CN108003240A - A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof - Google Patents
A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof Download PDFInfo
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- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4233—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-bacterial Ig
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Abstract
The present invention provides a kind of preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish, including mouse and rabbit is immunized after mariculture fish pathogenic bacteria are inactivated；Immunization laying hen after being mixed using 6 kinds of rabbit-anti cause of disease serum；Laying hen is screened, is collected, polishing purification obtains antiidiotype Yolk antibody crude product and the multi-joint antiidiotype Yolk antibody vaccine of seawater fish disease.Wherein, the mariculture fish pathogenic bacteria are Vibrio anguillarum, tarda, vibrio alginolyticus, vibrio parahaemolytious, Vibrio vulnificus, streptococcus.The multi-joint antiidiotype Yolk antibody vaccine of the present invention, can strengthen resistance of the fish to several fish diseases, a variety of seawater fish diseases are prevented；Oral route immunity inoculation fish can also be passed through；Prepare that antiidiotype Yolk antibody is simple and easy to do, materials are easy, sterilization processing is convenient, and preparation procedure is easy, and cost is low, and yield is high, meets national energy-saving environmental protection, the policy guidance of sustainable development.
Description
Technical field
The invention belongs to aquatic products vaccines arts, and in particular to a kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish
And preparation method thereof.
Background technology
China is the country of sea-farming fish crop maximum, and the annual quantity for cultivating ocean fish is up to 7,000,000,000 tails.It is close with cultivating
The increase of degree, water environment constantly deteriorate, and disease is also increasingly serious, every year because disease death marine cultured animal to country caused by
More than 100 hundred million economic loss.The disease for endangering China's marine cultured animal is mainly bacterial disease and virosis.Bacterial disease such as eel
Vibrios, tarda, vibrio parahaemolytious, Vibrio vulnificus, streptococcus, vibrio alginolyticus；Virosis such as irido virus, shrimp Leucoplakia
Poison and nervous necrosis virus etc., they are to cause the large quantities of dead arch-criminals of marine cultured animal.
All the time, mainly have for tackling the means of marine cultured animal disease and largely use antibiotic and chemical drugs
Thing, but long-term a large amount of uses of these medicines, have caused serious anti-medicine drug resistance, environmental pollution and medicament residue, finally
The health of the mankind is endangered by food chain.So many countries and the Chinese government just gradually forbid using antibiotic and chemicals
To prevent and cure diseases to marine cultured animal, large quantities of antibiotic and chemicals are included in by many countries and the Chinese government
Marine cultured animal list of banned substances.
Vaccine and biological agent meet it is environmentally safe, the features such as no drug resistance, noresidue, it has also become the world today is aquatic
The main product of Animal diseases prevention and control.The aquiculture animal vaccine that the whole world obtains production certification at present has kind more than 100, but
Most of is all traditional inactivated vaccine and attenuated live vaccine, and the major defect of these vaccines is that toxic side effect cannot be kept away completely
Exempt from.The fish vaccine that China obtains certification so far only has three products：Grass carp hemorrhage disease live-vaccine, tarda attenuated live epidemic disease
Seedling, and the multi-joint Anti-idiotype Antibody Vaccine of lefteye flounder fish.But so far both at home and abroad without the report of antiidiotype Yolk antibody vaccine
Road,
The preparation scheme of existing inactivated vaccine and attenuated live vaccine is：Largely expanded with fermentation tank or other methods first numerous
Grow cause of disease (germ or virus), then it is inactivated or is reduced with chemically or physically method its activity, inactivated vaccine
Or attenuated live vaccine.The major defect of this vaccine is the growth work that inactivation or method of attenuating can only eliminate or lower pathogen
Property, but the interior exotoxin that cause of disease contains in itself cannot be eliminated, deal with improperly and be easy to produce toxic side effect.
The content of the invention
In view of this, it is an object of the invention to provide a kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish
Preparation method, comprises the following steps：
1), by the bacterium solution of mariculture fish pathogenic bacteria median lethal dose concentration, adjuvant immunity mouse and family are added after inactivation
Rabbit；
2) serum of mouse and rabbit, is collected respectively, and adding adjuvant immunity after being mixed using 6 kinds of rabbit-anti cause of disease serum is laid eggs
Chicken；
3) laying hen, is screened, exempts from chicken using indirect elisa method detection height and produces egg, by resisting containing six kinds of pathogens
Idiotype Yolk antibody and potency is 10-4More than, it is classified as and adopts laying hen；
4) egg, separation yolk, broken removal foreign protein of laying hen, are collected, precipitated and separated takes supernatant, further removes
Lipoprotein after fat with obtaining antiidiotype Yolk antibody crude product；
5) antiidiotype Yolk antibody crude product is purified up to the multi-joint antiidiotype Yolk antibody vaccine of seawater fish disease.
