CN108003240B - Multi-connected anti-idiotype egg yolk antibody vaccine for marine cultured fish and preparation method thereof - Google Patents
Multi-connected anti-idiotype egg yolk antibody vaccine for marine cultured fish and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a mariculture fish concatenated anti-idiotype yolk antibody vaccine, which comprises the steps of inactivating pathogenic bacteria of mariculture fish and then immunizing a mouse and a rabbit; 6 kinds of rabbit pathogenic-resistant serum are mixed to immunize laying hens; screening laying hens, collecting, refining and purifying to obtain an anti-idiotype egg yolk antibody crude product and a seawater fish disease concatenated anti-idiotype egg yolk antibody vaccine. Wherein the marine fish pathogenic bacteria are vibrio anguillarum, edwardsiella, vibrio alginolyticus, vibrio parahaemolyticus, vibrio vulnificus and streptococcus. The concatenated anti-idiotype yolk antibody vaccine can enhance the resistance of seawater fish to various fish diseases and prevent the seawater fish diseases; the seawater fish can also be immunized by an oral way; the preparation of the anti-idiotype egg yolk antibody is simple and easy, the materials are easy to obtain, the aseptic treatment is convenient, the preparation process is simple and convenient, the cost is low, the yield is high, and the method accords with the national policy guidance of energy conservation, environmental protection and sustainable development.
Description
Technical Field
The invention belongs to the field of aquatic vaccines, and particularly relates to a concatenated anti-idiotype egg yolk antibody vaccine for marine cultured fishes and a preparation method thereof.
Background
China is the country with the largest output of the marine fishes, and the number of the cultured marine fishes reaches 70 hundred million fish tails every year. With the increase of the culture density, the water environment is continuously deteriorated, diseases are gradually serious, and the mariculture animals dead due to the diseases cause one billion economic losses to the country every year. The diseases harmful to marine culture animals in China mainly comprise bacterial diseases and virus diseases. Bacterial diseases such as Vibrio anguillarum, Edwardsiella, Vibrio parahaemolyticus, Vibrio vulnificus, Streptococcus, Vibrio alginolyticus; viral diseases such as iridovirus, shrimp white spot virus and nervous necrosis virus, etc., which are allergements leading to massive death of marine cultured animals.
In the past, the means for treating the diseases of the mariculture animals mainly uses antibiotics and chemical drugs in a large amount, but the long-term large-amount use of the drugs causes serious drug resistance, environmental pollution and drug residues, and finally harms the health of human beings through a food chain. Therefore, the use of antibiotics and chemical drugs for preventing and treating diseases of marine cultured animals is gradually prohibited by many countries and governments in China, and a large amount of antibiotics and chemical drugs are listed in the list of prohibited drugs for marine cultured animals by many countries and governments in China.
The vaccine and the biological preparation accord with the characteristics of no pollution to the environment, no drug resistance, no residue and the like, and become mainstream products for preventing and controlling aquatic animal diseases in the world nowadays. At present, more than 100 aquaculture animal vaccines are produced in batches all over the world, but most of the vaccines are traditional inactivated vaccines and attenuated live vaccines, and the main defect of the vaccines is that toxic and side effects cannot be completely avoided. The fish vaccine obtained by the wholesale literature in China so far only has three products: grass carp bleeding disease live vaccine, Edwardsiella attenuated live vaccine, and multiple anti-idiotype antibody vaccine of Paralichthys olivaceus. However, no anti-idiotype yolk antibody vaccine is reported at home and abroad so far,
the preparation scheme of the existing inactivated vaccine and attenuated live vaccine is as follows: the inactivated vaccine or the attenuated live vaccine is prepared by amplifying and propagating pathogens (germs or viruses) in a large quantity by using a fermentation tank or other methods, and then inactivating or reducing the activity of the pathogens (germs or viruses) by using a chemical or physical method. The main disadvantage of this vaccine is that the inactivation or attenuation method can only eliminate or reduce the growth activity of the pathogen, but cannot eliminate the endotoxin and endotoxin contained in the pathogen itself, and the toxic and side effects are easily caused by improper treatment.
