CN108558995B - Yolk antibody for preventing and treating novel goose astrovirus and preparation method thereof - Google Patents
Yolk antibody for preventing and treating novel goose astrovirus and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- 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
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a yolk antibody for preventing and treating novel goose astrovirus and a preparation method thereof, wherein the yolk antibody contains an anti-goose astrovirus antibody; the preservation number of the goose star-shaped virus strain is CCTCC NO: and V201808. The yolk antibody prepared by the invention has good safety, and no local or systemic adverse reaction caused by the yolk antibody occurs; but also can effectively prevent and/or treat the infection of the novel goose astrovirus, and has good commercial development prospect.
Description
Technical Field
The invention relates to the technical field of biological products for livestock, in particular to a yolk antibody for preventing and treating novel goose astrovirus and a preparation method thereof.
Background
Astrovirus (AStV) is a single-stranded positive-strand RNA virus which has no envelope, is spherical and has a diameter of 28-30nm, the genome of the virus has infectivity and a length of 6.4-7.9kb, and the infection of the virus has certain prevalence and can cause enteritis and diarrhea of human beings or animals, and some diseases are accompanied by vomit and abdominal pain. The astrovirus family can be divided into mammalian astrovirus and avian astrovirus according to different infection hosts, and the avian astrovirus can cause poultry to generate various diseases, and the clinical manifestations of the avian astrovirus are different due to different virus strains, virulence or infection hosts. Goose astrovirus (GAstV), which mainly attacks young goslings, has already occurred in a plurality of provinces, so that the death rate of the goslings is increased, and the healthy development of the Goose industry in China is greatly damaged.
In 2017, in 2-12 months, the gosling group in Shandong, Anhui, Jiangsu, Liaoning, Henan, Guangdong and the like in China outbreaks an infectious disease which is mainly characterized by gout. The disease mainly occurs to goslings of 5-20 days old, and the death rate can reach over 50 percent. The body cavities and joints of the sick goslings are seriously deposited with urate, the goslings are tired in lying and difficult to take, the growth of the goslings is slow, the feed-meat ratio is increased, the slaughtering qualification rate of the meat geese is obviously reduced, and the serious economic loss is caused to the meat goose breeding industry.
The research shows that the outbreak of the disease is caused by a novel goose astrovirus infection, no vaccine can be used for preventing the disease at present, and the conventional antiviral and antibacterial treatment method is ineffective.
The yolk antibody is an antibody which is extracted from an immunized egg and aims at a specific antigen, and is called yolk immunoglobulin IgG (egg yolk immunoglobulin), IgY for short, because the yolk only contains IgG antibodies. Egg yolk antibodies have many unique advantages over serum antibodies: (1) the method does not need to collect blood, and only needs to collect the yolk of the immune hyperimmune egg to purify so as to obtain the yolk antibody; (2) the product has good stability, heat resistance and acid resistance, and can still maintain certain activity at normal temperature; (3) the treatment effect is obvious, the specificity is strong, and the preparation method can be suitable for large-scale production; (4) safe, high-efficiency, no residue, mild and environment-friendly. However, as the disease is reported in China for the first time, no commercial vaccine and antibody are on the market at present, and the egg yolk antibody for the emergency prevention and early infection treatment of the novel goose astrovirus is not reported; moreover, the yolk contains a large amount of lipid, which brings inconvenience to the use, and the development and the application of the yolk antibody for preventing and treating the novel goose astrovirus are also restricted.
Disclosure of Invention
In view of the prior art, the invention aims to provide a yolk antibody for preventing and treating novel goose astrovirus, which is used for preventing and treating the novel goose astrovirus infection outbreak of goose groups, thereby making up the serious economic loss caused by the disease.
The invention also aims to provide a preparation method of the yolk antibody for preventing and treating the novel goose astrovirus. The preparation method is easy to operate and suitable for large-scale production; and the prepared yolk antibody has stable property, high purity, high titer, strong specificity, no drug residue, mildness and environmental protection, and has great application value for preventing and treating the novel goose astrovirus infection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a novel goose astrovirus separated from a diseased goose body with gout symptom, the preservation number is CCTCC NO: v201808, on the basis of the isolate, an inactivated vaccine capable of preventing and treating the novel goose astrovirus is developed, healthy laying hens are immunized with the inactivated vaccine at high strength, and then egg yolks after immunization are separated, and a yolkantibody is extracted and prepared. Specifically, the method comprises the following steps:
in a first aspect of the invention, the invention provides a yolk antibody for preventing and treating novel goose astrovirus, which contains an anti-goose astrovirus strain antibody; the preservation number of the goose star-shaped virus strain is CCTCC NO: and V201808.
