CN106620683A - Bicombinant oral microsphere vaccine used for fish and purpose thereof - Google Patents
Bicombinant oral microsphere vaccine used for fish and purpose thereof Download PDFInfo
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Abstract
The invention discloses a bicombinant oral microsphere vaccine used for fish, which comprises edwardsiella ictaluri microsphere vaccine and yersinia ruckeri microsphere vaccine The bicombinant oral microsphere vaccine can be used for effectively protecting infection of edwardsiella ictaluri and yersinia ruckeri for ictalurus punctatus, prevent edwardsiella ictaluri and yersinia ruckeri disease, and has the advantages of convenient usage and good application prospect.
Description
Technical field
The present invention relates to a kind of fishing bigeminy oral microsphere vaccine and application thereof.
Background technology
With the high speed development of culture fishery, intensive aquaculture model causes the frequent sudden and violent of various diseases
Send out, with the globalization of aquatic products trade, the prevalence of disease also becomes to be more prone to propagating.It is same with this
When production it is upper because drug dependence makes antibacterial produce drug resistance, bring drug residue, food safety, environment dirt
The problems such as dye.In addition sick fish ingests to decline even not take food and causes Drug therapy extremely difficult.Therefore vaccine
Use become the important means of disease prevention.At present majority aquaculture vaccines carry out immunity by injection,
There is workload big, easily fish body is caused stress the shortcomings of.Oral vaccine is exempted from as Fish are most promising
Epidemic disease mode, not only immune operation is convenient, and stress be little, and the use of bigeminy or multi-joint oral vaccine can also be same
When prevent the disease that causes of multiple pathogens.
Channel-catfish tardas disease and Y.rucker disease be Fen Bie You Channel-catfish tardas
E.ictaluri and Y.rucker Y.ruckeri cause disease, are two kinds of weights of Ietalurus Punetaus
Bacterial disease is wanted, the cultivation to Ietalurus Punetaus generates large effect, prevents it bigger
Breaking out for scope is particularly important.
At present with regard to Ban point Cha Wei Channel-catfish Channel-catfish tardas disease and Y.rucker disease bigeminy oral vaccine
Research have not been reported.
The content of the invention
In order to solve the above problems, the invention provides a kind of Ban point Cha Wei Channel-catfish Channel-catfish tardas disease and Shandong
Family name's yersinia disease bigeminy oral vaccine and its production and use.
Fishing of the present invention bigeminy oral microsphere vaccine, it wraps and includes Channel-catfish tardas microspheres vaccine and Lu Shi
Yersinia microspheres vaccine, wherein ， Channel-catfish tardas microspheres vaccine, Y.rucker microsphere
Vaccine is prepared as follows：
(1) Qu Channel-catfish tardas or Y.rucker, make bacterium solution, inactivation, obtain inactivation epidemic disease
Seedling；
(2) add sodium alginate to final concentration of 3.5% (w/v) in inactivated vaccine, mix, obtain mixed
Solution is closed, mixed solution is added drop-wise in liquid paraffin, wherein, the volume of mixed solution and liquid paraffin
For 2.4：7.6, emulsifying obtains emulsion；
(3) emulsion is added drop-wise to into the CaCl that equal-volume concentration is 7% (w/v)2In solution, add dense
Spend the CaCl for 0.05mol/L2Solution, stands, and removes upper oil phase and water, and microsphere is collected in centrifugation,
Cleaning, you can.
Preferably, the volume of Suo Shu Channel-catfish tardas microspheres vaccines and Y.rucker microspheres vaccine
Than for 1：1.
Preferably, in step (1), the bacterium solution Zhong ， Channel-catfish tardas or Y.rucker
Concentration be 4 × 109cfu/mL。
Preferably, in step (1), the mode of the inactivation is：Formalin is added to final concentration of
0.3% (w/v), 120r/min, inactivate 24h under the conditions of 28 DEG C.
Preferably, in step (2), emulsifying agent is added with the liquid paraffin, emulsifying agent is Span-80,
The concentration of emulsifying agent is 5.4% (w/v).
Preferably, in step (2), the mode of emulsifying is：2000r/min stirs 13min.
Preferably, in step (3), the time of standing is 1h.
Preferably, in step (3), the rotating speed of centrifugation is 8000r/min, and the time of centrifugation is 5min.
