CN1293066A - Concatenated antidiotype monoclonic antibody vaccine for fish diseases - Google Patents
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Abstract
A concatenated anti-idiotype monoclonic antibody vaccine for fish disease is prepared through immunizing mouse with eel vibrio, wound vibrio, alga-dissolving vibrio and delayed Edward siella, cell fusion, cloning, purifying, immunizing mouse, preparing highly specific monoclonic antibody with high effective volency, screening, and preparing said vaccine. Its advantages are high immunoprotection action to fish, no environmental pollution and no damage to human.
Description
The present invention relates to biological technical field
In recent years along with the fast development of China's sea-farming fishery, the particularly bacillary fish diseases that causes of fish diseases all causes the mortality of breed ocean fishes every year, has had a strong impact on the development of sea-farming fishery.To the prevention of fish diseases, also rest on the classical inactivated vaccine stage both at home and abroad so far.Existing 8 Fish vibrio inactivated vaccines and preparation method thereof obtain Japan, United States Patent (USP) in the world.Institute of oceanography, China Chinese Academy of Sciences South Sea has also successfully prepared ocean fishes Vibrio vulnificus inactivated vaccine at nineteen ninety-five.Do not see the report that other ocean fishes pathogenic bacteria successfully prepares in addition.Medically, the history in existing more than 200 year of the successful Application of vaccine, along with the development of medical science technology, the formation of vaccine is also brought in constant renewal in, the nucleic acid vaccine that has attenuation and inactivated vaccine, subunit vaccine, synthetic peptide vaccine, anti-idiotype antibody vaccine and newly-developed to get up.Anti-idiotype antibody has and the similar structure of the antigenic determinant of pathogen, and it is a kind of analogue antigen, and energy inducing self-body or allosome produce the antibody of opposing pathogen, make body obtain part or all of protective effect.This point is confirmed by a large amount of tests.The anti-idiotype antibody vaccine also has following unique advantage: 1. its can analogue antigen, but does not contain poisonous antigenic composition, and is nontoxic, safe and reliable; 2. not exist kind be difference to anti-idiotype antibody, need not consider kind to be problem during use.Through the update search of Internet terminal, there is no the report that the anti-idiotype antibody vaccine is applied to culture fish so far both at home and abroad.
The objective of the invention is to utilize biotechnology, produce multiple fish diseases bacterium anti-idiotype monoclonal antibodies, filter out β type anti-idiotype monoclonal antibodies, and simulate pathogenic bacteria and induce body to produce the antibody of disease-resistant bacterium, make body obtain part or all of protective effect.Above anti-idiotype monoclonal antibodies can be made into fish diseases anti-idiotype antibody vaccine, plays good immanoprotection action to culturing fish, and is free from environmental pollution, can not cause damage to the mankind by food chain.
Technical scheme of the present invention:
Vibrio anguillarum; Vibrio vulnificus; vibrio alginolyticus; slow love moral vibrio bacterial strain (providing) by Qingdao Marine University; carry out amplification cultivation; bacterium liquid lumbar injection immune mouse Ba/b/c mice after will cultivating again; get immunity back mouse boosting cell and SP2/0 myeloma cell and carry out cell fusion; the cell suspension of cell fusion is cultivated; detect positive colony with enzyme linked immunoassay method (ELISA method); to be defined as male hole with limiting dilution assay; carry out cloning; again with the hybridoma cell strain amplification cultivation of positive colony; lumbar injection is inoculated into the Ba/b/c mice and prepares the ascitic type monoclonal antibody; above ascitic type monoclonal antibody is tired with the ELISA method; subclass and cross matching; select the higher monoclonal antibody of tiring, ocean fishes is carried out protection test, selecting has the monoclonal antibody of higher protective rate to carry out purification to ocean fishes; with the amount of every mice 10～100 μ g, the lumbar injection immune mouse.Carry out cell fusion then, filter out the hybridoma cell strain of energy stably excreting anti-idiotype monoclonal antibodies,, be adjusted into 1 * 10 the cell strain amplification cultivation