In a second aspect of the present invention, there is provided a method for producing the above yolk antibody, comprising the steps of:
(1) the preservation number is CCTCC NO: v201808, wherein the goose astrovirus is used as a vaccine production strain to prepare an inactivated vaccine;
(2) injecting the prepared inactivated vaccine into an immunized laying hen to prepare a hyperimmune egg;
(3) the yolk antibody for preventing and treating the novel goose astrovirus is prepared by collecting yolk from the hyperimmune egg and performing primary inactivation, acidification extraction, secondary inactivation, rough filtration, sterilization filtration, concentration and tertiary inactivation.
Preferably, in step (1), the inactivated vaccine is prepared by the following method:
the preservation number is CCTCC NO: v201808, inoculating the goose-star virus into SPF chick embryos, and collecting allantoic fluid of dead chick embryos within 24-120h to obtain virus fluid; concentrating the virus liquid until the virus content is more than or equal to 105.0ELD50Inactivating the concentrated virus solution by formaldehyde, adding Tween-80, mixing to obtain a water phase, mixing white oil, aluminum stearate and Span-80 to obtain an oil phase, mixing the water phase and the oil phase according to the volume ratio of 1:1, and emulsifying to obtain the inactivated vaccine.
Preferably, in the step (2), the layer chicken is immunized by the prepared inactivated vaccine in 4 times, and each immunization is separated by 2 weeks. The immunization route is the neck subcutaneous or intramuscular injection of inactivated vaccine.
Preferably, in the step (3), the primary inactivation specifically comprises: mixing the yolk solution and water according to the volume ratio of 1 (0.5-1.5), uniformly stirring to obtain a yolk diluent, and preserving heat for 25-35 min at the temperature of 60-65 ℃.
Preferably, in the step (3), the acidifying extraction is specifically: and adding an acetate buffer solution with the volume of 2.5-3.5 times that of the yolk diluent into the yolk diluent, stirring, filtering and collecting filtrate. Further preferably, the pH value of the acetate buffer solution is 4.8-5.2; more preferably 5.0.
Preferably, in the step (3), the secondary inactivation specifically comprises: adding octanoic acid into the filtrate after acidification and extraction until the final concentration is 3.5-4.5%, stirring for 30-120min, and standing for 4-8 hours at 2-8 ℃ after stirring.
Preferably, in the step (3), the rough filtration specifically comprises: filtering with filter cloth, and filtering with filter membrane until the filtrate is clear. Further preferably, the filter cloth is a polypropylene 750B filter cloth.
Preferably, in the step (3), the sterilizing filtration is specifically: filter sterilized with a 0.22 μm filter.
Preferably, in the step (3), the concentration is specifically: concentrating by using a PES hollow fiber ultrafiltration membrane with the KD of 30-50KD at the temperature of 2-8 ℃ until the titer of the antibody is not lower than 1: 512.
Preferably, in step (3), the three inactivations are specifically: adding formaldehyde solution to make the final concentration of the formaldehyde solution be 0.1%, and inactivating at 37 deg.C for 16 h.
In a third aspect of the invention, the invention provides an application of the yolk antibody in preparation of a product for preventing and/or treating goose astrovirus infection. The preservation number of the goose astrovirus is CCTCC NO: and V201808.
The invention has the beneficial effects that:
(1) the novel goose star virus strain obtained by separating the diseased gosling body has excellent immunogenicity, can be stably passed in chick embryos, and has better protection for the infection of the novel goose star virus which is popular in the current place and takes gout as a main symptom.
(2) The preservation number is CCTCC NO: the goose astrovirus of V201808 is used as a vaccine production strain to prepare an inactivated vaccine to immunize laying hens, so that a yolk antibody can be efficiently obtained, the safety of the yolk antibody is good, and any local or systemic adverse reaction caused by the yolk antibody does not occur; but also can effectively prevent and treat the infection of the novel goose astrovirus, and has good commercial development prospect.
