CN100569285C - The application of immunostimulating complex in preparation fish immunity preparation - Google Patents
The application of immunostimulating complex in preparation fish immunity preparation Download PDFInfo
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- CN100569285C CN100569285C CNB2006100406222A CN200610040622A CN100569285C CN 100569285 C CN100569285 C CN 100569285C CN B2006100406222 A CNB2006100406222 A CN B2006100406222A CN 200610040622 A CN200610040622 A CN 200610040622A CN 100569285 C CN100569285 C CN 100569285C
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Abstract
The invention discloses the application of a kind of immunostimulating complex in preparation fish immunity preparation, described immune formulation is the oral immunity preparation or is the dipping bath immune formulation.The invention has the advantages that: oral immunity preparation or the dipping bath immune formulation made by immunostimulating complex ISCOMs not only can improve antibody horizontal, and can improve the lymphocytic propagation conversion capability of T, the induction of immunity protection.During the Isodose immunity, the antibody titer of ISCOMs group is apparently higher than no adjuvant group and ISM1312 adjuvant group.Same ISCOMs is used for oral immunity and not only can improves antibody horizontal, and can improve the lymphocytic propagation conversion capability of T, the induction of immunity protection.
Description
Technical field
The present invention relates to a kind of immunostimulating complex application in fish immunity, the particularly immunostimulating complex application in preparation fish immunity preparation.
Background technology
The immunization ways of aquatic animal vaccine mainly contains injection, dipping bath and oral three kinds of modes, though injecting immune can obtain good immune effect, owing to need to carry out at individuality one by one, therefore this mode is not adapted at using in large-scale the breed.
Aquatic animal especially Fish has the flourishing mucosa system that is made of body surface, gastrointestinal tract, the gill; the mucosa system is except participating in nonspecific immunity; can also mediate the specific immunity protection; but because Fish pipe intestinal digesting liquid is abundant; traditional vaccine is subjected to the influence of various enzymes in intestinal, pepsin and the body surface mucosa; effectively antigen losses is serious in oral administration or dipping bath immunologic process, and penetrance is not high, so effect is undesirable.Therefore how in oral or dipping bath immunologic process, to prevent effective antigen destroyed be the focus of Aquatic product vaccine research always.It also is the key technology that development oral type and the fishing of dipping bath type need to be resolved hurrily with vaccine.
Be degraded for fear of antigen, during research worker has been expected and the method for digestive enzyme, for example Mclean etc. (nineteen ninety) is used to protease inhibitor etc. to strengthen the oral immunity research of rainbow trout.Also having the antigenic measure of a kind of protection is preparation capsule-type vaccine; at present existing polytype capsule-type vaccine is in middle application; for example; Lillehaug etc. (1989) use freeze dried vibrio vaccine respectively the form peroral immunity rainbow trout of capsule form and antiacid material bag quilt; yet all undesirable with the immune effect of vaccine that these two kinds of forms are made, the vaccine that does not add any processing on the contrary has the certain protection effect on the contrary.This shows that antigenic ingestion efficiency also is the key factor that influences oral immunity, though capsule and antiacid material have been alleviated the gastrointestinal Digestive system to antigenic degraded, has also hindered antigenic absorption simultaneously.
In recent years, the research worker biotype oral vaccine capsule that begins one's study avoids antigen to be degraded.Mainly contain Salmonella, shigella, artemia etc. as capsular candidate's biology.As endotrophic antibacterial, highly cause weak Salmonella and shigella by the oral intestinal mucosa immuning tissue that can well purpose antigen be carried to the immune animal body, but antibacterial itself is as a kind of exogenous material, to animal body also is strong immunogenic, and the secondary that has influenced carrier is used; In addition, Salmonella is as the important indicator of water pollution, as the transmission carrier of fish vaccine with effective monitoring that may interference environment water quality.Salmonella a little less than these factors greatly reduce and highly cause and shigella are transmitted the practicality of carrier as fish vaccine.Yu Junhong etc. (calendar year 2001) carry two kinds of method oral immunization yarn fish fries such as method with the Vibrio anguillarum microcapsule vaccine directly to admix bait method and artemia, week back mattress counteracting toxic substances alive.The result shows, all cumulative mortalities of the bio-microcapsule vaccine group of carrying with artemia for (25%) far below matched group (95%).This immunization ways has bright development prospect, but also has certain deficiency, because Fish are many at edible this bait of fry phase, therefore has the risk of the anti-property crossed of immunity.Artemia carries vaccine also to be difficult to be prepared according to industrialization and standardized mode.Therefore prior art awaits people and develops the immune formulation that is prepared by immunologic stimulant.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part and a kind of fish immunity preparation by immunostimulating complex preparation is provided.
The application of immunostimulating complex in preparation fish immunity preparation, its structural feature is: described immune formulation is the oral immunity preparation or is the dipping bath immune formulation.
So-called oral immunity preparation refers to immunostimulating complex is made an addition to the immunity that gives vaccine in the feedstuff through the Fish gastrointestinal tract.So-called dipping bath immune formulation refers to immunostimulating complex is made an addition to breeding water body, through fish body surface, gill immunity.
Described oral immunity preparation is that immunostimulating complex and fish meal are stirred.
Described immunostimulating complex consists predominantly of pathogen antigen material, intercalator Quil A, cholesterol, lecithin.
QuilA is a kind of saponins material that goes out from plant extract, and the immunostimulating complex for preparing on this basis (ISCOMS) is compared the advantage that himself is arranged as a kind of form of oral vaccine with other types antigen.At first the Main Ingredients and Appearance QuilA of ISCOMs can directly act on microvillus, has loose intercellular and connects, and promotes the effect of pinocytosis, and these effects help to improve the antigenic penetrance of macromole, improve antigenic absorption; Secondly the immunostimulating complex that is interacted in solution by intercalator Quil A, cholesterol, lecithin and antigen and form can tolerate intestinal, pepsin, reduces effective antigenic loss in the absorption process, improves antigenic absorbtivity; The 3rd, the particular configuration of ISCOMs can be concentrated antigen in a large number on the surface, improves antigenic absorbtivity.At last, ISCOMs can induce interleukin, interferon to produce, activation t helper cell (Th cell), cytotoxic killer cell (CTL cell), bone-marrow-derived lymphocyte by stimulating MALT.Has the ability that produces comprehensive immunne response.Because ISCOMs can offer antigen by mucosal route, and can and have no side effect by oral administration, therefore be highly suitable in the development of Aquatic product vaccine and use.