Preferably, in step (3), the mode of cleaning is ethanol purge 3 times, then deionized water cleaning
3-4 time.
Present invention also offers aforesaid vaccine is preparing pre- anti-Channel-catfish tardas disease and/or Lu Shi yersinia genus
Purposes in Salmonella disease.
The present invention is with Ietalurus Punetaus Yuan Channel-catfish tardas and Y.rucker inactivated vaccine as antibacterial
Antigen, it is orally micro- that natural polymerses sodium alginate prepares sodium alginate bigeminy for potential carrier for oral vaccine delivery
Ball vaccine, and vaccine immunity protected effect is studied, from salivary lysozyme, antibody titer,
The protection effect of the aspect overall merit bigeminy vaccine such as expression of relative immunity protective rate and gene involved in immunity
Really, it is that just, safely effectively pre- antiplaque point pitches tail Channel-catfish Channel-catfish tardas disease and Lu Shi for production top
A kind of new aquaculture vaccines of Ademilson Salmonella disease exploitation.
Bigeminy microspheres vaccine of the present invention can be exempted from by Channel-catfish tardas and Lu Shi with effective protection Ietalurus Punetaus
The infection of yersinia, Yu Fang Channel-catfish tardas disease and Y.rucker disease, it is easy to use,
Application prospect is good.
Obviously, the above of the invention, according to the ordinary technical knowledge and customary means of this area,
Under the premise of without departing from above-mentioned basic fundamental thought of the invention, can also make other various ways modification,
Replace or change.
By the following examples the specific embodiment of form, remakes further to the above of the present invention
Detailed description.But this scope for being interpreted as above-mentioned theme of the invention should not be only limitted to Examples below.
All technologies realized based on the above of the present invention belong to the scope of the present invention.
Description of the drawings
The sodium alginate micro ball form (1000 ×) prepared after Fig. 1 process optimizations.a：Channel-catfish tardas
Microspheres vaccine；b：Y.rucker microspheres vaccine.
Fig. 2 sodium alginate micro ball particle diameter distributions.a：Channel-catfish tarda microspheres vaccines；b：Shandong
Family name's yersinia microspheres vaccine.
Release profiles are simulated under Fig. 3 different conditions outside sodium alginate microsphere.a：Channel-catfish tardas are micro-
Ball vaccine；b：Y.rucker microspheres vaccine.
Fig. 4 Ietalurus Punetaus the Total albumen content changes.Note：By each experimental group and matched group total protein
Content carries out significance difference analysis, and " * " represents significant difference (p<0.05), " * * " represents that difference extremely shows
Write (p<0.01).
Fig. 5 Ietalurus Punetaus serum total number born vigour changes.Note：By each experimental group with it is right
Significance difference analysis are carried out according to a group total number born vigor, " * " represents significant difference
(p<0.05), " * * " represents difference extremely significantly (p<0.01).
Fig. 6 Ietalurus Punetaus serum lysozyme level changes.Note：By each experimental group and matched group lysozyme
Vigor carries out significance difference analysis, and " * " represents significant difference (p<0.05), " * * " represents that difference extremely shows
Write (p<0.01).
Specific embodiment
The preparation of the bigeminy oral vaccine of the present invention of embodiment 1
1 antibacterial rejuvenation
Channel-catfish tarda E.ictaluri bacterium and Y.rucker Y.ruckeri are preserved by -40 DEG C
Bacterium is rule respectively in BHI, LB solid medium, 28 DEG C of culture 24h and 28 DEG C of Y.ruckeri of E.ictaluri
After culture 24h, dyeing microscopic examination is tested pure.Take two kinds of bacterium difference lumbar injection counteracting toxic substances health Ietalurus Punetaus.Treat
Fish after death, from the aseptic separation pathogen of fish body, cultivate respectively, then the inoculation of picking single bacterium colony by inoculation flat board
In broth bouillon, enter performing PCR identification, then counteracting toxic substances Ietalurus Punetaus again, continuous rejuvenation three times.