6/ ml, with every mice 0.5ml amount, lumbar injection is inoculated into the Ba/b/c mice, and preparation ascitic type anti-idiotype monoclonal antibodies adopts the ELISA method, and above monoclonal antibody is carried out subclass, tires and the evaluation of β type anti-idiotype monoclonal antibodies.The result shows, 4 strain monoclonal antibody 1D1,1E10,1H5,1H12 are arranged in the Vibrio anguillarum antiidiotype monoclonal antibody, 2 strain monoclonal antibody 1C9,4A5 are arranged in the Vibrio vulnificus antiidiotype monoclonal antibody, 2 strain monoclonal antibody 2F4,1B2 are arranged in the vibrio alginolyticus antiidiotype monoclonal antibody, 2 strain monoclonal antibody 1E5,1E11 are arranged in the Edwardsiella tarda antidiotypic monoclonal antibody, all can combine with ab1 (ascitic type monoclonal antibody) with antigen (pathogenic bacteria) competition specifically.With these monoclonal antibody immune mouses, can from mouse blood, detect the antibody of disease-resistant bacterium, illustrate that above monoclonal antibody is a β type anti-idiotype monoclonal antibodies.Mixed with above monoclonal antibody and incomplete Freund's adjuvant (1: 1～1: 2), lumbar injection immunity ocean fishes, matched group replaces monoclonal antibody with normal saline.After the immunity one month, infect ocean fishes, observe the protective effect of vaccine ocean fishes with lethal dose pathogenic bacteria lumbar injection.The result shows, has three strain monoclonal antibody 1E10,1D1,1H12 that the protective rate of ocean fishes is respectively 88.7%, 82.5% and 81.2% in the Vibrio anguillarum antiidiotype monoclonal antibody; There are 2 strain monoclonal antibody 1E11 and 1E5 that the protective rate of ocean fishes is respectively 60% and 20.8% in the Edwardsiella tarda antidiotypic monoclonal antibody; There are 2 strain monoclonal antibody 1B2 and 2F4 that the protective rate of ocean fishes is respectively 90% and 50% in the vibrio alginolyticus antiidiotype monoclonal antibody.Pick out the higher antiidiotype monoclonal antibody of protective rate and be mixed with the multi-joint antiidiotype monoclonal antibody of fish diseases bacterin preparation with certain proportion.
Embodiments of the invention:
(1) material
1. strain: the reference culture of Vibrio anguillarum, wound vibrio, vibrio alginolyticus, Edwardsiella tarda is provided by life institute of Qingdao Marine University.Above strain produces monoclonal antibody as immunogen.
The reference culture of vibrio alginolyticus, vibrio parahaemolytious, Vibrio vulnificus, Vibrio flurialis, Vibrio furnissii, Vibrio hollisae, vibrio mimicus, maiden vibrio, Maxwell vibrio is available from Beijing biological product company.The shigella flexneri of enterotoxigenic Escherichia coli, enteroinvasive E.Coli, enterohemorrhagic Escherichia coli, Shigella, Jue Shi shigella, shigella dysenteriae, Song Shi shigella are identified institute available from Chinese pharmaceutical biological product.Above strain is as the monoclonal antibody cross matching.
2. cell born of the same parents: the strain of SP2/0 murine myeloma cell, be so kind as to give by The Fourth Military Medical University cell engineering research center.
(2) experimental technique
1. preparation of immunogen antibacterial and deactivation
Vibrio anguillarum, vibrio alginolyticus, Vibrio vulnificus, Edwardsiella tarda, spend the night in 37 ℃ of incubators on bacteria culture media with plate loop method, physiological saline solution eluting lawn, and centrifugal 20 minutes of 3000rpm abandons supernatant, and the formaldehyde fixed of adding 1% is spent the night.Centrifugal 20 minutes of 3000rpm abandons supernatant, adds physiological saline solution, and after turbid numeration, 4 ℃ of refrigerators are preserved, in order to being used as immune original bacteria liquid.
2.Ba/b/c the immunity of mice
10 of the Ba/b/c female mices in 8 ages in week are with the antibacterial bacterium liquid (1 * 10 of above deactivation
8Individual/ml) with 0.5ml/ amount only, the lumbar injection immune mouse, the 7th, 14,28 day difference supplementary immunization after immunity for the first time, extracting spleen cell carried out cell fusion in the 31st day.
