CN104922662A - Preparation method of fish vaccine - Google Patents
Preparation method of fish vaccine Download PDFInfo
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- CN104922662A CN104922662A CN201510314655.0A CN201510314655A CN104922662A CN 104922662 A CN104922662 A CN 104922662A CN 201510314655 A CN201510314655 A CN 201510314655A CN 104922662 A CN104922662 A CN 104922662A
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Abstract
The invention provides a preparation method of a fish vaccine adopting nano chitosan as a carrier. Chitosan and electronegative tripolyphosphate can form nanoparticles in a quite mild condition through ion gelatification; a large amount of egg white can be wrapped in the nanoparticles through electrostatic interaction. According to the preparation method, an acetic acid/ sodium acetate buffer solution with the pH of 6 is adopted to dissolve chitosan, so that the pH of the solution is not too low, and the damage to the vaccine caused by too low pH in the blending process of fish vaccine liquid is reduced; particle gel is adopted to prepare chitosan nanoparticles for wrapping the fish vaccine; the preparation method is mild in reaction condition and simple, uses few devices, and has the advantages of being free of damage to antigen, and high in encapsulation efficiency.
Description
Technical field
The present invention relates to a kind of preparation method of vaccines for fish, particularly relating to a kind of take chitosan nanoparticles as the preparation method of the vaccines for fish of carrier.
Background technology
Vaccines for fish is by three kinds of approach immunity fish bodies: lumbar injection, immersion and oral.Because cultured area is large, injection need consume a large amount of labour forces, also keeps away unavoidable to the damage of Fish.Soak and then will consume heavy dose of vaccine, vaccine also can consume because of the institute of the various materials in breeding water body, deposit and produce huge economy and lose.And oral vaccine, although use the restriction of the size not being subject to time place and fish, destroyed after vaccine arrives gastrointestinal tract, again reduce the immune effect of vaccine.But nowadays oral vaccine is again that immune fish body is the most effective; the method the most easily broken through; vaccine novel thus starts flourish; for microspheres technology; it is that a kind of filmogen is overmolding to the technology of fine particle solid or liquid, feature be the capsule heart be hedged off from the outer world there is defencive function and be beneficial to the capsule heart storage transport.
Oral vaccine microsphere can not only cause whole body and mucosa-immune reaction, and administration safely, conveniently.At present, study more microsphere system and have polylactic acid microsphere, polyglycolic acid microsphere, PLGA microsphere and PLA-PEG copolymer microsphere.But there are some problems in these polymer supports: the combination of (1) carrier and antigen needs with an organic solvent, and organic solvent can cause antigen degeneration; (2) hydrophobic polymerizable objects system is belonged to, low to protein envelop rate; Antigen protein is in polymer support release, and burst effect is comparatively obvious; (3) polymer hydrolysis reduces solution pH value, makes antigen degeneration.Chitosan, except having good bioadhesive, biocompatibility, safety, by opening intercellular tight junction, can also strengthen the membrane permeability of medicine.In addition, chitosan microball is stable under sour environment, can not destroy antigen, only need mix in aqueous phase during antigen loaded, need not with organic solvent exposure.Because chitosan has above many premium properties, become the first choice of potential carrier for oral vaccine delivery.
Summary of the invention
The object of the present invention is to provide a kind of chitosan that uses as the preparation method of the vaccines for fish of carrier, can coated vaccine well, as vaccines for fish carrier, the method equipment needed thereby is few, and reaction condition is gentle, and cost is low, envelop rate is high, and the safety of product is good.
The present invention is achieved through the following technical solutions:
A preparation method for vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 0.5-1.5 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 2-5 ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 8-12 ml step (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips the sodium tripolyphosphate solution 3-8ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.3-0.6ml wherein again, continue to stir 8-15min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:5-8.
Containing free amine group in chitosan molecule, there are 1 pair of lone pair electrons in its nitrogen-atoms, make amino in alkalescence, so chitosan in conjunction with 1 hydrogen atom, can become positively charged electrolyte.Preparing in oral vaccine microsphere process, chitosan microball is by hydrogen bond, hydrophobic interaction and interionic interaction conjugated antigen protein, and have higher envelop rate, the compound alcohol adding special ratios can further improve envelop rate.
Chitosan energy and electronegative tripolyphosphate, under condition as mild as a dove, form nanoparticle by ionic gelation effect; And by electrostatic interaction, a large amount of protein encapsulation is entered in this nanoparticle.
The present invention use pH be 6 acetic acid/sodium acetate buffer dissolve chitosan, make the PH of solution can not be too low, reduce the too low destruction to vaccine of PH in the process of vaccines for fish medicinal liquid mixing.