It is prepared by 2 inactivated vaccines
The pure rear adjustment bacterial concentration of E.ictaluri experiences that will be enlarged by cultivating is 4 × 109Cfu/mL, so
Afterwards with the appropriate bacterium solution of conical flask subpackage and final concentration of 0.3% formalin is added in shaking table 120r/min,
28 DEG C of inactivation 24h.
The pure rear adjustment bacterial concentration of Y.ruckeri experiences that will be enlarged by cultivating is 4 × 109Cfu/mL, then
With the appropriate bacterium solution of conical flask subpackage and final concentration of 0.3% formalin is added in shaking table 120r/min,
28 DEG C of inactivation 24h.
Completely antibacterial physiological saline solution will be inactivated to wash 3 times, 4 DEG C of refrigerator is placed in and is saved backup.
3 sodium alginate micro ball vaccine preparation technology flow processs
Take appropriate sodium alginate to be sufficiently mixed with E.ictaluri inactivated vaccines, make alginic acid in mixed solution
Na concn is 3.5%, by mixed solution be slowly dropped to certain quantity of fluid paraffin (Span-80 containing emulsifying agent,
Emulsifier concentration is in 5.4%), to make the mixed solution be with the volume ratio of liquid paraffin in liquid paraffin
2.4:7.6,2000r/min stirring 13min, after fully emulsified, above-mentioned emulsion are added drop-wise to isopyknic
7% (M/V) CaCl2Calcification in solution forms microsphere, then by the CaCl of 0.05M2Aqueous solution is slowly poured into above-mentioned molten
Stand in liquid and remove after 1h upper oil phase and water, 8000r/min centrifugation 5min collect microsphere, clear with ethanol
Wash 3 times, then deionized water is cleaned 3-4 time, collects microsphere.
4. microspheres vaccine configuration of surface and granularmetric analyses
Take a small amount of microspheres solution on microscope slide, after covered with micro imaging system observation form simultaneously
Take pictures.600 microspheres are therefrom randomly selected, is measured using image analysis software Image-pro-pius 6.0
Size, SPSS softwares carry out statistical analysiss to particle diameter distribution situation.Particle diameter distribution represents with span,
Computing formula：
Span=(D90-D10)/D50
D in formula10, D50, D90Refer to the corresponding particle diameter in 10%, 50%, 90% place in particle-size accumulation scattergram respectively.
5. encapsulation efficiency is determined
The microsphere collected is resuspended, the centrifugation of 3mL microsphere suspensions is taken, the microsphere for obtaining is added to into appropriate 55
120r/min incubator overnights in mM sodium citrate solutions, fully dissolving.Solution 10000r/min is centrifuged again
10min, determines antibacterial OD values, according to formula with ultraviolet spectrophotometer by collected antibacterial at 600nm
Computational envelope efficiency：
Encapsulation efficiency (%)=(antibacterial OD values in the antibacterial OD values/theory microsphere in microsphere) × 100%
6. vaccine safety inspection
Take a small amount of microspheres vaccine and coat BHI flat boards, 48h is cultivated under the conditions of 28 DEG C, observation whether there is antibacterial
Growth.
40 tails health Ietalurus Punetaus are divided into into 4 groups, per group of 10 tails.Respectively by the microspheres vaccine for preparing with
1×109The concentration gavage of cfu/ tails, lumbar injection inoculation, arrange aseptic PBS gavages, lumbar injection control
Group, the incidence and survival rate of inoculation fish in observation statistics 14d.
7. in-vitro simulated release test
In order to detect stability of the microspheres vaccine under gastrointestinal conditions, with reference to Tian etc.[97]And Rodrigues
Deng[66]Method, configuration simulated gastric fluid (PBS solution of pH 2.0) and the simulated intestinal fluid (Tris-HCl of pH 9.0
Solution) detection microsphere stability.
With reference to Leal etc.[101]Method, take respectively the microspheres vaccine for preparing in right amount be scattered in 5ml simulation stomach
Liquid, simulated intestinal fluid and first transferring in simulated intestinal fluid after reaction 6h in simulated intestinal fluid gastric juice are continued instead
Should, react at 22 DEG C, take supernatant 500r/min respectively at 1,3,6,9,12,15,18,21 and 24h
Centrifugation 1min ultraviolet spectrophotometers determine the OD values of antibacterial at 600nm, and replication is averaged for 3 times
Value, according to formula the release rate of microspheres vaccine is calculated, and draws release profiles.