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
It is Vibrio anguillarum to state the mariculture fish pathogenic bacteria in step 1), tarda, vibrio alginolyticus, vibrio parahaemolytious, Vibrio vulnificus,
Streptococcus.
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
The method that mouse and rabbit are immunized in step 1) is stated as intraperitoneal injection or is subcutaneously injected, immune time is 3~4 times immune, every time
Interval 7~10 days；It is highly preferred that the inactivation bacterium amount that the mouse is immunized every time is 0.25 × 104~0.25 × 105.The rabbit
Immune inactivation bacterium amount is 0.5 × 104~0.5 × 105；Most preferably, the serum antibody titer of the immune mouse or rabbit reaches
To 10-4。
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
The immunization method for stating laying hen in step 2) is intramuscular injection, amount 0.5ml is immunized, antibody content is 10~50 μ g；Every 7~10
It is 1 time immune, is immunized 3 times altogether.
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
Step 3) screening laying hen is stated, exempts from chicken using indirect elisa method detection height and produces egg, if is containing six kinds of pathogens anti-only
Special type Yolk antibody and potency is 10-4More than.
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
Stating yolk tissue disruptive methods described in step 3) is：Yolk historrhexis is carried out with high pressure homogenizer, adds distilled water after crushing
Stir evenly.
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
It is the isoelectric point that the pH value of yolk solution is adjusted to lipoprotein to state foreign protein minimizing technology in step 3), and precipitation standing takes supernatant,
Bottom muddiness is partially placed into centrifuging and taking supernatant, after merging supernatant micro porous filtration further remove lipoprotein and fat, take filtrate, should
Filtrate is antiidiotype Yolk antibody semifinished product.
Preferably, in the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish of the present invention, institute
The purification process for stating anti-idiotype crude product in step 3) centrifuges for organic solvent deposit, taking precipitate；1~2 is handled repeatedly
It is secondary to take precipitation, it is dry or add appropriate distilled water to dissolve its sediment, resist up to the refined multi-joint antiidiotype yolk of seawater fish disease
Body vaccine.
The multi-joint antiidiotype Yolk antibody of seawater fish disease or seawater fish disease obtained present invention also offers the above method is more
Join antiidiotype Yolk antibody vaccine.
Compared with prior art, the present invention the present invention has the following advantages：
1. the present invention prepares a kind of multi-joint (being directed to more than six kinds seawater fish diseases) antiidiotype Yolk antibody vaccine, to connect
The immune mariculture fish of kind, to strengthen resistance of the fish to several fish diseases, prevents a variety of seawater fish diseases.Close at present
Reported in fish disease vaccine more, but most of is inactivated vaccine, attenuated live vaccine.There is grass carp hemorrhage disease live-vaccine in the country, more precious
Fish Wdwardsiella tarda attenuated live vaccine, the report of the multi-joint anti-idiotype monoclonal antibodies vaccine of lefteye flounder fish.It is but domestic and international so far
Have no report of the fish with antiidiotype Yolk antibody vaccine.
2. antiidiotype Yolk antibody vaccine, except through injection, immersion approach is immunized outside fish, moreover it is possible to is sprayed
Mariculture fish is immunized by oral route on to bait, it is time saving and energy saving by oral route immunity inoculation fish, it was immunized
Cheng Wuxu is any to cultivation fish progress to be stirred, and does not give cultivation any injury of fish tape.Different fingerlings, the different age of a fish can conveniently make
With.It is not reported both at home and abroad so far by the fish vaccine of oral route immunoprophylaxis.
3. when screening antiidiotype Yolk antibody, without killing livestock, without blood drawing, the high egg for exempting from chicken, section need to be only gathered
About resource, it is simple and easy to do.
4. preparing the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish, the vaccine of a production procedure production can be at the same time
Prevent a variety of seawater fish diseases, play and achieve many things at one stroke, save the effect of source synergy.Antiidiotype Yolk antibody vaccine is prepared, resource is rich
Richness, materials are easy, and sterilization processing is convenient, and preparation procedure is easy, and cost is low, and yield is high.Meet national energy-saving environmental protection, it is sustainable
The policy guidance of development.