3. cell fusion
(1) preparation of immune spleen cell
1. get the immune mouse spleen, put into the plate that fills the incomplete culture fluid of 5ml, place on the 200 purpose rustless steel copper mesh.Grind spleen with plunger, and wash steel mesh gently, splenocyte all is expressed in the solution by mesh with incomplete culture fluid in the plate.
2. splenocyte suspension is changed in the 50ml centrifuge tube, add incomplete culture fluid, mixing, centrifugal 5 minutes of 1000rpm, supernatant discarded to 30ml.
3. too many or too much for use full culture fluid with sedimentation cell centrifuge washing once (repeating step 2.) again, cell is overhang to 10ml, mixing is with the numeration of cell numeration plate.
(2) preparation of SP2/0 myeloma cell's suspension
1. the myeloma cell with 4 bottles of amplification cultivation collects in the 50ml centrifuge tube.
2. 1000rpm is centrifugal 5 minutes, supernatant discarded.
3. add the incomplete culture fluid of 30ml again, repeating step 2. again centrifuge washing once then cell is overhang to 10ml, mixing, the numeration.
(3) cell fusion
1. draw respectively and contain 1 * 10
8Individual splenocyte and suspension and contain 2-3 * 10
7Individual myeloma cell's suspension adds in the 50ml centrifuge tube, adds incomplete culture fluid to 30ml, fully mixing.
2. 1000rpm is centrifugal 7 minutes, supernatant discarded.
3. add 50% fusion agent PEG 0.7ml.
4. add the incomplete culture fluid of 25ml, make PEG dilution and lose the short effect of melting.
5. 800rpm is centrifugal 7 minutes, supernatant discarded.
6. add 20ml HAT culture fluid, the pressure-vaccum sedimentation cell overhangs it and mixing gently.
7. cell suspension being added to assist has in 96 well culture plates of feeder cells, and every hole 0.1ml contains 5%CO at 37 ℃
2Incubator in cultivate.
4. antibody test--indirect elisa method
(1) with known antigens inactivated bacterial liquid bag quilt.
(2) be added with the culture supernatant 100 μ l that clone in the long-living hole.
(3) enzyme-added mark monoclonal antibody Mus IgG antibody 100 μ l.
(4) add substrate solution 100 μ l, room temperature lucifuge colour developing 10～20 minutes.
(5) result of determination: measure the OD value under enzyme-linked immunosorbent assay instrument 492nm wavelength, the blank zeroing is if treat that gaging hole OD value more than or equal to 2.1 times of negative control holes, is positive colony.
5. cloning--limiting dilution assay
The hybridoma of waiting to do cloning in 96 orifice plates is blown and beaten the numeration of even back repeatedly with sample injector, take limiting dilution assay to carry out stepwise dilution then, be added to 96 orifice plates and cultivate.Observe clonal growth, label clone hole, and in time carry out positive detection.Forward the cell of positive monoclonal to 24 orifice plates and cultivate, treat that it is bred after some to change amplification culture in the cell bottle again over to.
6. hybridoma is frozen
To become the hybridoma of logarithmic growth to be collected into centrifuge tube, centrifugal 10 minutes of 1000rpm abandons supernatant, adds 1ml cryopreserving liquid mixing, is added to frozen pipe then, moves into the liquid nitrogen container long preservation at-70 ℃ for the temperature refrigerator back of spending the night.
7. ascitic type Monoclonal Antibody
The hybridoma of cultivating is blown and beaten, and centrifugal 10 minutes of 1000rpm collects supernatant and does the hypotype test, adds serum-free medium in sedimentary cell, and adjusting cell number is 1 * 10
6Individual/ml.Every Ba/b/c injected in mice 0.5ml, behind the inoculation hybridoma 7～14 days, draw neck dislocation to put to death mice, draw ascites, centrifugal, collect supernatant, after the packing-20 ℃ frozen standby.
