CN107982532B - Duck hepatitis A virus antigen-antibody complex vaccine and preparation method thereof - Google Patents
Duck hepatitis A virus antigen-antibody complex vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a duck hepatitis A virus antigen-antibody complex vaccine and a preparation method thereof, wherein the vaccine is a type 3 duck hepatitis A virus antigen and a duck egg yolk antibody in a volume ratio of 1: 50, compared with the prior art, the invention adds magnesium sulfate, sodium chloride and propolis into the type 3 duck hepatitis A virus antigen, and prepares duck egg yolk antigen by immunizing laying ducks with the antigen, and finally prepares the type 3 duck hepatitis A virus antigen-antibody compound vaccine by reasonably proportioning the egg yolk antibody and the type 3 duck hepatitis A virus antigen, and multiple tests show that the antigen-antibody compound prepared by the method has the binding efficiency and takes effect within 2 hours after the vaccine is injected, has no immunization blank period, and has high titer, good safety, high protection rate and long protection period.
Description
Technical Field
The invention relates to the technical field of vaccines for poultry, in particular to a duck hepatitis A virus antigen-antibody compound vaccine and a preparation method thereof.
Background
Duck hepatitis A is an acute and highly lethal infectious disease caused by duck hepatitis A virus of picornaviridae, and is one of the most major epidemic diseases harming ducklings at present. Duck viral hepatitis a is divided into three serotypes, namely: DHAV-1, DHAV-2, and DHAV-3. In recent years, the separation rate of the type 3 duck hepatitis A virus in clinic is close to 90 percent, and the duck hepatitis A virus is a main serotype popular in China at present.
The existing report proves that the duck virus A hepatitis can be developed in ducks of 2-39 days old, and the disease is mainly prevented by adopting a vaccine or a hyperimmune antibody at present. The vaccine immunization prevention has an immunization blank period, can not well protect the duck group with diseases in a small age of days, and has low protection rate. The action time of the hyperimmune antibody is short, the hyperimmune antibody can only last for about 7 days, and the effect is good in treatment, but the hyperimmune antibody is not good as a measure for immunoprophylaxis.
The antigen-antibody complex vaccine is also called immune complex vaccine, is a novel vaccine developed in the 90 s of the 20 th century, is formed by mixing specific hyperimmune serum with antigen according to a proper proportion, has the advantages of high safety and good immune effect, can break through the interference of maternal antibodies, is a few safe vaccines which can be inoculated in the hatching period at present, and is concerned in the prevention and control of animal epidemic diseases. The antibody in the antigen-antibody complex vaccine mainly plays a role in delaying the release of the virus, namely when the level of maternal antibodies is reduced to a certain level, the antibodies in the complex begin to release and replicate and generate immune protection, so that the safety and the immune effect of the antigen-antibody complex vaccine are better than those of the common vaccine.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a duck hepatitis A virus antigen-antibody complex vaccine, which aims to solve the technical problems of blank immunization period, short protection period and the like of the existing 3-type duck hepatitis A virus vaccine.
The invention is realized by the following technical scheme:
the invention provides a duck hepatitis A virus antigen-antibody complex vaccine, which comprises the following components in volume ratio of 1: 50 type 3 duck hepatitis A virus antigen and duck egg yolk antibody.
Further, the type 3 duck hepatitis A virus antigen contains 0.4-0.5% by mass of magnesium sulfate, 0.1-0.25% by mass of sodium chloride and 0.2-0.5% by mass of propolis, and has titer in the range of 106.0ELD50/0.2ml-107.0ELD500.2ml of the duck eggsThe yellow antibody is extracted from a hyperimmune duck egg immunized by the type 3 duck hepatitis A virus antigen.
