be: 800rpm.Start spray dryer, sodium alginate-antibacterial emulsion is directly sparged the above-mentioned 5%CaCl of containing with the speed of 7mL/min
2PBS solution in.After spraying finishes, continue to stir 20min, in case glutinous company the between sodium alginate microcapsule (SA-MS).The centrifugal 5min of 1000rpm washs microcapsule 3 times with the PBS liquid of sterilizing, and collects sodium alginate microcapsule.With collected sodium alginate microcapsule, be distributed in 0.2% the chitosan solution (chitosan solution and above-mentioned sodium alginate soln volume ratio are 1: 1) homogenizing refiner 11000rpm, effect 5min.The centrifugal 5min of 1000rpm, the PBS liquid with sterilization washs Aeromonas hydrophila calcium alginate-chitosan microcapsules (Ah-ACMS) 3 times then, collects microcapsule, promptly gets aeromonas hydrophila micro-capsular oral vaccine of the present invention.With above-mentioned Aeromonas hydrophila calcium alginate-chitosan microcapsules, be distributed in 8% the mannitol solution, freezing behind the mixing in-40 ℃ of refrigerators, lyophilization 15h again, capping packing.
The orthogonal design result of table 1Ah-ACMS preparation technology parameter
1.4 contain the preparation of the fish feed of aeromonas hydrophila micro-capsular oral vaccine
With water infiltration commercialization fish feed powder, it is stirred after in the pasty state.Prepared Aeromonas hydrophila sodium alginate-chitosan microcapsules oral vaccine in 1.3 is joined during feedstuff sticks with paste, stir.Calculate by 1% feedstuff of the fish body weight of throwing something and feeding every day and to add the microcapsule oral dose (fish should search for food 5.0 * 10 during immunity
7Cfu/g/ days bacterium amount).The compacting slivering is dried, and makes granule, and room temperature is preserved, and avoids moist.
(2) in-vitro evaluation of aeromonas hydrophila micro-capsular oral vaccine
Microcapsule form, diameter and distribution, bag to the Aeromonas hydrophila calcium alginate-chitosan microcapsules oral vaccine of preparation among the embodiment 1 in the present embodiment are tested and assessed respectively by efficient and principal characters such as carrying bacterium amount, in-vitro simulated release characteristic, Antigen Stability, safety and storage stability.
2.1 morphological observation
Get respectively before and after the lyophilizing Ah-ACMS a little, be scattered in the PBS liquid of pH 7.2, utilize Nikon eclipseTS100 type inverted microscope in * 200 times then, observe the particle diameter form down, and utilize Nikon DS-L digital micro imaging system acquisition image for * 400 times.Imaging results is seen Fig. 1-Fig. 3.
As can be seen from Figure 1, microcapsule is rounded substantially, the relative homogeneous of particle diameter, and the surface is more coarse, and tangible fold is arranged, and the visible many black points in microcapsule surface are not for being wrapped the antibacterial of quilt fully.
2.2 particle diameter mean size and distribution
Prepare microcapsule by the preparation technology after optimizing in 1.3, gather microcapsule image (* 400) by the method in 2.1, picked at random 800-1000 microsphere, utilize Jetta JEDA 801D morphology image analysis system to measure its diameter, utilize SPSS 11.5 statistics softwares that the diameter Distribution situation is carried out statistical analysis.Ah-ACMS microcapsule oral vaccine grading curve is seen Fig. 4.
As can be seen from Figure 4: the microcapsule diameter meansigma methods is 11.010 ± 0.145 μ m.Particle size distribution is more concentrated, and 70% microcapsule is distributed in 5-14 μ m, and 83% microcapsule diameter is less than 15 μ m, and basic symbols is rationally thought the size grade scale of microcapsule transmission system.
2.3 coating rate and drug loading are measured
Prepare each 100mL of SA-MS suspension that contains the full bacterium of deactivation and do not contain antibacterial respectively by the preparation technology after optimizing in 1.3.The centrifugal 5min of 500rpm collects supernatant respectively.Supernatant is middling speed qualitative filter paper (two circle boards of 15cm through diameter, model: 102) filter, collect two parts of filtrates respectively, with the SA-MS filtrate that does not contain antibacterial is blank, measures through ultraviolet spectrophotometer (Bio-Rad Smart Spec TM 3000) and contains the light absorption value of the SA-MS filtrate of the full bacterium of deactivation at 600nm.With the amount of bacteria of determining not wrap quilt in the filtrate (cfu).Weigh after the Ah-ACMS drying with equivalent (mg).According to following formula difference computational envelope rate (%) and year bacterium amount (cfu/mg).