Disclosure of Invention
In view of the above, the invention aims to provide a preparation method of a concatenated anti-idiotype yolk antibody vaccine for marine cultured fish, which comprises the following steps:
1) inactivating bacteria liquid with half lethal concentration of pathogenic bacteria of the marine fish, and adding an adjuvant to immunize mice and rabbits;
2) respectively collecting the serum of the mouse and the serum of the rabbit, mixing the serum with the anti-pathogenic serum of 6 rabbits, and adding an adjuvant to immunize the laying hens;
3) screening laying hens, detecting eggs laid by hyperimmune chickens by indirect ELISA, and detecting anti-idiotype yolk antibody containing six pathogenic bacteria with titer of 10-4The above lists are laying hens;
4) collecting eggs of laying hens, separating yolk, crushing to remove impurity proteins, layering precipitates, taking supernatant, and further removing lipoprotein and fat to obtain an anti-idiotype yolk antibody crude product;
5) purifying the crude anti-idiotype yolk antibody to obtain the concatenated anti-idiotype yolk antibody vaccine for the marine fish disease.
Preferably, in the preparation method of the concatenated anti-idiotype yolk antibody vaccine for the marine fish, the pathogenic bacteria of the marine fish in the step 1) are vibrio anguillarum, edwardsiella sp, vibrio alginolyticus, vibrio parahaemolyticus, vibrio vulnificus and streptococcus.
Preferably, in the preparation method of the multi-connected anti-idiotype yolk antibody vaccine for the marine fish, the method for immunizing the mice and the rabbits in the step 1) is intraperitoneal injection or subcutaneous injection, the immunization times are 3-4 times, and the interval is 7-10 days each time; more preferably, the amount of inactivated bacteria per immunization of the mouse is 0.25X 104～0.25×105. The inactivated bacteria amount of rabbit immunity is 0.5 × 104～0.5×105(ii) a Most preferably, the serum antibody titer of the immunized mouse or rabbit reaches 10-4。
Preferably, in the preparation method of the concatenated anti-idiotype yolk antibody vaccine for the marine cultured fish, the immunization method of the laying hens in the step 2) is intramuscular injection, the immunization amount is 0.5ml, and the content of the antibody is 10-50 mu g; the immunization is carried out 1 time every 7-10 days, and the total immunization is carried out 3 times.
Preferably, in the preparation method of the concatenated anti-idiotype yolk antibody vaccine for the marine fish, the step 3) of screening laying hens, and detecting whether eggs laid by hyperimmune chickens contain anti-idiotype yolk antibodies of six pathogenic bacteria with titer of 10 by using an indirect ELISA method-4The above.
Preferably, in the method for preparing the concatenated anti-idiotype yolk antibody vaccine for the mariculture fish, the method for breaking the yolk tissue in the step 3) comprises the following steps: crushing yolk tissue with a high-pressure homogenizer, adding distilled water after crushing, and stirring uniformly.
Preferably, in the preparation method of the concatenated anti-idiotype yolk antibody vaccine for mariculture fish, the method for removing the impurity protein in the step 3) comprises the steps of adjusting the pH value of the yolk solution to the isoelectric point of lipoprotein, standing for precipitation to obtain a supernatant, centrifuging the turbid part at the bottom to obtain the supernatant, combining the supernatants, performing microfiltration to further remove the lipoprotein and fat, and obtaining a filtrate, wherein the filtrate is a crude anti-idiotype yolk antibody product.
Preferably, in the preparation method of the concatenated anti-idiotype yolk antibody vaccine for the marine fish, the purification method of the anti-idiotype crude antibody in the step 3) is organic solvent precipitation centrifugation, and a precipitate is obtained; and repeatedly treating for 1-2 times, taking the precipitate, drying or adding a proper amount of distilled water to dissolve the precipitate, and thus obtaining the refined concatenated anti-idiotype yolk antibody vaccine for the seawater fish disease.
The invention also provides a concatenated anti-idiotype yolk antibody for seawater fish diseases or a concatenated anti-idiotype yolk antibody vaccine for seawater fish diseases, which is obtained by the method.