The invention also provides a preparation method of the duck hepatitis A virus antigen-antibody complex vaccine, which comprises the following steps:
(1) preparation of type 3 Duck hepatitis A Virus antigen
Inoculating 3-type duck hepatitis A virus immune allantoic sac to duck embryo, incubating at 37 deg.C, collecting duck embryo dead within 24-96 hr after inoculation, freezing at-20 deg.C for 0.5-1 hr, and respectively collecting allantoic fluid and embryo body; removing heads and limbs of embryo bodies, performing sufficient homogenization, centrifuging to obtain supernatant, mixing the supernatant with allantoic fluid, filtering through a 0.22-micron filter membrane, adding 0.4-0.5% by mass of magnesium sulfate, 0.1-0.25% by mass of sodium chloride and 0.2-0.5% by mass of propolis, and uniformly mixing to obtain a type 3 duck hepatitis A virus antigen;
(2) preparation of yolk antibody
Inactivating the antigen prepared in the step (1), immunizing the laying duck, and collecting the antibody with the titer of 1: taking the yolk and physiological saline according to the ratio of 1: 5 volume ratio, stirring evenly and filtering, then adding 40 volume percent of formaldehyde solution, penicillin solution and streptomycin solution into the filtrate in turn to ensure that the final volume concentration is 0.1% (v/v), 3000IU/mL of 2000-;
(3) preparation of vaccines
Mixing the antigen in the step (1) and the yolk antibody in the step (2) according to a volume ratio of 1: 50, mixing, and incubating at 37 deg.C for 1-2h to obtain vaccine.
Further, in the step (1), the type 3 duck hepatitis A virus for preparing the vaccine is type 3 duck hepatitis A virus SD15 strain which is separated, identified, stored and supplied by Anhui Qiangying Duck industry group Limited company, and the strain is delivered to China general microbiological culture Collection center of China institute of microbiology, Ministry of China, Ministry of sciences, No. 3, North Cheng Lu 1, Beijing, and Beijing, 11 months and 09 days in 2017, wherein the biological preservation number is CGMCC No. 14739; the virus strain is separated from liver of duck died by catalpo in Shandong catalpo meat duck farm in 2016 by Anhui Qiangying Duck group company, and has the biological characteristics that: the small RNA virus is 20-40 nanometers in size, can tolerate ether and chloroform, has certain thermal stability, has toxicity influenced by heating at 52 ℃ for 1 hour, can be proliferated in duck embryos and chicken embryos, and is better propagated in the duck embryos; compared with other conventional 3-type duck hepatitis A virus, the strain is used as a vaccine strain and has the following characteristics: advantages of immunogenicity: the immunogenicity is good, and the antibody titer is high after immunization; secondly, the binding capacity between the virus and the antibody in vitro is strong, and the strain can be effectively released to play a role after the IC vaccine is injected into the duck body; transport advantage: the titer can be kept for 1 year without influence under the conditions of cold chain transportation and cold storage at 4 ℃, and the frozen storage is not needed. The titer is not affected after the storage for 21 days at 37 ℃; and fourthly, the strain is the currently popular duck hepatitis 3 type strain, has higher homology with most of domestic separated strains, and is shown by a gene evolution tree to be in the same branch with most of the popular strains.
Further, in the step (1), the embryo body with the head and the limbs removed is fully homogenized, and then is centrifuged after repeated freeze thawing for 3 times.
Further, in the step (2), the method for inactivating the antigen is as follows: formaldehyde with a final concentration of 0.1% by volume is added to the antigen and treated at 37 ℃ for more than 40 h.
Further, in the step (2), the method for immunizing the laying ducks comprises the following steps: adopting a neck subcutaneous injection mode, 1 ml/piece; after 1 week, performing one-time intensified immunization, performing neck subcutaneous injection, wherein each injection is 1.5 ml; after 2 weeks, the immunization is strengthened once again, and the neck is injected subcutaneously, wherein the volume of the injection is 2ml per patient; immunization was then performed every 2 months, with subcutaneous injections into the neck at 1 ml/tube.
Further, in the step (2), the yolk is mixed with the normal saline solution, and is subjected to negative pressure suction filtration through 50-mesh and 100-mesh filter membranes in sequence, and then is mixed with the formaldehyde and the antibiotic solution.
Compared with the prior art, the invention has the following advantages: the invention provides a preparation method of a duck hepatitis A virus antigen-antibody complex vaccine, which comprises the steps of preparing a type 3 duck hepatitis A virus antigen, immunizing laying ducks with the antigen to prepare a hyperimmune antibody, and reasonably proportioning a yolk antibody and the type 3 duck hepatitis A virus antigen to prepare the type 3 duck hepatitis A virus antigen-antibody complex (IC) vaccine.
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.
Example 1
1. Material
The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used therein are commercially available products unless otherwise specified.
2. Method of producing a composite material
2.13 preparation of Duck hepatitis A Virus antigen (immunogen)
1) Seed poison for production: type 3 duck hepatitis A virus (SD15 strain) for vaccine production, isolated, identified, stored and supplied by Anhui Qiangying Duck group Co., Ltd, biological preservation No.: CGMCC No. 14739.