Described pathogen antigen material or be pathogen protein or for the polysaccharide antigenic substance.
Pathogen in the described pathogen protein is mainly the albumen that contains in antibacterial, virus, the parasitic disease substance.
The described fish that is used for immunity comprises fresh-water fishes and marine fish, is particularly useful for Anguillar japonica, Carnis Pseudosciaenae, tilapia.
Described fish immunity preparation to Fish oral, dipping bath is immune or be once immunity, or is secondary immunity, or is booster immunization.
Described secondary immunity is at initial immunity 7-14 days, use the same method again and dosage immunity is once again.
Described booster immunization is behind secondary immunity, used the same method again in 1-3 month and dosage immunity is once again.
When Fish stimulated at the first vaccine immunity of acceptance, under the specific antigen effect, the immunocyte that contains the specific antigen receptor was bred in vaccine, produces specific effector cell and specific antibody at specific antigen.From antigenic stimulation, to immunological effect takes place a process is arranged, therefore to the generation immunological effect for some time from immunity inoculation.Simultaneously because specific immunocyte quantity is less, thus response strength also a little less than.Behind initial immunity, optionally propagation has taken place in the immunocyte of body, produce immunological memory cell, when secondary immunity, a large amount of special immunocytes can be activated in a short time, therefore behind the secondary immunity, shorten dramatically to the time that produces immunological effect from immunity, the intensity of immunological effect and persistent period also significantly are better than initial immunity.Therefore the more relative imperfection with mammal of the immune system of Fish adopts the above immunity of secondary, can excite Fish to produce immunity better, keeps the sufficiently long immunity cycle.
Essence is for a better understanding of the present invention used respectively below by the immunostimulating complex (ISCOMs) of Aeromonas hydrophila β-hemA reorganization bacterium preparation and is adopted the oral methods immunity that the application of immunostimulating complex in preparing oral or immune formulation is described to the immunity of anguilla japonica adopting soaking method and with the multiple thing (ISCOMs) of immunostimulation of Aeromonas hydrophila β-hemA reorganization bacterium preparation to anguilla japonica.
One: the immunostimulating complex (ISCOMs) of Aeromonas hydrophila β-hemA reorganization bacterium preparation adopts the dipping bath method immunity to anguilla japonica
1. materials and methods
1.1 material
β-hemA is the Aeromonas hydrophila beta hemolysin, is a kind of albumen composition.After β-hemA reorganization bacterium inserts expression plasmid pCDNA3.0 by the beta hemolysin gene, obtain after importing engineering bacteria E.coli DH5 α again.Its expression product contains beta hemolysin.Main agents Quil A is available from Accurate chemical﹠amp; Scientific corporation company; Mega-10 is available from Huamei Bio-Engrg Co.; Cellproliferation ELISA, the Brdu test kit is available from Roche Holding Ag; Available from anguilla japonica field, permanently happy Hailin, Fujian Province, specification is every tail 50g for the examination anguilla japonica.
The TSB culture medium prescription
Soya peptone 3g, tryptone 17g, NaCl 15g, K
2HPO
42.5g glucose 0.5g adds distilled water to 1000ml, 121 ℃ of autoclaving 20min.
The TSA culture medium prescription
Soya peptone 3g, tryptone 17g, NaCl 15g, K
2HPO
42.5g, glucose 0.5g, Agar 15g.Add distilled water to 1000ml, 121 ℃ of autoclaving 20min.
1.2 method
1.2.1. antibacterial culturing
β-hemA bacterial classification inoculation in the plain agar culture medium flat plate, is cultivated 24h for 28 ℃, and picking list bacterium colony is inoculated in the TSB culture fluid of 10ml, and 28 ℃ of cultivations when OD value is 1.0, is got 5ml and are inoculated in the TSB culture medium of 500ml and cultivate 20h, gather in the crops.
1.2.2. extracellular products extracts
The centrifugal 30min of 10000rpm gets supernatant, adds ammonium sulfate to 60% saturation, and 4 ℃ are spent the night, the centrifugal 30min of 10000rpm, collecting precipitation, resuspended with 0.01M PBS (pH7.2), dialysis, every 4h changes liquid once, changes liquid 5～6 times, promptly gets and slightly carries extracellular products, and is standby in-20 ℃ of preservations.
1.2.3. the mensuration of protein content
Measure according to the Bradford method with DU Series7000 spectrophotometer (BECKMAN), record the A value at wavelength 595nm place, the reference standard curve draws proteic concentration.
1.2.4.ISCOMs preparation
Concentrate extracellular products to 4mg.mL
-1, with add behind the 1M citric acid treatment 2h Mega-10 to final concentration be 2%, 37 ℃ of cracking 4h, add Quil A to final concentration be 0.1%, add cholesterol then, lecithin (dissolving with chloroform in advance) to final concentration is 125 μ g.mL
-1, the ice-bath ultrasonic ripple is handled 8-10min, and the negative pressure of vacuum dechlorination is imitative, earlier with 0.01M citric acid treatment dialysis 4h, reuse 0.01MPBS (pH 7.2) 72h that dialyses, every 4h changes liquid once.
1.2.5. electron microscopic observation β-hemA-ISCOMs
Get testing sample 10 μ L and drip copper mesh,, on the JEM-1200EX Electronic Speculum, observe with the dyeing of 2% acetic acid uranium.
1.2.6.ISCOMs immune anguilla japonica
Immunization protocol is as shown in table 1, adopts secondary immunity, divides 9 groups to carry out, every group 15 tail, and 60min is soaked in immunity for the second time at every turn after 15 days, be about 30g for examination anguilla japonica specification, indoor culture, 24 ℃ of temperature are changed water 1/3 every day.Wherein the PCB representative contains the beta hemolysin gene recombination bacterium, and PC represents the engineering bacteria that does not contain beta hemolysin but contain the pCDNA3.0 empty plasmid, and ECPs represents extracellular products.BSA is a bovine serum albumin.PBS is the PH7.2 phosphate buffer, and concentration is 0.01M.