By the Ah-ACMS of this scheme preparation, envelop rate is (96.52 ± 0.17) % approximately, the envelop rate height, and antigen losses is few.It is high to carry the bacterium amount, approximately (2.41 ± 0.32) * 10
8Cfu/mg.Has the high envelop rate of desirable medicine carrying microcapsule, the characteristic of high drug load.
2.4 in-vitro simulated release test
Prepare pH=2.0PBS with deionized water, pH=7.0PBS and each 500mL of pH=10Tris-Hcl buffer.Get an amount of blank sodium alginate-chitosan microcapsules (ACMS (not containing antibacterial) and Ah-ACMS (containing antibacterial) suspension (containing the 0.2g microcapsule approximately) respectively, centrifugal, collect microcapsule, be resuspended in respectively in the above-mentioned three kinds of buffer of 20mL, magnetic force heated and stirred machine stirs, heating maintains about 25 ℃ solution temperature.Every is at regular intervals, with solution in the centrifugal 5min of 500rpm.Microcapsule is scattered in again in the fresh corresponding buffer of 20mL.Centrifugal gained supernatant through the middling speed qualitative filter paper (two circle boards, model: 102) filter, collect filtrate, the centrifugal 10min of 10000rpm, concentrated filtrate is to 1mL.With blank ACMS filtrate is blank, utilizes ultraviolet spectrophotometer to measure and has discharged amount of bacteria.Draw under the different pH microcapsule and release the bacterium time and discharge percentage curves, see Fig. 5 and Fig. 6 with accumulation.
As shown in Figure 5, microcapsule is acid similar with the release profiles under the neutrallty condition, and dispose procedure is slower, preceding 12 hours, release rate was less than 10%, and the microcapsule metamorphosis is not obvious, obvious swelling does not take place, the degraded that promptly prepared microcapsule can the gastric sour environment and the swelling action of neutral solution.And under alkali condition (pH=10), the 0-4h microcapsule is not obvious to the slow releasing function of antibacterial, and this may be relevant to the opposing of alkali liquor with the outer shell polysaccharide.4-12h, the release action of microcapsule is obviously accelerated, and the microcapsule expansion, and particle diameter increases, and illustrates that prepared Ah-ACMS is unstable under alkali condition.As shown in Figure 6, in the microcapsule pro-24 hours, the significantly prominent phenomenon of releasing being arranged.Behind the effect 120h, during pH=10,72% antibacterial release is arranged, and only have under the neutrallty condition about 33%, illustrate that this microcapsule has slow releasing function preferably.
2.5 vaccine antigen stability analysis
The antibacterial that collection discharges from lyophilizing Ah-ACM: get the about 0.2g of Ah-ACMS after the lyophilizing, in the citric acid three sodium solution (pH=10, NaoH regulates) of 20mL 3%, magnetic force heated and stirred machine stirs, heating, 37 ℃ of effect 5h.After the middling speed qualitative filter paper filters, the centrifugal 10min of 10000rpm, the resuspended antibacterial of PBS solution of pH=7 is to 10mL (concentration about 10
8Cfu/mL).
SDS-PAGE: before getting deactivation, (the formalin deactivation is according to method described in 1.2), lyophilizing microcapsule are discharged after the deactivation each 1mL of bacterium liquid, the centrifugal 1min of 10000rpm collects thalline.It is resuspended to add 100 μ L SDS-PAGE sample-loading buffers, boils 10min, the centrifugal 3min of 12000rpm, and supernatant is used for SDS-PAGE.Preparation contains the gel of 12% acrylamide.Behind the 80V voltage stabilizing electrophoresis 30min, about 120V electrophoresis 2h, when migrating to the glue lower edge, the bromophenol blue forward position finishes electrophoresis.From the Ah J that lives
1, deactivation Ah J
1And the Ah J that discharges among the Ah-ACMS
1The bacterium protein electrophoresis result as can be known, the basic zero difference of protein band before and after test is handled.
