CN107583047A - A kind of fish oral vaccine adjuvant and its formulation method - Google Patents
A kind of fish oral vaccine adjuvant and its formulation method Download PDFInfo
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Abstract
The invention discloses a kind of fish oral vaccine adjuvant, including following components：Anti- hydrochloric acid in gastric juice component, gastric juice constituents for suppressing, promote intestinal absorption component and polysaccharide parcel component；The anti-hydrochloric acid in gastric juice component is used to vaccine being adjusted to 6.5 7.5 in the local pH value of fish body intestines and stomach；The rush intestinal absorption component is used to assist delivery vaccine to be diffused into mucosa cells, including surfactant, oleic-acid or thickener；The polysaccharide parcel component is non-protein class polysaccharide.The fish oral vaccine adjuvant wraps up vaccine antigen using polysaccharide parcel component, effectively extends the release time of vaccine antigen；The anti-hydrochloric acid in gastric juice component and gastric juice constituents for suppressing of protection atmosphere are formed using vaccine, avoids denaturation of the vaccine antigen in intestines and stomach；It is aided with the absorption and utilization for promoting intestinal absorption component to promote vaccine antigen in fish enteron aisle again, penetrating power of the intestinal mucosa to vaccine antigen is improved, to improve the utilization rate of vaccine antigen and unit effect.
Description
Technical field
The present invention relates to fish bacterin preparation technical field, more particularly to a kind of fish oral vaccine adjuvant and its preparation
Method.
Background technology
Fish mucosal immune system has certain independence.The mucous membrane tissue (gill, skin, enteron aisle) of fish has abundant
The nonspecific defense factor and various immunocytes, it is not only to keep out first of barrier of cause of disease invasion, and with independent
The function of local immune response is completed, important theoretical foundation is provided for mucosa-immune inoculation.As fish systemic immunity is ground
That studies carefully progressively gos deep into, and the research of mucosa-immune is also taken seriously.Research shows that the hindgut of fish is thin in the presence of the epithelium for presenting antigen
Born of the same parents and macrophage, soluble and graininess antigen can be absorbed, therefore fish intestines are carry out mucosa-immune inoculation one
Individual active component.
Although fish are rudimentary vertebrates, but it still has more perfect immune system.Early in the 1930s just
Have been reported that fish have immune response, occurring " fish vaccine ", the time of application one of later antibiotic therewith turns into prevention disease
Major measure, until the seventies due to the chemotherapy resistance to the action of a drug, as bacterial resistance sex chromosome mosaicism shows after antibiotic usage, fish
Vaccine rises once again again, turns into an important branch of vaccinology in recent years.The development of fish vaccine is quite rapid, decades
Come, the species of vaccine is increasing, and mode classification is also varied, be divided into by immunization wayses vaccinate, soaking vaccine and mouth
Take vaccine；It is divided into Bacteria vaccine, subunit vaccine, recombinant vaccine and DNA vaccination etc. by vaccine antigen component type, according to antigen
Preparation method is divided into inactivated vaccine, attenuated vaccine, gene-deleted vaccine etc..The above vaccine is according to developing stage and using skill
Art can be using the reduction of fractions to a common denominator as traditional vaccine and the major class of new generation vaccine two.
Fish vaccine mainly has three kinds because of the own characteristic of fish, its inoculation method：When injection, second, immersion, third,
Orally.These three methods have respective advantage and disadvantage in actual applications：
1) injecting immune dosage is accurate, and induction immune response is all stronger than other several methods, if being aided with adjuvant can more improve
With extension immune response.But injecting immune is wasted time and energy, the stress reaction of fish is also big, to fry and less fish not
It is applicable.
2) the time saving convenience of immersion immunity, it is the effective method of administration for being only second to injection, and is applicable to be difficult to carry out
The fry of injection.But its assimilation effect is affected by various factors, and vaccine is not fully aware of into internal mechanism.Separately
Outside, in the research of DNA vaccination, it is very high to test DNA vaccination content in soak used, and need it is liposome embedded, thus
Cost is higher, using being restricted.
3) oral immunity is easy to operate to fish body not damaged, is not limited by time, place, fish body size.But due to fish
Before vaccine is absorbed, the digestive ferment in stomach decomposes vaccine the enteron aisle of class, cause vaccine degrade or inactivation, its absorption efficiency and
Bioavilability substantially reduces, and reaches the antigen concentration deficiency of target inoculation position, and is difficult to trigger effective immune response anti-
Should.Therefore, the application of oral vaccine needs to provide the guard method of vaccine antigen and effective immunologic adjuvant can be only achieved expection
Immune effect.
So far, very rare applicable oral vaccine occurs in fish production.In aquaculture, Joosten
The micro-capsule vibrio vaccine prepared Deng (1997), by testing the oral immunity of grass carp and trout, it was confirmed that micro-capsule vaccine is certain
Absorption of the fish body to antigen can be improved.Xiao Peng etc. (2007) orally emulsifies immune effect of the vaccine to turbot also by Vibrio anguillarum
Fruit is tested to have obtained same conclusion.These developments for being found to be oral vaccine provide help with exploitation.The heat studied at present
In terms of point is concentrated mainly on particulate, microcapsules, parcel microballoon.
But there is problems with existing oral vaccine：
1) generally existing granulation or the problem of packing technology complexity in terms of the granulation of vaccine and packing technology, and after pelletizing
High-temperature drying procedures easily make mycoprotein be denatured and lose partial immunity activity so that immune effect is poor, protective rate is low；
2) it is excessive to wrap up the number of plies, parcel material is not easy to be digested in intestines and stomach, is unfavorable for vaccine releasing in enteron aisle
Put；
3) wrapping up carrier and existing on the activity of vaccine influences, and what is used such as lapping is that high polymer material or albumen carry
Body, the solvent of high polymer material may use organic solvent, and the presence of organic solvent may destroy the immunogenicity of vaccine, and
Protion carrier easily produces non-specific antibody, influences immune effect.