Table 1ISCOMs immunization protocol (dividing 9 groups)
1.2.7 elisa (ELISA) detects the anguilla japonica serum antibody
1 group, 2 groups, 3 groups, 4 groups, 6 groups, 7 groups, 8 groups, 9 groups of experimental grouies (exempts from back 44d) every group before counteracting toxic substances got 4 tail anguilla japonicas and used ELISA and detect serum antibody.Reorganization bacterium PCB expression product (PCB ECPs) is by 12 μ g.mL
-1The concentration bag by 96 hole ELISA Plate, 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 2h that contains 2%BSA, the washing back adds anguilla japonica serum (1: 100), every hole 50 μ L, room temperature reaction 1h, PBS-T washing three times, (two is anti-, and 7E 2 mixes with 8H1 with 1: 1000 and dilutes to add the monoclonal antibody of mouse-anti anguilla japonica Ig then, this chamber preparation), every hole 50 μ L, room temperature reaction 1h, the same washing 3 times; The I gG (three is anti-, dilution in 1: 5000) that adds sheep anti mouse HRP labelling, room temperature reaction 1h, 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 2mol/LH
2SO
4Cessation reaction.Read the OD value with enzyme connection instrument Microplate Reader (BioRad Mode 1550) at the 495nm place.
1.2.8 anguilla japonica lymphocyte transformation test
To 1 group, 4 groups, 6 groups, 8 groups, 9 groups before counteracting toxic substances (exempt from back 44d) every group get four tail anguilla japonicas, under aseptic condition, take out spleen, the washing of the aseptic PBS of 0.01M (pH7.2) liquid squeezes out Cell sap, blows and beats cell dispersion with syringe.Spread and the isopyknic lymphocyte layering of Cell sap liquid in the centrifuge tube bottom, spread Cell sap above, 20, the centrifugal 10min of 00rpm, sucking-off middle white layer, 1640 liquid are resuspended, cell counting, making cell content is 3 * 10
6, adding the ConA final concentration is 5 μ g.mL
-1, splash into 96 holes trace cell plates respectively, every hole 100 μ l, every duplicate samples is done six parallel holes.The airtight cultivation of 27 ℃ biochemical incubator 3 days, every hole adds BrdU marking fluid 10 μ l, continue to cultivate 17h, the centrifugal 10min of 300g, abandoning supernatant, toast 15min in 60 ℃ of baking ovens, add FixDenat fixative 200 μ l/well, discard fixative after hatching 30min, add anti-BrdU-POD working solution 100 μ l/well, continue to hatch 90min, add lotion liquid 300 μ l/well, lotion three times adds substrate colour developing liquid at last, uses 1M H2SO425 μ l/well cessation reaction behind the reaction 17min.Blank is set, and blank has only culture medium, reads OD on 800 type microplate reader
450Value.
1.2.9. counteracting toxic substances protection experiment
Every group six tail anguilla japonica used TPS-30 bacterial strain lumbar injection counteracting toxic substances to each group counteracting toxic substances in the 45th day in once immune back, and counteracting toxic substances dosage is 5 * 10
7CFU (5 * LD
50).
2 results
2.1. anguilla japonica serum antibody
Once the immunity back is the 44th day, 1 group, 2 groups, 3 groups, 4 groups, 6 groups, 7 groups, 8 groups, 9 groups anguilla japonica serum antibody results as shown in Figure 1, through variance analysis, 1 group of serum antibody titer OD value be significantly higher than 2 groups with 3 groups (p＜0.05), 2 groups of serum antibody OD values are significantly higher than 3 groups (p＜0.05), 1 group of serum antibody OD value be significantly higher than 6 groups with 8 groups (p＜0.05).
2.4. anguilla japonica lymphocyte transformation test result
Immunity back 44d, 1 group, 4 groups, 6 groups, 8 groups, 9 groups anguilla japonica lymphocyte transformation test results as shown in Figure 2, through variance analysis, 1 group and 4 groups drench to change and are significantly higher than 6 groups, 8 groups and 9 groups (p＜0.05), do not have significant difference between wherein reaching 6 groups, 8 groups, 9 groups between 1 group and 4 groups.
2.1. immunoprotection result of the test
This research is 5 * 10 with concentration
7CFU (5 * LD
50) Aeromonas hydrophila TPS30 lumbar injection counteracting toxic substances, be dead peak period in the counteracting toxic substances 48h.Counteracting toxic substances no longer includes the phenomena of mortality and occurs after 1 week.The counteracting toxic substances result is as shown in table 2: through χ
2Check is compared difference all significantly (P＜0.05) for 7 groups with 1 group, 2 groups, 6 groups, 9 groups, compare significant difference (P＜0.05) with 2 groups, 6 groups, 9 groups for 1 group.
Table 2 contrast and immune anguilla japonica are attacked survival rate relatively to TPS30
3, conclusion
Confirm that by the test of anguilla japonica immunoprotection immunostimulating complex ISCOMs is used for the dipping bath immunity not only can improve antibody horizontal, and can improve the lymphocytic propagation conversion capability of T.The induction of immunity protection.During the Isodose immunity, the antibody titer of ISCOMs group is apparently higher than no adjuvant group and ISM1312 adjuvant group.At 30-120mg.L
-1In the concentration range, the working concentration positive correlation of immunoprotection level and vaccine.
Two: the immunostimulating complex (ISCOMs) of Aeromonas hydrophila β-hemA reorganization bacterium preparation adopts the oral methods immunity to anguilla japonica
1. materials and methods
1.1 material is identical with the material in the dipping bath method
1.2 method
1.2.1. antibacterial culturing
β-hemA bacterial classification inoculation in the plain agar culture medium flat plate, is cultivated 24h for 28 ℃, and picking list bacterium colony is inoculated in the TSB culture fluid of 10ml, and 28 ℃ of cultivations when OD value is 1.0, is got 5ml and are inoculated in the TSB culture medium of 500ml and cultivate 20h, gather in the crops.