Release rate (%)=(antibacterial OD values in the antibacterial OD values/microsphere in supernatant) × 100%
8. prepared by vaccine and vaccine feedstuff
It is prepared by bivalent inactivated vaccine：Concentration is 4 × 109The E.ictaluri and Y.ruckeri inactivation of cfu/mL
Vaccine presses 1:1 ratio (volume ratio) mix homogeneously makes bivalent inactivated vaccine, and 4 DEG C save backup.
It is prepared by bigeminy sodium alginate oral vaccine：Prepare E.ictaluri microspheres vaccines, Y.ruckeri micro-
Ball vaccine and sodium alginate sky microsphere, E.ictaluri, Y.ruckeri microspheres vaccine for preparing is adjusted
Whole antigen concentration 1 × 109Cfu/mL, by 1:1 ratio mix homogeneously makes bigeminy oral vaccine, and 4 DEG C of preservations are standby
With.
By the vaccine for preparing and empty microsphere respectively by the feedstuff after required immunizing dose (table 1) and crushing
Vaccine feedstuff is made in mixing reshaping, drying, is fed according to the 2% of body weight.
The immune programme for children of table 1
Note：Sodium alginate sky microsphere group immunizing dose M be equal to I group in microspheres vaccine quality；Experiment water temperature (22 ± 2) DEG C
9. serum collection and process
Respectively 6 are taken at random from each immune set of samples within the 1st, 2,3,4,5,6,7,8 weeks after immunity inoculation
Tail fish, is anaesthetized, tail vein blood with MS222 solution (1mg/L), per tail 0.2ml.Blood will be gathered
It is stored at room temperature 1h and 4h is kept in 4 DEG C of refrigerators, 4000r/min centrifugations l0min, receives under the conditions of 4 DEG C
Collection upper serum, -80 DEG C save backup.
10 immune index detections
10.1 the Total albumen content is detected
Using protein quantification (Coomassie Brilliant Blue) testing cassete (building up Bioengineering Research Institute purchased from Nanjing)
Determine the content of total protein in each group serum.
By the Coomassie brilliant blue stock solution in test kit and distilled water 1:4 are configured to working solution, now with existing
With.3 aseptic 4mL centrifuge tubes are taken, blank tube, standard pipe is respectively labeled as and is determined pipe, added successively
Enter 50 μ L distilled water, 50 μ L protein standard liquid (0.563g/L) and 50 μ L test serums, then often pipe adds
Enter 3mL biuret reagents, mix, stand 10min, wavelength 595nm, 1cm optical paths, distilled water is adjusted
Zero, survey each pipe OD values.Total protein content is calculated by following equation：
The Total albumen content (mg/mL)=(determining pipe OD values-blank tube OD values)/(standard pipe OD
Value-blank tube OD values) × protein standard substance concentration (mg/mL).
10.2 serum total number born (T-SOD) Activity determinations
Each group serum T-SOD is determined using T-SOD test kits (building up Bioengineering Research Institute purchased from Nanjing)
Enzyme activity.
2 centrifuge tubes are taken, control tube is respectively labeled as and is determined pipe, sequentially add a certain amount of distilled water
And test serum, then be separately added into after reagent 1,2,3,4 to every pipe, fully mix, 37 DEG C of water-baths
40min.2mL developers are often added after water-bath in pipe, is mixed, room temperature places 10min, and distilled water is adjusted
Zero, OD values are determined at wavelength 550nm.T-SOD vigor is calculated by following equation：
T-SOD vigor (U/mL)=(control tube OD value-determine pipe OD values)/(control tube OD value)
Extension rate before the extension rate × test sample of/50% × reaction system
10.3 serum lysozyme level is detected
Srum lysozyme activity determination, is measured using the microsphere mycopowder of lyophilizing by turbidity.First will
Test serum 10 times of dilutions of citrate buffer (pH5.5,0.02M), take 20 μ L and are added to U
In the orifice plate of type bottom 96, each sample has 3 repetitions.Then concentration is added to be 0.2mg/ml per hole
The μ L of microsphere mycopowder 200, determine initial absorbance under the absorbance of OD values 450nm rapidly, so
Afterwards the testing sample containing substrate is positioned over into 37 DEG C of calorstat incubation 1h, in identical 450nm absorbance
It is lower to determine final OD values.And using identical method, standard curve is done with Hen egg-white lysozyme, survey
Definite value should be within standard curve effective range.Bacteriolyze level represents with U/ml, 0.001/min tables
Show unit U.