(3) The preparation process of the egg yolk antibody is optimized, the egg yolk with high immunity is firstly inactivated initially after being collected, then acidified and extracted by using an acetate buffer solution, and then inactivated for the second time by using caprylic acid as an inactivating agent and an extracting agent, and then filtered, clarified, filtered and sterilized, finally inactivated for the third time by using formaldehyde after being concentrated by ultrafiltration, and finally the egg yolk antibody solution is obtained. The content of lipid in the final product is effectively reduced by the method of acidifying water and caprylic acid, the risk of adverse reaction is reduced, and meanwhile, the step of ultrafiltration concentration is helpful to ensure the antibody titer in the solution and improve the protection rate; higher antibody titer requires higher dilution times in future clinical application, so that the impurity content is indirectly reduced, and the safety is further improved.
Drawings
FIG. 1: the virus isolate N-AStV-SDPY of the invention is inoculated to goose embryo lesion: a, goose embryos in a normal growth state; and B is goose embryo lesion caused by the SDPY isolate.
FIG. 2: PCR identification results; in the figure, M is 2000bp Marker, lane 1 is blank control, lane 2 is AstV, lane 3 is AstV, lane 4 is AIV, lane 5 is NDV, lane 6 is ARV, lane 7 is GPV, lane 8 is GpoV, and lane 9 is GoCV.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, the gosling group in Shandong, Anhui, Jiangsu, Liaoning, Henan, Guangdong and the like in China outbreaks an infectious disease which is mainly characterized by gout in 2017. The symptoms of the disease are different from the prior astrovirus infection, and the symptoms of gout appear in goose flocks for the first time, so that the infectious disease is probably caused by the novel astrovirus. There is currently no drug or method available to effectively control this new astrovirus infection. The inactivated astrovirus vaccination is an effective measure for preventing and treating the infection and outbreak of the disease, but the premise is that a goose astrovirus strain with excellent immunogenicity needs to be separated and obtained.
As the astrovirus is an RNA virus and has a plurality of segments, different strains have certain differences in antigen structure, pathogenicity, cell culture characteristics and host specificity, and are easy to mutate during genetic evolution, so that the vaccine prepared by the traditional strains cannot well prevent and control the current epidemic goose astrovirus infection with gout as the main symptom.
The inventor of the application separates a novel virus N-AStV-SDPY from tissues such as the liver, the spleen, the kidney and the like of a gosling died from diseases, and identifies the novel virus N-AStV-SDPY as a goose astrovirus. The invention carries out whole genome sequencing on the strain N-AStV-SDPY, and carries out homology analysis and genetic evolution analysis with the astrovirus reported at present, and the result shows that the homology of each gene fragment of the strain N-AStV-SDPY is less than 70 percent; genetic evolution analysis shows that the strain N-AStV-SDPY is in a variation branch; the above results illustrate that: the strain N-AStV-SDPY is different from other astrovirus and is a new species of avian astrovirus. The novel goose astrovirus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: and V201808.
Because the vaccine immunization is mainly carried out in prevention, the time for generating antibodies after immunization is long, and timely and rapid treatment is difficult to carry out when the goose flock attacks diseases. Based on the method, the separated novel goose astrovirus is used for preparing vaccine immune layers, and the hyperimmune eggs are collected to prepare egg yolk antibodies for the emergency prevention and early infection treatment of the novel goose astrovirus.
In one embodiment of the invention, the yolk antibody for preventing and treating the novel goose astrovirus is prepared by the following method:
(1) taking the yolk of the novel goose-star virus high-immunity egg, and uniformly stirring to obtain yolk liquid;
(2) mixing the yolk liquid obtained in the step (1) with water according to a ratio of 1 (0.5-1.5) (V/V), uniformly stirring to obtain a yolk diluent, and preserving heat for 25-35 min at a temperature of 60-65 ℃;
(3) adding an acetate buffer solution with the volume of 2.5-3.5 times that of the yolk solution into the yolk diluent, stirring, filtering with a filter cloth, and collecting filtrate;
(4) adding octanoic acid into the filtrate obtained in the step (3) until the final concentration is 3.5-4.5% (V/V), stirring, and standing at 2-8 ℃ for 4-8 hours;
(5) filtering the solution after the placement in the step (4) until the filtrate is clear, and collecting the clear filtrate;
(6) and (4) filtering and sterilizing the clarified filtrate obtained in the step (5), adding formaldehyde to a final concentration of 0.1% (V/V), and inactivating at 35-37 ℃ for 12-20 hours.
