CN1140170C - Concatenated monoclonic antibody medicine for treating fish diseases - Google Patents
Concatenated monoclonic antibody medicine for treating fish diseases Download PDFInfo
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- CN1140170C CN1140170C CNB001139266A CN00113926A CN1140170C CN 1140170 C CN1140170 C CN 1140170C CN B001139266 A CNB001139266 A CN B001139266A CN 00113926 A CN00113926 A CN 00113926A CN 1140170 C CN1140170 C CN 1140170C
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- monoclonal antibody
- vibrio
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The present invention relates to a concatenated monoclonal antibody preparation for curing fish diseases, which belongs to the biological technical field. In the present invention, Vibrio anguillarum, Vibrio vulnficus, Vibrio alginolyticus and a tardive edwardsiella immune mouse carry out cell fusion, and then, are cloned; a monoclonal antibody with strong specificity and high valency is prepared, an anti-germ monoclonal antibody with high protective rate is sieved from the monoclonal antibody, so that the concatenated monoclonal antibody preparation for curing fish diseases is prepared to scale. The biological preparation has good preventing and curing action on fish diseases, simultaneously, does not pollute environment, and has no harm to humans by food chains either.
Description
One, affiliated field
The present invention relates to Monoclonal Antibody and applied technical field on the immunology.
Two, background technology
In recent years along with the fast development of China's mariculture fishery, fish disease is particularly more and more serious by bacterial fish disease, and annual mariculture fish Died Of Disease reaches 30%～40%, has caused serious economy loss.To culturing the control of fish disease, also rest on main employing chemicals and antibiotic starting stage both at home and abroad so far, the extensive application of chemicals can bring bad effect, and the one, cause the drug resistance of germ and lost efficacy, the 2nd, cause pollution to environment; Use antibiotic, also can cause harmful effect to the mankind by food chain.Fish disease mab treatment preparation does not appear in the newspapers so far.
Three, summary of the invention
Purpose of the present invention: utilize the Monoclonal Antibody technology on the immunology, produce the monoclone antibody of multiple fish disease germ, fish disease is had good preventing and therapeutic action, do not pollute the environment simultaneously, also can not work the mischief to the mankind by food chain.
Technical scheme of the present invention:
With Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, Wdwardsiella tarda (providing) by Qingdao Marine University, carry out amplification cultivation, bacterium liquid lumbar injection immune mouse Ba/b/c mouse after will cultivating again, get immunity back mouse boosting cell and SP2/0 bone marrow cell and carry out cytomixis, the cell suspension of cytomixis is cultivated, detect positive colony with the enzyme linked immunoassay method, to be defined as positive 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 mouse and prepares the ascitic type monoclonal antibody, above ascitic type monoclonal antibody is tired with the ELISA method, subclass and cross matching, selecting tires is 10
-5～1 * 10
-7The ascitic type monoclone antibody, germ with lethal dose mixes with 1: 1～1: 2 ratio respectively, infect ocean fish with lumbar injection then, control group replaces monoclonal antibody with physiological saline, observe the immune protective rate of monoclonal antibody to ocean fish, the result shows that 3 strain monoclonal antibody 4A6,3B2,4E12 are arranged in the anti-vibrio anguillarum monoclonal antibody, and they are respectively 80%, 60%, 50% to the protective rate of ocean fish; 2 strain monoclonal antibody 1A1,3E9 are arranged in the anti-Vibrio vulnificus monoclonal antibody, and they are respectively 87%, 69% to the protective rate of ocean fish; Two strain monoclonal antibody 3F7,1B4 are arranged in the anti-Wdwardsiella tarda monoclonal antibody, and they are respectively 80% and 60% to the protective rate of ocean fish; Pick out protective rate and be 60%～90% monoclonal antibody and be mixed with the multi-joint monoclonal antibody treatment of fish disease preparation by a certain percentage.
Characteristics of the present invention:
Utilizing the Monoclonal Antibody technology, is antigen with the bacterial strain of multiple ocean fish germ, the multi-joint monoclonal antibody treatment of preparation fish disease preparation.