Compared with the prior art, the invention has the following advantages:
1. the invention prepares a multi-connected (aiming at more than six seawater fish diseases) anti-idiotype yolk antibody vaccine which is used for immunizing seawater cultured fish so as to enhance the resistance of the seawater fish to various fish diseases and prevent the various seawater fish diseases. At present, fish disease vaccines are reported more, but most of the fish disease vaccines are inactivated vaccines and attenuated live vaccines. In China, live vaccines for grass carp bleeding diseases, live attenuated vaccines for edwardsiella tarda and multiple anti-idiotype monoclonal antibody vaccines for paralichthys olivaceus are reported. However, no report of the anti-idiotype yolk antibody vaccine for fish is found at home and abroad so far.
2. The anti-idiotype yolk antibody vaccine can immunize seawater fish by injection and soaking, can also be sprayed on bait to immunize seawater fish by oral administration, and immunizes the seawater fish by oral administration, thereby saving time and labor, avoiding any turning over of the cultured fish in the immunization process, and avoiding any damage to the cultured fish. Can be conveniently used for different fish species and different fish ages. The fish vaccine inoculated and immunized by oral administration is not reported at home and abroad so far.
3. When the anti-idiotype egg yolk antibody is screened, the killing is not needed, the blood drawing is not needed, and only the eggs of the hyperimmune chickens are collected, so that the resource is saved, and the method is simple, convenient and easy to implement.
4. The multi-connected anti-idiotype yolk antibody vaccine for the marine cultured fish is prepared, and the vaccine produced by one production process can prevent various marine fish diseases simultaneously, thereby achieving the effects of multiple purposes and resource saving and synergism. The preparation of the anti-idiotype egg yolk antibody vaccine has the advantages of rich resources, easily obtained materials, convenient aseptic treatment, simple preparation process, low cost and high yield. The method conforms to the national policy guidance of energy conservation, environmental protection and sustainable development.
Detailed Description
Bacterial source and numbering list related to the invention
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Preparation of seawater fish disease immunogen
According to the research report of offshore pathogenic bacteria in China, main pathogenic bacteria in China on the coastal region include vibrio anguillarum, edwardsiella, vibrio alginolyticus, vibrio parahaemolyticus, vibrio vulnificus and streptococcus. The above-mentioned standard bacterial strains of marine fish pathogenic bacteria are purchased from international and domestic cell banks, amplified and cultured, and tested for toxicity, and the concentration of half lethal dose of each pathogenic bacteria is respectively calculated. The median lethal dose of six pathogenic bacteria is 1 × 104～1×105/ml。
2. Preparation of seawater fish disease antibody
Immunization: and (2) fixedly inactivating the bacterial liquid with the half lethal dose concentration of various pathogenic bacteria by using formaldehyde fixing liquid, wherein the volume of Freund incomplete adjuvant is 1:1, respectively immunizing a mouse and a rabbit after mixing, injecting 0.5ml of immunogen into the abdominal cavity of each mouse, injecting 1ml of immunogen into the subcutaneous cavity of each rabbit, immunizing 3-4 times at intervals of 7-10 days, wherein the inactivated bacteria amount of each immunization of each mouse is 0.25 multiplied by 104～0.25×105. The inactivated bacteria amount of each time of immunization of rabbit is 0.5 × 104～0.5×105。
And (3) detecting the titer of the antibody: and 3-5 days after the second immunization, respectively collecting blood of each mouse and each rabbit (the mouse cuts the tail to take blood, and the rabbit draws blood from the ear vein), centrifuging to take serum, and detecting the antibody and titer of each pathogenic bacterium in the serum by an ELISA method. If the antibody titer reaches 10-4The above mice and rabbits were bled and sera were collected seven days after the third immunization. If the antibody titer does not reach 10-4And then the fourth immunization is carried out, or the animals are eliminated and replaced by new animals for re-immunization.
Collecting and storing the antibody: the antibody titer reaches 10-4The mice and rabbits can be bled to collect serum. Mice were bled by removing the eyeball after breaking the cervical marrow. Rabbits were bled by carotid cannulation. Serum was collected after centrifugation. Subpackaging and storing in a refrigerator at-25 deg.C. Two antisera were prepared for each pathogen, one was mouse anti-pathogenic serum (used for anti-idiotypic egg yolk antibodies and their primary screening for layer hens) and the other was rabbit anti-pathogenic serum (used as immunogen for anti-idiotypic antibody preparation).
