CN110577596A - Egg yolk antibody for resisting chicken coccidiosis and preparation method and device thereof - Google Patents
Egg yolk antibody for resisting chicken coccidiosis and preparation method and device thereof Download PDFInfo
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- CN110577596A CN110577596A CN201910920572.4A CN201910920572A CN110577596A CN 110577596 A CN110577596 A CN 110577596A CN 201910920572 A CN201910920572 A CN 201910920572A CN 110577596 A CN110577596 A CN 110577596A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a yolk antibody and a preparation method and a device thereof, wherein by utilizing the test object of the invention, the Eimeria tenella, the Eimeria maxima and the Eimeria necatrix are rejuvenated and proliferated, the coccidia are mixed according to equal proportion, then the antigen is prepared by an ultrasonic crushing method, the obtained antigen solution is respectively and uniformly mixed with equal volume of Freund's complete adjuvant and Freund's incomplete adjuvant, then 3 times of immunization is carried out on laying hens, and after 3 times of immunization, the yolk antibody in the collected egg yolk is extracted, purified, identified and evaluated; the research of different yolk powder preparation processes is respectively carried out, the preparation effect of freeze-dried powder is obviously higher than that of a powder spraying technology, the yolk and diluent proportion relationship, the freeze-drying temperature curve and other parameter conditions in the preparation process are relatively low in cost, the preparation efficiency is high, the loss rate of the antibody is only 10%, the antibody titer is high, the antibody titer is highly reduced after high-temperature powder spraying, and the method is obviously superior to the method for preparing yolk powder by the high-temperature powder spraying technology.
Description
Technical Field
the invention relates to the technical field of egg yolk antibody preparation for preventing chicken coccidiosis, in particular to an egg yolk antibody for resisting chicken coccidiosis and a preparation method and a device thereof.
background
Chicken coccidiosis (Avian coccidiosis) is caused by Eimeria (Eimeria:)Eimeria) One or more coccidia of (a) is parasitic protozoa disease caused by parasitizing in intestinal epithelial cells of chicken. The disease is distributed globally, and causes great economic loss for the chicken industry. In 1870, Rivolta and Eimer report that coccidian parasitizes in intestinal tracts of chickens for the first time, and the coccidiosis in chickens is one of the most frequent, serious and difficult diseases to prevent in intensive chicken farms in China at present. For a long time, the main means for preventing and treating chicken coccidiosis is to apply chemical drugs, but coccidiosis is extremely easy to generate drug resistance, and the phenomenon that coccidiosis still occurs during the drug application period is sometimes caused. In recent years, with the improvement of living standard of people, the drug residue is rightThreats to human health are of increasing concern. In addition, the development and application of coccidian vaccines have many key problems, and the immune prevention of chicken coccidiosis is difficult to popularize and apply in a short time. The countries of the european union have banned the use of anticoccidial chemical synthesis drugs as feed additives, and china plans to add anticoccidial drugs to feed in 2020. Recently, the yolk antibody is gradually paid attention to animal disease resistance, and the yolk antibody not only has the effect of killing coccidia, but also can promote the growth of chicks, so that the yolk antibody is expected to have wide development prospect in prevention and treatment of coccidiosis in chickens in the future. In a word, the egg yolk antibody has good application prospect in the prevention and treatment process of chicken coccidiosis, can replace antibiotics or chemical drugs, reduces the drug resistance of coccidia, and is a novel green and environment-friendly chemical drug substitute.
The yolk antibody IgY is characterized by comprising the following components: has good stability, heat resistance, acid resistance, alkali resistance, ion resistance and certain enzymolysis resistance. Studies have shown that IgY is resistant to digestion by trypsin and chymotrypsin in the gut of young animals. The nature of the poultry immune system dictates that small doses of antigen can be effective, and thus large quantities of high titer specific antibodies can be obtained over a longer period of time with a small amount of antigen. The laying hens lay eggs 280/egg, and the IgY content is about 100-150 mg/egg, so that the egg yolk antibody produced by the laying hens has great production potential. In addition, the preparation of the specific IgY antibody also has the advantages of economy, easy operation, large yield and the like. However, the existing method for preparing and purifying the yolk antibody still needs to be improved, and the high-temperature spraying method is easy to cause the activity inactivation, the function loss and the like of the yolk antibody.