Western blot: after electrophoresis finishes, earlier glue is taken out, once with washed with de-ionized water.Cutting and big filter paper and pvdf membrane such as glue to be transferred, and pvdf membrane soaked 5min in methanol.Filter paper is put up successively according to the order of (+) filter paper-film-glue-filter paper (-) after using the transfer printing buffer moistening, puts into transfer printing instrument (must get rid of bubble when placing each layer), adopts half-dried transfer method that it is transferred on the pvdf membrane.After transfer printing finished, pvdf membrane spent the night with 4 ℃ of sealings of 5% skimmed milk.One resists the rabbit anti-serum (1: 2000 times of dilution) for preparing for the J-1 strain Ah tropina with deactivation.Two anti-are the goat anti-rabbit antibody of horseradish peroxidase-labeled (1: 2000 times of dilution), develop the color with diaminobenzidine (DAB).
Western blot testing result as can be known, the antibacterial that from Ah-ACMS, discharges can with the anti-Ah serum of rabbit generation immunoreation, and band is clear.Illustrate that microcapsule preparation technology is gentle, the antigenicity to tropina does not cause obvious destruction.
2.6 safety evaluatio test
Choose 10 6 age in week Kunming be healthy mice, male female half and half.By 10 times of (Ah-ACMS 0.05g/ contains antibacterial 10 of normal immunizing dose
10Cfu) per os medication, one week of continuous irrigation stomach.The normal raising observed all interior mice poisoning symptom, a death condition and cutd open the pathological changes situation that liver, kidney are observed in inspection.
Observe and find, irritate stomaches after one week by 10 times of (the about 0.05g/ of Ah-ACMS) per os of normal immunizing dose, the mice performance is active, does not have poisoning or death condition and takes place, and cuts open inspection and finds that the test mice Liver and kidney does not all have enlargement or necrosis.Show that in the microcapsule preparation process generation has toxic material, both are as antigen vectors safety, reliable.
2.7 storage stability test
Ah-ACMS vaccine with preparation is sub-packed in the 5mL cillin bottle, seals after the lyophilizing, respectively at-20 ℃, 4 ℃, preserves down for 25 ℃.And in the 0th, 3,6,9 the end of month samplings detects, and leading indicators such as the outward appearance of microcapsule vaccine, dissolubility, size, antigenicity, slow-releasing are measured.Evaluate the Antigen Stability in different time points of vaccine with the Western blot result of the agglutination of the Ah that from Ah-ACMS, discharges and the anti-Ah positive serum of rabbit and Ah whole bacterial protein.Discharge the slow-releasing of used time evaluation microcapsule with 50% antibacterial., the results are shown in Table 2.
Table 2Ah-ACMS storage stability test
As known from Table 2, Ah-ACMS vaccine freeze-drying preparation is at different somes Check-Out Time, and outward appearance is the white loose powder, favorable solubility in water, no obvious sediment.Under different temperature conditions, though the particle diameter of microcapsule has expansion in various degree, difference is remarkable (P＞0.05) not, and mean diameter is all less than 13 μ m, and basic symbols is rationally thought the particle diameter requirement of microcapsule oral vaccine.Discharge antibacterial all can with the anti-Ah positive serum of rabbit generation agglutination, western blot band is clear, shows that bacterioprotein still has good immunogenicity.Along with the growth of storage life, 50% antibacterial discharges needed time decreased, the prominent property the released enhancing of microcapsule, but still have the good slow release effect.But the Ah-ACMS vaccine of being developed storage-stable 9 months at least under condition of different temperatures, stable in properties under the room temperature has been broken away from the dependence to cold chain, has reduced storage and cost of transportation.
Embodiment 3Ah-ACMS oral vaccine immune protective effect
, raise in the glass jar of 300L as animal pattern with the pond crucian carp of 120-150g, raise pond crucian carp, utilize the continuous oxygenation of oxygen charging pump with the tap water behind the aeration.The commercial pellet of regularly throwing something and feeding changes water and cleaning solid metabolite.Carassius auratus after feed is normal, begins test through week domestication.Verify the immunological effect of Ah-ACMS oral vaccine of the present invention according to following scheme to Carassius auratus.
3.1 immune programme for children
The test fish is divided into six groups, and 40 every group, fasting 24h before the immunity carries out immunity according to table 3 scheme.Oral matched group at every turn by the fish body weight 1% respectively at the 8:00 point, 12:00 point, 16:00 point are thrown something and fed and are contained the feed granules of vaccine; Oral and the injection matched group employing Ah inactivated vaccine of Ah, the injection group is by the lumbar injection immunity; The blank group general goods fish meal of throwing something and feeding.In process of the test, pay close attention to the situation of searching for food of Carassius auratus.Water temperature is about 18-25 ℃.