3. Preparation of anti-idiotype yolk antibody for seawater fish disease
Preparation of immunogen: antibodies of 6 rabbits against marine fish pathogens (antibody titer of 10)-4) Mixing the mixture in equal volume, mixing the mixture with an equal volume of Freund incomplete adjuvant, and injecting 0.5ml of immune layer chicken into each layer chicken, wherein the amount of each pathogenic bacteria antibody is about 10-50 mu g. The immunization is carried out once every 7-10 days, and the immunization is carried out three times.
Screening laying hens: and 3-5 days after the second immunization, collecting eggs laid by the laying hens on the same day, and detecting whether various pathogenic bacteria anti-idiotypic antibodies exist in the yolk of the eggs and the titer of the antibodies by using an ELISA method. If the yolk contains 6 kinds of anti-idiotypic antibodies of pathogenic bacteria and the titer reaches 10-4In the above, the laying hens are listed as the objects for collecting eggs to prepare the anti-idiotype yolk antibody, and the laying hens which do not reach the standard are not listed as the objects for collecting eggs. The eggs are sold to customers as usual, and the food safety problem does not exist. The standard for judging whether the yolk contains the anti-idiotype antibody of the pathogenic bacteria is as follows: whether the antibody can react with antibodies generated by different organisms with antigen specificity; whether it competes with the antigen for binding to the antigen-specific antibody; whether it can induce different organisms to produce antigen-specific antibodies.
Collecting eggs: collecting eggs of laying hens meeting the egg collecting standard on the third day after the third immunization, continuously collecting the eggs for 20 days, supplementing immunization once again after 20 days if necessary, and continuously collecting the eggs for 20 days until the laying hens are eliminated.
Yolk separation: cleaning eggs with tap water, soaking in 75% alcohol for 10 min for sterilization, air drying, transferring into clean room, and manually separating yolk.
Breaking egg yolk tissues: the yolk tissue was disrupted by a high pressure homogenizer (60 Pa). Adding 10-40 times of distilled water after crushing, and uniformly stirring.
Removing the hybrid protein: the pH value of the yolk solution is adjusted to the isoelectric point (pH 5.2-6.0) of each lipoprotein by using hydrochloric acid and sodium hydroxide solution, so that turbid precipitates of the yolk solution are generated. The solution is kept stand in a refrigerator at 4-8 ℃ overnight. The egg yolk solution was precipitated for the contaminating proteins. And sucking the supernatant after precipitation by using a suction pipe on the next day, and centrifuging the turbid part at the bottom in a centrifugal machine of 3500-4000 r/min for 20-40 minutes and then taking the supernatant. Mixing the two supernatants, filtering with microporous filter (pore size of 0.22 μm) to remove lipoprotein and fat, and collecting filtrate which is crude product of anti-idiotype yolk antibody.
Purification of anti-idiotype yolk antibodies: separating and purifying the anti-idiotype egg yolk antibody by an organic solvent precipitation method, adding cold alcohol into the anti-idiotype antibody crude extract to enable the concentration of the anti-idiotype antibody crude extract to reach 50-60%, fully stirring to enable the anti-idiotype antibody crude extract to be turbid and precipitated, centrifuging for 20-30 min by a 4000r/min centrifugal machine, and taking a precipitate. Adding distilled water to restore the original volume, then adding cold alcohol to make the concentration reach 25-30%, fully stirring to make it turbid and precipitate, and centrifuging in the same way to obtain precipitate. Then distilled water is added to restore the original volume. Adding alcohol to make the concentration reach 20-25%, fully stirring to make it turbid and precipitate, centrifuging at the same time, collecting precipitate, oven-drying the precipitate at 37 deg.C, or adding appropriate amount of distilled water to make the precipitate dissolve to obtain refined concatenated anti-idiotype yolk antibody vaccine for seawater fish disease, with yield of 25-45%.