Disclosure of Invention
The invention aims to provide a yolk antibody for resisting chicken coccidiosis and a preparation method and a device thereof.
The invention solves the technical problems through the following technical scheme:
a method for preparing an egg yolk antibody, comprising: immunizing laying hens with antigen prepared based on coccidia, and extracting yolk antibody from egg yolk of laying hens.
Optionally, the coccidia comprise at least 3 and more of the following:
Eimeria tenella, Eimeria maxima and Eimeria necatrix.
Optionally, the immunizing layer chicken with the coccidian-based antigen comprises:
Purifying oocysts of Eimeria tenella, Eimeria maxima and Eimeria necatrix, and adjusting the concentration of oocysts to 1 × 108Carrying out ultrasonic crushing for multiple times for the mixed solution of each ml, wherein the working time of the ultrasonic crushing is 3 seconds, each time interval is 8 seconds, and the total time is 30 minutes;
mixing the antigen solution after ultrasonic crushing with immunopotentiator in equal volume, and performing intramuscular injection immunization on 200-day-old laying hens, wherein the antigen solution is 1 × 106Each individual/feather was immunized 1 time every 10 days for a total of 5 times.
Optionally, the extracting yolk antibodies from egg yolk comprises:
Collecting eggs immunized for 5 times, removing egg white, adding distilled water according to the volume ratio of 1:7 for dilution, and fully stirring; standing at 4 deg.C to precipitate lipid, collecting supernatant, centrifuging at low temperature with ultracentrifuge at rotation speed of 10000rmp/min for 25min, and collecting supernatant.
Optionally, the method further includes:
Collecting supernatant, adding saturated ammonium sulfate solution to final concentration of 50%, standing for 30min, centrifuging at 8000rmp/min, and collecting precipitate;
mixing a saturated ammonium sulfate solution and a carbonate buffer solution with the concentration of 0.1mol/L, pH of 7.6 according to the ratio of 1:2 to obtain a mixed solution, adding the precipitate into the mixed solution, uniformly mixing and standing for 30 minutes to obtain the final concentration of 30%, removing the supernatant, and dissolving the precipitate in a Phosphate Buffer Solution (PBS) with the concentration of 0.1mol/L, pH of 7.6 to obtain the purified antibody.
Optionally, the extracting yolk antibodies from egg yolk comprises:
Removing supernatant from eggs, subpackaging egg yolk in a glass ware under aseptic condition, and freezing in a dryer at low temperature to obtain freeze-dried egg yolk antibody powder.
optionally, the extracting yolk antibodies from egg yolk comprises:
Removing supernatant from ovum gallus Domesticus, diluting with 60% sterilized water, stirring, freezing in-40 deg.C freeze-drying machine for 4 hr, and freezing at-15 deg.C for 15 hr; the obtained freeze-dried substance is transferred to 10 ℃ and placed for 1.5 hours, and finally transferred to 36 ℃ and dried for 4 hours, so as to obtain the freeze-dried yolk antibody powder.
In order to achieve the above object, there is also provided a yolk antibody produced by the production method according to any one of the above aspects.
In order to realize the purpose, the application of the yolk antibody in preparing a preparation for preventing and treating chicken coccidiosis is also provided.
To achieve the above object, there is also provided an apparatus for producing an egg yolk antibody, which is used for carrying out the method of any one of the above objects.
