CN1292794C - Preparation of immuno-stimulation composition for hemolycin in monad - Google Patents
Preparation of immuno-stimulation composition for hemolycin in monad Download PDFInfo
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- CN1292794C CN1292794C CNB031528309A CN03152830A CN1292794C CN 1292794 C CN1292794 C CN 1292794C CN B031528309 A CNB031528309 A CN B031528309A CN 03152830 A CN03152830 A CN 03152830A CN 1292794 C CN1292794 C CN 1292794C
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- hemolysin
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Abstract
The present invention relates to the fields of the molecular biology and the immunology, particularly to a preparation technology of immune immuno-stimulation composition for hemolysin in aeromonas hydrophila, which comprises the steps: aeromonas hydrophila strains are cultured so that a culture solution is obtained; supernatant liquid is obtained after the culture solution is centrifuged, and is treated by saturated ammonium sulfate, precipitates are heavily suspended and dialyzed by PBS after centrifugation, and then suspension, namely toxin, is obtained; the toxin is concentrated to 1 to 3 mg/ml and then is treated for 1 to 3 hours by adding organic acid, Mega-10 is added in the toxin until the final concentration of the toxin is 2%, the toxin is treated for 4 hours at 37 DEGC and is mixed with a Quil A adjuvant until the final concentration is 0.05 to 0.2%, cholesterol and lecithin are added in the toxin until the final concentration is 125 mu g*mL-1, the toxin is treated by organic acid and then is dialyzed for 4 hours, and a suspension, namely a immune stimulation composition, is obtained. The immune stimulation composition has the advantages of stable immune effect and obvious synergism, and the immune stimulation composition can generate strong and lasting immune response so that the strong immune protecting force generated by immune animals generate is not stimulated.
Description
Technical field
The present invention relates to molecular biology and field of immunology, the preparation technology of the immunostimulating complex of hemolysin in particularly a kind of Aeromonas hydrophila.
Background technology
Aeromonas hydrophila (Aeromonas hydrophila, Ah) belong to the vibrionaceae Aeromonas, be people, Fish, amphibian, reptiles and mammiferous pathogenic bacterium, therefore the diseases such as diarrhoea that can cause fish bacterial septicemia, batrachia red leg disease and people cause a large amount of economic losses every year.
The virulence factor of Aeromonas hydrophila comprises adhesion factor on extracellular toxin, exoenzyme, the film, S layer albumen, pili, transferrins and outer membrane protein etc.The extracellular toxin of Ah pathogenic strain is important virulence factor, and its research is quite deep.Different research worker are given extracellular toxin with different addresses, as the HEC toxin of Lu Cheng equality report, and the enterotoxin of report such as Rose, the hemolysin of reports such as the gas lysin of report such as Bukley and Asao.Hemolysin is vital virulence factor, and it has hemolytic, intestinal toxicity and cytotoxic effect, and animal subjects such as anguilla japonica, Carassius auratus, white mice are had independently pathogenic effects, is important protective antigen.
Aeromonas hydrophila disease immunoprophylaxis research has become the focus of science of fish disease research, and scientific achievement now has Aeromonas hydrophila deactivation whole-bacterial-vaccine, and it is applied in main epidemic-stricken area, has obtained certain effect.But because Aeromonas hydrophila serotype is numerous, immunogenicity there are differences between bacterial strain, while Aeromonas hydrophila antigenic structure complexity, be not easy to preparation connection Seedling, full vaccine antigenicity after deactivation often morphs, moreover full vaccine granule is big, and absorbed probability is less, more than all multifactor popularizing of full bacterium inactivated vaccine that restricted.
Existing genetic engineering subunit vaccine has kept the advantage of traditional subunit vaccine, as: advantages such as safety is good, and effectively immunizing composition is single-minded, has the suitability for industrialized production of being easy to simultaneously, and production cost is low, but immunogenicity is not strong.In subunit seedling, use adjuvant can significantly strengthen the immunogenicity of subunit vaccine, conventional adjuvants such as the at present clinical aluminium hydroxide that uses, propolis improve immunogenic limited in one's ability more, and Freund adjuvant possesses stronger potentiation, but its toxicity, side effect to animal is big, uses and be inhibited in clinically.
Summary of the invention
The objective of the invention is to overcome weak point of the prior art, a kind of, immune effect strong to the Aeromonas hydrophila immunogenicity is stable, toxicity, side effect is little and provide, and is convenient to prepare the preparation technology of the immunostimulating complex of multiple vaccines.