Preferably, the novel goose astrovirus hyperimmune egg in the step (1) is an egg produced by immunizing hens for 4 times by using the novel goose astrovirus inactivated vaccine, and the immunization interval is 2 weeks each time; further preferably, the immunization route is the subcutaneous or intramuscular injection of a novel goose astrovirus inactivated vaccine to the neck.
Preferably, in the high-immunity yolk liquid of the novel goose astrovirus in the step (1), the titer of the novel goose astrovirus antibody is not less than 1: 1024.
Preferably, in the step (1), eggshell sterilization is carried out on the high-immunity egg, and then yolk is collected; more preferably, the disinfection method comprises the steps of soaking and disinfecting the eggshells for 15min by using 1% benzalkonium bromide solution, and spraying 75% alcohol to the surfaces of the eggshells after the eggshells are dried.
Preferably, the water in step (1) is injection water which is kept at 80 ℃ for 30min and then cooled to 65 ℃ or below.
Preferably, the pH value of the acetate buffer solution in the step (2) is 4.8-5.2, and more preferably 5.0.
Preferably, the stirring in the step (3) is continued for 30-120min, and more preferably for 90 min.
Preferably, the filter cloth in the step (3) is a polypropylene 750B filter cloth.
Preferably, the stirring in the step (4) is continued for 30 to 120min, more preferably 90 min.
Preferably, the filtration in the step (5) is performed by filtering with a filter cloth and then filtering with a filter membrane until the filtrate is clear; further preferably, the filter cloth is a polypropylene 750B filter cloth.
Preferably, the pore size of the filter used for the filter sterilization in step (6) is 0.22. mu.m.
Preferably, the titer of the novel goose astrovirus antibodies in the clear filtrate after the sterilization in the step (6) is not less than 1: 512.
Preferably, after degerming in the step (6), ultrafiltration concentration is carried out, and then formaldehyde is added for inactivation; preferably, the ultrafiltration concentration is realized by a PES hollow fiber ultrafiltration membrane with the KD of 30-50 KD; further preferably, the concentration is performed at 2 to 8 ℃.
According to the technical scheme, after the egg yolk of the hyperimmune egg is collected, preliminary inactivation is firstly carried out, then acidification and extraction are carried out by using an acetate buffer solution, then octanoic acid is used as an inactivating agent and an extracting agent for secondary inactivation, filtration clarification and filtration sterilization are carried out on the basis, finally, tertiary inactivation is carried out by using formaldehyde after ultrafiltration concentration, and finally the egg yolk antibody solution is obtained. The content of lipid in the final product is effectively reduced by the method of acidifying water and caprylic acid, the risk of adverse reaction is reduced, and meanwhile, the step of ultrafiltration concentration is helpful to ensure the antibody titer in the solution and improve the protection rate; higher antibody titer requires higher dilution times in future clinical application, so that the impurity content is indirectly reduced, and the safety is further improved.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer. In the quantitative test in the embodiment of the invention, three times of repeated experiments are set, and the results are averaged; in the examples of the present invention,% is by mass unless otherwise specified.
Example 1: discovery and identification of novel goose astrovirus
1. Materials and methods
1.1 collection and treatment of pathological materials:
taking tissues of livers, spleens, kidneys and the like of the dead gosling geese, adding sterile normal saline according to the proportion of 1:4 for homogenization treatment, repeatedly freezing and thawing the homogenate liquid in a refrigerator for 3 times, centrifuging at 6000r/min for 15min, taking supernate, filtering bacteria by a 0.22 mu m filter, and storing at-20 ℃ for later use.
1.2 main reagents:
goose embryos are purchased from Shandong chat in a goose farm, RNA extraction kit is purchased from Beijing K.K. Shijieke Co., Ltd, ordinary agarose gel recovery kit is purchased from Beijing Quanjin Co., reverse transcription kit is purchased from Baozhikang K.K., Ltd, 2 × Es Taq MasterMix is purchased from Beijing K K.K. Shijieke Co., Ltd, and DL2000Marker is purchased from Baozhikang K.K. Co., Ltd.
1.3 primer design:
a pair of specific primers for amplifying 601bp fragments is designed according to all astrovirus gene sequences on a gene sequence database Genbank established by the national center for biotechnology information, and is synthesized by Beijing Ongkoku New Biotechnology Co.