1.2.2. extracellular products extracts
The centrifugal 30min of 10000rpm gets supernatant, adds ammonium sulfate to 60% saturation, and 4 ℃ are spent the night, the centrifugal 30min of 10000rpm, collecting precipitation, resuspended with 0.01M PBS (PH7.2), dialysis, every 4h changes liquid once, changes liquid 5～6 times, promptly gets and slightly carries extracellular products, and-20 ℃ of preservations are standby.
1.2.3. the mensuration of protein content
Measure according to the Bradford method with DU Series7000 spectrophotometer (BECKMAN), record the A value at wavelength 595nm place, the reference standard curve draws proteic concentration.
1.2.4.ISCOMs preparation
Concentrate extracellular products to 4mg.mL
-1, with add behind the 1M citric acid treatment 2h Mega-10 to final concentration be 2%, 37 ℃ of cracking 4h, add Quil A to final concentration be 0.1%, add cholesterol then, lecithin (dissolving with chloroform in advance) to final concentration is 125 μ g.mL
-1, the ice-bath ultrasonic ripple is handled 8-10min, and the negative pressure of vacuum dechlorination is imitative, earlier with 0.01M citric acid treatment dialysis 4h, reuse 0.01MPBS (pH 7.2) 72h that dialyses, every 4h changes liquid once.
1.2.5. electron microscopic observation β-hemA-ISCOMs
Get testing sample 10 μ L and drip copper mesh,, on the JEM-1200EX Electronic Speculum, observe with the dyeing of 2% acetic acid uranium.
1.2.6.ISCOMs peroral immunity anguilla japonica
One time immunization experiment adopts once immunity, divides 3 groups to carry out, every group 18 tail, and grouping and immunization method are as shown in table 3.Be about 30g for examination anguilla japonica specification, indoor culture, 24 ℃ of temperature are changed water 1/3 every day.Wherein PCB ECPs representative contains beta hemolysin gene recombination bacterium extracellular products, the ISCOMs that PCBECPs ISCOMs representative is made by PCB ECPs.PBS is the PH7.2 phosphate buffer, and concentration is 0.01M.
An oral immunity grouping of table 3: β-hemA-ISCOMs
| Grouping | Immunization method | Mantissa |
| 1PCB ECPs ISCOMs | The continuous 10d of the oral PCB ECPs of every tail every day ISCOMs protein content 50ug | 18 |
| 2PCB ECPs group | The continuous 10d of the oral PCB ECPs of every tail every day protein content 50ug | 18 |
| The 3PBS group | Every day every tail oral with |
18 |
Immunity back 10d and 20d get 4 tail anguilla japonicas respectively for every group and carry out the lymphocyte transformation experiment, and concrete lymphocyte transformation experimental technique is with example 1.Immunity back 30d carries out counteracting toxic substances protection experiment, uses Aeromonas hydrophila TPS-30 with 1.5 * 10
7CFU.ml
-1Concentration lumbar injection counteracting toxic substances, observe a week continuously.The counteracting toxic substances survival rate of PCB ISCOMs group is 44.4%, the counteracting toxic substances survival rate 12.5% of PCB group, the counteracting toxic substances survival rate 12.5% of matched group.
Secondary immunity is adopted in the secondary immunity experiment, divides 2 groups to carry out, every group 7 tail, and grouping and immunization method are as shown in table 4.Be about 30g for examination anguilla japonica specification, indoor culture, 24 ℃ of temperature are changed water 1/3 every day.Wherein PCB ECPs representative contains beta hemolysin gene recombination bacterium extracellular products, the ISCOMs that PCB ECPsISCOMs representative is made by PCB ECPs.PBS is the PH7.2 phosphate buffer, and concentration is 0.01
Table 4: β-hemA-ISCOMs secondary oral immunity grouping
| Grouping | Immunization method | Mantissa |
| 1PCB ECPs ISCOMs | The oral PCB ECPs of every tail every day ISCOMs protein content 50ug, continuous 7d is at interval behind the 7d, again with the protein content continuous oral 7d of 50ug | 7 |
| The 2PBS group | Every day every tail oral with |
7 |
Begin back 44d in immunity, get 2 tail anguilla japonicas for every group, gather serum and splenocyte, carry out the detection of serum antibody titer and the detection of spleen lymphocyte proliferation conversion capability, concrete grammar is with example 1.Begin back 45d in immunity and carry out counteracting toxic substances protection experiment, use Aeromonas hydrophila TPS-30 with 1.3 * 10
7CFU.ml
-1Concentration lumbar injection counteracting toxic substances, observe a week continuously.
2 results:
2.1 immunization experiment result
In immunization experiment; immunity back 10d; 20d ISCOMs group lymphopoiesis conversion capability is significantly higher than non-ISCOMs group and matched group (P＜0.05) (table 5) (Fig. 3); the immune protective rate of immunity back 30d conforms to the lymphopoiesis conversion capability; the immune protective rate of ISCOMs group is 44.4%; the immune protective rate of PCB group is 12.5%, and the immune protective rate of matched group is 12.5%, significant difference (P＜0.05).
Table 5: OD450 value behind the once immune spleen lymphocyte proliferation
| Grouping | PCB ISCOMs group | The PCB group | The PBS group |
| 10d | 0.590 | 0.443 | 0.461 |
| 20d | 0.122 | 0.068 | 0.064 |
2.2 secondary immunity experimental result
Secondary immunity begins back 44d, and the serum antibody titer of ISCOMs group and lymphocyte transformation ability are significantly higher than the PBS group, and secondary immunity begins the counteracting toxic substances of back 45d, and the immune protective rate of ISCOMs group can reach 100% (Fig. 4).
3, conclusion
Confirm that by the test of anguilla japonica immunoprotection ISCOMs is used for oral immunity not only can improve antibody horizontal, and can improve the lymphocytic propagation conversion capability of T, the induction of immunity protection.
In sum, the present invention has following advantage compared to existing technology:
Immunostimulating complex I SCOMs is used for the dipping bath immunity not only can improve antibody horizontal, and can improve the lymphocytic propagation conversion capability of T, the induction of immunity protection.During the Isodose immunity, the antibody titer of ISCOMs group is apparently higher than no adjuvant group and ISM1312 adjuvant group.Same ISCOMs is used for oral immunity and not only can improves antibody horizontal, and can improve the lymphocytic propagation conversion capability of T, the induction of immunity protection.