In summary, screening a kind of non-protein class lapping and one can protect oral vaccine antigen from intestines and stomach
Destroy, accelerate vaccine antigen absorbed intact so as to induce the adjuvant prescription of preferable immune response, formulate a set of simple to operation
Oral vaccine preparation technology it is very necessary.
The content of the invention
For overcome the deficiencies in the prior art, an object of the present invention is that provide one kind preferably can be absorbed by fish
Fish oral vaccine adjuvant.
The second object of the present invention is the formulation method for providing above-mentioned fish oral vaccine.
An object of the present invention adopts the following technical scheme that realization：
A kind of fish oral vaccine adjuvant, including following components：Anti- hydrochloric acid in gastric juice component, gastric juice constituents for suppressing, promote enteron aisle
Absorbent components and polysaccharide parcel component；
The anti-hydrochloric acid in gastric juice component is used to vaccine being adjusted to 6.5-7.5 in the local pH value of intestines and stomach；The rush enteron aisle is inhaled
Component is received to be used to assist delivery vaccine to be diffused into mucosa cells, including surfactant, oleic-acid or thickener；The polysaccharide bag
It is non-protein class polysaccharide to wrap up in component.
Further, fish oral vaccine adjuvant includes following components by weight：The anti-hydrochloric acid in gastric juice component of 3-6 parts, 2-5 parts
Gastric juice constituents for suppressing, 2-5 parts promote intestinal absorption component and 2-6 parts polysaccharide parcel component.
Further, the anti-hydrochloric acid in gastric juice component is Bronsted alkali.
Further, the anti-hydrochloric acid in gastric juice component is sodium acid carbonate, sodium carbonate, sodium citrate, calcium phosphate, calcium carbonate, carbonic acid
It is more than one or both of magnesium, magnesium hydroxide, magnesium phosphate, magnesia and polymer hyaluronic acid with negative electrical charge.
Further, the gastric juice constituents for suppressing is plant-derived pepsin inhibitor, hemoglobin, egg white egg
In vain, more than one or both of proteasome or EDTA.
Further, the rush intestinal absorption component includes saponin, sodium salicylate, sodium lauryl sulfate, oleic acid, sub- oil
One kind in acid, bile salts, olein, carbomer, lecithin, lysolecithin, sorbitan and phospholipid or
It is two or more.
Further, the polysaccharide parcel component includes poly-D-lysine, poly-ornithine, chitosan, alginic acid, collagen
It is more than one or both of albumen, agarose and sulfate fiber.
The second object of the present invention adopts the following technical scheme that realization：
A kind of formulation method of fish oral vaccine, with vaccine antigen, anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing, rush
Intestinal absorption component and polysaccharide parcel component are raw material, are comprised the following steps：
1) anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing and rush intestinal absorption component are mixed, adds edible vegetable oil to stir
Uniformly；
2) material for obtaining step 1) is added slowly in fish feed, mixing；
3) add water to stir to thick polysaccharide parcel component, add vaccine antigen, be mixed；
4) material that step 3) obtains is mixed with the material that step 2) obtains, stirred；
5) material for obtaining step 4) is dried.
Further, the anti-hydrochloric acid in gastric juice component is used to vaccine being adjusted to 6.5-7.5 in the local pH value range of intestines and stomach；
The rush intestinal absorption component is used to assist delivery vaccine to be diffused into mucosa cells, including surfactant, oleic-acid or thickening
Agent；The polysaccharide parcel component is non-protein class polysaccharide.
Further, the types of spawn of the vaccine antigen be Streptococcusagalactiae, Streptococcus iniae, Aeromonas hydrophila,
Vibrio parahaemolytious, tarda, point-like pseudomonad, Pseudomonas fluorescens, columnar fiber bacterium, grass carp hemorrhage fever virus, brocade
In carp herpesviral any one or it is two or more；The state of the vaccine antigen is inactivation antigen, subunit antigen or subtracted
It is more than one or both of virus live vaccine antigen.
Further, step 5), in the abundant dryness in the sun in ventilation.
Compared with prior art, the beneficial effects of the present invention are：
1) fish oral vaccine adjuvant provided by the invention, except being resisted vaccine using the low polysaccharide parcel component of sensitization
Original parcel in the inner, effectively extends the release time of vaccine antigen；Using vaccine formed protection atmosphere layer anti-hydrochloric acid in gastric juice component and
Gastric juice constituents for suppressing, avoid denaturation of the vaccine antigen in intestines and stomach；It is aided with again and promotees intestinal absorption component to promote epidemic disease
Absorption and utilization of the seedling antigen in fish enteron aisle, penetrating power of the intestinal mucosa to vaccine antigen is improved, to improve vaccine antigen
Utilization rate and unit effect；
2) formulation method provided by the invention, by first inhaling anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing and rush enteron aisle
Receive after component carries out homogeneous with edible vegetable oil and mixed again with feed, using polysaccharide wrap up component add water coagulate the soup after by vaccine
Antigen is wrapped up, and vaccine antigen is entered fish digestive tube under multilayer protection from the inside to the outside, is made fish to vaccine antigen
Absorbability more preferably.
Brief description of the drawings
Fig. 1 is the result of the test figure of detection example 1；
Fig. 2 is the double crush syndrome method standard curve of detection example 2；
Fig. 3 is the result of the test figure of detection example 2, wherein, curve is followed successively by high dose group, middle dose group, low from top to bottom
Dosage group, blank vaccine immunity group and absolute blank control group.
Fig. 4 is the result of the test figure of detection example 4.
Embodiment
Below, with reference to accompanying drawing and embodiment, the present invention is described further, it is necessary to which explanation is, not
Under the premise of afoul, new implementation can be formed between various embodiments described below or between each technical characteristic in any combination
Example.
The present invention provides a kind of fish oral vaccine adjuvant, it is characterised in that including following components：Anti- hydrochloric acid in gastric juice component, stomach
Digestive ferment constituents for suppressing, promote intestinal absorption component and polysaccharide parcel component；
The anti-hydrochloric acid in gastric juice component is used to vaccine being adjusted to 6.5-7.5 in the local pH value of intestines and stomach；The rush enteron aisle is inhaled
Component is received to be used to assist delivery vaccine to be diffused into mucosa cells, including surfactant, oleic-acid or thickener；The polysaccharide bag
It is non-protein class polysaccharide to wrap up in component.