An upstream primer: 5 '-TGG TGG TGY TTY CTC AAR A-3';
a downstream primer: 5 '-GYC KGT CAT CMC CRT ARC A-3'.
Note: y, K, M, R is a degenerate base. Wherein Y represents C/T; k represents G/T; m represents A/C; r represents A/G.
1.4 virus isolation:
aseptically inoculating the obtained tissue sample treatment liquid into 13-day-old goose embryos in an allantoic cavity way, wherein each goose embryo is 0.2 mL; and a negative control was set with sterile physiological saline and incubated at 37 ℃. Embryo irradiation is carried out twice every day, dead goose embryos within 24 hours are discarded, and continuous observation is carried out for 6 days. The dead goose embryos are treated at 4 ℃ overnight, allantoic fluid is aseptically collected, and the change of embryo bodies is observed. The tissue treatment fluid is blindly transmitted for three generations for subsequent detection.
1.5PCR identification:
1.5.1RNA extraction:
extracting virus RNA from the virus liquid obtained in step 1.4 according to the instruction of the RNA extraction kit, and storing at-20 ℃ for later use.
1.5.2 reverse transcription to obtain cDNA:
the reverse transcription kit used was PrimeScript having a commercial number of RR036A from Baozo (Dalian) Co., LtdTMRT Master Mix, 5 XPrimeScript RT Master Mix X2uL, Total RNA-2 uL of the sample to be tested extracted in step 1.5.1 were added in sequence to 200 uLPCR reaction tube, RNase Free dH was used2O was supplemented to 10. mu.l system. Placing in a PCR instrument for reaction at 37 deg.C for 15 min; after 5s at 85 ℃ the cells were stored at 4 ℃.
1.5.3PCR amplification:
amplification was performed using a 20 μ L system: template cDNA × 2 μ L, upstream and downstream primers × 1 μ L, 2 × Es TaqMasterMix × 10 μ L, using ddH2O was supplemented to 20. mu.L system. Mixing, instantly separating, reacting in PCR instrument at 95 deg.C for 5min, then at 95 deg.C for 45s, 68 deg.C for 30s, at 72 deg.C for 45s for 30 cycles, and storing at 4 deg.C after 10min at 72 deg.C.
2. As a result:
2.1 Virus isolation:
the collected mixed tissue homogenate of the positive liver and the kidney is processed, and 13-day-old goose embryos are inoculated in an aseptic manner by a chorioallantoic cavity way, wherein each goose embryo is 0.2 mL; and a negative control was set with sterile physiological saline and incubated at 37 ℃. Goose embryo death occurred 2-5 days after inoculation of goose embryos (see figure 1), and the blank control goose embryos of the same batch were normal.
2.2PCR identification:
the products amplified by PCR with the primers showed a specific band corresponding to the expected size of 601bp after electrophoresis in 1% agarose gel (FIG. 2). Indicating the presence of the novel astrovirus (AstV) in the cell culture of the isolate. In addition, the conventional goose-source virus detection of the isolated strain comprises the following steps: avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), reovirus (ARV), Goose Parvovirus (GPV), goose polyoma virus (GpoV) and goose circovirus (GoCV), no other viral contamination was detected (fig. 2).
Finally, a stable strain is obtained through separation, and the strain is named as N-AStV-SDPY. The whole genome sequence of the strain N-AStV-SDPY is determined by adopting a second-generation sequencing technology, and the whole genome sequence of the strain is shown as SEQ ID NO. 1. Homology alignments and genetic evolutionary analyses are then performed with astrovirus as published in the prior art. The results show that the homology of each gene fragment of the strain N-AStV-SDPY is less than 70 percent; genetic evolution analysis shows that the strain N-AStV-SDPY is in a variation branch; the above results illustrate that: the strain N-AStV-SDPY is different from other astrovirus and is a new species of avian astrovirus.
The virus is preserved, and the preservation number is CCTCC NO: and V201808.
Example 2: preparation of inactivated vaccine
(1) Virus propagation procedure: inoculating the novel goose star virus strain separated in the example 1 into healthy SPF (specific pathogen free) chick embryos of 9 days old according to the dose of 0.2 ml/egg, discarding dead embryos within 24 hours, collecting allantoic fluid of dead chick embryos within 24-120 hours, and storing at-20 ℃.