8. the evaluation of monoclonal antibody
The anti-vibrio anguillarum monoclonal antibody screens the positive cell strain of 8 strains energy stably excreting monoclonal antibody, called after 2H2,3B2,3D2,3A3,3C4,4A6,4E3 and 4E12 respectively.Wherein 2H2,3D2,3B2 and 4E12 are IgG3 through subgroup identification; 4E3 is IgG
2a3A3,3C4,4A6 are IgG
1Through titration, it is 1 * 10 that these monoclonal antibodies are tired
-5～1 * 10
-7
Anti-Vibrio vulnificus monoclonal antibody screens the hybridoma cell strain of 12 strains energy stably excreting monoclonal antibody, called after 1B12,3E9,3F11,3E3,2G8,3D5,1E11,3H4,2F1,1A1,2F6 and 1G1.Through subgroup identification, wherein 2F6 is IgG
3, 1B12,3E3,3H4,1A1 be IgG
2a2G8,3D5,2F1 are IgG
1Through titration, tiring of above monoclonal antibody is 1 * 10
-5～1 * 10
-7
Anti-vibrio alginolyticus monoclonal antibody screens in the hybridoma of 8 strains energy stably excreting monoclonal antibody, called after 2E3,2E4,2G2,2G8,2D11,3A10,3B12 and 4C7.Through subgroup identification, wherein 2E3 is IgG
3, 2G2,2D11,3A10 are IgG
2a2E4,3B12 are IgG
2b2G8,4C7 are IgG
1Through the evaluation of tiring, tiring of above monoclonal antibody is 1 * 10
-6～1 * 10
-7
Anti-Wdwardsiella tarda monoclonal antibody screens 10 strain monoclonal antibody hybridoma cell strains, respectively called after 1B4,4D3,4D5,3F7,5A11,5H5,6B1l, 6E1,6E6 and 6H3.Through subgroup identification, wherein 1B4,3F7,5A11,5H5,6B11 are IgG
1, 6El is IgG
2b4D3,4D5,6E6,6H3 are IgG
3Through titration, tiring of above monoclonal antibody is 1 * 10
-6～1 * 10
-7
(3) preparation of antiidiotype monoclonal antibody (ab2)
1. animal immune: above monoclonal antibody is carried out purification, then with the lumbar injection immune mouse, immunity for the first time: 500 a μ l/ mice (100 μ g/ antibody proteins/only).
Preceding 3 days of the 2nd, 4,6 weeks after the immunity first time and cell fusion, respectively in kind each supplementary immunization is once.
2. cell fusion:
(1) preparation of immune spleen cell
1. get the immune mouse spleen, put into the plate that fills the incomplete culture fluid of 5ml, place on the 200 purpose rustless steel copper mesh.Grind spleen with plunger, and wash steel mesh gently, splenocyte all is expressed in the solution by mesh with incomplete culture fluid in the plate.
2. splenocyte suspension is changed in the 50ml centrifuge tube, add incomplete culture fluid, mixing, centrifugal 5 minutes of 1000rpm, supernatant discarded to 30ml.
3. too many or too much for use full culture fluid with sedimentation cell centrifuge washing once (repeating step 2.) again, cell is overhang to 10ml, mixing is with the numeration of cell numeration plate.
(2) preparation of SP2/0 myeloma cell's suspension
1. the myeloma cell with 4 bottles of amplification cultivation collects in the 50ml centrifuge tube.
2. 1000rpm is centrifugal 5 minutes, supernatant discarded.
3. add the incomplete culture fluid of 30ml again, repeating step 2. again centrifuge washing once then cell is overhang to 10ml, mixing, the numeration.
(3) cell fusion
1. draw respectively and contain 1 * 10
8Individual splenocyte and suspension and contain 2-3 * 10
7Individual myeloma cell's suspension adds in the 50ml centrifuge tube, adds incomplete culture fluid to 30ml, fully mixing.
2. 1000rpm is centrifugal 7 minutes, supernatant discarded.
3. add 50% fusion agent PEG 0.7ml.
4. add the incomplete culture fluid of 25ml, make PEG dilution and lose the short effect of melting.
5. 800rpm is centrifugal 7 minutes, supernatant discarded.
6. add 20ml HAT culture fluid, the pressure-vaccum sedimentation cell overhangs it and mixing gently.