Compared with the prior art, the invention has the following advantages: the method comprises the steps of rejuvenating and proliferating eimeria tenella, eimeria maxima and eimeria necatrix, mixing the eimeria tenella, eimeria maxima and eimeria necatrix, crushing by ultrasonic waves to prepare antigen for immunizing laying hens, and extracting purified egg yolk antibody from egg yolk.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing a yolk antibody against chicken coccidiosis according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of an IgY electrophoretic analysis result according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing the growth and decline of antibody titer in yolk after immunization according to an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the effect of different processing methods on the antibody titer in egg yolk according to the embodiment of the present invention.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
referring to fig. 1, a chicken coccidia yolk antibody preparation process is shown, which comprises:
S101, E.tenella, E.maxima and E.necatrix proliferation and rejuvenation;
S102, Eimeria tenella, Eimeria maxima and Eimeria necatrix antigen preparation;
S103, extracting, purifying, identifying and evaluating the antibody titer of the immunized yolk antibody;
S104, preparing chicken coccidian egg yolk antibody powder.
Specifically, the preparation method of the yolk antibody comprises the following steps: immunizing a laying hen with an antigen prepared based on coccidia; yolk antibody is extracted from egg yolk of laying hen.
Specifically, the coccidia comprises at least 3 and more than 3 of the following coccidia: eimeria tenella, Eimeria maxima and Eimeria necatrix.
Immunizing a laying hen with an antigen prepared based on coccidia, comprising: purifying oocysts of Eimeria tenella, Eimeria maxima and Eimeria necatrix, and adjusting the concentration of oocysts to 1 × 108Carrying out ultrasonic crushing for multiple times for the mixed solution of each ml, wherein the working time of the ultrasonic crushing is 3 seconds, each time interval is 8 seconds, and the total time is 30 minutes; mixing the antigen solution after ultrasonic crushing with immunopotentiator in equal volume, and performing intramuscular injection immunization on 200-day-old laying hens, wherein the antigen solution is 1 × 106Each individual/feather was immunized 1 time every 10 days for a total of 5 times.
extracting yolk antibodies from egg yolk, comprising: collecting eggs immunized for 5 times, removing egg white, adding distilled water according to the volume ratio of 1:7 for dilution, and fully stirring; standing at 4 deg.C to precipitate lipid, collecting supernatant, centrifuging at low temperature with ultracentrifuge at rotation speed of 10000rmp/min for 25min, and collecting supernatant.
In practical application, the method further comprises the following steps: collecting supernatant, adding saturated ammonium sulfate solution to final concentration of 50%, standing for 30min, centrifuging at 8000rmp/min, and collecting precipitate; mixing a saturated ammonium sulfate solution and a carbonate buffer solution with the concentration of 0.1mol/L, pH of 7.6 according to the ratio of 1:2 to obtain a mixed solution, adding the precipitate into the mixed solution, uniformly mixing and standing for 30 minutes to obtain the final concentration of 30%, removing the supernatant, and dissolving the precipitate in 0.1 mol/LpH7.6 Phosphate Buffer Solution (PBS) to obtain the purified antibody.
In addition, the yolk antibody is extracted from the yolk of the egg, and comprises the following components:
Removing supernatant from eggs, subpackaging egg yolk in a glass ware under aseptic condition, and freezing in a dryer at low temperature to obtain freeze-dried egg yolk antibody powder. The method specifically comprises the following steps: removing supernatant from ovum gallus Domesticus, diluting with 60% sterilized water, stirring, freezing in-40 deg.C freeze-drying machine for 4 hr, and freezing at-15 deg.C for 15 hr; the obtained freeze-dried substance is transferred to 10 ℃ and placed for 1.5 hours, and finally transferred to 36 ℃ and dried for 4 hours, so as to obtain the freeze-dried yolk antibody powder.
in order to achieve the above object, there is also provided a yolk antibody produced by the production method according to any one of the above aspects.
In order to realize the purpose, the application of the yolk antibody in preparing a preparation for preventing and treating chicken coccidiosis is also provided.
To achieve the above object, there is also provided an apparatus for producing an egg yolk antibody, which is used for carrying out the method of any one of the above objects.