(2) Concentrating the virus liquid: concentrating the harvested virus liquid to 1/3 of the original volume by using an ultrafiltration concentrator at the temperature of 2-8 ℃, performing sterile inspection according to the appendix of the current Chinese veterinary pharmacopoeia, performing sterile growth, and reserving a sample for testing the toxicity value. The virus content of the virus liquid is more than or equal to 10 per 0.1ml5.0ELD50The concentrated allantoic fluid is then inactivated.
(3) Inactivation: the concentrated virus solution was introduced into an inactivation tank, and 10% formaldehyde solution was added to make the final concentration 0.1%. The formaldehyde solution is added and then introduced into another inactivation tank to avoid that viruses adhered near the tank opening can not contact the inactivator. And (3) performing sealed inactivation at 37 ℃ for 24 hours (counted by the temperature in the tank reaching 37 ℃), stirring once every 4-6 hours, and after inactivation, storing at 2-8 ℃ for later use.
(4) Preparing an inactivated vaccine:
1) preparing an oil phase: 94 parts of white oil for livestock and 2 parts of aluminum stearate are taken, placed in an oil phase preparation tank, heated to 80 ℃, added with Span-806 parts until the temperature reaches 115 ℃, kept for 30min and cooled for later use.
2) Preparation of an aqueous phase: and adding 96 parts of virus liquid which is qualified after inactivation and is detected into 4 parts of sterilized Tween-80, starting a homogenizer to stir for 20-30 min, and completely dissolving the Tween-80.
3) Emulsification: the proportion of the oil phase and the water phase is 1:1(V/V), the oil phase is firstly led into a colloid mill, stirred at 2500 rpm, slowly added into the water phase, and emulsified for 5 minutes at 10000 rpm after the addition is finished. Adding thimerosal in 0.01 wt% of the vaccine before stopping emulsification, and mixing. After emulsification, 10ml of the mixture was centrifuged at 3000r/min for 15 minutes, and an anhydrous phase precipitated at the bottom of the tube.
After sterile subpackaging, capping and storing at 2-8 ℃.
The prepared inactivated vaccine is subjected to the following steps: quality inspection of dosage form, centrifugal stability, viscosity and sterility is carried out by referring to pharmacopoeia of the people's republic of China (2015 edition).
The results were: the preparation form of the inactivated vaccine prepared by the invention is water-in-oil (W/O); the centrifugal stability, viscosity and sterility test were in accordance with the regulations of the pharmacopoeia of the people's republic of China (2015 edition).
Example 3: preparation of novel goose astrovirus egg yolk antibody
1. Immunizing the laying hens by using the inactivated vaccine:
the novel goose-star virus inactivated vaccine prepared in example 2 is used for immunizing laying hens, 2.0ml of inactivated vaccine is injected subcutaneously into the neck of each chicken for the first time, the second immunization is carried out after 14 days, 2.0ml of inactivated vaccine is injected subcutaneously into the neck of each chicken, the third immunization is carried out 14 days after the second immunization, 2.0ml of inactivated vaccine is injected subcutaneously into the neck of each chicken, the boosting immunization is carried out 14 days after the third immunization, 2.0ml of inactivated vaccine is injected subcutaneously into the neck of each chicken, and the new goose-star virus neutralizing antibody titer is measured by collecting egg yolk 14 days after the boosting immunization and is not less than 1: 1024.
2. Egg yolk antibody production:
(1) egg shell disinfection: collecting high immunity eggs, and soaking in 1% benzalkonium bromide solution for 15 min. Taking out, naturally airing or blow-drying, and spraying 75% alcohol to disinfect the surface of the eggshell for later use.
(2) Yolk separation: a mechanical egg beating mode is adopted, egg white, blastoderm and frenulum are fully removed during egg beating, and egg yolk is collected.
(3) Inactivation I: stirring the collected yolk thoroughly to make the yolk into uniform paste, starting a peristaltic pump, pumping the yolk liquid into an interlayer reaction tank, adding injection water (the injection water is sterilized at 80 ℃ for 30min and cooled to below 65 ℃) with the same volume as the yolk, stirring uniformly, and keeping the temperature at 60-65 ℃ (inactivating) for 30 min.