7. cell suspension being added to assist has in 96 well culture plates of feeder cells, and every hole 0.1ml contains 5%CO at 37 ℃
2Incubator in cultivate.
3. anti-idiotype antibody detects--double antibodies sandwich ELISA method
(1) with the disease-resistant bacteria antibody coated elisa plate of the rabbit behind the purification
(2) be added with culture supernatant 100 μ l in the hole of clonal growth
(3) enzyme-added mark monoclonal antibody Mus IgG antibody 100 μ l
(4) add substrate solution 100 μ l, room temperature lucifuge colour developing 10～20 minutes
(5) result of determination: measure the OD value under enzyme-linked immunosorbent assay instrument 492nm wavelength, the blank zeroing is if treat that gaging hole OD value more than or equal to 2.1 times of negative control holes, is positive colony.
4. cloning
The hybridoma of waiting to do cloning in 96 orifice plates is blown and beaten the numeration of even back repeatedly with sample injector, take limiting dilution assay to carry out stepwise dilution then, be added to 96 orifice plates and cultivate.Observe clonal growth, label clone hole, and in time carry out positive detection.Forward the cell of positive monoclonal to 24 orifice plates and cultivate, treat that it is bred after some to change amplification culture in the cell bottle again over to.
5. hybridoma is frozen
To become the hybridoma of logarithmic growth to be collected into centrifuge tube, centrifugal 10 minutes of 1000rpm abandons supernatant, adds 1ml cryopreserving liquid mixing, is added to frozen pipe then, is-70 ℃ of immigration liquid nitrogen container long preservation of spending the night for temperature refrigerator.
6. the preparation of ascitic type anti-idiotype monoclonal antibodies
The hybridoma of cultivating is blown and beaten, and centrifugal 10 minutes of 1000rpm collects supernatant and does straight type test, adds serum-free medium in sedimentary cell, and adjusting cell number is 1 * 10
6Individual/ml.Every Balb/c injected in mice 0.5ml, behind the inoculation hybridoma 7～14 days, draw neck dislocation to put to death mice, draw ascites, centrifugal, collect supernatant, after the packing-20 ℃ frozen standby.
7. the hypotype and the evaluation of tiring
(1) the Vibrio anguillarum anti-idiotype monoclonal antibodies screens 7 strain positive colonies altogether, respectively called after: 1E10,1H12,2H12,1H5,1D1,2B12,2F12.Identify through hypotype: 1E10 belongs to IgG
3, 2H12 and 1H12 belong to IgG
2a, 1H5,1D1,2B12,2F12 are IgG
2bThrough titration, more than the scope of tiring of anti-unique monoclonal antibody is 1 * 10
-4～1 * 10
-6
(2) the Edwardsiella tarda antidiotypic monoclonal antibody screens 6 strain positive colonies altogether, respectively called after 2C8,1E5,2F1,2B3,1E11,1B11.Through subgroup identification 2C8,2F1 and 1B11 is IgG
2b1E11,2B3,1E5 are IgG3.Through titration, tiring of above anti-idiotype antibody can reach 1 * 10
-5～1 * 10
-6
(3) Vibrio vulnificus antiidiotype monoclonal antibody screens 5 strain positive colonies altogether, respectively called after 1C9,4A5,3A12,1C11,3B7.Through subgroup identification 1C9,3A12 is IgG
2b, 4A5 is IgG
2a, 1C11,3B7 be IgG3.Through titration, tiring of above antibody is 1 * 10
-4～1 * 10
-5
(4) vibrio alginolyticus antiidiotype monoclonal antibody screens 5 strain positive colonies altogether, respectively called after 1H2,2F4,1D10,1H5,1B2.Through subgroup identification, 1H2 is IgG
2a, 2F4 is IgG
2b, 1D10 and 1B2 are IgG3,1H5 is IgG1.Through titration, tiring of above monoclonal antibody is 1 * 10
-4～1 * 10
-5
8. the evaluation of β type anti-idiotype antibody
(1) suppresses to measure demonstration through competition, Vibrio anguillarum anti-idiotype monoclonal antibodies 1D1,1E10 and 1H12 can effectively suppress antigen and combine with Ab1, suppression ratio reaches more than 70%, and suppression ratio reduces with the decline of Ab2 concentration, the above three strain monoclonal antibodies Ab1 epi-position identical with antigen recognition is described, can be effectively and antigenic competition Ab1 binding site, illustrate that they are β type Vibrio anguillarum anti-idiotype monoclonal antibodies.