Embodiment
Strain source according to the present invention and number table
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Based on the reality in the present invention
Apply example, those of ordinary skill in the art's all other embodiments obtained without making creative work, all belong to
In the scope of protection of the invention.
Embodiment 1
1. the preparation of seawater fish disease immunogene
According to the survey report of China coastal seas pathogenic bacteria, China coast Main Pathogenic Bacteria has Vibrio anguillarum, Ai Dehuashi
Bacterium, vibrio alginolyticus, vibrio parahaemolytious, Vibrio vulnificus, streptococcus.From the world, domestic cell bank buys above seawater fish morbid bacterium
Reference culture, by its amplification cultivation, survey poison, calculate the concentration of the median lethal dose of every kind of pathogen respectively.Six kinds of germs
Median lethal dose be 1 × 104~1 × 105/ml。
2. the preparation of seawater fish disease antibody
It is immune：By the bacterium solution of various pathogen median lethal dose concentration, fix and inactivate through formaldehyde fixer, and Freund is endless
Full adjuvant volume 1：Mouse and rabbit is immunized after 1 mixing respectively, 0.5ml immunogenes are immunized in every intraperitoneal injection of mouse, and rabbit is every
Immune 1ml immunogenes are only subcutaneously injected, are immunized 3~4 times altogether, every minor tick 7~10 days, the inactivated bacteria that every mouse is immunized every time
Measure as 0.25 × 104~0.25 × 105.The each immunological sterilization bacterium amount of rabbit is 0.5 × 104~0.5 × 105。
Antibody titer detects：Second it is immune after the 3rd~5 day, gathering the blood of each mouse and rabbit respectively, (mouse cuts tail
Blood is taken, rabbit is drawn blood from ear vein), centrifuging and taking serum, with every kind of cause of disease bacteria antibody and potency in ELISA method detection serum.Such as
Fruit antibody titer reaches 10-4Serum is collected in mouse and rabbit above, the immune rear bloodletting in 7th day of third time.If antibody titer
Do not reach 10-4, then carry out being immunized for the 4th time, or eliminate, the animal renewed is immunized again.
Collect antibody and preservation：Antibody titer reaches 10-4Mouse and rabbit, you can serum is collected in bloodletting.Mouse passes through
Eyeball bloodletting is plucked after disconnected neck marrow.Rabbit is intubated bloodletting by arteria carotis.Serum is taken after centrifugation.- 25 DEG C of refrigerators of postposition are dispensed to preserve.
Every kind of pathogen all prepares two kinds of antiserums, and one kind is that mouse anti-microbial pathogen serum (is used for antiidiotype Yolk antibody and its production
The primary dcreening operation of laying hen), another kind is rabbit-anti cause of disease serum (immunogene prepared as anti-idiotype).
3. the preparation of seawater fish disease antiidiotype Yolk antibody
The preparation of immunogene：By the antibody (antibody titer 10 of 6 kinds of rabbit-anti fish pathogen-4) mixing in equal volume, then
After isometric incomplete Freund's adjuvant mixing, intramuscular injection immunization laying hen, every laying hen injects 0.5ml, containing every kind of
The amount of cause of disease bacteria antibody is about 10~50 μ g.It was immunized once, is immunized altogether three times every 7~10 days.
The screening of laying hen：Second immune 3rd~5 day latter, and the egg of production, uses ELISA method detection on the day of collection laying hen
It whether there is various pathogen anti-idiotypes and its potency in its yolk.If resist respectively containing 6 kinds of pathogens in yolk
Idiotype antibody, and potency is up to 10-4More than, which is listed in pair that collection egg prepares antiidiotype Yolk antibody
As the laying hen for not reaching this standard is not listed in the object of collection egg then.Its egg is sold to client as usual and is eaten, and is not present
Food-safety problem.Judge whether the standard of the anti-idiotype containing pathogen is in yolk：Can be with antigentic specificity
The antibody response that different bodies produce；Whether can be with the antibody of antigenic competition combination antigentic specificity；Whether can induce not of the same race
Body produces the antibody of antigentic specificity.
The collection of egg：Meet the laying hen of collection egg standard, start within 3rd day to gather egg after third time is immune,
After continuous acquisition 20 days, 20 days, if it remains desirable, supplementary immunization once, is then continuing collection 20 days, until laying hen quilt again
Eliminate.