Purpose of the present invention can realize through following scheme.
The preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila, its main points are to have following steps:
1, provide a kind of Aeromonas hydrophila bacterial strain,
2, in culture medium, this bacterial strain carried out amplification culture and culture fluid,
3, from culture fluid, extract toxin,
(1) provide a kind of centrifuge,
(2) in centrifuge to behind the medium centrifugal supernatant,
(3) with the toxin in the saturated ammonium sulfate concentrated supernatant, in centrifuge, get precipitation after centrifugal,
(4) will precipitate with PBS resuspended, and the dialysis, obtain suspension and be toxin,
4, prepare immunostimulating complex according to following order,
(1) toxin be concentrated into add organic acid treatment 1～3h behind 1～3mg/ml for the first time,
(2) adding Mega-10 is 2% to final concentration, processing 4h under 37 ℃,
(3) adding Quil A adjuvant to final concentration is 0.05～0.2%,
(4) adding cholesterol, lecithin to final concentration are that 125 μ g/mL get compositions,
(5) add for the second time the 4h that dialyses after organic acid treatment compositions, the reuse 0.01M PBS 72h that dialyses obtains suspension and is immunostimulating complex.
Hemolysin also claims hemolytic factor, is the vital virulence factor that Aeromonas hydrophila produces pathogenicity, and it has hemolytic, intestinal toxicity and cytotoxic effect.And immunostimulating complex herein is meant that hemolysin combines the product that generates with adjuvant, can directly use as vaccine.Preparation technology to this immunostimulating complex makes description below below:
The bacterial strain of one Hygrophilous monad at first will be provided, and this bacterial strain can be the bacterial strain that nature exists, and can also be the reorganization bacterium that obtains by the DNA recombinant technique.Present technique field personnel all can obtain Aeromonas hydrophila by conventional route.
Its two, the bacterial strain that is provided is carried out amplification culture so that prepare a large amount of culture fluid that contains this Aeromonas hydrophila.The cultivation of this bacterial strain can adopt strain culturing technology well-known to those skilled in the art to carry out.
They are three years old, culture fluid is centrifugal and obtain supernatant in centrifuge, mainly contain hemolysin in this supernatant, or also contain other effective immunizing composition, and adopt saturated ammonium sulfate to handle supernatant is in order to make the toxin protein in the supernatant can partly obtain concentrating and purifying, and then from supernatant, separate toxin (i.e. precipitation or antigen) with centrifuge, precipitate at last with the PBS dialysis and remove toxin after suspension after ammonium sulfate, the dialysis is purification.
They are four years old, toxin is concentrated into adds organic acid behind 1～3mg/ml and handle 1～3h, it is in order to make toxin protein be exposed its hydrophobic region by acidification that organic acid is handled, handling and add Quil A adjuvant to final concentration through Mega-10 is 0.05～0.2%, then add cholesterol, lecithin to final concentration is 125 μ g/mL, help forming the stable shape structure of winning over by any means like this, handle back dialysis 4h with organic acid again and, obtain suspension and be immunostimulating complex with the PBS 72h that dialyses.
The present invention adopts the processing technology of the special immunostimulating complex that never is applied to Aeromonas hydrophila, make toxin protein be exposed its hydrophobic region with the organic acid processing by acidify, make its easier combining be immunostimulating complex with Quil A adjuvant, like this, under Electronic Speculum, observe immunostimulating complex and have typical cage trellis grain structure, thereby its antigenic determinant more can fully expose, thus the humoral immunization of activating immune system (B cell), cellular immunization (CD effectively
4 +The T cell) and cytotoxic T cell (CD
8 +The T cell) all three kinds of approach, so immune effect is stable, potentiation is strong, this effect is that employed genetic engineering subunit vaccine or full bacterium inactivated vaccine are beyond one's reach in the prior art.
Simultaneously; it can produce strong again and lasting immune response; the immunoprotection phase is long; test is found after deliberation; it is higher more than 10 times with antigen or protein body than single to be loaded with the inductive antibody titer of various antigenic immunostimulating complexs; and used antigen dose reduces 10 times, illustrates that the antigen that embeds in the immunostimulating complex has good immunogenicity.
And the present invention does not adopt Freund adjuvant, thereby the phenomenon too big to the stimulation of immunity receptor can not occur this immunostimulating complex yet and use as vaccine the time.The immune toxicity, side effect of this immunostimulating complex is also less simultaneously.