(2) Edwardsiella tarda antidiotypic monoclonal antibody 1E5 and 1E11 can suppress combining of antigen and Ab1 strongly, and suppression ratio reaches more than 60%, and suppression ratio reduces with the reduction of its concentration.The Ab1 epi-position that this 2 strain monoclonal antibody is identical with antigen recognition is described, effectively with the binding site of antigenic competition Ab1.Illustrate that they are β type Edwardsiella tarda antidiotypic monoclonal antibodies.
9. be that immunogen is induced the antibody (ab3) that produces disease-resistant bacterium with anti-idiotype monoclonal antibodies (ab2)
(1) be immunogen immune Ba/b/c mice with ascitic type ab2, each immunity amount and supplementary immunization number of times are with the preparation of ab2.The 4th immunity extractd the eyeball of mouse blood-letting after 5 days, collected antiserum (Ab3).
(2) detection-indirect elisa method of ab3,
1) with known antigens inactivated bacterial liquid bag quilt.
2) be added with culture supernatant 100 μ 1 that clone in the long-living hole.
3) enzyme-added mark monoclonal antibody Mus IgG antibody 100 μ l.
4) add substrate solution 100 μ l, room temperature lucifuge colour developing 10～20 minutes.
5) result of determination: measure the OD value under enzyme-linked immunosorbent assay instrument 492nm wavelength, the blank zeroing is if treat that gaging hole OD value more than or equal to 2.1 times of negative control holes, is positive colony.
(3) induce the anti-blood (ab3) that contains anti-vibrio anguillarum in the Mus serum of generation by Vibrio anguillarum anti-idiotype monoclonal antibodies 1E10,1H5,1D1 and 2H12, the OD value is respectively a matched group more than 2 times, and it is tired is 1: 100～1: 1000.
(4) induce in the Mus serum of generation by Edwardsiella tarda antidiotypic monoclonal antibody 1E5 and 1E11 and contain anti-Wdwardsiella tarda antibody.It is tired is 1: 10～1: 200.
10. the anti-idiotype monoclonal antibodies vaccine is to the immanoprotection action of Paralichthys olivaceus
(1) method
Antiidiotype monoclonal antibody and incomplete Freund mix at 1: 1; lumbar injection immunity Paralichthys olivaceus ocean fishes; every endnote is penetrated 0.05m1 (containing monoclonal antibody 10～20 μ g); after the immunity one month; pathogenic bacteria lumbar injection with lethal dose is attacked Paralichthys olivaceus, observes the protective effect of anti-idiotype monoclonal antibodies vaccine to Paralichthys olivaceus.
The computing formula of protective rate: immune protective rate=(experimental group survival rate-matched group survival rate)/matched group mortality rate * 100%
(2) result
1. Vibrio anguillarum antiidiotype monoclonal antibody 1D1,1E10,1H12 are respectively 82.5%, 88.7% and 81.2% to the protective rate of Paralichthys olivaceus.
2. Edwardsiella tarda antidiotypic monoclonal antibody 1E11 and 1E5 are to the protective rate difference 60.2% and 20.8% of Paralichthys olivaceus.
3. anti-unique monoclonal antibody 2F4,1B2 of vibrio alginolyticus is respectively 50.2% and 90.0% to the protective rate of Paralichthys olivaceus.
4. Vibrio vulnificus antiidiotype monoclonal antibody Va1, Va2 are respectively 31.6% and 15.2% to the protective rate of Paralichthys olivaceus.
More than the ocean fishes protective rate is reached anti-idiotype monoclonal antibodies more than 60%, can be used as ocean fishes antiidiotype monoclonal antibody immune formulation.