10.4 micro-agglutinations experiment detection antibody level of serum
10.4.1 it is prepared by antigen
Prepare with 2 inactivated vaccines, by E.ictaluri, Y.ruckeri inactivation, adjustment bacterial concentration is
3×108cfu/mL。
10.4.2 the determination of optimum reaction condition
The selection of optimal antigen concentration：Antigen is pressed into 1 with PBS:2,1:4,1:8 doubling dilutions, with blood
Microagglutination test is carried out clearly.
The selection of optimal reaction temperature：It is anti-under the conditions of micro-agglutination plate is respectively placed in into 37 DEG C and 28 DEG C
Should, overnight, impact of the observing response temperature to reacting.
10.4.3 experimental procedure and criterion
50 μ L PBS are added in 96 orifice plate 1-12 holes, then 50 μ L blood to be measured is added in the 1st hole
Clearly, blow and beat 8-10 time, take 50 μ L and add the 2nd hole, successively doubling dilution, to the 10th hole, discards
50 μ L, increase serum, as antigen control, does not then add the antigen of optium concentration to the hole of end 2 in every hole
50 μ L, while setting the positive, negative control, mix and are reacted under optimal reaction temperature, overnight, see
Examine record result.
Result judgement standard：
*：Supernatant is clarified, and hole bottom center has a large amount of densification agglutinate structure aggregations, and edge has a large amount of solidifying
Collection piece；
+++：Supernatant is relatively clarified, and hole bottom center has more agglutinate structure, and there is more coagulation at edge
Piece；
++：Bottom hole portion has to be precipitated in flakes, different from antigen control hole, and there is a small amount of coagulation piece at edge；
+：Bottom hole portion agglutinate structure spot distribution, edge micro-agglutination vestige；
-：Bottom hole portion is similar to antigen control hole without obvious agglutinate structure, and edge is without coagulation vestige；
Agglutination titer with the highest antibody extension rate of appearance " ++ " as antibody
10.5 challenge test and immune protective effect are determined
10.5.1 measure of the Channel-catfish tardas to Ietalurus Punetaus median lethal dose(LD 50) LD50
With physiological saline solution doubling dilution E.ictaluri bacterial suspensions, by test Ietalurus Punetaus point
For 5 groups, per group of 10 tails.Per corresponding dilution of bacteria 0.2ml of tail lumbar injection.7-14 is observed after counteracting toxic substances
My god, record test fish falls ill and death condition.LD50 is calculated with karber's method.
10.5.2 measure of the Y.rucker to Ietalurus Punetaus median lethal dose(LD 50) LD50
In 2.6.1 methods Y.ruckeri is calculated to Ietalurus Punetaus median lethal dose(LD 50) LD50.
10.5.3 challenge test
The 29th day after immunity, challenge test is carried out, with 50 × LD50 concentration counteracting toxic substances, lumbar injection
0.2mL/ tails.Ith, E.ictaluri counteracting toxic substances are used for II group；IIIth, Y.ruckeri counteracting toxic substances are used for IV group；Ⅴ、
VI group is distinguished counteracting toxic substances with two kinds of bacterium, records after counteracting toxic substances the morbidity of each group fish and death condition in 14d, is separately taken
The liver of dying fish body, kidney carry out connecing bacterium culture, determine Species of Pathogens.And calculate relative protection ratio
(Relative Percent Survival, RPS), is calculated by following equation：
RPS (%)=[(matched group mortality rate-immune group mortality rate)/matched group mortality rate] × 100%
10.6 challenge test and immune protective effect are determined
The 29th day after immunity, challenge test is carried out, with 50 × LD50 concentration counteracting toxic substances, lumbar injection
0.2mL/ tails.Per group is used respectively E.ictaluri, Y.ruckeri counteracting toxic substances, each in 14d after record counteracting toxic substances
The morbidity of group fish and death condition, separately taking the liver of dying fish body, kidney carries out connecing bacterium culture, determines pathogen
Species.And relative protection ratio (Relative Percent Survival, RPS) is calculated, by following public affairs
Formula is calculated：
RPS (%)=[(matched group mortality rate-immune group mortality rate)/matched group mortality rate] × 100%
3 results
3.1 microspheres vaccine configuration of surface and granularmetric analyses
Sodium alginate micro ball vaccine balling-up of the present invention is good (Fig. 1), even particle size distribution (Fig. 2) ， Channel-catfish
Tarda microspheres vaccine particle diameter (8.88 ± 1.26) μm, span 0.38.Y.rucker
Microspheres vaccine particle diameter (8.76 ± 1.73) μm, span 0.47.