Therefore, the method has the advantages of simple operation, low cost, high preparation efficiency and high antibody titer. Using the test subjects of the present invention, Eimeria tenella, Eimeria maxima and Eimeria necatrix were rejuvenated and proliferated, and then Eimeria tenella was addedCoccidia, Eimeria maxima and Eimeria necatrix in equal proportions (1X 10)5Oocysts /), preparing an antigen by an ultrasonic crushing method, respectively mixing the obtained antigen solution with equivalent volume of Freund's complete adjuvant and Freund's incomplete adjuvant, sequentially immunizing laying hens for 3 times, and extracting, purifying, identifying and evaluating the antibody titer of the collected egg yolk antibody after 3 times of immunization; the research of different yolk powder preparation processes is respectively carried out, the preparation effect of freeze-dried powder is obviously higher than that of a powder spraying technology, the yolk and diluent proportion relationship, the freeze-drying temperature curve and other parameter conditions in the preparation process are relatively low in cost, the preparation efficiency is high, the loss rate of the antibody is only 10%, the antibody titer is high, the antibody titer is highly reduced after high-temperature powder spraying, and the method is obviously superior to the method for preparing yolk powder by the high-temperature powder spraying technology.
The invention is realized by the following modes: a preparation method of trivalent yolk antibody comprises rejuvenating and proliferating Eimeria tenella, Eimeria maxima and Eimeria necatrix. Then mixing the Eimeria tenella, the Eimeria maxima and the Eimeria necatrix, carrying out ultrasonic crushing to prepare antigen immune layer chicken, and then extracting purified yolk antibody from the yolk of the egg.
1.1 preparation of immunogen and immune layer chicken
Purifying Eimeria tenella, Eimeria maxima and Eimeria necatrix oocysts, and adjusting the concentration of oocysts to 1 × 108And (3) carrying out ultrasonic crushing on the mixed solution per ml, wherein the working time is 3 seconds, the interval is 8 seconds, and the total time is 30 minutes. Mixing the antigen with the same volume with novel immunopotentiator (Freund's complete adjuvant or Freund's incomplete adjuvant), and immunizing layer chicken of 1 × 10 days old with intramuscular injection6Each individual/feather was immunized 1 time every 10 days for a total of 5 times.
1.2 preliminary separation of yolk antibody by water washing
Collecting 5-immune eggs, removing egg white, adding distilled water according to the volume ratio of 1:7 for dilution, and fully stirring; standing at 4 deg.C to precipitate lipid thoroughly. And (3) obviously layering the diluent, taking the upper layer liquid, centrifuging by using a low-temperature ultracentrifuge at the rotating speed of 10000rmp/min for 25min, and collecting the supernatant.
1.3 purification of IgY by saturated ammonium sulfate method
Collecting supernatant, slowly adding saturated ammonium sulfate solution to final concentration of 50%, avoiding generating bubbles as much as possible, standing for 30min, and centrifuging at 8000rmp/min to obtain precipitate. The mixture was slowly added to a mixture of saturated ammonium sulfate solution and 0.1mol/L carbonate buffer solution (pH 7.6) (ratio: 1: 2), mixed and left to stand for 30 minutes to a final concentration of 30%, the supernatant was removed, the precipitate was dissolved in 0.1mol/L Phosphate Buffer Solution (PBS) pH7.6, and the purified antibody was identified by SDS-PAGE electrophoresis, and the result is shown in FIG. 2, which represents the antibody purification result, wherein M: a protein Marker; 1: purified IgY. The purified IgY was identified to have a band around 66 kD, which is consistent with the expected results.
1.4 establishment of chicken coccidian yolk antibody indirect ELISA method (including S103)
after the immunized eggs are purified by saturated ammonium sulfate, the prepared egg yolk antibody is detected by adopting an indirect ELISA method, the optimal dilution of coccidian antigen is determined by adopting a matrix titration method, meanwhile, the dilution concentration, the reaction time and other parameters of an enzyme-labeled secondary antibody are determined, and the established method is used for detecting the egg yolk antibody to be detected.
1.4.1 preparation of coating antigens
The difference between the preparation of the immunogen and the method for immunizing the laying hens in the step 1.1 is that the antigen concentration is adjusted to be 0.9 mg/mL.