Advantage of the present invention:
Concatenated monoclonic antibody medicine for treating fish diseases of the present invention reaches more than 80% the ocean fish protective rate; result of treatment is good; do not pollute the environment; can not cause damage to the mankind; and the prepared hybridoma cell strain that can produce ocean fish disease-specific monoclonal antibody can infinitely increase by the biological effect device; realize the specific monoclonal antibody industrialized production, have very high society and economy benefit.
Four, embodiment:
Embodiments of the invention:
(1) material
1, bacterial classification: the reference culture of Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, Edwardsiella tarda is provided by life institute of Qingdao Marine University.Above bacterial classification produces monoclone antibody as immunogene.
The reference culture of vibrio alginolyticus, vibrio parahaemolytious, Vibrio vulnificus, Vibrio flurialis, Vibrio furnissii, Vibrio hollisae, vibrio mimicus, maiden vibrios, Maxwell vibrios is available from Beijing biological products company.Intestines toxicity large intestine Du bacterium, enteroinvasive E.Coli, enterohemorrhagic Escherichia coli, the shigella flexneri of Shigella, Jue Shi Shigella, shigella dysenteriae, Song Shi Shigella are identified institute available from Chinese pharmaceutical biological product.Above bacterial classification is done the monoclone antibody cross matching.
2, cell line: the strain of SP2/O 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 immunogene bacterium and deactivation
Vibrio anguillarum, vibrio alginolyticus, Vibrio vulnificus, Edwardsiella tarda are incubated on the bacteria culture media with plate streaking, spend the night in 37 ℃ of incubators, and stroke-physiological saline solution wash-out lawn, 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 stroke-physiological saline solution, and after turbid numeration, 4 ℃ of refrigerators are preserved, in order to being used as immune original bacteria liquid.
2, the immunity of Ba/b/c mouse
10 of the Ba/b/c mouse in 8 ages in week are with the bacterium 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 cytomixis in the 31st day.
3, cytomixis
(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 stainless 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/O bone marrow cell suspension
1. the myeloma cell with 4 bottles of amplifications 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) cytomixis
1. draw respectively and contain 1 * 10
8Individual splenocyte is examined suspension and is contained 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 PEG0.7ml.
4. add the incomplete culture fluid of 25ml, make PEG dilution and lose the short effect of melting.
5. 800rmp is centrifugal 7 minutes, supernatant discarded.
6. add the 20mlHAT 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 culture supernatant 100 μ l in the hole of clonal growth.
(3) enzyme-added mark monoclonal antibody mouse 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 492 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, record 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 enlarged 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 after-70 ℃ of low temperature refrigerators spend 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 the cell of precipitation, 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 mouse, draw ascites, centrifugal, collect supernatant ,-20 ℃ of refrigerators are frozen standby after the packing.
8, the evaluation of monoclone antibody
The anti-vibrio anguillarum monoclonal antibody screens the positive cell strain of 8 strains energy stably excreting monoclone antibody, called after 2H2,3B2,3D2,3A3,3C4,4C6,4E3 and 4E12 respectively.Wherein 2H2,3B2,3D2 and 4E12 are IgG through subgroup identification
34E3 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 monoclone antibody, called after 1B12,3E9,3F11,3E3,2G8,3D5,1E11,3H4,2F1,1A1,2F6 and 1G1.Through subgroup identification, wherein 2F6 is IgG
31B12,3E3,3H4,1A1 are IgG
2a2G8,3D5,2F1 are IgG
1Through titration, it is 1 * 10 that above monoclonal antibody is tired
-6～1 * 10
-7
Anti-vibrio alginolyticus monoclonal antibody screens the hybridoma cell strain 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 Ig
G32G2,2D11,3A10 are IgG
2a2E4,3B12 are IgG
2b2G8,4C7 are IgG
1Through titration, it is 1 * 10 that above monoclonal antibody is tired
-6～1 * 10
-7
Anti-Wdwardsiella tarda monoclonal antibody screens the hybridoma cell strain of 10 strain monoclonal antibodies, respectively called after 1B4,4D3,4D5,3F7,5A11,5H5,6B11,6E1,6E6 and 6H3.Through subgroup identification, wherein 1B4,3F7,5A11,5H5,6B11 are IgG
16E1 is IgG
2b4D3,4D5,6E6,6H3 are IgG
3Through titration, it is 1 * 10 that above monoclonal antibody is tired
-6～1 * 10
-7
9, the immunoprotection of ocean fish is tested
(1) with bacterium liquid (5LD
50) (volume ratio was mixed, and with the amount lumbar injection lefteye flounder of 0.1ml/ bar, every injection of antibodies amount is 100 μ gt/ bars in 1: 1 with the ascitic type monoclone antibody.Control group is that same bacterium liquid mixes with physiological saline at 1: 1, observes the survival rate of lefteye flounder in 5 days.The protective rate computing formula:
Protective rate (%)=(experimental group survival rate-control group survival rate)/control group lethality * 100%
(2) result
To the sick treatment of ocean fish protection test, from the monoclone antibody of four kinds of germs, screened the monoclonal antibody that ocean fish is had better protective rate respectively through monoclone antibody.