4. Detection of concatenated anti-idiotype egg yolk antibody of marine fish disease
1) Detecting the content of the anti-idiotype yolk antibody:
the content of the anti-idiotype yolk antibody vaccine produced in the embodiment 1 of the invention is detected by a quantitative ELISA method, and the result shows that the content is more than 7-8 mg/ml.
The specific procedure is as follows:
a coating buffer (0.05MpH9.6 carbonate coating buffer) is used to coat the yolk antibody standard
And the yolk antibody prepared by the invention is respectively expressed by 2n(multiple of square) dilution to n12。
And B, respectively and sequentially adding the egg yolk antibody standard substance which is serially diluted by 2 times and is diluted by 12 grades and the anti-idiotype egg yolk antibody of the invention into reaction pores of an ELISA plate, wherein each 12 pores is added with 100 mu l of the egg yolk antibody, and incubating for 1 hour at 37 ℃.
And C, discarding the liquid in the small hole, washing 3 times with the washing buffer solution for 3 minutes each time, and patting dry.
D, adding an antibody of the rabbit anti-yolk antibody (diluted by an antibody diluent to be 1:1000), and incubating at 37 ℃ for 0.5-1 hour, wherein each well is 100 mu l.
E repeating the procedure C.
HRP-labeled goat anti-rabbit IgG antibody (1:1000 dilution) was added to F at 37 ℃ in 100. mu.l/well
And (5) incubating for 0.5-1 hour.
G repeat procedure C.
Adding a color developing agent (containing 0.045% of hydrogen peroxide and 10-20 mg of phthalic acid-citric acid buffer with pH 5.0), and developing for 15-20 minutes at normal temperature in a dark place, wherein each well contains 100 mu l of the color developing agent.
And I, on an ELISA detector, after a blank control group is zeroed at the position of 490nm, measuring the OD value of each hole, and if the OD value of the hole to be measured is more than or equal to 2.1 times of that of a control hole, determining that the hole is positive, and determining that the negative control hole is colorless.
The detection results are shown in table 1:
TABLE 1 OD value table for different dilutions of the anti-idiotype yolk antibody according to the invention
Note: the original concentration of the standard solution was 1 mg/ml.
K results: the OD values of the standard egg yolk antibody and each level of dilution of the anti-idiotype egg yolk antibody prepared by the invention are shown in table 1, a standard curve is drawn by curve expert software, the R value reaches more than 0.98, and the content of the anti-idiotype egg yolk antibody in the product is 7-8 mg/ml.
2) Anti-idiotype yolk antibody titer detection
The titer of the anti-idiotype yolk antibody produced by the invention is detected by an indirect ELISA method, and the result shows that the titer is 1 multiplied by 10-4～1×10-5。
The specific procedure is as follows:
a six mice were treated with coating buffer (0.05MpH9.6 carbonate coating buffer) against seawater
Pathogen antibodies were diluted 1: 1000. Then adding the antibodies into small holes of an ELISA plate respectively, adding 10 small holes for each antibody, and incubating for 1-2 hours at 37 ℃ with each hole of 100 mu l.
B the mouse anti-pathogenic antibodies in the wells were discarded and washed 3 times with 3 minutes each time with wash buffer.
C10-fold serial dilution (10) of an anti-idiotype antibody according to the invention with an antibody dilutionn) Dilution of 10 stages at all times (10)10). Different dilutions of anti-idiotype yolk antibody were added to the microplate in sequenceIn the wells, 6 wells (corresponding to the antibodies of the mice with six pathogenic bacteria) are added to each dilution, 60 wells are added to 10 dilutions, 100 μ l of each dilution is placed in each well, and incubation is carried out at 37 ℃ for 0.5-1 hour.
And D, repeating the procedure B.
E, adding 100. mu.l of rabbit anti-yolk antibody (diluted 1:1000 with antibody diluent) per well, and incubating at 37 ℃ for 0.5-1 hour.
F, repeating the procedure B.
G, adding an HRP-labeled monoclonal antibody rabbit IgG antibody (diluted 1:1000), placing 100 mu l of the antibody in each hole, and incubating for 0.5-1 hour at 37 ℃.
H, repeating the procedure B.