Yolk separates：Egg is cleaned with tap water, 75% alcohol is put into and soaks 10 minutes sterilizings, pull out and dry
Afterwards, clean room is passed to, manually separates yolk.
Yolk historrhexis：Yolk historrhexis is carried out with high pressure homogenizer (60Pa).After broken plus 10~40 times are distilled
Water stirs evenly.
Foreign protein removes：The pH value of yolk solution is adjusted to the isoelectric point of various lipoprotein with hydrochloric acid and sodium hydroxide solution
(PH5.2~6.0), make yolk solution produce turbidity and precipitation.By 4 DEG C~8 DEG C refrigerator overnights of above solution left standstill.Make yolk solution
Foreign protein precipitation.Second day supernatant drawn with suction pipe after precipitation, bottom muddiness are partially placed into 3500~4000r/min centrifugations
Machine centrifugation 20~take supernatant after forty minutes.After two kinds of supernatants are mixed grease removal egg is further removed through micro porous filtration (0.22 μm of aperture)
White and fat, takes filtrate, which is antiidiotype Yolk antibody semifinished product.
The purifying of antiidiotype Yolk antibody：Antiidiotype Yolk antibody is isolated and purified with organic solvent precipitation method, anti-
In idiotype antibody crude extract plus cold alcohol makes its concentration reach 50~60%, and being sufficiently stirred makes its turbidity and precipitation, in 4000r/
Min 20~30min of centrifuge, taking precipitate.Distilled water is added to return to original volume, then plus cold alcohol reaches its concentration
To 25~30%, being sufficiently stirred makes its turbidity and precipitation, is ibid centrifuged, taking precipitate.Again plus distilled water makes it return to original
Carry out volume.Alcohol is added its concentration is sufficiently stirred up to 20~25%, is made its turbidity and precipitation, ibid centrifuged, collect precipitation
Thing, sediment are dried in 37 DEG C of incubators, or add appropriate distilled water to dissolve its sediment, refined seawater fish disease it is multi-joint anti-only
Special type Yolk antibody vaccine, yield are 25~45%.
4. the detection of the multi-joint antiidiotype Yolk antibody of ocean fish disease
1) antiidiotype Yolk antibody content detection：
The content of the antiidiotype Yolk antibody vaccine produced with the Enzyme-Linked Immunosorbent Assay detection embodiment of the present invention 1, as a result shows
It is 7~more than 8mg/ml to show its content.
Specific procedure is as follows：
A uses coating buffer solution (0.05MpH9.6 carbonate colors are by buffer solution) by Yolk antibody standard items
The Yolk antibody prepared with the present invention is respectively with 2n(square multiple) is diluted to n12。
B is serially diluted for 2 times by more than, totally 12 grades of diluted Yolk antibody standard items and antiidiotype Yolk antibody of the present invention
Sequentially be added to respectively in ELISA Plate reaction aperture, every kind of 12 hole, adds 100 μ l per hole, put 37 DEG C be incubated 1 it is small when.
C discards the liquid in aperture, is washed 3 times, 3 minutes every time, patted dry with lavation buffer solution.
D adds the antibody of rabbit-anti Yolk antibody (to be diluted to 1 with antibody diluent:1000), per 100 μ l of hole, 37 DEG C of incubations are put
0.5~1 it is small when.
E repetitive routines C.
F adds the goat anti-rabbit igg antibody (1 that HRP is marked:1000 dilutions), per 100 μ l of hole, put 37 DEG C
When incubation 0.5~1 is small.
G repetitive routines C.
H adds color developing agent (phosphoric acid-lemon acid buffering of the PH5.0 pacified containing 0.045% hydrogen peroxide and 10~20mg neighbours benzene two
Agent), per 100 μ l of hole, develop the color 15~20 minutes under room temperature lucifuge.
I is on ELISA detectors, in 490nm by strong point, after blank control group zeroing, each hole OD values is surveyed, if treating gaging hole OD
It is the positive that value, which is greater than or equal to 2.1 times of control wells, and negative control hole is colourless.
Testing result such as table 1：
The OD value tables of the antiidiotype Yolk antibody difference dilution factor of the present invention of table 1
Note：The original concentration of standard items liquid is 1mg/ml.
K results：Yolk antibody standard items and the present invention prepare the OD values such as table of antiidiotype Yolk antibody dilution factors at different levels
1, standard curve is drawn by curve expert software, R values reach more than 0.98, and antiidiotype Yolk antibody contains in product of the present invention
Measure as 7~8mg/ml.