2) Preparation of SD15 strain venom: diluting SD15 strain for production with normal saline solution 200 times (V/V), inoculating 11-day-old duck embryo into allantoic cavity, incubating at 37 deg.C, and examining embryo every 4 h. Collecting the duck embryos which die within 24-96 hours after inoculation, placing at-20 ℃ for 0.5 hour, collecting allantoic fluid and embryo bodies respectively, and removing heads and limbs from the embryo bodies. Fully homogenizing embryo bodies with heads and limbs removed, repeatedly freezing and thawing for 3 times, centrifuging at 5000r/min for 30min, taking supernatant, mixing with the allantoic fluid collected previously, filtering through a 0.22 micron filter membrane, adding 0.4% by mass of magnesium sulfate, 0.1% by mass of sodium chloride and 0.2% by mass of propolis, uniformly mixing to obtain SD15 strain virus fluid, namely type 3 duck hepatitis A virus antigen, subpackaging the SD15 strain virus fluid in sterile glass bottles, and transferring to a refrigeration house at 4-8 ℃ for storage.
3) SD15 strain venom titer determination: sterilizing physiological salt for SD15 strain virus liquidDiluting with water 10 times, and taking 10 times-5、10-6、10-7、10-8Diluting with 4 pieces of solution, inoculating 5 SPF duck embryos of 11-12 days old into allantoic cavity, each 0.2 ml. Meanwhile, a normal saline control is set, and each embryo is 0.2 ml. The inoculation was followed by incubation at 37 ℃ and examination of the embryos every 4h for 168 hours. Calculating ELD by using death of duck embryo, edema and thickening of allantoic membrane and embryo body lesion as infection50. The potency of the venom should be 106.0ELD50/0.2ml～107.0ELD50Between 0.2ml, the titer measured in this example was 106.3ELD50/0.2ml。
4) Sterile test of SD15 strain venom: the SD15 strain virus liquid is taken, and bacteria and mycoplasma inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, which is in accordance with the pharmacopoeia regulations.
2.2 preparation of yolk antibody
1) Adding 0.1% (volume ratio) of formaldehyde into the SD15 strain virus solution, inactivating at 37 deg.C for 40 hr, and immunizing laying duck with 1 ml/per bottle of neck subcutaneous injection; after 1 week, performing one-time intensified immunization, performing neck subcutaneous injection, wherein each injection is 1.5 ml; after 2 weeks, the immunization is strengthened once again, and the neck is injected subcutaneously, wherein the volume of the injection is 2ml per patient; immunization was then performed every 2 months, with subcutaneous injections into the neck at 1 ml/tube. From 10 days after 3 times of immunization, collecting duck eggs every 3 days for antibody titer detection, and mixing yolk with sterilized normal saline (121 ℃, high-pressure steam sterilization for 20 minutes) according to the ratio of 1: 5, diluting according to a volume ratio, wherein the agar diffusion titer of the diluted solution is 1: and when the time is more than 64, collecting and obtaining the hyperimmune duck egg which is used for preparing the duck hepatitis A egg yolk antibody of type 3.
2) Highly exempt from duck's egg disinfection: cleaning impurities on the surface of the collected high-immunity duck eggs with tap water, soaking the duck eggs in 0.15% poisoning solution for 3-5 minutes, and airing;
3) separating egg yolk: opening the eggshell under aseptic condition, sufficiently removing egg white, frenulum and placenta, and collecting egg yolk;
4) diluting and stirring: under the aseptic condition, the yolk is mixed with sterilized normal saline according to the ratio of 1: diluting at the ratio of 5(V/V), and stirring for 10 minutes at 3000-4000 r/min;
5) and (3) filtering: under the aseptic condition, the yolk and the sterilized normal saline solution in the step 4 are sequentially filtered by 50-mesh and 100-mesh filter membranes under negative pressure; after the filtration is finished, adding 40% formaldehyde solution, penicillin solution and streptomycin solution into the filtrate in sequence to ensure that the final volume concentrations are respectively: 0.1% (v/v), 2000IU/mL, after all addition, fully stirring.
6) Reaction: and (3) placing the semi-finished product of the antibody added with the formaldehyde and the antibiotics in the step 5) at 37 ℃ for reacting for 1h to obtain the egg yolk antibody, and transferring the egg yolk antibody to a refrigerator at 4-8 ℃ for storage for later use.