1.4.2 establishment of Indirect ELISA method
(1) Screening for optimal dilution of antigen:
Diluting the coating antigen by 1:50, 1:100, 1:200, 1:400 and 1:800 times respectively, and coating overnight; the plates were washed four times with PBST, blocked with 5% skimmed milk powder and incubated for 2h at 37 ℃. PBST buffer solution is washed for 3 times, purified yolk antibody diluted by 1:50, 1:100, 1:200, 1:400 and 1:800 times is added respectively, and the mixture is incubated for 1h at 37 ℃; washing with PBST buffer solution for 3 times, adding 1:700 horseradish peroxidase labeled rabbit anti-chicken secondary antibody, and incubating at 37 ℃ for 1 h; according to OD450 nmThe P (positive)/N (negative) ratio is calculated, the maximum ratio being the best oneAnd (4) coating conditions. The results show that the P/N value is optimal when the antigen is diluted 1:100 times, so that the optimal antigen coating concentration is selected as 1:100 times.
(2) Screening of enzyme-labeled secondary antibody concentration and reaction time
Diluting the rabbit anti-chicken labeled by horseradish peroxidase with 1g/L BSA solution to 1: 5000, 1: 10000, 1: 15000 and 1: 20000 times as the second antibody, incubating at 37 ℃ for 30min, 60 min, 90min and 120 min, and determining the optimal reaction condition of the second antibody. Adding 50 mu L of chromogenic base solution TMB/hole after finishing the secondary antibody reaction, adding 2mol/L of sulfuric acid solution to terminate the reaction when the secondary antibody reacts for 5min, 10min, 15min and 20min at 37 ℃ in a dark place respectively, and determining the optimal chromogenic time.
1.5 detection of antibody levels at different stages of immunization Using the established ELISA method
purified yolk antibody (0.0090 mg/mL) was added in 100. mu.L per well overnight at 4 ℃. The plates were washed 4 times with PBST, blocked with 5% skimmed milk powder and incubated at 37 ℃ for 2 h. The plate was washed 3 times with wash solution, 100. mu.L of primary antibody diluted in multiple aliquots was added to each well, and incubated at 37 ℃ for 1 h. The plate was washed 3 times with washing solution, and a secondary HRP-labeled antibody was added thereto at 100. mu.L/well, followed by incubation at 37 ℃ for 1 h. Washing the plate for 4 times with washing solution, adding TMB developing solution, 100 μ L/well, and reacting for 10min in dark. The reaction was terminated by adding 50. mu.L/well of 2mol/L sulfuric acid solution, and OD was measured450mmThe value of (c). The results are shown in FIG. 3, which shows the law of antibody growth after immunization: after the five-immunization 10 days, the egg yolk antibody level is in an ascending trend, the highest value is reached after the five-immunization 60 days, and the highest titer of the antibody is 1: 25600. Then there is a short time of a large drop followed by a steady rise to a certain level. In addition, the results also show that the high titer of the yolk antibody after immunization can be maintained for more than three months, which greatly improves the yield of the yolk antibody.
1.6 preparation of anti-coccidiosis trivalent yolk antibody freeze-dried powder
1.6.1 establishment of yolk powder Freeze drying technique (including S104)
Removing supernatant from eggs, subpackaging egg yolks in glassware under aseptic condition, quickly freezing in a dryer at low temperature, and freeze-drying and screening the eggs according to the following parameters: adding 60% sterilized water of egg yolk liquid, diluting, stirring, quickly freezing in a freeze dryer at-40 deg.C for 4 hr, and freezing at-15 deg.C for 15 hr; transferring the lyophilized extract to 10 deg.C for 1.5 hr, and transferring to 36 deg.C for 4 hr to obtain yolk powder. And (4) filling the collected freeze-dried egg yolk antibody powder into a sealing bag, and sealing and storing.
1.6.2 high temperature spray powder control test
In the test, after yolk lipid is precipitated, 8% maltodextrin serving as a protective agent is added into supernatant filtration, fully and uniformly stirred, and then spray drying is carried out, wherein the conditions are as follows: the air inlet temperature is 150-.