Present invention simultaneously provides a kind of formulation method of fish oral vaccine, with vaccine antigen, anti-hydrochloric acid in gastric juice component, peptic digest
Enzyme level component, rush intestinal absorption component and polysaccharide parcel component are raw material, are comprised the following steps：
1) anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing and rush intestinal absorption component are mixed, adds edible vegetable oil to stir
Uniformly；
In the step, by anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing and promote intestinal absorption component using edible vegetable oil
It is uniformly dispersed, to form protection atmosphere；
2) material for obtaining step 1) is added slowly in fish feed, mixing；
In the step, the material and the weight of fish feed that step 1) obtains are than preferably 6-10:100；The proportioning model
In enclosing, the stability of protection atmosphere on the one hand can be effectively ensured, on the other hand can ensure the utilization rate of fish feed, Bu Huizao
Into gastricism；
3) add water to stir to thick polysaccharide parcel component, add vaccine antigen, be mixed；
The step is that the vaccine enwrapped granule of unit is formed using vaccine antigen as core, that is, is formed and concentrate application point；
4) material that step 3) obtains is mixed with the material that step 2) obtains, stirred；
The step will protect atmosphere with concentrating application point to be combined, and obtain not only good to vaccine antigen protectiveness but also there is no harm in
The vaccine feed for hindering feed digestion to absorb；
5) material for obtaining step 4) is dried.
As further scheme, further, the fish oral vaccine adjuvant includes following components by weight：
The anti-hydrochloric acid in gastric juice component of 3-6 parts, 2-5 part gastric juices constituents for suppressing, 2-5 parts promote intestinal absorption component and 2-6 parts polysaccharide parcel component.
In the present invention, anti-hydrochloric acid in gastric juice component is Bronsted alkali, and its main function is that protection atmosphere is formed on vaccine surface,
The intestines and stomach pH value of part is 6.5-7.5 where making vaccine antigen, is inactivated with reducing vaccine antigen to be influenceed by digestive juice；It is excellent
Choosing, the anti-hydrochloric acid in gastric juice component is selected from but is not limited to sodium acid carbonate, sodium carbonate, sodium citrate, calcium phosphate, calcium carbonate, carbonic acid
It is more than one or both of magnesium, magnesium hydroxide, magnesium phosphate, magnesia and polymer hyaluronic acid with negative electrical charge.
In the present invention, the gastric juice constituents for suppressing is gastric juice inhibitor, wherein digestive enzyme inhibitor according to
Structure includes protide and non-protein class.Digestive enzyme inhibitor has selectivity, can select only effectively to suppress certain fungi
The sprouting of sporozoite and the growth of mycelia；And do not remained after fish take, to human body fanout free region.Preferably, the present invention is used
Gastric juice constituents for suppressing can specifically be selected from, but not limited to, plant-derived pepsin inhibitor, hemoglobin, egg white egg
In vain, more than one or both of proteasome or EDTA.
In the present invention, absorption and utilization of the vaccine antigen in fish enteron aisle can be promoted by promoting intestinal absorption component, make vaccine
Antigen causes effective immune response at the middle hindgut position of fish；Preferably, the rush intestinal absorption component is selected from but not limited
In saponin, sodium salicylate, sodium lauryl sulfate, oleic acid, linoleic acid, bile salts, olein, carbomer, lecithin,
It is more than one or both of lysolecithin, sorbitan and phospholipid.
In the present invention, there is slow-release function after the polysaccharide parcel component parcel vaccine antigen, package structure is in fish stomach
It will not be destroyed in portion's sour environment, and gradually be discharged in the alkaline environment of fish enteron aisle, be formed and concentrate application point, beneficial to vaccine
The absorption and utilization of antigen；Polysaccharide parcel component is natural material, will not produce allergic reaction in vivo, and can in vivo by
Gradually degrade, wrap up component using polysaccharide is wrapped in feed granules surface by vaccine, ensures that fish take in vaccine while ingesting,
The utilization rate of vaccine is improved, while does not also interfere with fish and feed is digested and assimilated.Preferably, the polysaccharide parcel component
Selected from but be not limited in poly-D-lysine, poly-ornithine, chitosan, alginic acid, collagen, agarose and sulfate fiber
One or more.
In the present invention, comprising anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing, rush intestinal absorption component and polysaccharide parcel group
Point, mutually acted synergistically between each component, quaternity is indispensable, while the feed granules of carrier are transported as vaccine, both
Fish can be induced to feed, adhered to again beneficial to the parcel of vaccine antigen.After the vaccine feed wrapped enters the alimentary canal stomach of fish
Fish stomach where changing vaccine under the synergy of anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing and polysaccharide parcel component is local
Sour environment, ensure that the integrality of vaccine antigen, polysaccharide component is under the conditions of the pH of enteron aisle after reaching fish body enteron aisle position
Start to discharge vaccine antigen, while most vaccine antigen is passed through enteron aisle table by the effect for promoting intestinal absorption component
Mucocutaneous membrane system, into fish body blood circulation, so as to induce immune response.
Embodiment 1
A kind of vaccines for fish oral vaccine adjuvant, it is made up of following raw materials with mass parts：3 parts of anti-hydrochloric acid in gastric juice components；2 parts of stomaches
Digestive ferment constituents for suppressing；2 parts of rush intestinal absorption components；2 parts of polysaccharide wrap up component.
Its moderate resistance hydrochloric acid in gastric juice component includes 1 part of sodium citrate, 2 parts of Carbon Dioxide calcium；The gastric juice constituents for suppressing includes
1.5 parts of ovalbumin, 0.5 part of EDTA；The rush intestinal absorption component includes 1 part of linoleic acid, 1 part of carbomer；The polysaccharide bag
Component is wrapped up in as 1 part of alginic acid, 1 part of poly-D-lysine.