3.2 microspheres vaccine encapsulation efficiencies
Parallel verified experimental result stability ， Channel-catfish tarda microspheres vaccines are averagely encapsulated under same process
Efficiency is 96.37%.
Y.rucker sodium alginate micro ball vaccine, its average encapsulation efficiency is 94.51%.
3.3 microspheres vaccine safety examinations
Take Shi Liang Channel-catfish tardas, Y.rucker inactivated vaccine and microspheres vaccine respectively to coat
BHI flat boards, respectively set 3 repetitions, 48h observations are cultivated under the conditions of 28 DEG C, without bacterial growth.
By prepare 2 kinds of microspheres vaccines with 1 × 109Cfu/ tail gavages, the healthy speckle of lumbar injection inoculation
Cha Wei Channel-catfish, arrange inoculation fish in aseptic PBS lumbar injections matched group, 14d and do not show any abnormalities, equal nothing
Morbidity or it is dead (table 2,3), it was demonstrated that vaccine safety is good.
The Channel-catfish tarda microspheres vaccine safety experiment results of table 2
The Y.rucker microspheres vaccine safety experiment result of table 3
The in-vitro simulated release of 3.4 microspheres vaccines
In simulated gastric fluid (pH 2.0), the extremely low ， Channel-catfish tarda microspheres vaccines of release rate of microsphere
10.7% and 12.9% is respectively with Y.rucker microspheres vaccine 24h preparations；When microsphere exists
When in simulated intestinal fluid (pH 9.0), the phenomenon of burst release (23% and 26%) of starting is occurred in that, then continued
Quickly release, 24h preparations are respectively 87% and 89% (Fig. 3).
In order to more accurately simulating vaccine it is oral after release behavior in fish gastrointestinal tract environment, microsphere is first
First 6h is processed in simulated gastric fluid, transfer in simulated intestinal fluid and continue with, Channel-catfish tardas are micro-
Ball vaccine 24h preparations 89%.Through the process of simulated gastric fluid, when microsphere is transferred to simulated intestinal fluid
Afterwards, faster rate of release, 18h preparations 83.4% are shown, and is processed in simulated intestinal fluid
Microsphere 18h preparations be 80%.
Y.rucker microspheres vaccine is processed in simulated gastric fluid and is transferred in simulated intestinal fluid after 6h,
24h preparations 92%, are transferred to 18h preparations 87.9% in simulated intestinal fluid, far above only existing
The microsphere 18h preparations 70% processed in simulated intestinal fluid.
3.5 the Total albumen content are detected
Fig. 4 is shown in each group Ietalurus Punetaus the Total albumen content change after immunity.The 1st week after immunity,
Except bivalent inactivated vaccine group, each test group the Total albumen content significantly raises (p<0.01), in reality
That what is tested reached peak value in the 2nd week each experimental group the Total albumen content week, and bigeminy microspheres vaccine is basic, normal, high
Still it is significantly higher than matched group (p within dosage group to the 7th week<0.05)；Bivalent inactivated vaccine is only the 2nd, 3,
5 weeks (ps extremely notable with matched group difference<0.01), remaining cycle difference is notable (p ＞ 0.05)；It is empty
Microsphere group total serum protein experiment be significantly higher than matched group within the 1st, 2 weeks, drop quickly to afterwards with it is right
According to group difference not significant level (p ＞ 0.05).The total egg of bigeminy microspheres vaccine group serum during whole experiment
Bai Hanliang is generally higher than other each groups；During whole experiment, matched group the Total albumen content is relatively steady
It is scheduled on 50-55mg/mL.