1.6.3 evaluation of antibody Activity in lyophilized powder of trivalent yolk antibody against Coccidia
The lyophilized powder is subjected to indirect ELISA (enzyme-linked immunosorbent assay) method to detect the antibody level of the lyophilized powder, the loss condition of the egg yolk antibody before and after lyophilization is evaluated, and high-temperature powder spraying is used as a control test. Through the detection of the established indirect ELISA method, the antibody activity in the freeze-dried egg yolk powder is still higher and is 90% of the activity in egg yolk liquid, while the activity of the egg yolk powder prepared by adopting a high-temperature powder spraying mode is only 30% of the activity of the antibody in the egg yolk liquid, and the result is shown in figure 4, which represents the evaluation of the effect of the coccidian egg yolk antibody powder prepared by different processes.
1.7 evaluation of the Effect of the yolk antibody prepared by the invention on treating coccidiosis in chicks
The 18-day-old broilers were randomly divided into 6 groups, namely a white control group (no infection and no treatment), a red control group (no treatment), a negative yolk control group (normal yolk infection), a drug treatment group, a hyperimmune yolk treatment group a and a group b, wherein 22 broilers are respectively selected. All groups received 1X 10 except the white control group4And (3) carrying out a pest attack test on one or more test groups, starting treatment on the test groups 3 days after pest attack, wherein the hyperimmune yolk therapy group A is orally administered with 10ml of yolk liquid, the group B is 5ml of yolk liquid, and the drug treatment group receives 0.25 g/one concentration treatment. And respectively calculating the survival rate, the average weight gain, the number of oocysts discharged per gram of excrement, the lesion reduction rate, the coccidian resistance index and the like as indexes, and comprehensively evaluating the effect of the yolk antibody on treating the coccidian infection of the chicken. The results are as follows:
(1) In terms of relative weight gain:
As can be seen from table 1, the average weight gain of the test chickens of the other egg yolk antibody addition groups and the drug treatment group is significantly increased compared with the red control group and the negative egg yolk treatment group, wherein the weight gains of the drug (chlorpromazine hydrochloride)) treatment group and the hyperimmune egg yolk treatment group a are the most, the relative weight gains are 83.30% and 106%, respectively, and the difference is significant compared with the other groups ((P <0.05), so that the passive immune protection effect of the chicks caused by the addition of IgY is better, and the difference is not great compared with the treatment effect of the drug salinomycin.
TABLE 1 Effect of yolk antibody on the weight gain Effect of Coccidia-infected chickens
| Group of | average weight gain (g) | Relative weight gain (%) |
| White control group | 11.02±9.89bc | 100 |
| Red control group | -13.06±15.77adef | 0 |
| Negative yolk control group | -6.29±17.35adf | 0 |
| Hyperimmune yolk therapy group a | 11.71±14.24bc | 106 |
| hyperimmune yolk therapy group b | 4.94±12.48b | 44.70 |
| Drug treatment group | 9.18±18.80bc | 83.30 |
Note: the difference is significant (P <0.05) for different letters within the same column; the same letter indicates no difference (P > 0.05).
(2) In terms of rate of oocyst reduction:
As shown in Table 2, except for the blank control group, the other groups all had different numbers of oocysts to be discharged, wherein the red control group and the negative control group discharged the largest amount of oocysts, and the yield of oocysts of the other test groups was decreased to different degrees. The oocyst reduction rates of the hyperimmune yolk therapy group a, the drug therapy group and the group b are respectively 99.99%, 99.98% and 97.40%, and the oocyst yield is greatly reduced compared with that of the red control group.