A kind of formulation method of fish oral immunity adjuvant is：Adjuvant gastric juice constituents for suppressing, promote intestinal absorption component
Need to be sufficiently stirred mixing with anti-2 parts of hydrochloric acid in gastric juice component edible vegetable oil before use, be stirred, make with conventional feed again after mixing
Fully with feed mix.Polysaccharide wraps up component using preceding needing individually to add water stirring and dissolving, need to be slowly added to, be dissolved to homogeneous and glue
Thick shape is preferred, the addition control of polysaccharide parcel component to make its final volume specific concentration be 0.01%, after preparing again with epidemic disease
Seedling antigen mixes parcel and arrives above-mentioned feed surface.
Embodiment 2
A kind of vaccines for fish oral vaccine adjuvant, it is made up of following raw materials with mass parts：3 parts of anti-hydrochloric acid in gastric juice components；3 parts of stomaches
Digestive ferment constituents for suppressing；2 parts of rush intestinal absorption components；3 parts of polysaccharide wrap up component.
Wherein, anti-hydrochloric acid in gastric juice component includes 1 part of sodium citrate, 1 part of Carbon Dioxide calcium, 1 part of anhydrous calcium chloride；The peptic digest
Enzyme level component includes 2 parts of ovalbumin, 1 part of EDTA；The rush intestinal absorption component includes 1 part of sodium salicylate, bile salts
1 part；The polysaccharide parcel component is 2 parts of alginic acid, 1 part of poly-D-lysine.
The formulation method of the fish oral vaccine comprises the following steps：By adjuvant gastric juice constituents for suppressing, promote enteron aisle suction
Mixing need to be sufficiently stirred before with anti-3 parts of hydrochloric acid in gastric juice component edible vegetable oil by receiving component, be carried out again with conventional feed after mixing
Stirring, it is allowed to fully mix with feed.Polysaccharide wraps up component using preceding needing individually to add water stirring and dissolving, need to be slowly added to, dissolving
It is preferred to homogeneous viscous shape, the addition control of polysaccharide parcel component prepares to make its final volume specific concentration be 0.05%
Mix parcel with vaccine antigen again afterwards and arrive above-mentioned feed surface.
Embodiment 3
A kind of adjuvant prescription available for vaccines for fish oral immunity, it is made up of following raw materials with mass parts：4 parts of anti-stomaches
Acid constituents；3 parts of gastric juice constituents for suppressing；3 parts of rush intestinal absorption components；5 parts of polysaccharide wrap up component.
Its moderate resistance hydrochloric acid in gastric juice component includes 3 parts of calcium carbonate, 1 part of sodium acid carbonate；The gastric juice constituents for suppressing includes bean powder 2
Part, 1 part of proteasome；The rush intestinal absorption component includes 1 part of sodium salicylate, 2 parts of bile salts；The polysaccharide wraps up component
For 3 parts of chitosan, 2 parts of poly-ornithine.
A kind of formulation method of fish oral immunity adjuvant is：Adjuvant gastric juice constituents for suppressing, promote intestinal absorption component
Need to be sufficiently stirred mixing with anti-4 parts of hydrochloric acid in gastric juice component edible vegetable oil before use, be stirred, make with conventional feed again after mixing
Fully with feed mix.Polysaccharide wraps up component using preceding needing individually to add water stirring and dissolving, need to be slowly added to, be dissolved to homogeneous and glue
Thick shape is preferred, the addition control of polysaccharide parcel component to make its final volume specific concentration be 0.1%, after preparing again with vaccine
Antigen mixes parcel and arrives above-mentioned feed surface.
Embodiment 4
A kind of adjuvant prescription available for vaccines for fish oral immunity, it is made up of following raw materials with mass parts：5 parts of anti-stomaches
Acid constituents；4 parts of gastric juice constituents for suppressing；5 parts of rush intestinal absorption components；6 parts of polysaccharide wrap up component.
Its moderate resistance hydrochloric acid in gastric juice component includes 2 parts of sodium acid carbonate, 3 parts of polymer hyaluronic acid；The gastric juice constituents for suppressing
Include 2 parts of bean powder, 2 parts of proteasome；The rush intestinal absorption component includes 2 parts of saponin, 3 parts of bile salt；The polysaccharide bag
Component is wrapped up in as 3 parts of chitosan, 3 parts of sulfate fiber.
A kind of formulation method of fish oral immunity adjuvant is：Adjuvant gastric juice constituents for suppressing, promote intestinal absorption component
Need to be sufficiently stirred mixing with anti-4 parts of hydrochloric acid in gastric juice component edible vegetable oil before use, be stirred, make with conventional feed again after mixing
Fully with feed mix.Polysaccharide wraps up component using preceding needing individually to add water stirring and dissolving, need to be slowly added to, be dissolved to homogeneous and glue
Thick shape is preferred, the addition control of polysaccharide parcel component to make its final volume specific concentration be 0.5%, after preparing again with vaccine
Antigen mixes parcel and arrives above-mentioned feed surface.
Embodiment 5
A kind of adjuvant prescription available for vaccines for fish oral immunity, it is made up of following raw materials with mass parts：6 parts of anti-stomaches
Acid constituents；5 parts of gastric juice constituents for suppressing；5 parts of rush intestinal absorption components；6 parts of polysaccharide wrap up component.
Its moderate resistance hydrochloric acid in gastric juice component includes 4 parts of sodium acid carbonate, 2 parts of calcium carbonate；The gastric juice constituents for suppressing includes egg white
1 part of albumen, 4 parts of bean powder；The rush intestinal absorption component includes 3 parts of bile salts, 2 parts of sodium lauryl sulfate；The polysaccharide bag
Component is wrapped up in as 3 parts of alginic acid, 3 parts of agarose.