Screen 3 strain monoclonal antibody 4A6,3B2,4E12 in the anti-vibrio anguillarum monoclone antibody.They are respectively 80%, 60%, 50% to the treatment immune protective rate of ocean fish.
Anti-Vibrio vulnificus monoclone antibody screens 2 strain monoclonal antibody 3E9,1A1, and they are respectively 87%, 89% to the protective rate of ocean fish.
Anti-Wdwardsiella tarda Dan Zhongkang screens 2 strain monoclonal antibody 3F7,1B4, and they are respectively 80% and 60% to the protective rate of ocean fish.
Do not screen the monoclonal antibody that ocean fish is had protective effect in the anti-vibrio alginolyticus monoclonal antibody, but screen a strain 2D11 suckling mouse is had fine protective effect, its protective rate reaches 90%.
10, preparation finished product
With the 4A6 in the anti-vibrio anguillarum monoclonal antibody, the 3E9 in the anti-Vibrio vulnificus monoclonal antibody, the 3F7 monoclonal antibody in the anti-Wdwardsiella tarda monoclonal antibody mixes with 1: 1: 5 ratio, makes the multi-joint monoclonal antibody treatment of fish disease preparation.
Claims (1)
1, concatenated monoclonic antibody medicine for treating fish diseases is characterized in that:
Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, Wdwardsiella tarda are carried 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 bone marrow cell and carry out cytomixis, the cell suspension of cytomixis is cultivated, detect positive colony with the enzyme linked immunoassay method, to be defined as positive 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 mouse and prepares the ascitic type monoclonal antibody again; Above ascitic type monoclonal antibody is tired with the ELISA method, subclass and cross matching, selecting tires is 10
-5～1 * 10
-7The ascitic type monoclone antibody; Mix in 1: 1～1: 2 the ratio and the germ of lethal dose respectively; infect ocean fish with lumbar injection then; ocean fish is carried out protection test, select protective rate to ocean fish and be 60%～90% monoclonal antibody, be mixed with concatenated monoclonic antibody medicine for treating fish diseases in 1: 1: 5 ratio.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001139266A CN1140170C (en) | 2000-09-20 | 2000-09-20 | Concatenated monoclonic antibody medicine for treating fish diseases |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001139266A CN1140170C (en) | 2000-09-20 | 2000-09-20 | Concatenated monoclonic antibody medicine for treating fish diseases |
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|---|---|
| CN1292993A CN1292993A (en) | 2001-05-02 |
| CN1140170C true CN1140170C (en) | 2004-03-03 |
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| CNB001139266A Expired - Fee Related CN1140170C (en) | 2000-09-20 | 2000-09-20 | Concatenated monoclonic antibody medicine for treating fish diseases |
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| CN1311073C (en) * | 2005-04-08 | 2007-04-18 | 东北农业大学 | Anti-swine infectious enterogastritis recombinant N protein hybridoma cell strain |
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Granted publication date: 20040303 Termination date: 20190920 |