2) antiidiotype Yolk antibody bioactivity
With indirect elisa method, the potency of antiidiotype Yolk antibody produced by the invention is detected, its potency of the results show is
1×10-4~1 × 10-5。
Specific procedure is as follows:
A uses coating buffer solution (0.05MpH9.6 carbonate colors are by buffer solution) by six kinds of mouse resistant to sea water
Cause of disease bacteria antibody carries out 1:1000 dilutions.Then it is added separately in ELISA Plate aperture, every kind of antibody adds 10 apertures,
Per 100 μ l of hole, put 37 DEG C be incubated 1~2 it is small when.
B discards the mouse anti-microbial pathogen antibody in aperture, is washed 3 times, every time 3 minutes with lavation buffer solution.
C carries out 10 times to the anti-idiotype of the present invention with antibody diluent and is serially diluted (10n), 10 grades are diluted always
(1010).Different dilution factor antiidiotype Yolk antibodies are sequentially added in ELISA Plate aperture, every grade of dilution factor adds 6 holes (with six kinds
Pathogen mouse antibodies are corresponding), 10 grades of dilution factors add 60 apertures altogether, per 100 μ l of hole, put 37 DEG C be incubated 0.5~1 it is small when.
D repeats B programs.
E adds the antibody of rabbit-anti Yolk antibody (to dilute 1 with antibody diluent:1000), per 100 μ l of hole, 37 DEG C is put and is incubated 0.5
~1 it is small when.
F repeats B programs.
G adds the monoclonal antibody rabbit igg antibody (1 that HRP is marked:1000 dilutions), per 100 μ l of hole, put 37 DEG C be incubated 0.5~1 it is small when.
H repeats B programs.
I adds developer, and (PH5.0 phosphoric acid-citric acid containing 0.045% hydrogen peroxide liquid and 10~20mg o-phenylenediamines delays
Fliud flushing) per 100 μ l of hole, develop the color 15~20 minutes in room temperature lucifuge.
J adds terminate liquid (10.6% sulfuric acid solution), per 50 μ l of hole.
K is on ELISA detectors, at 490nm wavelength, after blank control zeroing, each hole OD values is surveyed, if treating gaging hole OD values
More than or equal to negative control hole, 2.1 times are the positive.
Respectively join the potency such as following table of anti-idiotype in multi-joint antiidiotype Yolk antibody
Respectively join the bioactivity result table of anti-idiotype in multi-joint antiidiotype Yolk antibody prepared by 2 present invention of table
As a result：Table 2 shows each connection anti-idiotype in the multi-joint anti-idiotype of seawater fish disease prepared by the present invention
Potency is 10-4~10-5。
3) antiidiotype Yolk antibody effect detects
A tests fish：600 tail of lefteye flounder fish, (5~7cm, 4~5 monthly ages) are divided into 4 groups, 150 tail of each group.
B is immunized：
I group (150 tail) is tested to be carried out vaccine inoculation with intraperitoneal injection and be immunized.After vaccine and Freund's incomplete adjuvant are mixed, warp
The immune ocean fish of intraperitoneal injection, 50 μ l (being 3.75 μ g containing amount of vaccine) are penetrated per endnote.
To test II group (150 tail), immersion immunity, its vaccine immunity amount is 3 times of injection,
The vaccine of 1.69mg is dissolved into 13.5 liters of seawater, is stirred evenly, is then put into 150 tail ocean fishes
Soaked 30 minutes in seawater containing vaccine, if a collection of density is excessive to divide 2~3 batches of progress.30
Drag in Hui Chi after minute and normally raise.
Test III group (150 tail) to be immunized by oral route, vaccine is dissolved in physiological saline, is sprayed onto fish bait
On, make bait into half wet condition, throw into and fish is fed in water, immune vaccine amount and immersion immunity are suitable, but carry out in three times, during interval
Between be one week.Control group (150 tail), it is without any processing, compare.Carry out attacking poison after normally putting in a suitable place to breed one month after immune.
C strains prepare：After 6 kinds of strain amplification cultivations, poison is surveyed.