2.33 preparation of Duck hepatitis A Virus antigen-antibody Complex (IC) vaccine
1) Respectively mixing SD15 strain virus liquid and yolk antibody according to the ratio of 1: 30. 1: 40. 1: 50. 1: 60 volume ratio was mixed in a sterile container.
2) Stirring: after mixing, the mixture was stirred at 2000r/min for 10 minutes to mix the two well.
3) And (3) incubation: and (3) incubating the stirred vaccine at 37 ℃ for 1-2 hours to ensure that the antibody and the antigen fully react to obtain the antigen-antibody compound vaccine.
4) And (4) sterile inspection: the prepared vaccine is taken, and bacteria and mycoplasma inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, which is in accordance with the pharmacopoeia regulations.
5) Subpackaging and storing: the prepared vaccine is subpackaged in a sterilized glass bottle (sterilized by high pressure steam at 121 ℃ for 20 minutes) under the aseptic condition, a rubber plug is covered, an aluminum cover is sealed, a label is attached, and the prepared vaccine is stored at 4 ℃.
2.4 verification of immune Effect
2.4.1 antigen antibody binding assays
The incubated vaccine was mixed with physiological saline in a ratio of 1: diluting at a ratio of 1(v/v), inoculating susceptible 11-12 days old duck embryos, each embryo being 0.2ml, examining embryos every 4h, and continuously observing for 168 h. And (3) observing the death condition of the duck embryo 24-168 hours after inoculation, and observing whether the dead duck embryo body has lesion or not after the observation period is finished, wherein the results are shown in the following table:
table 1: verification of antigen-antibody binding effect under different proportions
2.4.2 safety testing
10 white feather meat ducks of 1-5 days old are injected with 1.0ml of vaccine subcutaneously in each neck, 5 controls are set at the same time, and 2.0ml of sterilized normal saline is injected subcutaneously in each neck. Feeding under the same condition, continuously observing for 14 days, recording the ingestion, drinking and clinical conditions of the test ducks, and observing whether any local and systemic adverse reactions caused by the vaccines exist or not, as shown in the following table 2:
table 2: safety test results of antigen-antibody complexes
| Antigen antibody ratio | Number of inoculations | Adverse reaction |
| 1：30 | 10 | 6 |
| 1：40 | 10 | 2 |
| 1：50 | 10 | 0 |
| 1：60 | 10 | 0 |
| Blank control | 5 | 0 |
2.4.3 immunogenicity test
Using 5 white feather meat ducks of 1-5 days old in each group, injecting vaccine 0.5 ml/duck subcutaneously in each neck, taking 5 ducks of the same day old as immunization as a control, taking blood and separating serum of all ducks 7 days after immunization, determining type 3 duck hepatitis A antibody titer by using a Qiongzhang method, wherein the subcutaneous injection titer in each neck is not less than 10 after blood taking6.0ELD500.2ml of duck hepatitis A virus type 3 (SD15 strain) strain 0.5 ml/piece, and observed continuously for 7 days to record death and mental state of duck group.
Table 3: immune duck agar-agar antibody detection result
Table 4: test results of supply toxicity protection
| Group of | Number of meat ducks | Dosage of offensive toxin | Virus liquid potency | Number of dead ducks | Mortality rate | Rate of protection |
| 1：30 | 5 | 0.5 ml/piece | >106.0ELD50/0.2ml | 4 | 80％ | 20％ |
| 1：40 | 5 | 0.5 ml/piece | >106.0ELD50/0.2ml | 3 | 60％ | 40％ |
| 1：50 | 5 | 0.5 ml/piece | >106.0ELD50/0.2ml | 0 | 0％ | 100％ |
| 1：60 | 5 | 0.5 ml/piece | >106.0ELD50/0.2ml | 0 | 0％ | 100％ |
| Blank control | 5 | 0.5 ml/piece | >106.0ELD50/0.2ml | 5 | 100％ | 0％ |
2.4.4 results
From the test results, in the preparation process of the antigen-antibody complex, the immune effect of the vaccine is directly influenced by the proportion of the antigen and the antibody, the antigen and the antibody are accurately matched, if the antigen is excessive, the live virus has high efficacy and can damage an immune object, and if the antibody is excessive, the concentration of the antigen-antibody complex formed by combination is low, and the free antibody in the complex is excessive, so that the immune action time is shortened.