Description of drawings
Fig. 1 is dipping bath immunity anguilla japonica serum antibody titer figure
Fig. 2 is that the commentaries on classics block diagram is drenched in the dipping bath immunity
The once immune lymphopoiesis of Fig. 3 A right and wrong ISCOMs group oral immunity transforms situation
Fig. 3 B is that the once immune lymphopoiesis of ISCOMs group oral immunity transforms situation
Fig. 4 is an oral immunity secondary immunity design sketch
Fig. 5 A is this research department is about 40nm tool typical case cage grating texture feature with the size of Aeromonas hydrophila outer membrane protein preparation ISCOMs
This research department of Fig. 5 B is about the ISCOMs of 40nm tool typical case cage grating texture feature with the size of Aeromonas hydrophila reorganization hemolysin preparation
The specific embodiment
Below in conjunction with embodiment the present invention is described in more detail.
The immunostimulating complex that the Vibrio vulnificus tropina is made (ISCOMs) is applied to the anguilla japonica oral immunity
1. materials and methods
1.1 material
The strain FJ03-X2 that makes this product separates the blood of suffering from the vibriosis European eel from Fujian in mid-June, 2003, biochemical identification and molecular biology method through strictness are accredited as biological 1 type Vibrio vulnificus, and lumbar injection infects the LD to the European eel of 20-30g/ tail
50Be about 7.1 * 10
3The CFU/ tail.This strain is that the Fujian Province Agriculture Science Academy, Institute of Biotechnology is separated, identifies, preserved.Main agents Quil A is available from Accurate chemical﹠amp; Scientific corporation company; Mega-10 is available from Huamei Bio-Engrg Co.; From anguilla japonica field, permanently happy Hailin, Fujian Province, specification is every tail 50g for the examination anguilla japonica.
The TSB culture medium prescription
Soya peptone 3g, tryptone 17g, NaCl 15g, K
2HPO
42.5g glucose 0.5g adds distilled water to 1000ml, 121 ℃ of autoclaving 20min.
The TSA culture medium prescription
Soya peptone 3g, tryptone 17g, NaCl 15g, K
2HPO
42.5g, glucose 0.5g, Agar 15g.Add distilled water to 1000ml, 121 ℃ of autoclaving 20min.
1.2 method
1.2.1 the pre-amplification and the amplification culture of recovery strain: qualified after testing recovery strain is inoculated in the triangular flask that fills 100mlTSB, 28 ℃ of constant-temperature shaking culture 24h, 100rpm is inoculated in the fresh autoclaved TSB culture fluid with the 1% pre-culture that increases, and cultivates 30h with similarity condition.The culture broth of dress 1L in the conical flask of 2L,
1.2.2 bacteria inactivation:
Collect antibacterial in the aseptic plastic bucket (Plastic Drum is sterilized in advance with 70% ethanol, should use in 48 hours) of 20L, adding final concentration is the formalin of 0.3% (V/V), after ambient temperature overnight leaves standstill, puts 4 ℃ of refrigerators, thoroughly deactivation in the 48h.Steriling test: fully shake up antibacterial, sterilization rifle head is drawn 0.2ml, and superclean bench upper berth flat board is placed 28 ℃ of constant incubators and cultivated 48h, sterilizes completely that culture does not have bacterium colony to occur, and blank dull and stereotyped the contrast established in test.
1.2.3 the centrifugal 30min of centrifugal collection: 6000-8000rpm collects, concentrates antibacterial.The Biomass of antibacterial (weight in wet base) should be 15-17g.L
-1The antibacterial of centrifugal collection is resuspended with deactivation culture supernatant or sterile saline, is settled to 50ml (concentrating 20 times) behind every liter of vaccine centrifugal.
1.2.4 the ultrasonic disruption of antibacterial
The ultrasonic disruption instrument is JY98-3D, and the diameter of hyperacoustic ultrasonic probe (being called horn again) is 20mm, and ultrasonic power is made as 800W, and action routine is: 10s at interval, action time 5s, effect total time is the 6min/ wheel.The ultrasonic disruption instrument carries out cleaning sterilization with 70% ethanol, treats cracking bacterium liquid pre-freeze, pre-moltenly carries out ultrasonication when leaving a small amount of ice crystal, and the effect volume is the concentrated bacterium liquid of special-purpose ultrasonic cup 250ml.Act on 3 altogether and take turns, the omnidistance time is 18min, and the practical function time is 6min.The output initial power power that pointer shows is about 720W, and the power of final stage can be near the value of setting (800W).
The sterilization PBS that uses behind the ultrasonic treatment adjusts bacterioprotein concentration to 25mg.ml for the first time
-1
1.2.5 Vibrio vulnificus tropina ISCOMs vaccine production.
In the tropina of ultrasonic degradation, add 0.10% (W/V) QuilA, 125 μ g.ml
-1Lecithin and cholesterol (final concentration).Because lecithin and cholesterol all are Organic substances not soluble in water, earlier substance dissolves in two is made into 10mg.ml in chloroform when preparation lecithin and cholesterol
-1Storage liquid, 4 ℃ standby.
The thalline crude protein is chilled to 4 ℃ after adding the substrate of the ISCOMs that QuilA, lecithin and cholesterol constitute in proportion in advance, places in the ultrasonic disruption instrument.Program is set to: interval 10s, and ultrasonication 5s, the omnidistance time is 18min.Temperature is not higher than 60 ℃.Under cleaning condition, the ISCOMs of supersound process is collected in the 2L conical flask behind the mixing, measure ISCOMs protein concentration (seeing Appendix 5), adjust the protein concentration of ISCOMs to 30mg.ml with sterilization PBS
-1Obtain goods after twice ultrasonic is handled and be Vibrio vulnificus tropina ISCOMs ,-20 ℃ frozen standby.
1.2.6 with the immune anguilla japonica of Vibrio vulnificus tropina immunostimulating complex (ISCOMs)
By fish body weight 20ml every day dosage spice per ton, (for example feedstuff throwing rate is calculated by 1%, and then the per kilogram feedstuff adds Vibrio vulnificus tropina ISCOMs 5ml to be made for oral immunity preparation of the present invention.The using method of this oral immunity preparation is: continuous oral 5d, obey 5d after two weeks at interval again.1d adds oral by half amount, 2d begins full dose and adds oral.