A kind of formulation method of fish oral immunity adjuvant is：Adjuvant gastric juice constituents for suppressing, promote intestinal absorption component
Need to be sufficiently stirred mixing with anti-5 parts of hydrochloric acid in gastric juice component edible vegetable oil before use, be stirred, make with conventional feed again after mixing
Fully with feed mix.Polysaccharide wraps up component using preceding needing individually to add water stirring and dissolving, need to be slowly added to, be dissolved to homogeneous and glue
Thick shape is preferred, the addition control of polysaccharide parcel component to make its final volume specific concentration be 0.02%, after preparing again with epidemic disease
Seedling antigen mixes parcel and arrives above-mentioned feed surface.
Performance detection and effect assessment
Detect example 1.Tilapia mossambica feeds vaccine feed and contrasted with stomach pH value after blank feed
The purpose of this test example is to compare the pH value whether checking vaccine adjuvant formula can change fish stomach, is protected so as to play
Protect the effect of vaccine antigen.
Experiment every group of 5 tails, is divided into 2 groups, one group of feeding blank is commercially available expanded using body weight 20g or so healthy Tilapia mossambica
Tilapia feed is as blank feed, fish oral vaccine feed that one group of feeding embodiment 1 obtains, and feeding volume is by body weight
3%-5% is calculated, and 0.5h, 1.5h, 2.5h, 5.5h, 6.5h after the completion of feeding, every group takes two Tilapia mossambicas, dissection, determines stomach
The pH value of solution, contrast the difference of two test group stomach pH value.
After blank feed and the feeding of vaccine feed, the pH value of the Tilapia mossambica stomach in different time changes such as Fig. 1 institutes
Show, its pH value changes greatly, 0.5h or so after feeding, and the pH of two groups is 5.5, with the extension of time, the gradual quilt of feed
Digestion, the pH changes in 0.5-2.5h of blank feed group are little, but rapid decline afterwards, when being reduced to 3,6.5h to pH during 5.5h
PH is down to 2, and this explanation is that stomach has emptied, and feed enters in enteron aisle.And vaccine feed group pH in 0.5-2.5h becomes in rising
Gesture, 7 are risen to by 5.5, tend to decline again afterwards, but because the adjustment effect of all adjuvant components in vaccine, pH be not violent
Decline, to pH during 6.5h still 6 or so.
Detect example 2.The suppression experiment of gastric juice constituents for suppressing protection streptococcus subunit vaccine antigenic
The tail of healthy Tilapia mossambica 100 that body weight is 30 ± 5g is chosen in this experiment, is randomly divided into 5 groups, every group of 20 tail fishes, with hammer
Bacterium subunit is vaccine antigen, and using the fish oral vaccine that embodiment 1 obtains as middle dose group, i.e., the amount of oral adjuvant is 3
The anti-hydrochloric acid in gastric juice component of part；2 parts of gastric juice constituents for suppressing；2 parts of rush intestinal absorption components；2 parts of polysaccharide wrap up component；Its moderate resistance hydrochloric acid in gastric juice
Component includes 1 part of sodium citrate, 1 part of Carbon Dioxide calcium；The gastric juice constituents for suppressing includes 1.5 parts of ovalbumin, EDTA
0.5 part；The rush intestinal absorption component includes 1 part of linoleic acid, 1 part of carbomer；Polysaccharide parcel component be 1 part of alginic acid,
1 part of poly-D-lysine, is halved as low dose group using the usage amount of gastric juice constituents for suppressing, with gastric juice constituents for suppressing
Usage amount is double to be used as high dose group.Wherein, streptococcus subunit vaccine antigenic uses the Sip albumen of Prokaryotic expression, purification, tool
Body group see the table below.
The suppression experiment packet transaction of the gastric juice constituents for suppressing of table 1
Wherein, absolute blank control group gavages isometric distilled water, and blank vaccine immunity group gavages to be pressed down without gastric juice
The sip protein antigen of component processed.
After each dosage gastric juice constituents for suppressing and other fixed amount adjuvant components and Sip protein encapsulations, stream is fabricated to
Body bait, each group packet more than are fed, and 1.0mL/ tails, every group of 20 tails gavage, and gavage 1 time, absolute blank group is given
Volume distilled water.1h, 2h, 3h, 4h, 5h, 6h after gavaging respectively, each time point take 3 tail fishes, and Tilapia mossambica is put to death and dissected,
Middle hindgut, cuts off along intestinal tube before clip, scrapes intestinal wall mucus into EP pipes with disposable medicinal sterile wood chip, is often added in pipe
10min is centrifuged in 4 DEG C with 3500rpm/min rotating speed after 0.5mL sterile salines, upper strata intestinal juice is collected, -20 DEG C
Preserve, prepare measure streptococcus Sip protein contents.
The gastric juice constituents for suppressing protection parcel streptococcus in Tilapia mossambica intestines and stomach is detected using sandwich ELISA method
The situation of antigen.This method is immune using one section of polypeptide (amino acid sequence 351-370) of Sip proteantigens sequence (434aa)
New zealand white rabbit and the antibody that produces are as capture antibody, by the use of the antibody of the anti-Sip albumen of mouse as detecting antibody, sheep anti mouse
HRP establishes sandwich ELISA detection method as enzyme mark system：
1) preparation of method antigen
Sip proteantigens：Bacillus coli expression LrrG-Sip albumen, from the Zhujiang River aquatic products research of Chinese aquatic science institute
Institute (Lu Maixin/can be small beautiful), antigen concentration is 0.37mg/mL (20mM Tris HCl, 100mM NaCl, pH=8).
Polypeptide antigen：Shanghai Qiangyao Biotechnology Co., Ltd. synthesize (20mg, purity ＞ 95%), polypeptide respectively with keyhole
Hemocyanin (KLH), ovalbumin (OVA), thyroprotein (TRD) coupling, more peptide coupling antigens are prepared into, through band of dialysing
After (Molecular weight cut of 10000) dialysis 24h, UV detector 280nm identifies that concentration is 0.5mg/
ML (PBS, pH=7.2).