The present invention utilizes particle to prepare the chitosan nanoparticles of coated vaccines for fish, and reaction condition is gentle, and preparation method is simple, uses equipment few, has and does not destroy antigen, advantage that envelop rate is high.
concrete enforcement
Embodiment 1: a kind of preparation method of vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 1 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 3ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 10ml step (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips the sodium tripolyphosphate solution 5ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.5ml wherein again, continue to stir 10min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:6.
Measuring envelop rate with Coomassie Brilliant Blue is 95.6%.
Embodiment 2: a kind of preparation method of vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 0.5 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 2 ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 8 ml steps (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips the sodium tripolyphosphate solution 3ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.3 ml wherein again, continue stirring 8 min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:5.
Measuring envelop rate with Coomassie Brilliant Blue is 91.2%.
Embodiment 3: a kind of preparation method of vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 1.5 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 5 ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 12 ml steps (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips the sodium tripolyphosphate solution 8ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.6ml wherein again, continue to stir 15min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:8.
Measuring envelop rate with Coomassie Brilliant Blue is 92.3%.
Embodiment 4: a kind of preparation method of vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 0.4 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 6 ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 6 ml steps (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips sodium tripolyphosphate solution 9 ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.2 ml wherein again, continue stirring 16 min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:4.
Measuring envelop rate with Coomassie Brilliant Blue is 34.3%.
Embodiment 5: a kind of preparation method of vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 1 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 3ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 10ml step (2), limit edged stirs, mixing speed is 800r/min, dropwise drip the sodium tripolyphosphate solution 5ml of 2mg/ml more wherein, solution becomes opalescence suspension, continues to stir 10min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
Measuring envelop rate with Coomassie Brilliant Blue is 87.5%.
As can be seen from above-described embodiment, the envelop rate of embodiment 1-3, all more than 90%, higher than embodiment 5, illustrates that adding of compound alcohol can improve envelop rate.
Claims (3)
1. a preparation method for vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 1 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 3ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 10ml step (2), limit edged stirs, mixing speed is 800r/min, dropwise drip the sodium tripolyphosphate solution 5ml of 2mg/ml more wherein, solution becomes opalescence suspension, continues to stir 10min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product.
2. a preparation method for vaccines for fish, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 0.5-1.5 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 2-5 ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 8-12 ml step (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips the sodium tripolyphosphate solution 3-8ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.3-0.6ml wherein again, continue to stir 8-15min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:5-8.
3. the preparation method of a kind of vaccines for fish as claimed in claim 2, comprises the steps:
(1) get sodium acetate 54.6g, add 1mol/L acetum 20ml and dissolve, then be diluted with water to 500ml, the acetic acid/sodium acetate buffer of obtained pH=6.0;
(2) acetic acid/sodium acetate buffer using step (1) obtained is as dissolution with solvents chitosan, and obtained concentration is the chitosan solution of 2mg/ml; In the chitosan solution of 2mg/ml, add Tween80 again, make the mass fraction of Tween80 in chitosan solution be 1 ﹪;
(3) getting 3ml vaccines for fish medicinal liquid and 3ml concentration is that the metabisulfite solution of 50mmol/L mixes, join in the obtained chitosan solution of 10ml step (2), limit edged stirs, mixing speed is 800r/min, more dropwise drips the sodium tripolyphosphate solution 5ml of 2mg/ml wherein, and solution becomes opalescence suspension, dropwise drip compound alcohol 0.5ml wherein again, continue to stir 10min, carry out ultrasonic disperse, obtain dispersion liquid;
(4) dispersion liquid step (3) obtained again with the rotating speed high speed centrifugation 20min of 10000r/min, get centrifugal after precipitation distilled water wash, then to washing after precipitation in add NaCl and distilled water, the mass fraction making NaCl is 0.9%, to obtain final product;
In described compound alcohol, each volume feed is ethanol: 1,2-PD=1:6.
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| CN201510314655.0A CN104922662A (en) | 2015-06-10 | 2015-06-10 | Preparation method of fish vaccine |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101199505A (en) * | 2007-12-20 | 2008-06-18 | 沈阳药科大学 | Verapamil liposome and preparing method thereof |
| CN101773637A (en) * | 2009-01-09 | 2010-07-14 | 苏州知微堂生物科技有限公司 | Broad-spectrum antiviral nano traditional Chinese medicine and preparation method thereof |
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- 2015-06-10 CN CN201510314655.0A patent/CN104922662A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101199505A (en) * | 2007-12-20 | 2008-06-18 | 沈阳药科大学 | Verapamil liposome and preparing method thereof |
| CN101773637A (en) * | 2009-01-09 | 2010-07-14 | 苏州知微堂生物科技有限公司 | Broad-spectrum antiviral nano traditional Chinese medicine and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| L LI ET AL: "Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in black seabream Acanthopagrus schlegelii Bleeker to protect from Vibrio parahaemolyticus", 《JOURNAL OF FISH DISEASES》 * |
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Application publication date: 20150923 |