Immunostimulating complex is not only the person of presenting of vaccine antigen, but also can present immunostimulation, produces strong and lasting immune response, thereby can also be as adjuvant.This is of the present invention one unexpected effect.
The invention reside in
Used organic acid is a citric acid.
And then
The concentration of adding citric acid is 0.25M for the first time.
The concentration of adding citric acid is 0.01M for the second time.
The use of citric acid can expose the hydrophobic region of toxin protein (being antigen), to make things convenient for the immunologically competent cell in the immunity receptor---and T cell and B cell recognition antigen also combine with antigen.It is the citric acid treatment toxin protein of 0.25M that the inventor adopts concentration; 0.01M citric acid treatment compositions; it carries out immunity with immunostimulating complex after adjuvant combines to Anguillar japonica; with the immunostimulating complex group of dosage aspect the humoral immunoresponse(HI) than Freund adjuvant group with do not add the adjuvant group and have higher protective rate; can reach 100%; the Freund adjuvant group can reach 87.5%, does not have only 50% and add the adjuvant group.
The present invention also is
Used organic acid is the glycine buffer of pH2.5.
Organic acid of the present invention for example can also be a malic acid etc. for can make toxin protein expose the organic acid of its hydrophobic region.
Provide that a kind of that Aeromonas hydrophila is carried out the step of amplification culture is as follows:
1, with inoculation in the plain agar culture medium,
2, cultivate the 24-48 hour culture of winning at 29 ℃,
3, the single bacterium colony of picking from first culture is inoculated in the 5ml LB culture fluid,
4, cultivated 24-48 hour at 29 ℃, when OD value is 1.0, gets 1ml and be inoculated in respectively in the 100ml TSB culture medium and cultivated 30 hours, gather in the crops.
For a kind of cultivation of bacterial strain commonly used, a lot of records is arranged in the prior art, difference only is the number of incubation time, the height of cultivation temperature, and adopt which kind of culture.The effect that the inventor selects above-mentioned processing step that Aeromonas hydrophila is cultivated is fine, and the carrying out of more convenient subsequent technique.
The present invention is also that further the concrete steps of obtaining the supernatant that contains toxin from culture fluid are:
In centrifuge to behind the centrifugal 30min of culture fluid 10000rpm supernatant.
Adopt saturated ammonium sulfate to handle supernatant and make toxin globulin in the supernatant can partly obtain concentrating and the technology routine techniques of purification and solid-liquid separation in all on the books, and the present invention provides a kind of especially and handles supernatant with the saturated ammonium sulfate gradient, and gets sedimentary concrete steps after centrifugal in centrifuge:
(1) saturation of adding ammonium sulfate to 20% in supernatant, 4 ℃ left standstill 4 hours, and the centrifugal 30min of 10000rpm abandons precipitation in centrifuge, the supernatant of winning,
(2) saturation of adding ammonium sulfate to 60% in first supernatant, 4 ℃ left standstill 4 hours, the centrifugal 30min of 10000rpm in centrifuge, collecting precipitation.
And then
To precipitate and use PBS resuspended, and dialysis, changed liquid once in per 4 hours, change 5-6 back of liquid results suspension and be toxin, and standby in-20 ℃ of preservations.
Purpose of the present invention can also realize through following scheme.
On the basis of above-mentioned disclosed technical scheme, can also adopt following steps simultaneously:
Compositions is handled 8～10min with the ice-bath ultrasonic ripple earlier, and then the reuse organic acid is handled the back dialysis.
The ice-bath ultrasonic ripple is handled the binding ability that can increase adjuvant and toxin protein, to increase the immune effect of immunostimulating complex.
In sum, the present invention has following advantage compared to prior art: can make antigenic determinant fully expose and combine with adjuvant through technology provided by the present invention and make immunostimulating complex, this immunostimulating complex is the humoral immunization of activating immune system (B cell), cellular immunization (CD effectively
4 +The T cell) and cytotoxic T cell (CD
8 +The T cell) all three kinds of approach, so immune effect is stable, potentiation is remarkable, can produce strong and lasting immune response, makes to be exempted from animal and produce strong immune protective efficiency, and can not produce stimulation to immunity receptor.
Description of drawings
Fig. 1 is the immunostimulating complex Electronic Speculum figure of Aeromonas hydrophila hemolysin.