TABLE 2 yolk antibody treatment effect on oocyst production in coccidial infected chickens
| Group of | Oocyst yield (volume) | relative oocyst yield (%) | Oocyst reduction ratio (%) |
| White control group | 0 | 0 | 0 |
| Red control group | 1.45x107 | 100 | 0 |
| Negative yolk control group | 9.30x106 | 66.2 | 33.80 |
| Hyperimmune yolk therapy group a | 1.50x104 | 0.01 | 99.99 |
| Hyperimmune yolk therapy group b | 3.70x105 | 2.60 | 97.40 |
| Drug treatment group | 2.70x104 | 0.02 | 99.98 |
(3) In the aspect of pathological conditions
As shown in Table 3, each chicken in each group was infected with 1X 10 of the virus4After sporulation of oocysts, the red control group had the most serious lesions, the average lesion score reached 3.13, and the caecum lesions of the hyperimmune yolk treated group a and the drug treated group were obviousSignificant relief (P)<0.05), the reduction rate of pathological changes respectively reaches 40 percent and 60 percent, but the drug treatment group has certain advantages. The lesions in the other groups were not significantly reduced.
TABLE 3 Scoring of chicken lesions in each group
| group of | lesion scoring | lesion reduction ratio (%) | |
| White control group | 0 | 0 | |
| Red control group | 3.13±1.533be | 0 | |
| negative yolk control group | 2.75±0.707e | 12 | |
| Hyperimmune yolk therapy group a | 1.88±0.835a | 40 | |
| hyperimmune yolk therapy group b | 2.13±0.991 | 32 | |
| Drug treatment group | 1.25±0.707ab | 60 |
Note: the difference is significant (P <0.05) for different letters within the same column; the same letter indicates no difference (P > 0.05).
(4) Evaluation of anticoccidial index
As shown in table 4, the ACI of each test group was higher than that of the red control group as a whole, wherein the ACI of the hyperimmune yolk treated group a and the drug treated group were higher, 187.30 and 170.80, respectively, while the ACI of the hyperimmune yolk treated group b was relatively lower, and the ACI of the red control group and the negative yolk control group were the lowest. As for the ACI index, the group A and the drug treatment group have better immune protection effect, and the rest groups have poorer immune protection effect.
TABLE 4 comparison of anticoccidial index ACI for each group
| Group of | Survival rate | Relative rate of weight gain | Value of the disease | oocyst value | ACI |
| White control group | 100% | 100% | 0 | 0 | 200 |
| Red control group | 77% | 0 | 31.3 | 40 | 5.7 |
| Negative yolk control group | 86% | 0 | 27.5 | 30 | 28.5 |
| Hyperimmune yolk therapy group a | 100% | 106 | 18.7 | 0 | 187.3 |
| Hyperimmune yolk therapy group b | 100% | 44.7 | 21.2 | 1 | 123.5 |
| drug treatment group | 100% | 83.3 | 12.5 | 0 | 170.8 |
mixing the amplified and purified live oocysts of eimeria polytricha according to a certain proportion to form immunogen, performing intramuscular injection immunization on 240-day-old laying hens, after 5 times of immunization, extracting egg yolk antibodies from the immunized eggs at each stage by adopting a saturated sulfuric acid method, and optimizing the optimal water dilution times and the optimal pH value for extracting IgY, thereby establishing the method for extracting and purifying the IgY from the eggs. Then, the purity of the purified anti-coccidian IgY is determined by an SDS-PAGE electrophoresis method; the established indirect ELISA method is used for dynamically monitoring the level of the antibody of the egg; screening freeze-drying parameters to establish a chicken coccidian egg yolk antibody powder preparation technology. The results show that the antibody level in the yolk after the oocyst 5 immunization gradually rises, and the antibody level decreases in a small amplitude within a short time, and reaches a peak value after 60 d. SDS-PAGE analysis shows that the purity of the purified yolk antibody reaches 98 percent, and the titer can reach 1: 25600/mg. The activity survival rate of the antibody in the chicken coccidian egg yolk antibody powder prepared by the freeze-drying technology is more than 95 percent and is obviously superior to that of the high-temperature powder spraying technology. The prepared chicken coccidium yolk antibody has obvious effect of treating chicken coccidium, lays a solid foundation for developing and producing the chicken coccidium resistant yolk antibody and the application thereof, and provides a new means for solving the problems of drug resistance and serious drug residue caused by clinical treatment of coccidium chemical drugs.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A preparation method of a yolk antibody for resisting chicken coccidiosis is characterized by comprising the following steps: immunizing laying hens with antigen prepared based on coccidia, and extracting yolk antibody from egg yolk of laying hens.