3.6 serum total number born Activity determinations
After to the immunity of each group Ietalurus Punetaus, serum total number born vigour changes situation is shown in figure
5.Started each group T-SOD vigor from the 1st week in rising trend, and the 1st week enzyme activity of each experimental group is equal
It is significantly higher than matched group (p<0.01).Peak value occurs in the 2nd week, begins to decline afterwards, bigeminy microsphere
Vaccine low dose group to the 7th week serum T-SOD vigor is still significantly higher than matched group (p<0.05), and
Whole experiment 2-7 is all, and low dose group serum T-SOD vigor is above other experimental grouies and control
Group,；It is significantly higher than matched group (p within the middle and high dosage group of bigeminy microspheres vaccine to the 6th week<0.01).Two
Connection inactivated vaccine caused rapidly T-SOD vigor to raise after immunity, but decrease speed is fast, to the 5th week
When with matched group difference not significantly (p ＞ 0.05)；Empty microsphere group serum T-SOD during whole test
Vigor is below vaccine group, is only significantly higher than matched group (p at the 1st, 2,3 weeks<0.01), afterwards
It is reduced to matched group difference not significantly (p ＞ 0.05).During whole test, matched group serum always surpasses
Superoxide dismutase vigor is relatively stable in 118-124U/mL.
3.7 serum lysozyme content detection
Fig. 6 is shown in each group Ietalurus Punetaus serum lysozyme level change after immunity.From the beginning of the 1st week, respectively
There is significance and raises (P in test group serum lysozyme level<0.01).Test the 1st, 2 weeks bigeminy
Microspheres vaccine high dose group and bivalent inactivated vaccine serum lysozyme level are significantly higher than other groups (P<
0.01) after, but reaching peak value at the 2nd week, bigeminy microspheres vaccine is low, middle dose group antalzyme activity shows
Write higher than other experimental grouies and matched group (P<0.01), to the 7th week, bigeminy microspheres vaccine is low, middle dose
Amount group antalzyme activity still with matched group difference extremely significantly (P<0.01)；Bivalent inactivated vaccine group lysozyme
Also begin to raise after vigor immunity, but relative microspheres vaccine group, the reduction of inactivated vaccine group antalzyme activity is more
Hurry up；Empty microsphere group antalzyme activity is below other experimental grouies during testing, and high-caliber enzyme activity is insisted on
The continuous time is also shorter, to when the 5th week with matched group difference not significantly (p ＞ 0.05).In whole experimental period
Between, matched group serum lysozyme level is relatively stable in 105-111U/mL.
3.8 micro-agglutinations experiment detection antibody level of serum
Micro-agglutination experimental result is carried out under optimum reaction condition and is shown in Table 4,5.Yi Channel-catfish tardas
For coagulation experimental antigen when：The basic, normal, high dosage group of bigeminy microspheres vaccine and bivalent inactivated vaccine group exist
Specific antibody is can detect that when the 3rd week, but antibody titer is relatively low, only 1:2, antibody horizontal afterwards
Rise, the basic, normal, high dosage group of bigeminy microspheres vaccine reached peak value at the 5th week, respectively 1:32、
1:16、1:8, reduce afterwards, the basic, normal, high dosage group of bigeminy microspheres vaccine still can be examined when the 8th week
Specific antibody titres are measured for 1:2.Bivalent inactivated vaccine group detected specificity at the 3rd, 4,5 weeks
Antibody, but level is all relatively low, only 1:2, remaining cycle fails to detect specific antibody.It is empty micro-
Set of balls and matched group detect (table 4) without specific antibody.
During with Y.rucker as coagulation experimental antigen：The basic, normal, high dosage group of bigeminy microspheres vaccine
And bivalent inactivated vaccine group detected specific antibody at the 4th week, but antibody titer is relatively low, afterwards
Antibody horizontal rises, and the basic, normal, high dosage group of bigeminy microspheres vaccine reached peak value at the 5th week, respectively
For 1:16、1:8、1:8.To bigeminy microspheres vaccine when the 8th week is low, middle dose group remains to detect low water
Flat specific antibody.Bivalent inactivated vaccine detected specific antibody at the 4th, 5,6 weeks, potency
It is up to 1:4.Empty microsphere group and matched group detect (table 5) without specific antibody.