Fig. 2 is the ELISA serum antibody titer sketch map after the immunity for the first time.
Fig. 3 is the ELISA serum antibody titer sketch map after the immunity for the second time.
The specific embodiment
Below in conjunction with embodiment the present invention is described in more detail.
Embodiment 1:
One, material requested
(1) main agents
Quil A is available from inspection dimension health technology company limited in Beijing; Mega-10 is available from Huamei Bio-Engrg Co..
(2) strain
Aeromonas hydrophila L-316 is by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute fish diseases center isolation identification and make up its β-hemA bacterium of recombinating.
(3) laboratory animal
Anguillar japonica is available from permanently happy Anguillar japonica field, Fujian Province, specification: about 200g.
Two, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila has following steps:
1, extract the reorganization bacterium of Aeromonas hydrophila L316,
2, in culture medium, this bacterial strain carried out amplification culture and culture fluid,
(1) with inoculation in the plain agar culture medium,
(2) cultivate the 24-48 hour culture of winning at 29 ℃,
(3) the single bacterium colony of picking from first culture is inoculated in the 5ml LB culture fluid,
(4) cultivated 24-48 hour at 29 ℃, when OD value is 1.0, gets 1ml and be inoculated in respectively in the 100ml TSB culture medium and cultivated 30 hours, gather in the crops.
3, from culture fluid, extract toxin,
(1) provide a kind of centrifuge,
(2) in centrifuge to behind the centrifugal 30min of culture fluid 10000rpm supernatant,
(3) handle supernatant with saturated ammonium sulfate and in centrifuge, get precipitation after centrifugal,
1. add the saturation of ammonium sulfate to 20% in supernatant, 4 ℃ left standstill 4 hours, and the centrifugal 30min of 10000rpm abandons precipitation in centrifuge, the supernatant of winning,
2. add the saturation of ammonium sulfate to 60% in first supernatant, 4 ℃ left standstill 4 hours, the centrifugal 30min of 10000rpm in centrifuge, collecting precipitation.
(4) will precipitate with PBS resuspendedly, dialysis was changed liquid once in per 4 hours, and change liquid 5-6 time back and gather in the crops suspension and be toxin, and standby in-20 ℃ of preservations,
4, prepare immunostimulating complex according to following order,
(1) toxin be concentrated into add 0.25M citric acid treatment 2h behind the 2mg/ml,
(2) add Mega-10 to final concentration be 2%, 37 ℃ of cracking 4h,
(3) adding Quil A adjuvant to final concentration is 0.1%,
(4) add cholesterol (dissolving with chloroform in advance) then, lecithin to final concentration is that 125 μ g/mL get compositions, the ice-bath ultrasonic ripple is handled 8-10min, the negative pressure of vacuum dechlorination is imitative,
(5) with the 4h that dialyses after the 0.01M citric acid treatment compositions, the reuse 0.01M PBS 72h that dialyses obtains suspension and is immunostimulating complex.
Three, measure the haemolysis valency of toxin
The toxin 0.01mol/LPBS of-20 ℃ of preservations is made doubling dilution on micro plate, add V-type 96 hole agglutination plates, every hole adds 25 μ L, and 1% rabbit erythrocyte that adds equivalent is then put 37 ℃ 1 hour, 4 ℃ result of determination after 2 hours, is the haemolysis valency with 50% hemolytic high dilution.
The result who measures toxin haemolysis valency is 17.56HU/ml.
Four, toxin protein assay
Press the Bradford method with DU Series 7000 spectrophotometers (BECKMAN) and measure, measure the A value at wavelength 595nm place, the reference standard curve draws proteic concentration.
Measurement result: toxin protein content is 3299ug.
Five, the electron microscopic observation of immunostimulating complex
Get testing sample 10ul and drip copper mesh,, on the JEM-120EX Electronic Speculum, observe with the dyeing of 2% Sodium phosphotungstate.
The result as shown in Figure 1.
Six, adopt immunostimulating complex that Anguillar japonica is carried out immunity
Immunity for the first time divides six groups and carries out every group 15 tail.
I: hemolysin (PCB), 40ug tail
-1
I: hemolysin (PCB), 40ug tail
-1
II: hemolysin (PCB) and Freund adjuvant mixed in equal amounts, 40ug tail
-1
III: immunostimulating complex, 20ug tail
-1
IV: immunostimulating complex, 40ug tail
-1
V: immunostimulating complex, 80ug tail
-1
The VI:PBS matched group, 0.1ml;
Immunization ways: intramuscular injection, every tail 0.1-0.2ml.Get 5 tails and carry out second time immunity for every group after 15 days, immunization route and dosage are with immune identical for the first time.