Adding a display agent (0.045% hydrogen peroxide liquid and 10-20 mg o-phenylenediamine in phosphoric acid-citric acid buffer solution with pH 5.0) into each well, and developing for 15-20 minutes at normal temperature in a dark place.
Stop buffer (10.6% sulfuric acid solution) was added to J, 50. mu.l per well.
And K, detecting the OD value of each hole after blank control is zero at the wavelength of 490nm on an ELISA detector, and if the OD value of the hole to be detected is more than or equal to 2.1 times of that of the negative control hole, determining that the hole to be detected is positive.
The potency of each of the concatemeric anti-idiotype yolk antibodies is shown in the following table
TABLE 2 Table of the results of the potency test of each of the concatameric anti-idiotypic yolk antibodies prepared according to the invention
As a result: table 2 shows that the titer of each combined anti-idiotype antibody in the combined anti-idiotype antibodies for seawater fish diseases prepared by the invention is 10-4～10-5。
3) Anti-idiotype yolk antibody potency assay
A, experimental fish: the flounder 600 tails (5-7 cm, 4-5 months old) are divided into 4 groups, and each group has 150 tails.
B, immunization:
experiment I group (150 tails) were immunized by intraperitoneal injection. Mixing the vaccine and incomplete adjuvant, and injecting 50 μ l (containing 3.75 μ g vaccine) per tail of immunized sea fish via intraperitoneal injection.
Experiment II (150 pieces) is soaked for immunization, the vaccine immunization amount is 3 times of that of the vaccine for injection,
dissolving 1.69mg vaccine into 13.5L seawater, stirring, and adding 150 tail sea fish
Soaking in seawater containing the vaccine for 30 minutes, and if the density of one batch is too high, the soaking can be carried out by 2-3 batches. 30
After minutes, the mixture is fished back to the pool for normal feeding.
Experiment III (150 tails) was immunized orally by dissolving the vaccine in normal saline, spraying it on fish bait to make it semi-wet, and feeding fish in water with the vaccine amount equivalent to the immersion immunity but three times with an interval of one week. Control group (150 tailed), no treatment, control. After immunization, the vaccine is normally bred for one month and then is detoxified.
C, strain preparation: after 6 strains are amplified and cultured, the toxicity is measured.
And (3) virus detection program: the concentration of each strain of germ stock solution is detected by a plate counting method, and then 5-grade dilution is carried out by a 10-time serial dilution method. And (4) carrying out intraperitoneal injection on the sea fish with the counteracting poison by using bacterial liquids with different dilution concentration levels (100 mu l of each injection), and calculating the half lethal dose of various germs on the sea fish. The half lethal dose concentration of various bacteria is used for carrying out toxicity attacking tests on the marine fishes. The median lethal dose of each strain is shown in Table 3.
TABLE 3 test results of half lethal dose of various bacteria
As a result: table 3 shows that the median lethal dose of six pathogenic bacteria to the experimental marine fish is 106/ml～104/ml。
Counteracting toxic substances: the fish of all levels were challenged with 5-half times the lethal dose concentration of each strain, 20 tails of each group of fish were intraperitoneally injected with 100 μ l each tail, observed for 7 days, the number of deaths of each group of fish was counted, and the protection rate of the vaccine against marine fish was calculated as shown in table 4.
Table 4 shows that the protective rates of the injection type vaccine to six pathogenic bacteria are 75-85% respectively; the protective rate of the soaking type vaccine to the attack of six pathogenic bacteria is 55 to 65 percent; the protection rate of the oral vaccine against six pathogenic bacteria is 75-85%.