2. The method of claim 1, wherein the coccidia comprise at least 3 and more of:
Eimeria tenella, Eimeria maxima and Eimeria necatrix.
3. The method of claim 1, wherein immunizing a layer chicken with coccidian-based antigen comprises:
Rejuvenating and proliferating Eimeria tenella, Eimeria maxima and Eimeria necatrix, purifying oocysts of Eimeria tenella, and adjusting the concentration of oocysts to 1 × 108Carrying out ultrasonic crushing for multiple times for the mixed solution of each ml, wherein the working time of the ultrasonic crushing is 3 seconds, each time interval is 8 seconds, and the total time is 30 minutes;
Mixing the antigen solution after ultrasonic crushing with immunopotentiator in equal volume, and performing intramuscular injection immunization on 200-day-old laying hens, wherein the antigen solution is 1 × 106Each individual/feather was immunized 1 time every 10 days for a total of 5 times.
4. The method of claim 3, wherein the extracting yolk antibodies from egg yolk comprises:
Collecting eggs immunized for 5 times, removing egg white, adding distilled water according to the volume ratio of 1:7 for dilution, and fully stirring; standing at 4 deg.C to precipitate lipid, collecting supernatant, centrifuging at low temperature with ultracentrifuge at rotation speed of 10000rmp/min for 25min, and collecting supernatant.
5. the method of claim 4, further comprising:
Collecting supernatant, adding saturated ammonium sulfate solution to final concentration of 50%, standing for 30min, centrifuging at 8000rmp/min, and collecting precipitate;
Mixing a saturated ammonium sulfate solution and a carbonate buffer solution with the concentration of 0.1mol/L, pH of 7.6 according to the ratio of 1:2 to obtain a mixed solution, adding the precipitate into the mixed solution, uniformly mixing and standing for 30 minutes to obtain the final concentration of 30%, removing the supernatant, and dissolving the precipitate in a Phosphate Buffer Solution (PBS) with the concentration of 0.1mol/L, pH of 7.6 to obtain the purified antibody.
6. The method of claim 1, wherein the extracting yolk antibodies from egg yolk comprises:
Removing supernatant from eggs, subpackaging egg yolk in a glass ware under aseptic condition, and freezing in a dryer at low temperature to obtain freeze-dried egg yolk antibody powder.
7. The method of claim 1, wherein the extracting yolk antibodies from egg yolk comprises:
Removing supernatant from ovum gallus Domesticus, diluting with 60% sterilized water, stirring, freezing in-40 deg.C freeze-drying machine for 4 hr, and freezing at-15 deg.C for 15 hr; the obtained freeze-dried substance is transferred to 10 ℃ and placed for 1.5 hours, and finally transferred to 36 ℃ and dried for 4 hours, so as to obtain the freeze-dried yolk antibody powder.
8. A yolk antibody produced by the production method according to any one of claims 1 to 6.
9. Use of the yolk antibody of claim 8 in the preparation of a preparation for preventing and treating chicken coccidiosis.
10. A device for producing an egg yolk antibody, wherein the device is used for carrying out the method according to any one of claims 1 to 6.
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| CN111471107A (en) * | 2020-04-27 | 2020-07-31 | 中国农业大学 | Anti-coccidiosis multivalent recombinant protein yolk antibody and preparation method and application thereof |
| CN111551744A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Newcastle disease virus N protein IgY antibody colloidal carbon detection test paper and application thereof |
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| CN101186647A (en) * | 2007-12-13 | 2008-05-28 | 成都乾坤动物药业有限公司 | Method for preparing chicken coccidiosis high immunity yolk antibody |
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| CN111471107A (en) * | 2020-04-27 | 2020-07-31 | 中国农业大学 | Anti-coccidiosis multivalent recombinant protein yolk antibody and preparation method and application thereof |
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