It is same as the prior art that present embodiment is not stated part
The application of immunostimulating complex (ISCOMs) on the marine fish oral immunity that embodiment 2 sea water pathogen tropinas are made
1. materials and methods
1.1 material
The strain that manufacturing this product of manufacturing this product is used is Carnis Pseudosciaenae vibrio parahaemolytious (wVp), vibrio alginolyticus (Val33) and Vibro harveyi (Vha), by Fujian Province Agriculture Science Academy, Institute of Biotechnology keeping, evaluation and supply.Main agents QuilA is available from Accurate chemical﹠amp; Scientific corporation company; Mega-10 is available from Huamei Bio-Engrg Co.; For trying Carnis Pseudosciaenae, snapper from Fujian Province's Xiapu County.
1%NaCl nutrient broth medium prescription
Peptone 10g, extracted beef powder 3g, NaCl 10g adds distilled water to 1000ml, pH7.3 ± 0.2,121 ℃ autoclaving 20min.
1%NaCl nutrient agar prescription
Peptone 10g, extracted beef powder 3g, Agar 15g, NaCl 10g adds distilled water to 1000ml, pH7.3 ± 0.2,121 ℃ autoclaving 20min.
1.2 method
1.2.1 the amplification culture of bacterial strain
From ultra cold storage freezer, take out to produce and use strain, under 0 ℃, aseptic condition, open fast and protect kind of a vial closure, inoculating loop picking upper strata ice crystal, be seeded in the 100ml 1%NaCl nutrient broth fluid medium, 28 ℃, 100rpm cultivates 20h or is inoculated in flat board (1%NaCl Nutrient agar) and cultivates 20h.Get 500 μ l and cultivate bacterium liquid, be inoculated in the aseptic 1%NaCl nutrient broth liquid medium of 1L prepared fresh (the aseptic conical flask of 2L), 28 ℃, 100rpm constant-temperature shaking culture 30h calculates the bacterium number, puts 4 ℃ of preservations.
The deactivation of thalline
Under the aseptic condition amplification culture bacterium liquid is inoculated in the aseptic plastic bucket of 10L, adds final concentration and be 0.3% formalin, 28 ℃ leave standstill 12h after, be positioned over 4 ℃ of refrigerator 48h.Fully shake up bacterium liquid behind the 48h, aseptic absorption 0.2ml bacterium liquid coating 1%NaCl nutrient agar panel, coating process sterile working.Coated 1%NaCl nutrient agar panel is placed 28 ℃ of constant incubator 24h and is cultivated, and sterilizing completely, culture should not have visible bacterium colony to appear on the 1%NaCl nutrient agar panel.
1.2.3 the collection of thalline
Deactivation is centrifugal 30min of bacterium liquid 6000rpm or the centrifugal 15min of 10000rpm completely, collects the deactivation thalline.The sterile saline washing of the thalline usefulness original bacteria liquid volume 3.3% of centrifugal collection, resuspended, concentrated, the thalline after concentrating quantitatively is 9 * 10 with sterile saline
10CFU.ml
-1
1.2.4 tropina preparation
Ultrasonic degradation prepares the thalline crude protein: the ultrasonic disruption instrument is JY98-3D, and hyperacoustic ultrasonic probe diameter is 20mm.Under the aseptic condition, the antibacterial concentrated solution is displaced to ultrasonic disruption instrument cup special (with the sterilization of 70% alcohol wash), the antibacterial concentrated solution of every glass of packing 250ml.It is indoor to have the cup special of dress antibacterial resuspended liquid to be positioned over Ultrasound Instrument, 20mm place to the resuspended liquid level of antibacterial is fixing with the ultrasound probe bottom of alcohol disinfecting, close Ultrasound Instrument chamber door, (working time 5s, blanking time 10s, work number of times 32-48 time, ultrasonic power is made as 720W to set the ultrasonic disruption parameter.), starting the ultrasonication program, the tropina for preparing behind the ultrasonic degradation places 4 ℃ of low temperature standby.
1.2.5 the preparation of sea water pathogen tropina ISCOMs
The tropina that will obtain through ultrasonic disruption, under room temperature condition successively with QuilA, the 125 μ g.ml of 0.1% (W/V)
-1Lecithin, 125 μ g.ml
-1Cholesterol mixes, and repeats 1.2.4 ultrasonication program, and the goods that obtain are end article.
1.2.6 the immune marine fish made from sea water pathogen tropina of immune complex stimulus complex (ISCOMs)
The tropina of vibrio parahaemolytious wVp2, vibrio alginolyticus Val33 and Vibro harveyi Vha is made the ISCOMs trigeminy vaccine with 1: 1: 1 ratio, and vaccine thalline content is 9 * 10
10CFU.ml
-1, before vibrio parahaemolytious, vibrio alginolyticus and Vibro harveyi onset peak period, the Carnis Pseudosciaenae (young adult fish) of sea area cage culture is carried out field oral immunity experiment.Make oral immunity preparation (as feedstuff throwing rate is 5%, and then the per kilogram feedstuff adds this vaccine 1ml) by fish body weight 50ml every day ISCOMs trigeminy vaccine dosage spice per ton.The instructions of taking of this oral immunity preparation is: continuous oral 7 days, obeyed again 7 days after the week at interval.First day oral by half amount interpolation, and the beginning full dose was added oral in second day.
It is same as the prior art that present embodiment is not stated part
Embodiment 3: the application of the immunostimulating complex that Aeromonas hydrophila is made (ISCOMs) on the anguilla japonica oral immunity
1. materials and methods
1.1 material
The t bacteria PS30 that makes this product is provided by fresh water institute money east, Zhejiang, and ZN1 is that biotechnology research institute in academy of agricultural sciences, Fujian Province separated the European eel from Zhouning County, Fujian Province trouble Aeromonas hydrophila septicemia in 2004.Above strain is by Fujian Province Agriculture Science Academy, Institute of Biotechnology keeping, evaluation and supply.Main agents QuilA is available from Accurate chemical﹠amp; Scientific corporation company; Mega-10 is available from Huamei Bio-Engrg Co.; For trying anguilla japonica from Fujian Province's Changle city.