Table 3 test grouping and immune programme for children
3.2 the serum agglutinating antibody is tired
According to the time point in the table 3, from tail vein sample of blood, drawn.Spend the night in 4 ℃ of placements, after solidifying, 3000rpm, centrifugal 5min, separation of serum, frozen in-20 ℃, to be checked.Utilize the glass plate agglutination test of improvement to measure.Be summarized as follows: get 50 μ L immune group (or matched group) serum with 50 μ L PBS solution (pH 7.2) doubling dilutions, (concentration is about 10 to add the heat-inactivated Aeromonas hydrophila suspension of 50 μ L then
9Cfu/mL), behind the mix homogeneously, place wet box, spend the night under the room temperature, observe the bacterial agglutination situation in microscopically (* 40 times).Tiring of antibody can cause that antibacterial liquid agglutinative maximum serum diluting multiple takes place represents.The results are shown in Figure 7.
Second week beginning behind the initial immunity, the serum agglutinating antibody appears in each group in succession, the agglutinating antibody level of oral group of microcapsule (group I, II, III) and full bacterium injection group (group V) apparently higher than blank group (group VI) and full fungus oral group (group IV) (P＜0.05, n=3).Group V head exempts from back 14d and begins to occur agglutinating antibody, and 28-35d antibody reaches peak value, and agglutinating antibody is apparently higher than other each groups, and downward trend appears in antibody during 42d; Group I, II slowly rise at 14-35d, peak about 35d, slightly descend during 42d.And group is during III42d, and agglutinating antibody is still on the rise.Group IV is in low-level state with the agglutinating antibody of group VI always, and humoral immunity level is low.
3.3 macrophage phagocytic activity
3.3.1 the separation of head-kidney macrophage
In two weeks of back of immunity for the third time,, from the Carassius auratus head-kidney, separate macrophage with reference to people's such as Braun-Nesje method.The sterile working opens the abdominal cavity, the clip head-kidney.Wash 2-3 time with cold Hank ' s liquid, go up grinding with asepsis injector post core in 200 order stainless (steel) wire cells sieve, collect filtering single cell suspension, the centrifugal 15min of 1500rpm, Hank ' s liquid washed cell twice is abandoned supernatant, and cell precipitation (includes the 100IU/mL mycillin with the L-15 culture fluid, 10% hyclone, the 10IU/mL heparin) resuspended.Single cell suspension utilize discontinuous gradient centrifugation (Percoll gradient, Sigma. density: 1.08-1.07), 4 ℃, 400g, centrifugal 40min.Collect the macrophage layer, utilize L-15 culture fluid washed twice, abandon supernatant, utilize L-15 culture fluid (containing 10% hyclone) re-suspended cell, utilize the cell counting count board counting.Detect cell viability with trypan blue liquid.Adjusting cell concentration is 1.0 * 10
6Individual/mL.
3.3.2 macrophage kills bacterial activity and detects
Physiological saline solution is regulated new Aeromonas hydrophila (the Ah J that cultivates
1) concentration to 1.0 * 10
7Cfu/mL gets the macrophage suspension (1.0 * 10 of bacterium liquid 100 μ L and 100 μ L
6Cells) mixing in 96 porocyte culture plates adds the fresh Carassius auratus serum of 50 μ L then, behind the mixing, hatches 2 hours under 28 ℃ of water-baths, and rolling makes a movement once in a while therebetween.After 2 hours, get above-mentioned mixed liquor of 0.2mL and 0.8mL physiological saline solution mixing, to discharge not by the Aeromonas hydrophila of the work of macrophage phagocytic.Bacterium liquid behind doubling dilution, is got 200 μ L tiling LB solid culture plate, 28 ℃ of following overnight incubation, statistics bacterium colony number is calculated as follows the macrophage phagocytic rate.
Immunity back each activate the phagocytic capacity of organizing of two weeks is relatively seen Fig. 8 for the third time.Through variance analysis, macrophage has marked difference (P＜0.05) to the kill capability of Ah bacterium liquid alive between group I, II, III, V, is significantly higher than group VI and group IV, and the macrophage phagocytic ability of group III is the strongest, reaches (45.25 ± 1.98) %.But group IV macrophage does not have significant difference (P＞0.05) between the kill capability of antibacterial and blank group (organizing VI).