Survey malicious program：Each strain germ original liquid concentration is detected with plate count, then with 10 times of serial dilutions, carries out 5
Level dilution.Malicious ocean fish (penetrating 100 μ l per endnote) is attacked with the bacterium solution intraperitoneal injection of different diluted concentration ranks, calculates various germs
To the median lethal dose of ocean fish.Challenge test is done to ocean fish with the half lethal dose concentration of various bacterium.Various bacterium median lethal doses are shown in
Table 3.
The various bacterium median lethal dose result of the test tables of table 3
As a result：Table 3 shows that six kinds of pathogenic bacteria are respectively 10 to the median lethal dose for testing ocean fish6/ ml~104/ml。
Attack poison：Fishes at different levels are carried out with 5 times of median lethal dose concentration of various strains to attack poison, every group of 20 tail of fish, per tail abdomen
Chamber injects 100 μ l, observes 7 days, the death toll for each group fish that counts and calculates vaccine and is shown in Table 4. to the protective rate of ocean fish
Table 4 shows that the protective rate that injection-type vaccine attacks six kinds of germs poison is respectively 75%~85%；Soak type vaccine pair
The protective rate that six kinds of germs attack poison is 55%~65%；Oral type vaccine six kinds of germs are attacked the protective rate of poison for 75%~
85%.
Table 4 attacks malicious method vaccine to ocean fish protective rate
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (11)
1. a kind of preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish, comprises the following steps：
1), by the bacterium solution of mariculture fish pathogenic bacteria median lethal dose concentration, adjuvant immunity mouse and rabbit are added after inactivation；
2) serum of mouse and rabbit, is collected respectively, and adjuvant immunity laying hen is added after being mixed using 6 kinds of rabbit-anti cause of disease serum；
3) laying hen, is screened, exempts from chicken using indirect elisa method detection height and produces egg, if is containing six kinds of pathogens anti-only
Special type Yolk antibody and potency；
4) egg, separation yolk, broken removal foreign protein of laying hen are collected, precipitated and separated takes supernatant, further removes grease removal egg
In vain with obtaining antiidiotype Yolk antibody crude product after fat；
5) antiidiotype Yolk antibody crude product is purified up to the multi-joint antiidiotype Yolk antibody vaccine of seawater fish disease.
2. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, the mariculture fish pathogenic bacteria in the step 1) are Vibrio anguillarum, and tarda, vibrio alginolyticus, vibrio parahaemolytious, creates
Hinder vibrios, streptococcus.
3. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, the method for mouse and rabbit is immunized in the step 1) as intraperitoneal injection or is subcutaneously injected, immune time is immune 3~4
It is secondary, every minor tick 7~10 days.
4. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 3, its feature
It is, the inactivation bacterium amount that the mouse is immunized every time is 0.25 × 104~0.25 × 105.The inactivation bacterium amount of the rabbit immunization is
0.5×104~0.5 × 105。
5. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 3, its feature
It is, the serum antibody titer of the immune mouse or rabbit reaches 10-4。
6. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, the immunization method of laying hen is intramuscular injection in the step 2), amount 0.5ml is immunized, antibody content is 10~50 μ g；Often
It is 1 time immune every 7~10 days, it is immunized 3 times altogether.
7. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, the method for step 3) the laying hen screening is that ELISA method detection height exempts from the anti-uniqueness that the egg of chicken production contains 6 kinds of pathogens
Type antibody, and potency is 10-4More than.
8. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, yolk tissue disruptive methods are described in the step 4)：Yolk historrhexis is carried out with high pressure homogenizer, is added after crushing
Distilled water stirs evenly.
9. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, foreign protein minimizing technology is the isoelectric point that the pH value of yolk solution is adjusted to lipoprotein in the step 4), and precipitation is stood
Take supernatant, bottom muddiness is partially placed into centrifuging and taking supernatant, after merging supernatant micro porous filtration further remove lipoprotein and fat, take
Filtrate, the filtrate are antiidiotype Yolk antibody semifinished product.
10. the preparation method of the multi-joint antiidiotype Yolk antibody vaccine of mariculture fish according to claim 1, its feature
It is, the purification process of anti-idiotype crude product centrifuges for organic solvent deposit in the step 4), taking precipitate；Locate repeatedly
Reason takes precipitation for 1~2 time, dry or add appropriate distilled water to dissolve its sediment, up to the multi-joint antiidiotype of seawater fish disease is refined
Yolk antibody vaccine.
11. according to claim 1~10 any one the method obtain the multi-joint antiidiotype Yolk antibody of seawater fish disease or
The multi-joint antiidiotype Yolk antibody vaccine of seawater fish disease.
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