2) preparation of method antibody
Capture Antibody preparation：Experimental animal is healthy new zealand white rabbit 3 (wherein 1 is blank control), body weight
1.4-1.5kg, purchased from southern medical courses in general big experiment animal center, its serum is detected before being immunized as feminine gender.During initial immunity, coupling is taken
The good μ L of polypeptide 200 add sterile saline to be diluted to 1mL, then with Freund's complete adjuvant with 1：1 ratio mixing, use are micro
Emulsifier fully mixes emulsification (emulsifying effectiveness is advisable to drip the 5min indiffusion in cold water).During booster immunization, coupled peptide is dilute
The method of releasing is same as above, so with incomplete Freund's adjuvant with 1：1 ratio is emulsified, and emulsifying effectiveness is same as above.First immunisation, subcutaneous note
Penetrate the polypeptide antigen emulsified and subcutaneously divide 4 site injections, blank control group injection equivalent Freund's adjuvant in nape part (buttocks)
With the emulsion of physiological saline；Second of immune, incomplete Freund's adjuvant emulsification after being spaced 2 weeks；2 weeks third times are spaced again to exempt from
Epidemic disease, dosage, formulation, injecting method are exempted from two.Before immune, three exempt from rear 7d, 14d, 21d in ear venous blood collection 1-2mL
Serum is separated, potency is detected with agar gel diffusion test.When serum antibody titer reaches 212, to the rabbit row arteria carotis after being immunized
Bloodletting, sterile blood sampling, rabbit blood place 2h at room temperature, after its solidification, put 4h in 4 DEG C of refrigerators, are centrifuged with 3000rpm/min
10min, serum is separated, -20 DEG C save backup.
Detect Antibody preparation：Clean level Kunming small white mouse female mice 5, purchased from Nanfang Medical Univ's Experimental Animal Center, 12
Week old, 20 ± 1g of body weight.Breeding observing one week before experiment, every mouse first carry out canthus blood sinus with 100 μ L plastics pipette tips and take blood
(150 μ L) is placed in 1.5mL EP pipes, is statically placed in room temperature 2h, 3000rpm/min centrifugation 10min, is collected serum, -20 DEG C of preservations.
Mouse takes blood second day, is first injected intraperitoneally, and just exempts from the LrrG-Sip albumen (50 μ g/ only) of Freund's complete adjuvant emulsification, and two
The LrrG-Sip albumen (50 μ g/ are only) that incomplete Freund's adjuvant emulsification is immunized in abdominal cavity two is carried out after week, abdominal cavity is carried out again after three weeks
The LrrG-Sip albumen (50 μ g/ are only) of incomplete Freund's adjuvant emulsification is immunized, small white mouse canthus blood sinus takes blood after four weeks, separates blood
Clearly, antibody titer is detected using indirect ELISA.
3) foundation of double-antibodies sandwich ELISA
With pig anti-rabbit immunoglobulin (U.S. KPL, 0.5mg dried frozen aquatic products, deionized water dissolving (1mg/mL), according to 5 μ g/
ML concentration is coated with, and 4 DEG C overnight, are then closed with 1%BSA/PBS, and 37 DEG C are closed 1h, standby after washing 3 times.Press polypeptide rabbit
More anti-(the 30min) → LrrG-Sip standard proteins/in source or Tilapia mossambica intestinal contents supernatant (30min) → mouse to be checked resist
The experiment of more anti-(the 30min) → rabbit-anti mouse secondary antibodies (HRP) (30min) of LrrG-Sip → substrate colour developing (10min) → color development stopping
Program, LrrG-Sip polypeptide antibody optimal reaction concentration is determined using square formation method and LrrG-Sip resists most suitable working concentration, root more
Determined according to These parameters, determine end reaction program.
Finally determine that the more anti-extension rate in rabbit source is 1 according to square formation titration：2000, measuring samples press 1:50、1:
100、1:200、1:400 4 dilutions, the anti-Sip protein antibodies 1 of mouse：5000 dilutions, rabbit-anti mouse HRP 1：4000 dilutions.According to upper
State step detection 2.3.1 in different time points collect enteron aisle mucus sample, while Sip protein standard substances set 1000ng/mL,
Seven concentration gradients of 100ng/mL, 10ng/mL, 1ng/mL, 0.1ng/mL, 0.01ng/mL, 0ng/mL.According to Sip Protein Detections
As a result standard curve is drawn, draws equation, calculates the content of Sip albumen in sample.
Standard curve is drawn according to Sip albumen various concentrations standard items, sees Fig. 2.Draw linear equation Y=0.1661X
(R2=0.9922), Sip protein contents=Y/0.1661 × extension rate (ng/mL) in sample.
The range of linearity is taken to fall the casing slime sample extension rate 1 in standard curve：200 be best sample extension rate.
With this multiple dilute sample, by taking gastric juice constituents for suppressing middle dose group as an example, testing result specifically see the table below.
Sip determining the protein quantity in the different time sampled point Tilapia mossambica enteron aisle of table 2
From the result of table 2, the Sip protein contents measured in the 1-5h after gavaging in enteron aisle gradually rise, and illustrate through stomach
The amount that portion reaches the Sip albumen in enteron aisle also gradually rises, and this explanation Sip albumen is not disappeared in Tilapia mossambica stomach by pepsin
Change and destroy, that is, prove that gastric juice constituents for suppressing can effectively suppress pepsin and play a role.
Different tests group gavages rear Sip determining the protein quantity as shown in figure 3, the gastric juice constituents for suppressing pair of various dose
The inhibitory action of pepsin is different, and blank vaccine immunity group without gastric juice constituents for suppressing because causing Sip albumen in stomach
Major part is degraded, therefore relatively low in the content that intestines measures.Gastric juice constituents for suppressing high dose group is compared with middle dose group
There is no marked difference to the inhibitory action of pepsin.