The TSB culture medium prescription
Soya peptone 3g, tryptone 17g, NaCl 15g, K
2HPO
42.5g glucose 0.5g adds distilled water to 1000ml, 121 ℃ of autoclaving 20min.
The TSA culture medium prescription
Soya peptone 3g, tryptone 17g, NaCl 15g, K
2HPO
42.5g, glucose 0.5g, Agar 15g.Add distilled water to 1000ml, 121 ℃ of autoclaving 20min.
1.2 method
1.2.1 the amplification culture of bacterial strain
Take out to produce from ultra cold storage freezer and use strain, under 0 ℃, aseptic condition, open fast and protect kind of a vial closure, inoculating loop picking upper strata ice crystal is seeded in the 100mlTSB fluid medium, and 28 ℃, 100rpm cultivates 20h or is inoculated in the dull and stereotyped cultivation of TSA 20h.Get 500 μ l and cultivate bacterium liquid, be inoculated in the TSB fluid medium of 1L prepared fresh (the aseptic conical flask of 2L), 28 ℃, 100rpm constant-temperature shaking culture 30h calculates the bacterium number, puts 4 ℃ of preservations.
The deactivation of thalline
Under the aseptic condition amplification culture bacterium liquid is inoculated in the aseptic plastic bucket of 10L, adds final concentration and be 0.3% formalin, 28 ℃ leave standstill 12h after, be positioned over 4 ℃ of refrigerator 48h.Fully shake up bacterium liquid behind the 48h, aseptic absorption 0.2ml bacterium liquid coating 1%NaCl nutrient agar panel, coating process sterile working.Coated TSA flat board is placed 28 ℃ of constant incubator 24h and is cultivated, and sterilizing completely, culture should not have visible bacterium colony to appear on the 1%NaCl nutrient agar panel.
1.2.3 the collection of thalline
Deactivation is centrifugal 30min of bacterium liquid 6000rpm or the centrifugal 15min of 10000rpm completely, collects the deactivation thalline.The sterile saline washing of the thalline usefulness original bacteria liquid volume 3.3% of centrifugal collection, resuspended, concentrated, the thalline after concentrating quantitatively is 9 * 10 with sterile saline
10CFU.ml
-1
1.2.4 tropina preparation
Ultrasonic degradation prepares the thalline crude protein: the ultrasonic disruption instrument is JY98-3D, and hyperacoustic ultrasonic probe diameter is 20mm.Under the aseptic condition, the antibacterial concentrated solution is displaced to ultrasonic disruption instrument cup special (with the sterilization of 70% alcohol wash), the antibacterial concentrated solution of every glass of packing 250ml.It is indoor to have the cup special of dress antibacterial resuspended liquid to be positioned over Ultrasound Instrument, 20mm place to the resuspended liquid level of antibacterial is fixing with the ultrasound probe bottom of alcohol disinfecting, close Ultrasound Instrument chamber door, (working time 5s, blanking time 10s, work number of times 32-48 time, ultrasonic power is made as 720W to set the ultrasonic disruption parameter.), starting the ultrasonication program, the tropina for preparing behind the ultrasonic degradation places 4 ℃ of low temperature standby.
1.2.5 the preparation of Aeromonas hydrophila tropina ISCOMs
The TPS-30 tropina and the ZN1 tropina mixed in equal amounts that will obtain through ultrasonic disruption, under room temperature condition successively with QuilA, the 125 μ g.ml of 0.1% (W/V)
-1Lecithin, 125 μ g.ml
-1Cholesterol mixes, and repeats 1.2.4 ultrasonication program, and the goods that obtain are end article, and vaccine protein content is 31mg.ml
-1
1.2.6 with Aeromonas hydrophila tropina ISCOMs immunity anguilla japonica
The two valency Seedlings of the ISCOMs that Aeromonas hydrophila TPS-30, ZN1 make in this seminar are at previous month of Aeromonas hydrophila onset peak period (2005.4), carry out field oral immunity experiment to throwing the anguilla japonica that Seedling cultures then.(as feedstuff throwing rate is 1% to make oral immunity preparation of the present invention by the two valency Seedling dosage spices of fish body weight 50ml every day ISCOMs per ton, then the per kilogram feedstuff adds this vaccine 5ml, as feedstuff throwing rate is 2%, and then the per kilogram feedstuff adds this vaccine 2.5ml, by that analogy).The using method of this oral immunity preparation is: continuous oral 7 days, obeyed again 7 days after the week at interval.First day oral by half amount interpolation, and the beginning full dose was added oral in second day.
It is same as the prior art that present embodiment is not stated part.
Embodiment 4
To be applied to anguilla japonica in the dipping bath mode according to the immunostimulating complex (ISCOMs) that the Vibrio vulnificus tropina of embodiment 1 method preparation is made.
The ISCOMs vaccine that Vibrio vulnificus is made is previous month of Vibrio vulnificus onset peak period, carries out field dipping bath immunization experiment to throwing the anguilla japonica that Seedling cultures then.Add immunostimulating complex by the amount of 100 milliliters in water per ton and make dipping bath immune formulation of the present invention.The using method of this dipping bath immune formulation is, continues to change water after 1 hour, finishes once immunity.
It is same as the prior art that present embodiment is not stated part
Embodiment 5
The application of immunostimulating complex (ISCOMs) in the immunity of marine fish dipping bath that to make according to the sea water pathogen tropina of embodiment 2 preparation
The tropina of vibrio parahaemolytious wVp2, vibrio alginolyticus Val33 and Vibro harveyi Vha is made the ISCOMs trigeminy vaccine with 1: 1: 1 ratio, and vaccine thalline content is 9 * 10
10CFU.ml
-1, before vibrio parahaemolytious, vibrio alginolyticus and Vibro harveyi onset peak period, the Carnis Pseudosciaenae (young adult fish) of sea area cage culture is carried out field dipping bath immunization experiment.Add corresponding immunostimulating complex by water 100 ml vols per ton and make dipping bath type immune formulation, the using method of this dipping bath type immune formulation is: continue to change water after 1 hour, finish once immunity.The amount of pressing 100 milliliters in water per ton after two weeks is again added at interval, continues to change water after 1 hour, finishes secondary immunity.