The micro-agglutination detection antibody potency of table 4
Note：Micro-agglutination is tested with Channel-catfish tardas as antigen；"-" represents feminine gender, i.e. serum and obvious coagulation does not occur with antigen
The micro-agglutination detection antibody potency of table 5
Note：Micro-agglutination is tested with Y.rucker as antigen；"-" represents feminine gender, i.e. serum and obvious coagulation does not occur with antigen
3.9 immune protective rate
During whole experiment, before counteracting toxic substances, there are not the exception feelings such as death in experiment Ietalurus Punetaus
Condition.Each experimental group presses the immune cycle (7d) of one, 2% oral immunity feedstuff of body weight, the 29th after immunity
It carries out counteracting toxic substances.As a result ， Yong Channel-catfish tarda challenge viral dosage groups and matched group, bigeminy microsphere epidemic disease are shown
The basic, normal, high dosage group of Seedling and bivalent inactivated vaccine group obtain respectively anti-Channel-catfish tardas relative immunity
Protective rate is 75.0%, 67.9%, 60.7% and 39.3%；Empty microsphere group protective rate is 10.7%, normal to raise
Material matched group after counteracting toxic substances in 14 days, mortality rate 93.3% (table 6).
With Y.rucker challenge viral dosage group and matched group, the basic, normal, high dosage of bigeminy microspheres vaccine
Group and bivalent inactivated vaccine group obtain respectively anti-Y.rucker relative immunity protective rate
65.5%th, 69.0%, 65.5% and 34.5%；；Empty carrier group protective rate is 10.3%, chow diet control
Group after counteracting toxic substances in 14 days, mortality rate 96.7% (table 7)
Ietalurus Punetaus mortality rate and relative immunity protective rate after the counteracting toxic substances of table 6
Ietalurus Punetaus mortality rate and relative immunity protective rate after the counteracting toxic substances of table 7
To sum up, bigeminy microspheres vaccine of the present invention can be exempted from by Channel-catfish tardas with effective protection Ietalurus Punetaus
With the infection of Y.rucker, Yu Fang Channel-catfish tardas disease and Y.rucker disease, use
Convenient, application prospect is good.
Claims (10)
1. a kind of fishing bigeminy oral microsphere vaccine, it is characterised in that：Its Bao Kuo Channel-catfish tarda microsphere
Vaccine and Y.rucker microspheres vaccine, wherein, Channel-catfish tarda microspheres vaccines, Lu Shi
Ademilson Salmonella microspheres vaccine is prepared as follows：
(1) Qu Channel-catfish tardas or Y.rucker, make bacterium solution, inactivation, obtain inactivation epidemic disease
Seedling；
(2) add sodium alginate to final concentration of 3.5% (w/v) in inactivated vaccine, mix, obtain mixed
Solution is closed, mixed solution is added drop-wise in liquid paraffin, wherein, the volume of mixed solution and liquid paraffin
For 2.4：7.6, emulsifying obtains emulsion；
(3) emulsion is added drop-wise to into the CaCl that equal-volume concentration is 7% (w/v)2In solution, add dense
Spend the CaCl for 0.05mol/L2Solution, stands, and removes upper oil phase and water, and microsphere is collected in centrifugation,
Cleaning, you can.
2. vaccine according to claim 1, it is characterised in that：Suo Shu Channel-catfish tarda microsphere epidemic diseases
Seedling is 1 with the volume ratio of Y.rucker microspheres vaccine：1.
3. vaccine according to claim 1, it is characterised in that：In step (1), the bacterium solution
The concentration of Zhong ， Channel-catfish tardas or Y.rucker is 4 × 109cfu/mL。
4. vaccine according to claim 1, it is characterised in that：In step (1), the inactivation
Mode be：Formalin is added to final concentration of 0.3% (w/v), 120r/min, is gone out under the conditions of 28 DEG C
24h living.
5. vaccine according to claim 1, it is characterised in that：In step (2), the liquid
Emulsifying agent is added with paraffin, emulsifying agent is Span-80, and the concentration of emulsifying agent is 5.4% (w/v).
6. vaccine according to claim 1, it is characterised in that：In step (2), the side of emulsifying
Formula is：2000r/min stirs 13min.
7. vaccine according to claim 1, it is characterised in that：In step (3), standing when
Between be 1h.
8. vaccine according to claim 1, it is characterised in that：In step (3), centrifugation turn
Speed is 8000r/min, and the time of centrifugation is 5min.
9. vaccine according to claim 1, it is characterised in that：In step (3), the mode of cleaning
Clean 3-4 time for ethanol purge 3 times, then deionized water.
10. the vaccine described in claim 1-9 any one is preparing Yu Fang Channel-catfish tardas disease and/or Shandong
Purposes in family name's yersinia disease.
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Cited By (2)
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