Detect example 3.Promote the effect test that intestinal absorption component absorbs to promoting antigen
Experiment is divided into two groups, from body weight 50g or so healthy Tilapia mossambica, every group of 10 tails.With the commercially available conventional Rofe of blank
The feeding of fish expanded pellet diet is basic feed, and test group one is to contain 1 × 10 with what embodiment 1 obtained8Cfu/g vaccine antigen and life
The HRP (4.0ng/5ml, 5mL/ tail) of thing element (biotin) mark fish oral vaccine feed is fed, and test group two is
Test control group, feeding are the contrast vaccine feed without rush intestinal absorption component with the difference of embodiment 1, contained in vaccine feed
There is the HRP (4.0ng/5ml, 5mL/ tail) that biotin (biotin) marks.To contain vaccine antigen (1 × 108Cfu/g it is) and biological
The HRP (4.0ng/5mL/ tails) of element mark parcel fluid bait is blank vaccine immunity group；Two groups of fishes use syringe and filling
Taking syringe needle to be gavaged, gavage above two feed respectively, 30min, 60min, 90min, 120min are taken a blood sample after gavaging,
Standing separation serum, the activity of HRP enzymes is detected using ELISA method.ELISA concrete operation steps are as follows：
1) coating of ELISA Plate：Streptavidin is diluted to 5 μ g/mL coated elisa plates with coating buffer, 100 μ L/ holes, 37 DEG C
After being incubated 1h, 4 DEG C of coatings wash coating plate overnight, with 1* cleaning solutions, 250 μ L/ holes, wash 5 times, dry；
2) closing of ELISA Plate：150 μ L/ holes confining liquids, 37 DEG C of closing 60min, wash 250 μ L/ holes with 1* cleaning solutions, wash
Wash 5 times, dry, 4 DEG C save backup；
3) detect：The serum of different time collection is pressed 1:50、1:100、1:200 times of dilutions, 100 μ L/ holes sample-adding, each
Sample repeats 2 holes of detection, is washed after being incubated at room temperature 30min, washs coating plate with 1* cleaning solutions, 250 μ L/ holes, washs 5 times, get rid of
It is dry, substrate TMB colour developings are then added, 100 μ L/ holes, lucifuge colour developing 10min, add terminate liquid terminating reaction, 50 μ L/ holes.
OD450Nm readings.
According to OD450Reading result, the average enzymatic activity of each sample is calculated, from following table result.Test control group
Absorption of the immune group vaccine antigen in enteron aisle is seldom, gavages the OD that rear 30min is measured450Value is only 0.351 (serum 1:50 times
During dilution), the absorption of the immune group vaccine antigen of test group one in enteron aisle is quite notable, gavages the OD that rear 30min is measured450
Value is up to 1.220 (serum 1:During 50 times of dilutions), extension detected value over time gradually reduces, after illustrating that antigen enters in vivo
Progressively it is absorbed and used, two groups of comparing results are visible, and vaccine antigen can be effectively facilitated in intestines by promoting intestinal absorption component
Absorb.
The rush antigen assimilation effect that table 3 promotees intestinal absorption component compares
Detect example 4.Oral vaccine each component is used alone on influence caused by vaccine immunity antibody
The tail of healthy Tilapia mossambica 250 that body weight is 20 ± 5g is chosen in this experiment, is randomly divided into 5 groups, every group of 50 tail fishes, every group of feeding
Hello the vaccine feed containing different oral adjuvant components, feeding volume are calculated by the 3%-5% of fish body weight, are fed daily twice, continuously
Feeding 7 days.Vaccine feed feeds conventional blank feed after terminating.Oral immunity terminates rear 7d, 14d, 21d, 28d, 35d, 42d points
Do not take a blood sample, separate serum, adopt 5 tail fishes every time.Then the antibody production of different tests group is detected with ELISA method.
The fish oral vaccine feed that test group 5 is obtained with embodiment 1 is fed, i.e., comprising 3 parts of anti-hydrochloric acid in gastric juice components；2
Part gastric juice constituents for suppressing；2 parts of rush intestinal absorption components；2 parts of polysaccharide wrap up component.Its moderate resistance hydrochloric acid in gastric juice component includes citric acid
1 part of sodium, 2 parts of Carbon Dioxide calcium；The gastric juice constituents for suppressing includes 1.5 parts of ovalbumin, 0.5 part of EDTA；The rush intestines
Road absorbent components include 1 part of linoleic acid, 1 part of carbomer；The polysaccharide parcel component is 1 part of alginic acid, 1 part of poly-D-lysine.
Coating antigen used in experiment is streptococcus killed vaccine antigen, and vaccine antigen concentration is 1 × 10 in feed8cfu/g。
The preparation difference of other each group oral vaccine feeds is：Test group 1 is only with 3 parts of anti-hydrochloric acid in gastric juice components and vaccine
Antigen is stirred mixing with feed；Test group 2 is only stirred with 2 parts of gastric juice constituents for suppressing and vaccine antigen with feed
Mix；Test group 3 only promotees intestinal absorption component and vaccine antigen with 2 parts and is stirred mixing with feed；Test group 4 is with 3 parts
Anti- hydrochloric acid in gastric juice component, 2 parts of gastric juice constituents for suppressing, 2 parts of rush intestinal absorption components and vaccine antigen and feed are stirred mixing.
Antibody level detection ELISA concrete operation steps are as follows：
1) coating of ELISA Plate and closing：Sip albumen is diluted to 1 μ g/mL coated elisa plates with coating buffer, 100 μ L/ holes,
After 37 DEG C are incubated 1h, 4 DEG C of coatings wash coating plate overnight, with 1* cleaning solutions, 250 μ L/ holes, wash 5 times, dry；Then seal up
The μ L/ holes of liquid 150 are closed, 37 DEG C of closing 60min, 250 μ L/ holes is washed with 1* cleaning solutions, washs 5 times, dry, 4 DEG C save backup.
2) detect：The serum of different time collection is pressed 1:50 times of dilutions, 100 μ L/ holes, are incubated at room temperature 30min, are washed with 1*
Liquid washing coating plate is washed, 250 μ L/ holes, washs 5 dryings.Then 1 is added:The rabbit-anti fish IgM antibody of 1000 times of dilutions, 100 μ
L/ holes, 30min is incubated at room temperature, washs coating plate with 1* cleaning solutions, 250 μ L/ holes, wash 5 times, dry.Add 1:2000 times
The goat-anti rabbit HRP of dilution, 30min is incubated at room temperature, washs coating plate with 1* cleaning solutions, 250 μ L/ holes, wash 5 times, dry.Then
Substrate TMB colour developings are added, 100 μ L/ holes, lucifuge colour developing 10min, add terminate liquid terminating reaction, 50 μ L/ holes.OD450Nm readings.