Protective rate measurement result after the immunity sees Table 1:
Protective rate behind table 1 secondary immunity
| Protective rate after the immunity for the first time | Protective rate after the immunity for the second time | |||||
| Counteracting toxic substances mantissa | Dead mantissa | Protective rate | Counteracting toxic substances mantissa | Dead mantissa | Protective rate % | |
| I II III IV V VI | 8 8 8 8 8 8 | 4 3 2 1 2 8 | 4/8 5/8 6/8 7/8 6/8 0/8 | 5 5 5 5 5 5 | 3 2 1 0 0 5 | 40 60 80 100 100 0 |
Seven, the Anguillar japonica after immunity for the first time and the immunity is for the second time extracted toxin and carries out elisa (ELISA)
The toxin that is extracted is by 50ugmL
-1The amount bag by 96 hole ELISA Plate (Xiamen Mao Yuan plastic molding and processing plant), blank wraps quilt with PBS, every hole 50 μ L, 4 ℃ are spent the night.Remove solution in the hole, with the PBS-T room temperature sealing that contains 2%BSA 2 hours, the washing back adds blood serum sample to be checked (making doubling dilution since 1: 100), every hole 50 μ L, room temperature reaction 1 hour, PBS-T washing three times, the monoclonal antibody (3D11 and 3H1 mix the back with dilution in 1: 1000) that adds mouse-anti Anguillar japonica Ig then, every hole 50 μ L, room temperature reaction 1 hour, the same washing 3 times; The IgG (dilution in 1: 1000) that adds sheep anti mouse HRP labelling, room temperature reaction 1 hour, the same washing 3 times, reuse distilled water flushing 1 time.Add the reaction substrate OPD of 50 μ L at last, develop the color behind the room temperature 10-15min, use 2molL
-1H
2SO
4Cessation reaction.Read the OD value with enzyme connection instrument Microplate Reader (BioRad Mode 1550) at the 495nm place.
The ELISA experimental result is seen Fig. 2 and Fig. 3.
This test to expose its hydrophobic region, combines it with citric acid treatment Aeromonas hydrophila hemolysin easilier with Quil A.In the immunostimulating complex group, use 20ug, 40ug, three dosage groups of 80ug; 40ug dosage group best results wherein, protective rate reaches 100%, and the serum antibody titer of 80ug dosage group is identical with the 40ug group; but protective rate is identical with the 40ug group, also reaches 100%.II group (Freund adjuvant) is basic identical with the serum antibody titer that the III group is produced, but its protective rate is lower than immunostimulating complex group, illustrates that immunostimulating complex can produce higher protective rate under low antibody titer.
Embodiment 2:
Citric acid in the step of the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila of embodiment 1 is replaced being used to prepare the immunostimulating complex of Aeromonas hydrophila with the glycine buffer of pH2.5;
Toxin is concentrated into the glycine buffer that adds pH2.5 behind the 1mg/ml handles 1h;
Adding Quil A adjuvant to final concentration is 0.05%;
Remaining experiment material and experimental procedure are identical with embodiment 1; experiment effect: 40ug dosage group best results; protective rate reaches 100%; taking second place is 80ug dosage group, and as seen adopting glycine to handle immunostimulating complex that toxin protein and citric acid treatment toxin protein obtained, to be used for immunity receptor is carried out the effect of humoral immunization basic identical.
Most preferred embodiment:
The preparation technology's of the immunostimulating complex of hemolysin in the Aeromonas hydrophila of embodiment 1 step following steps are revised as:
Toxin is concentrated into adds 0.25M citric acid treatment 3h behind the 3mg/ml;
Adding Quil A adjuvant to final concentration is 0.2%;
Remaining experiment material and experimental procedure are identical with embodiment 1, and experiment effect is identical with embodiment 2.
It is same as the prior art that various embodiments of the present invention are not stated part.