It is same as the prior art that present embodiment is not stated part
Embodiment 6
The application of immunostimulating complex (ISCOMs) in the immunity of anguilla japonica dipping bath that to make according to the Aeromonas hydrophila of embodiment 3 preparation
The two valency Seedlings of the ISCOMs that Aeromonas hydrophila TPS-30, ZN1 are made are previous month of Aeromonas hydrophila onset peak period, carry out field dipping bath immunization experiment to throwing the anguilla japonica that Seedling cultures then.Add corresponding immunostimulating complex by water 100 ml vols per ton and make dipping bath type immune formulation.The using method of this immune formulation is: continue to change water after 1 hour, finish once immunity.The amount of pressing 100 milliliters in water per ton after two weeks is again added at interval, continues to change water after 1 hour, finishes secondary immunity.
It is same as the prior art that present embodiment is not stated part.
Embodiment 7
The application of immunostimulating complex (ISCOMs) in anguilla japonica oral immunity, dipping bath immunity that Aeromonas hydrophila lipopolysaccharide composition is made
Aeromonas hydrophila TPS-30 and ZN1 are seeded in the 100mlTSB fluid medium, and 28 ℃, 100rpm cultivates 20h or is inoculated in the dull and stereotyped 20h of cultivation of TSA.Get 500 μ l and cultivate bacterium liquid, be inoculated in the TSB fluid medium of 1L prepared fresh (the aseptic conical flask of 2L), 28 ℃, 100rpm constant-temperature shaking culture 30h, centrifugal 30min of 6000rpm or the centrifugal 15min of 10000rpm collect the antibacterial thalline, 0.02M/LTris-HCl (PH7.6-7.8), the washed twice standardize solution, 1g antibacterial (weight in wet base) is resuspended in the pure water of 3ml approximately, adds preheating 68 ℃ of saturated phenol, constantly stirring and evenly mixings of equal-volume 95%.About 15min, be cooled to 10-15 ℃, 10000r/min4 ℃ of centrifugal collection, the upper strata is the saturated water of phenol, the centre is a Denatured protein, the stratum is the water-saturated phenol phase, beats easily the upper strata, and the stratum needs twice of minimum extracting repeatedly, 4 ℃ of fully dialysis, obtain gelatin-like LPS, make ISCOMs, make anguilla japonica oral immunity preparation and dipping bath immune formulation by the method for embodiment 3.
It is same as the prior art that present embodiment is not stated part.
Embodiment 8
The application of immunostimulating complex (ISCOMs) in the Carnis Pseudosciaenae immunity that vibrio parahaemolytious and vibrio alginolyticus polysaccharide antigen are made
Vibrio parahaemolytious wVp2, vibrio alginolyticus Val33 are inoculated in respectively in the 100ml 1%NaCl nutrient broth fluid medium, and 28 ℃, 100rpm cultivates 20h or is inoculated in flat board (1%NaCl Nutrient agar) and cultivates 20h.Get 500 μ l and cultivate bacterium liquid, be inoculated in the aseptic 1%NaCl nutrient broth liquid medium of 1L prepared fresh (the aseptic conical flask of 2L), 28 ℃, 100rpm constant-temperature shaking culture 30h.Boiling water boils 10min, and the centrifugal collection thalline of desk centrifuge 10000r/min composition, normal saline are resuspended in the sterile saline of 0.2ml at last with method washing 3 times.Add E.C. 3.4.21.64 (25mg/ml) 5 μ l, mixing is put 60 ℃ of about 1h of constant water bath box, the centrifugal 10min of 10000/min, and the application of sample rifle is gently drawn supernatant, makes ISCOMs by the method for embodiment 2.The oral immunity preparation and the dipping bath immune formulation that are used for Carnis Pseudosciaenae.
What adopt among the above embodiment is Aeromonas hydrophila, Vibrio vulnificus, vibrio alginolyticus, the immunostimulating complex that vibrio parahaemolytious breathes out the preparation of Vickers mycoprotein utilizes oral, the method of dipping bath is carried out, can certainly be with Aeromonas hydrophila wherein, Vibrio vulnificus, vibrio alginolyticus, vibrio parahaemolytious breathes out Vickers mycoprotein composition or polysaccharide composition virus, parasitic albumen composition or polysaccharide composition prepare virus immunity as raw material stimulates complex or parasite immunostimulating complex, through admixing feedstuff or dissolving in the water, make corresponding oral immunity preparation or dipping bath immune formulation.
Claims (5)
1. the application of immunostimulating complex in preparation fish immunity preparation, it is characterized in that: described immune formulation is the oral immunity preparation or is the dipping bath immune formulation that described immune formulation is the immune formulation that the immunostimulating complex of Aeromonas hydrophila β-hemA reorganization bacterium preparation is made.
2. the application of immunostimulating complex according to claim 1 in preparation fish immunity preparation is characterized in that: described oral immunity preparation is for stirring immunostimulating complex and fish meal.
3. the application of immunostimulating complex according to claim 1 in preparation fish immunity preparation, it is characterized in that: described dipping bath immune formulation is for to add immunostimulating complex in the entry.
4. the application of immunostimulating complex according to claim 1 in preparation fish immunity preparation, it is characterized in that: the fish oral, that dipping bath is immune that is applicable to described immunostimulating complex comprises fresh-water fishes and marine fish.
5. the application of immunostimulating complex according to claim 4 in preparation fish immunity preparation is characterized in that: oral, the dipping bath of described immunostimulating complex is immune to be particularly useful for Anguillar japonica, Carnis Pseudosciaenae, tilapia.
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| CN102210862B (en) * | 2011-06-01 | 2013-06-12 | 中国人民解放军南京军区福州总医院 | Livin-based immune-stimulating complex, preparation method thereof and application thereof |
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| CN108030918B (en) * | 2017-12-21 | 2021-12-24 | 福建省农业科学院生物技术研究所 | Immunostimulating compound and preparation method and application thereof |
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| WO2001076623A1 (en) * | 2000-04-12 | 2001-10-18 | Astrazeneca Ab | Iscom polypeptide delivery system for helicobacter pylori antigens |
| CN1583171A (en) * | 2003-08-22 | 2005-02-23 | 福建省农业科学院畜牧兽医研究所 | Preparation of immuno-stimulation composition for hemolycin in monad |
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