With test group 1-4 prepare vaccine feed immunity Tilapia mossambica, i.e., respectively with comprise only independent component or individually help
Agent component has the vaccine feed feeding Tilapia mossambica after seedling of missing, and each test group antibody level has notable difference.Can by Fig. 4 results
See, test group 1-3 produces without potent antibodies substantially, illustrates to lack anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing, promotees intestinal absorption
The vaccine antigen of component is easy to be destroyed by gastrointestinal tract environment and can not produced effective immune anti-after entering fish body intestines and stomach
Should.Although test group 4 does not lack above-mentioned several components, part protection vaccine antigen can be played a part of, it is more due to lacking
The parcel of sugared composition, it is extremely limited into the amount of antigen in fish enteron aisle, though therefore immune antiboidy have a generation, it is horizontal relatively low, and
Duration is short.Test group 5 includes all adjuvant components and vaccine antigen of oral vaccine, and vaccine antigen can be in each component
The lower middle hindgut for smoothly entering Tilapia mossambica of synergy, so as to cause effectively immune response, produces corresponding immune antiboidy.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It is appreciated that other embodiment.
Claims (10)
1. a kind of fish oral vaccine adjuvant, it is characterised in that including following components：Anti- hydrochloric acid in gastric juice component, gastric juice suppression group
Divide, promote intestinal absorption component and polysaccharide parcel component；
The anti-hydrochloric acid in gastric juice component is used to vaccine being adjusted to 6.5-7.5 in the local pH value of intestines and stomach；The rush intestinal absorption group
Divide and be used to assist delivery vaccine to be diffused into mucosa cells, including surfactant, oleic-acid or thickener；The polysaccharide parcel group
It is divided into non-protein class polysaccharide.
2. fish oral vaccine adjuvant as claimed in claim 1, it is characterised in that the anti-hydrochloric acid in gastric juice component is Bronsted
Alkali.
3. fish oral vaccine adjuvant as claimed in claim 1, it is characterised in that the anti-hydrochloric acid in gastric juice component be sodium acid carbonate,
Sodium carbonate, sodium citrate, calcium phosphate, calcium carbonate, magnesium carbonate, magnesium hydroxide, magnesium phosphate, magnesia and the poly with negative electrical charge
It is more than one or both of body hyaluronic acid.
4. fish oral vaccine adjuvant as claimed in claim 1, it is characterised in that the gastric juice constituents for suppressing is plant
It is more than one or both of source property pepsin inhibitor, hemoglobin, ovalbumin, proteasome or EDTA.
5. fish oral vaccine adjuvant as claimed in claim 1, it is characterised in that the rush intestinal absorption component is gleditsia sinensis
Glycosides, sodium salicylate, sodium lauryl sulfate, oleic acid, linoleic acid, bile salts, olein, carbomer, lecithin, haemolysis ovum
It is more than one or both of phosphatide, sorbitan and phospholipid.
6. fish oral vaccine adjuvant as claimed in claim 1, it is characterised in that the polysaccharide parcel component is that poly relies ammonia
It is more than one or both of acid, poly-ornithine, chitosan, alginic acid, collagen, agarose and sulfate fiber.
7. the fish oral vaccine adjuvant as described in claim any one of 1-6, it is characterised in that fish oral vaccine adjuvant bag
Include following components by weight：The anti-hydrochloric acid in gastric juice component of 3-6 parts, 2-5 part gastric juices constituents for suppressing, 2-5 parts promote intestinal absorption group
Divide and 2-6 parts polysaccharide wraps up component.
A kind of 8. formulation method of fish oral vaccine, with vaccine antigen, anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing, rush intestines
Road absorbent components and polysaccharide parcel component are raw material, are comprised the following steps：
1) anti-hydrochloric acid in gastric juice component, gastric juice constituents for suppressing and rush intestinal absorption component are mixed, adds edible vegetable oil to stir；
2) material for obtaining step 1) is added slowly in fish feed, mixing；
3) add water to stir to thick polysaccharide parcel component, add vaccine antigen, be mixed；
4) material that step 3) obtains is mixed with the material that step 2) obtains, stirred；
5) material for obtaining step 4) is dried.
9. formulation method as claimed in claim 8, it is characterised in that the anti-hydrochloric acid in gastric juice component is used for vaccine in intestines and stomach office
The pH value in portion is adjusted to 6.5-7.5；The rush intestinal absorption component is used to assist delivery vaccine to be diffused into mucosa cells, including table
Face activating agent, oleic-acid or thickener；The polysaccharide parcel component is non-protein class polysaccharide.
10. formulation method as claimed in claim 8, it is characterised in that the types of spawn of the vaccine antigen is agalasisa hammer
Bacterium, Streptococcus iniae, Aeromonas hydrophila, vibrio parahaemolytious, tarda, point-like pseudomonad, Pseudomonas fluorescens, post
In shape fiber bacterium, grass carp hemorrhage fever virus, Koi herpesvirus any one or it is two or more；The state of the vaccine antigen
More than one or both of inactivation antigen, subunit antigen or attenuated live vaccine antigen.
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| CN111273007A (en) * | 2020-03-13 | 2020-06-12 | 内江师范学院 | Kit and detection method for rapidly detecting fish Edwardsiella ictaluri |
| CN114053399A (en) * | 2021-11-18 | 2022-02-18 | 广东渔跃生物技术有限公司 | Tilapia mossambica streptococcus oral vaccine and preparation method thereof |
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| CN114053399A (en) * | 2021-11-18 | 2022-02-18 | 广东渔跃生物技术有限公司 | Tilapia mossambica streptococcus oral vaccine and preparation method thereof |
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