Claims (10)
1, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila is characterized in that having following steps:
(1) provide a kind of Aeromonas hydrophila bacterial strain,
(2) in culture medium, this bacterial strain carried out amplification culture and culture fluid,
(3) from culture fluid, extract toxin,
1. a kind of centrifuge is provided,
2. in centrifuge to behind the medium centrifugal supernatant,
3. handle supernatant with saturated ammonium sulfate and in centrifuge, get precipitation after centrifugal,
4. it is resuspended to precipitate the molten PBS of using, and dialysis, and obtain suspension and be toxin,
(4) prepare immunostimulating complex according to following order,
1. add for the first time organic acid treatment 1～3h after toxin being concentrated into 1～3mg/ml,
2. adding Mega-10 is 2% to final concentration, processing 4h under 37 ℃,
3. adding Quil A adjuvant to final concentration is 0.05～0.2%,
4. add cholesterol, lecithin to final concentration is that 125 μ g/mL get compositions,
(5) add for the second time the 4h that dialyses after organic acid treatment compositions, the reuse 0.01M PBS 72h that dialyses obtains suspension and is immunostimulating complex.
2, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1 is characterized in that, used organic acid is a citric acid.
3, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 2 is characterized in that, the concentration of adding citric acid is 0.25M for the first time.
4, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 2 is characterized in that, the concentration of adding citric acid is 0.01M for the second time.
5, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1 is characterized in that, used organic acid is the glycine buffer of pH2.5.
6, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1 is characterized in that the concrete steps of Aeromonas hydrophila being carried out amplification culture are as follows:
(1) with inoculation in the plain agar culture medium,
(2) cultivate the 24-48 hour culture of winning at 29 ℃,
(3) the single bacterium colony of picking from first culture is inoculated in the 5ml LB culture fluid,
(4) cultivated 24-48 hour at 29 ℃, when OD value is 1.0, gets 1ml and be inoculated in respectively in the 100ml TSB culture medium and cultivated 30 hours, gather in the crops.
7, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1, it is characterized in that the concrete steps of obtaining the supernatant that contains toxin from culture fluid are: in centrifuge to behind the centrifugal 30min of culture fluid 10000rpm supernatant.
8, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1 is characterized in that, handles supernatant with saturated ammonium sulfate, and gets sedimentary concrete steps after centrifugal in centrifuge:
(1) saturation of adding ammonium sulfate to 20% in supernatant, 4 ℃ left standstill 4 hours, and the centrifugal 30min of 10000rpm abandons precipitation in centrifuge, the supernatant of winning,
(2) saturation of adding ammonium sulfate to 60% in first supernatant, 4 ℃ left standstill 4 hours, the centrifugal 30min of 10000rpm in centrifuge, collecting precipitation.
9, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1 is characterized in that, will precipitate with PBS resuspended, and dialysis, changed liquid once in per 4 hours, and changed 5-6 back of liquid results suspension and be toxin, and standby in-20 ℃ of preservations.
10, the preparation technology of the immunostimulating complex of hemolysin in the Aeromonas hydrophila according to claim 1 is characterized in that, compositions is handled 8～10min with the ice-bath ultrasonic ripple earlier, and then the reuse organic acid is handled the back dialysis.
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| CN100569285C (en) * | 2006-05-16 | 2009-12-16 | 福建省农业科学院生物技术研究所 | The application of immunostimulating complex in preparation fish immunity preparation |
| SK287315B6 (en) | 2006-06-02 | 2010-06-07 | Biotika, A. S. | A method for polymyxin B isolation from fermented soil |
| SK287293B6 (en) | 2006-06-15 | 2010-05-07 | Biotika, A. S. | A method for fermentation of polymyxin B by means of productive microorganism Bacillus polymyxa |
| CN108524927A (en) * | 2018-05-17 | 2018-09-14 | 广州普麟生物制品有限公司 | A kind of grass carp hemorrhage disease inactivated vaccine and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1112021A (en) * | 1994-05-16 | 1995-11-22 | 石家庄市特种养殖发展公司 | Aluminium hydroxide vaccine for prevention of aerohydrophilic monadosis and its preparation method |
| CN1112022A (en) * | 1994-05-16 | 1995-11-22 | 石家庄市特种养殖发展公司 | High immune serum for prevention of aerohydrophilic monadosis and its preparation method |
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|---|---|---|---|---|
| CN1112021A (en) * | 1994-05-16 | 1995-11-22 | 石家庄市特种养殖发展公司 | Aluminium hydroxide vaccine for prevention of aerohydrophilic monadosis and its preparation method |
| CN1112022A (en) * | 1994-05-16 | 1995-11-22 | 石家庄市特种养殖发展公司 | High immune serum for prevention of aerohydrophilic monadosis and its preparation method |
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