3.4 the mensuration of the median lethal dose(LD 50) of Carassius auratus
The LB fluid medium is cultivated acquisition 2.4 * 10
9The Ah J of cfu/mL
1Bacterium liquid dilutes 10 successively by 10 multiple proportions
-1-10
-4Doubly, get each dilution bacterial suspension and inoculate 10 tail Carassius auratuss in the abdominal cavity respectively, every tail 0.5mL.Establish the blank group simultaneously, lumbar injection PBS, 0.5mL/ tail.Death toll respectively organized in record, utilizes the Reed-Mueneh method to calculate Ah J
1Bacterium is 1.26 * 10 to the median lethal dose(LD 50) of Carassius auratus
6The cfu/ tail
3.5 the mensuration of relative protective rate
Immunity is two weeks later, get 30 tail Carassius auratuss, the about 50 * LD of lumbar injection respectively from each group
50(promptly 6.3 * 10
7The cfu/ tail).Observed 7 days continuously behind the counteracting toxic substances, write down death condition day by day.From dead fish internal organs organ bacterial isolate bacterium, and pathogen identified.Calculate and respectively organize relative protective rate (RPS).
Dead peak appears in 24-56h behind the counteracting toxic substances, no longer occurs dead after 5 days at counteracting toxic substances.All dead Carassius auratus Ah detect and all are positive.Each is organized relative protective rate and sees the following form 4.The relative protective rate of ACMS high dose group, ACMS low dose group, ACMS adjuvant group, Ah injection matched group is respectively 39.3%, 32.1%, 50.0%, 82.1%.Adjuvant has significantly strengthened the immune effect of microcapsule oral vaccine, makes oral vaccine improve 17.9% to the protection efficient of Carassius auratus.
Table 4 is respectively organized Carassius auratus to Ah J
1Mortality rate and relative protective rate that strain is attacked
The present invention utilizes improvement spraying-ionomer technology to prepare aeromonas hydrophila micro-capsular oral vaccine, and technology is simple, and the preparation condition gentleness is farthest protected antigen active.The microcapsule oral vaccine is that the phagocytosis by antigen presenting cells such as phagocyte has targeting, and the size of microcapsule has direct influence to engulfing of microcapsule in the body.The difficult point of a maximum of the present invention is by suitable preparation technology microcapsule diameter to be controlled in the scope that can be caught by antigen presenting cell.It is generally acknowledged, the particle diameter of mammiferous microcapsule oral vaccine is being ideal less than 10 μ m and high antigen bearing capacity and stability, but people such as Romalde report, it utilizes the sodium alginate Ge Shi Lactococcus microcapsule oral immunity Squaliobarbus ourriculus of the about 30 μ m of diameter of spray drying method for preparation to obtain good immune effect.The size of the microcapsule that the present invention is prepared is between 10.009-15.068 μ m, and the particle diameter of best preparation technology's gained microcapsule is 11.010 ± 0.145 μ m.Because behind the Fish oral immunity, antigenic catch and process such as offer it be unclear that can be thought prepared vaccine Pass Test requirement substantially.
Prepared Ah-ACMS is only stable under fish gastric sour environment, and slowly discharges in intestinal, could constantly stimulate the immunocyte in the intestinal, obtains satisfied immunoreation.According to Rodrigues, tilapia is after diet, and the pH of its gastric is about 2.0, and the pH in its intestinal is about 10.0, and food is about 12h at the intravital mean residence time of Fish.Prepared microcapsule is stronger to acid (pH=2) and neutral (pH=7) condition resistance, under alkalescence (pH=10) condition, shows the prominent property released.Can predict that this transmission system should have the effect of antigen protection and slow release simultaneously in Fish digestibility system.