TABLE 4 protective ratio of vaccine against marine fish by challenge
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A preparation method of a mariculture fish concatenated anti-idiotype yolk antibody vaccine comprises the following steps:
1) inactivating the bacteria liquid with half lethal dose concentration of pathogenic bacteria of the marine fish, and then adding an adjuvant to immunize mice and rabbits, wherein the inactivated bacteria dose of each immunization of the mice is 0.25 multiplied by 104～0.25×105The inactivated bacteria amount for rabbit immunization is 0.5 × 104～0.5×105The serum antibody titer of the immunized mouse or rabbit reaches 10-4；
2) Respectively collecting the serum of a mouse and the serum of a rabbit, mixing the serum with 6 rabbit pathogenic-resistant sera, and adding an adjuvant to immunize a laying hen, wherein the immunization method of the laying hen is intramuscular injection, the immunization amount is 0.5ml, and the antibody content is 10-50 mu g; immunizing for 1 time every 7-10 days, and immunizing for 3 times;
3) screening laying hens, and detecting whether eggs laid by the hyperimmune chickens contain anti-idiotype yolk antibodies and titer of six pathogenic bacteria by using an indirect ELISA method;
4) collecting eggs of laying hens, separating yolk, crushing to remove impurity proteins, layering precipitates, taking supernatant, and further removing lipoprotein and fat to obtain an anti-idiotype yolk antibody crude product;
5) purifying the anti-idiotype yolk antibody crude product to obtain a concatenated anti-idiotype yolk antibody vaccine for seawater fish diseases;
the pathogenic bacteria of the marine fish are vibrio alginolyticus with the number of ATCC 33838, vibrio anguillarum with the number of ATCC 19106, edwardsiella tarda with the number of ATCC 23657, vibrio parahaemolyticus with the number of ATCC 7802T, vibrio vulnificus with the number of ATCC33147 and streptococcus with the number of MCCC1A 10884.
2. The method for preparing the concatenated anti-idiotype yolk antibody vaccine for the mariculture fish of claim 1, wherein the method for immunizing the mice and the rabbits in the step 1) is intraperitoneal injection or subcutaneous injection, and the immunization times are 3-4 times, and the interval is 7-10 days each time.
3. The method for preparing the concatenated anti-idiotype yolk antibody vaccine for the mariculture fish as claimed in claim 1, wherein the method for screening the laying hens in the step 3) is to detect that the eggs laid by the hyperimmune chickens contain anti-idiotype antibodies of 6 pathogenic bacteria by an ELISA method, and the titer is 10-4The above.
4. The method for preparing the concatenated anti-idiotype yolk antibody vaccine for the mariculture fish according to claim 1, wherein the disruption method in the step 4) is as follows: crushing yolk tissue with a high-pressure homogenizer, adding distilled water after crushing, and stirring uniformly.
5. The method for preparing the multi-connected anti-idiotype yolk antibody vaccine for the mariculture fish according to claim 1, wherein the method for removing the hybrid protein in the step 4) comprises the steps of adjusting the pH value of the yolk solution to the isoelectric point of lipoprotein, standing for precipitation, taking the supernatant, centrifuging the turbid part at the bottom, taking the supernatant, combining the supernatants, performing microfiltration to further remove the lipoprotein and fat, and taking the filtrate, wherein the filtrate is a crude product of the anti-idiotype yolk antibody.
6. The method for preparing the concatenated anti-idiotype yolk antibody vaccine for the marine fish according to claim 1, wherein the crude anti-idiotype antibodies in step 5) are purified by organic solvent precipitation centrifugation to obtain a precipitate; and repeatedly treating for 1-2 times, taking the precipitate, drying or adding a proper amount of distilled water to dissolve the precipitate, and thus obtaining the refined concatenated anti-idiotype yolk antibody vaccine for the seawater fish disease.
7. The concatenated anti-idiotype yolk antibody vaccine for marine fish as prepared by the method of any one of claims 1 to 6, or the concatenated anti-idiotype yolk antibody vaccine for marine fish.
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| CN109306018A (en) * | 2018-11-01 | 2019-02-05 | 西安德轩驰生物科技有限公司 | A kind of growth hormone release inhibiting hormone immunogen complex and its method for preparing growth hormone release inhibiting hormone antiidiotype Yolk antibody |
| CN110724196A (en) * | 2019-11-19 | 2020-01-24 | 西安咸辅生物科技有限责任公司 | Preparation method of anti-idiotype egg yolk antibody |
| CN112175076A (en) * | 2020-07-24 | 2021-01-05 | 中山大学附属第一医院 | Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection |
| CN112175077A (en) * | 2020-08-10 | 2021-01-05 | 中山大学附属第一医院 | Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection |
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