Example 2
The embodiment provides a preparation method of a duck hepatitis A virus antigen-antibody complex vaccine, which comprises the following steps:
(1) inoculating 3-type duck hepatitis A virus immune allantoic sac to duck embryo, incubating at 37 deg.C, collecting duck embryo dead in 24-96 hr after inoculation, freezing at-20 deg.C for 1 hr, and respectively collecting allantoic fluid and embryo body; removing heads and limbs of embryo bodies, fully homogenizing, centrifuging, taking supernatant, mixing the supernatant with allantoic fluid, filtering through a 0.22-micron filter membrane, adding 0.5% by mass of magnesium sulfate, 0.25% by mass of sodium chloride and 0.5% by mass of propolis, and uniformly mixing to obtain the type 3 duck hepatitis A virus antigen, wherein the other steps are the same as those in example 1. Meanwhile, the control group A is set without adding magnesium sulfate, sodium chloride and propolis to obtain the control group A antigen.
(2) Preparing a yolk antibody: inactivating the antigen in the step (1) by formaldehyde, then immunizing the laying duck, and collecting the antibody with the titer of 1: taking the yolk and physiological saline according to the ratio of 1: 5 volume ratio, stirring evenly and filtering, then adding 40 percent volume ratio of formaldehyde solution, penicillin solution and streptomycin solution into the filtrate in turn to ensure that the final volume concentration is 0.1 percent (v/v), 3000IU/mL and 3000IU/mL respectively, mixing evenly, placing at 37 ℃ for reaction for 1h to obtain the egg yolk antibody, and other steps are the same as the step of example 1. Meanwhile, setting a control group B, immunizing laying ducks by using type 3 duck hepatitis A virus (SD15 strain) for vaccine preparation, and preparing a control group B egg yolk antibody;
(3) preparing a vaccine: mixing the antigen of the step (1) and the antibody of the step (2) according to a volume ratio of 1: 50 mixing, 2000r/min, stirring for 10 min, and incubating at 37 deg.C for 2 hr to obtain vaccine. The vaccine is tested for bacteria and mycoplasma according to the appendix of the current Chinese veterinary pharmacopoeia, which is in accordance with the pharmacopoeia regulations.
(4) Antigen-antibody binding assays and safety assays
Antigen-antibody binding assay: vaccine and normal saline were mixed at a ratio of 1: diluting at a ratio of 1(v/v), inoculating susceptible 11-12 days old duck embryos, each embryo being 0.2ml, examining embryos every 4h, and continuously observing for 168 h. And (5) observing the death condition of the duck embryo 24-168 hours after inoculation, and observing whether the dead duck embryo body has a lesion or not after the observation period is finished.
And (3) safety inspection: 5 white feather meat ducks of 1-5 days old are injected with 1.0ml of vaccine subcutaneously in each neck, 5 controls are set at the same time, and 2.0ml of sterilized normal saline is injected subcutaneously in each neck. Feeding under the same condition, continuously observing for 14 days, recording the ingestion, drinking and clinical conditions of the test ducks, and observing whether any local and systemic adverse reactions caused by the vaccines exist
The results are that except that 1 duck embryo body lesion appears in the control group B immunized by the type 3 duck hepatitis A virus (SD15 strain) for vaccine preparation, all other things are normal, which indicates that the safety performance of the vaccines prepared in the embodiment and the control group A, B meets the requirements, but the combination effect of the hyperimmune antibody obtained by the immunization of the type 3 duck hepatitis A virus (SD15 strain) and the hyperimmune antibody obtained by the immunization directly by the prepared antigen is better and safer.
(5) Immunogenicity testing
Using 5 white feather meat ducks of 1-5 days old in each group, injecting vaccine 0.5 ml/duck subcutaneously in each neck, taking 5 ducks of the same day old as immunization as a control, taking blood and separating serum of all ducks 7 days after immunization, determining type 3 duck hepatitis A antibody titer by using a Qiongzhang method, wherein the subcutaneous injection titer in each neck is not less than 10 after blood taking6.0ELD500.2ml of duck hepatitis A virus type 3 (SD15 strain) strain 0.5 ml/single duck hepatitis A virus, which was observed continuously for 7 days, and on 7 days, the death and mental state of the duck group were recorded, and after 7 days, the injection was performed again, and on 14 days, the death and mental state of the duck group were recorded, and the results are shown in the following table:
table 3: immune duck agar-agar antibody detection result
Table 4: test results of supply toxicity protection
The results show that the titer of antibodies of the vaccine of the embodiment and the control group A, B for the duck hepatitis A virus type 3 immunization is not lower than 1:64, but compared with the control group A, B, the titer of antibodies of the embodiment can reach 1: 256. the challenge test result shows that the immune protection rate of the vaccine of the embodiment in 14 days is 100%, while the control A, B shows that meat ducks die in the second challenge, which indicates that the vaccine of the embodiment has a longer immune protection effect. The meat ducks of the blank control group all died, and the protection rate was 0%.