In sum, the present invention is the wall material with sodium alginate and chitosan, utilizes the improvement spraying to prepare good biocompatibility, the aeromonas hydrophila micro-capsular oral vaccine of stable in properties with the ionomer technology.(1) by the important technological parameters such as bacterial concentration, sodium alginate concentration, spray velocity and mixing speed that influence microcapsule diameter being carried out the quadrature screening, determined optimum production preparation parameter; (2) according to the prepared microcapsule of optimizing of technology, the profile rounding, mean diameter is 11.010 ± 0.145 μ m, size is more even, meets antigen presenting cell such as phagocyte and engulfs the required particle diameter requirement of microcapsule.Antigen coated rate is up to (96.52 ± 0.17) %, and antigen losses is few; Drug loading is about (2.41 ± 0.32) * 10
8It is higher that cfu/mg, microcapsule carry the bacterium amount, easily stimulates body to produce immunne response, meets desirable microcapsule standard; (3) prepared microcapsule is stable under the simulation gastrointestinal conditions, and its antibacterial discharges and has the soda acid dependency, and prominent property released and slow releasing function are obvious, and continuable released antigen constantly induces body to produce immunne response; (4) preparation condition gentleness, production technology is simple, and cost is low, and safety is good, is easy to industrialization;
In influencing the multiple factor of vaccines for fish effect, immunization route is to the appreciable impact of vaccine protection efficient.In fact, but vaccination approach appreciable impact vaccine antigen composition arrives the efficient of fish immunity effective-site.Proved already that the aeromonas hydrophila inactivated vaccine intramuscular injection can obtain immune effect preferably, and the immersion immunity effect is taken second place.For handle has the hindgut that immunogenic antigen is delivered to can stimulate fish to produce immunne response, this has researched and developed calcium alginate-chitosan microcapsules antigen transmission system.The microcapsule vaccine of developing Ah is infected certain immunoprotection (protecting 39.3% relatively) can be provided, adjuvant IMS1312 makes the relative protective rate of microcapsule vaccine be raised to 50.0% by 32.1%, illustrates that this oral adjuvant can strengthen the immunogenicity of microcapsule oral vaccine.The oral attack to viable bacteria of the direct spice of the full bacterium of deactivation can not provide protection (protective rate is-3.6% relatively), may be antibacterial destroyed or degraded of its epitope major part before arriving Carassius auratus intestinal back segment, and the antigen uptake amount is few.And the microcapsule transmission system of developing can be resisted gastric acid and protease to antigenic destruction, and in addition, the immunostimulant of the targeting of microcapsule, slow-releasing and wall material (sodium alginate, chitosan) all can make its immunogenicity be protected.
Claims (4)
1. aeromonas hydrophila micro-capsular oral vaccine is comprising calcium alginate-chitosan controlled-releasing microcapsule and be encapsulated in deactivation Aeromonas hydrophila in the described microcapsule.
2. aeromonas hydrophila micro-capsular oral vaccine as claimed in claim 1, envelop rate (96.52 ± 0.17) % of wherein said calcium alginate-chitosan controlled-releasing microcapsule parcel deactivation Aeromonas hydrophila carries bacterium amount (2.41 ± 0.32) * 10
8Cfu/mg.
3. aeromonas hydrophila micro-capsular oral vaccine as claimed in claim 1 or 2, the particle diameter of wherein said calcium alginate-chitosan controlled-releasing microcapsule are 11.01 μ m.
4. aeromonas hydrophila micro-capsular oral vaccine as claimed in claim 1 or 2, wherein said Aeromonas hydrophila is the J-1 strain, belong to Aeromonas hydrophila (Aeromonas hydrophila), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 6th, 2009, and its preserving number is CGMCC NO.3220.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010125067 CN101862308A (en) | 2010-03-16 | 2010-03-16 | Aeromonas hydrophila micro-capsular oral vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010125067 CN101862308A (en) | 2010-03-16 | 2010-03-16 | Aeromonas hydrophila micro-capsular oral vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101862308A true CN101862308A (en) | 2010-10-20 |
Family
ID=42954372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010125067 Pending CN101862308A (en) | 2010-03-16 | 2010-03-16 | Aeromonas hydrophila micro-capsular oral vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101862308A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102139102A (en) * | 2011-04-01 | 2011-08-03 | 上海海洋大学 | Preparation method of microcapsule vaccines capable of resisting infection of aeromonas hydrophila |