11. preparation finished product
With the 1E10 in the Vibrio anguillarum antiidiotype monoclonal antibody, the 1E11 in slow Edward's antiidiotype monoclonal antibody, the 1B2 monoclonal antibody in the algae vibrio antiidiotype monoclonal antibody of washing is mixed with 1: 2: 1 ratio, makes the multi-joint antiidiotype monoclonal antibody of fish diseases bacterin preparation.
Advantage of the present invention:
Concatenated antidiotype monoclonic antibody vaccine for fish diseases of the present invention reaches more than 60% the protective rate of ocean fish, anti-uniqueness Type monoclonal antibody vaccine is except having ocean fish the good immunoprotection, and it also possesses the advantage of following uniqueness: namely do not contain poisonous resisting Ultimate constituent can not cause any infringement to ocean fish, and not exist kind be difference to anti-idiotype in addition, need not consider during use Planting is problem, and the scope of application is very wide, very useful to the development of mariculture industry.
Claims (1)
1. the multi-joint anti-idiotype monoclonal antibodies bacterin preparation of fish diseases is characterized in that:
Just Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, Wdwardsiella tarda carry out amplification cultivation, bacterium liquid lumbar injection immune mouse Ba/b/c after will cultivating again, get immunity back mouse boosting cell and SP2/0 myeloma cell and carry out cell fusion, the cell suspension of cell fusion is cultivated, detect positive colony with enzyme linked immunoassay method (ELISA method), to be defined as male hole with limiting dilution assay, carry out cloning, with the hybridoma cell strain amplification cultivation of positive colony, lumbar injection is inoculated into the Ba/b/c mice and prepares the ascitic type monoclonal antibody again; Above ascitic type monoclonal antibody is tired with the ELISA method, and the higher monoclonal antibody of tiring is selected in subclass and cross matching, ocean fishes is carried out protection test, selecting has the monoclonal antibody of higher protective rate to carry out purification to ocean fishes, with the amount of every mice 10～100 μ g, and the lumbar injection immune mouse; Carry out cell fusion then, filter out the hybridoma cell strain of energy stably excreting anti-idiotype monoclonal antibodies, cell amplification is cultivated, be adjusted into 1 * 10
6/ ml, with every mice 0.5ml amount, lumbar injection is inoculated into the Ba/b/c mice, and preparation ascitic type anti-idiotype monoclonal antibodies adopts the ELISA method, and above monoclonal antibody is carried out subclass, tires and the evaluation of β type anti-idiotype monoclonal antibodies; Mixed with above monoclonal antibody and incomplete Freund's adjuvant (1: 1～1: 2), lumbar injection immunity ocean fishes, picking out has the antiidiotype monoclonal antibody of higher protective rate to be mixed with the multi-joint antiidiotype monoclonal antibody of fish diseases bacterin preparation with certain proportion to ocean fishes again.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB00113924XA CN1173741C (en) | 2000-09-20 | 2000-09-20 | Concatenated antidiotype monoclonic antibody vaccine for fish diseases |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB00113924XA CN1173741C (en) | 2000-09-20 | 2000-09-20 | Concatenated antidiotype monoclonic antibody vaccine for fish diseases |
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| CN1293066A true CN1293066A (en) | 2001-05-02 |
| CN1173741C CN1173741C (en) | 2004-11-03 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102166350A (en) * | 2011-04-13 | 2011-08-31 | 中国水产科学研究院黄海水产研究所 | Flounders quintuplet inactivated vaccine and preparation method thereof |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
-
2000
- 2000-09-20 CN CNB00113924XA patent/CN1173741C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102166350A (en) * | 2011-04-13 | 2011-08-31 | 中国水产科学研究院黄海水产研究所 | Flounders quintuplet inactivated vaccine and preparation method thereof |
| CN102166350B (en) * | 2011-04-13 | 2012-11-21 | 中国水产科学研究院黄海水产研究所 | Flounders quintuplet inactivated vaccine and preparation method thereof |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
| CN108003240B (en) * | 2017-12-04 | 2020-06-05 | 西安德轩驰生物科技有限公司 | Multi-connected anti-idiotype egg yolk antibody vaccine for marine cultured fish and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN1173741C (en) | 2004-11-03 |
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