The above is a detailed embodiment and a specific operation process of the present invention, which are implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above-mentioned examples.
Claims (7)
1. The duck hepatitis A virus antigen-antibody complex vaccine is characterized by comprising the following components in a volume ratio of 1: 50 and a duck egg yolk antibody, wherein the duck egg yolk antibody is obtained by extracting a hyperimmune duck egg immunized by an inactivated duck hepatitis A virus type 3 antigen, the duck hepatitis A virus type 3 is a duck hepatitis A virus type 3 SD15 strain, and the biological preservation number is CGMCC No. 14739.
2. The duck hepatitis A virus antigen-antibody complex vaccine as claimed in claim 1, wherein the type 3 duck hepatitis A virus antigen contains magnesium sulfate 0.4-0.5% by mass, sodium chloride 0.1-0.25% by mass and propolis 0.2-0.5% by mass, and has a potency of 106.0ELD50/0.2ml-107.0ELD50/0.2ml。
3. A method for preparing the duck hepatitis a virus antigen-antibody complex vaccine as claimed in claims 1-2, comprising the steps of:
(1) preparation of type 3 Duck hepatitis A Virus antigen
Inoculating 3-type duck hepatitis A virus immune allantoic sac to duck embryo, incubating at 37 deg.C, collecting duck embryo dead within 24-96 hr after inoculation, freezing at-20 deg.C for 0.5-1 hr, and respectively collecting allantoic fluid and embryo body; removing heads and limbs of embryo bodies, performing sufficient homogenization, centrifuging to obtain supernatant, mixing the supernatant with allantoic fluid, filtering through a 0.22-micron filter membrane, adding 0.4-0.5% by mass of magnesium sulfate, 0.1-0.25% by mass of sodium chloride and 0.2-0.5% by mass of propolis, and uniformly mixing to obtain a type 3 duck hepatitis A virus antigen;
(2) preparation of yolk antibody
Inactivating the antigen prepared in the step (1), immunizing the laying duck, and collecting the antibody with the titer of 1: taking the yolk and physiological saline according to the ratio of 1: 5 volume ratio, stirring evenly and filtering, then adding 40 volume percent of formaldehyde solution, penicillin solution and streptomycin solution into the filtrate in turn to ensure that the final volume concentration is 0.1% (v/v), 3000IU/mL of 2000-;
(3) preparation of vaccines
Mixing the antigen in the step (1) and the yolk antibody in the step (2) according to a volume ratio of 1: 50, mixing, and incubating at 37 deg.C for 1-2h to obtain vaccine.
4. The method for preparing a duck hepatitis A virus antigen-antibody complex vaccine according to claim 3, wherein in the step (1), the embryo body with the head and the limbs removed is homogenized sufficiently, and then is subjected to repeated freeze thawing for 3 times and then centrifugation.
5. The method for preparing a duck hepatitis A virus antigen-antibody complex vaccine according to claim 3, wherein in the step (2), the antigen inactivation method comprises: formaldehyde with a final concentration of 0.1% by volume is added to the antigen and treated at 37 ℃ for more than 40 h.
6. The method for preparing a duck hepatitis A virus antigen-antibody complex vaccine according to claim 3, wherein in the step (2), the method for immunizing the laying duck comprises the following steps: adopting a neck subcutaneous injection mode, 1 ml/piece; after 1 week, performing one-time intensified immunization, performing neck subcutaneous injection, wherein each injection is 1.5 ml; after 2 weeks, the immunization is strengthened once again, and the neck is injected subcutaneously, wherein the volume of the injection is 2ml per patient; immunization was then performed every 2 months, with subcutaneous injections into the neck at 1 ml/tube.
7. The method for preparing a duck hepatitis A virus antigen-antibody complex vaccine as claimed in claim 3, wherein in step (2), the egg yolk is mixed with a normal saline solution, and after negative pressure suction filtration through 50 mesh and 100 mesh filter membranes in sequence, the egg yolk is mixed with a formaldehyde and antibiotic solution.
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