| WO2013166681A1 (en) * | 2012-05-10 | 2013-11-14 | 聚和国际股份有限公司 | Composition for producing microcapsules and medicine made thereof |
| 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 |
| CN105695372A (en) * | 2016-04-18 | 2016-06-22 | 华中农业大学 | High pathogenicity Aeromonas hydrophila and application |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181635A (en) * | 2007-12-10 | 2008-05-21 | 河北师范大学 | Method for preparing pathogenicity hydrosphere unit cell bacterium killed vaccine |
| CN100566751C (en) * | 2007-05-29 | 2009-12-09 | 江苏省家禽科学研究所 | The preparation method of poultry cholera microcapsule vaccine |
-
2010
- 2010-03-16 CN CN 201010125067 patent/CN101862308A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100566751C (en) * | 2007-05-29 | 2009-12-09 | 江苏省家禽科学研究所 | The preparation method of poultry cholera microcapsule vaccine |
| CN101181635A (en) * | 2007-12-10 | 2008-05-21 | 河北师范大学 | Method for preparing pathogenicity hydrosphere unit cell bacterium killed vaccine |
Non-Patent Citations (2)
| Title |
|---|
| 《水利渔业》 20011230 陈超然 鱼用疫苗的研究现状 2.2节内容 1-3 第21卷, 第5期 2 * |
| 《现代生物医学进展》 20081230 寿旭锋 壳聚糖/海藻酸钠微囊在中药提取物缓释制剂中的应用 2.2节内容 1-3 第8卷, 第5期 2 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102139102A (en) * | 2011-04-01 | 2011-08-03 | 上海海洋大学 | Preparation method of microcapsule vaccines capable of resisting infection of aeromonas hydrophila |
| WO2013166681A1 (en) * | 2012-05-10 | 2013-11-14 | 聚和国际股份有限公司 | Composition for producing microcapsules and medicine made thereof |
| CN104302279A (en) * | 2012-05-10 | 2015-01-21 | 聚和国际股份有限公司 | Composition for producing microcapsules and medicine made thereof |
| 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 |
| CN105695372A (en) * | 2016-04-18 | 2016-06-22 | 华中农业大学 | High pathogenicity Aeromonas hydrophila and application |
| CN105695372B (en) * | 2016-04-18 | 2018-05-08 | 华中农业大学 | A kind of highly pathogenic Aeromonas hydrophila and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102533618B (en) | Lactobacillus plantarum CCFM8724 and application thereof | |
| CN105163752B (en) | Clay-ware and application thereof | |
| CN101144062B (en) | Lactobacillus casei strain and application for products thereof in bird immunity | |
| CN104689310B (en) | A kind of Aeromonas hydrophila and Aeromonas veronii bigeminy oral slow-releasing microsphere vaccine and preparation method | |
| KR101467903B1 (en) | The preparing method of immune improving agents | |
| CN109666609A (en) | A kind of Rhodococcus ruber fermentation process and its application as adjuvant in animal vaccine | |
| CN101862308A (en) | Aeromonas hydrophila micro-capsular oral vaccine | |
| Xu et al. | Immunomodulatory effects of chicken egg yolk antibodies (IgY) against experimental Shewanella marisflavi AP629 infections in sea cucumbers (Apostichopus japonicus) | |
| Sun et al. | The CpG ODNs enriched diets enhance the immuno-protection efficiency and growth rate of Chinese mitten crab, Eriocheir sinensis | |
| CN104305168A (en) | A preparing method of lactobacillus rhamnosus particle food relieving alcoholic liver injury | |
| CN102294026A (en) | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof | |
| CN101947213B (en) | Method for preparing microcapsules from peptidoglycan in lactic acid bacterial cell walls | |
| CN108721616B (en) | A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof | |
| CN104288250B (en) | A kind of liquid vitamin premixed feed preparation of anti-piglet isospora and its preparation method and application | |
| CN101991847A (en) | Triple inactivated vaccine against dairy cattle mastitis and preparation method thereof | |
| CN104087559B (en) | A kind of infectious bursa of Fabricius virus, inactivated vaccine and preparation method thereof | |
| CN101606581A (en) | A kind of green feed additive and preparation method thereof | |
| CN105920593A (en) | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product | |
| KR20180016360A (en) | How to treat bacterial infections in aquaculture | |
| CN102286408B (en) | Bacillus amyloliquefaciens WH3, and preparation method and application thereof | |
| CN105597097A (en) | Preparation method of compound IgY for resisting mycotoxin, bacterial exotoxin and pathogen as well as preparation thereof | |
| CN101507816A (en) | Riemerlla anatipestifer bivalent inactivated vaccine and preparation method thereof | |
| CN108685933B (en) | Improve the traditional Chinese medicine ingredients prescription and its preparation method and application of pig immunosuppressive condition | |
| JP3649787B2 (en) | Prophylactic agent for enterococcal infection in fish and use thereof | |
| CN106798920B (en) | A kind of compound immunological adjuvant and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20101020 |