(4) Acidifying and extracting: firstly, adding an acetic acid buffer solution with the pH value of 5.0, which is 3 times the volume of the original yolk, into an isolated reaction tank, then adding the yolk solution, starting a stirrer, and fully stirring for 30 minutes.
(5) And (3) inactivation II: adding caprylic acid with the final concentration (V/V) of 4% as an inactivating agent and an extracting agent into the egg yolk liquid, violently stirring for 90min, and standing for 4-8 hours at the temperature of 2-8 ℃.
(6) Coarse filtration: after filtration through a polypropylene 750B filter cloth, the filtrate was filtered through a cartridge filter until clear.
(7) And (3) degerming and filtering: filter sterilized with a 0.22 μm filter. Storing at 2-8 ℃ for no more than 14 days. And simultaneously sampling and detecting the neutralizing antibody titer of the novel goose astrovirus.
(8) Concentration: if the detected neutralizing antibody titer of the novel goose astrovirus is lower than 1:512, performing ultrafiltration concentration by a proper multiple by using a concentration membrane with the KD of 30-50KD at the temperature of 2-8 ℃ on the yolk antibody subjected to sterilization and filtration, wherein the neutralizing antibody titer of the novel goose astrovirus after concentration is not lower than 1: 512.
(9) Inactivation III: introducing the concentrated solution into an inactivation tank, metering 10% of formaldehyde solution, starting a stirrer to stir so as to fully mix the formaldehyde solution, wherein the final concentration (V/V) of the formaldehyde solution is 0.1%, and inactivating the formaldehyde solution for 16 hours at 37 ℃.
(10) Subpackaging and storing: and (3) sub-packaging the inactivated filtrate into sterile glass bottles under the aseptic condition, covering a rubber plug, rolling an aluminum cover, sticking a label, and preserving for later use at the preservation temperature of-20 ℃.
Example 4: quality test of yolk antibody
1. And (3) safety inspection:
2.0ml of the egg yolk antibody prepared in the embodiment 3 of the invention is injected into 10 susceptible goslings (all novel goose astrovirus maternal antibodies are less than 1:4) with 1-3 days of age, and the egg yolk antibody is proved to be good in safety after observation for 14 days when the egg yolk antibody is totally healthy and survived.
2. And (4) sterile inspection:
the results were obtained according to the pharmacopoeia of the people's republic of China (2015 edition): has no contamination of bacteria, mycoplasma and exogenous virus.
3. Efficacy test (immune challenge method):
5 days old susceptible gosling (the novel goose astrovirus maternal antibodies are all less than 1:4)20 are randomly divided into A, B four groups of 10. Wherein A is 0.5ml of yolk antibody prepared in example 3 injected subcutaneously or intramuscularly in neck of the immunized group; and B is a control group for counteracting toxic substances, and 0.5ml of physiological saline is injected subcutaneously or intramuscularly. And (5) isolated breeding.
After 16 hours, A, B two groups of gosling were injected intramuscularly with 0.5ml each of the new goose astrovirus (preservation number: CCTCC NO: V201808) diluted 10 times with normal saline. After the challenge, the goslings are observed for 10 days, and the morbidity and the mortality of each group of goslings are recorded.
As a result: 10/10 protection after group A toxin challenge; the disease started after 12 hours of the group B virus challenge, and 9 deaths occurred within 10 days.
Example 5: application of yolk antibody
1. Application in prevention of novel goose astrovirus
30 susceptible goslings (novel goose astrovirus maternal antibodies are all less than 1:4) with 3-5 days of age are taken as test objects and are randomly divided into three groups of 10. Wherein:
test groups: injecting 0.5ml of the yolk antibody prepared in example 3 into neck subcutaneously or intramuscularly;
control group: immunizing laying hens with the currently marketed avian astrovirus inactivated vaccine, preparing a yolk antibody according to the method of the embodiment 3, and injecting the yolk antibody into the neck subcutaneously or intramuscularly, wherein each 0.5ml of the yolk antibody is injected into the neck subcutaneously or intramuscularly;
blank control group: the physiological saline is injected subcutaneously or intramuscularly, 0.5ml each.
The three groups of goslings are separately fed, and after 16 hours, the new goose astrovirus which is diluted by 10 times by normal saline is respectively injected into muscles, and each volume is 0.5 ml. After the challenge, the goslings are observed for 10 days, and the morbidity and the mortality of each group of goslings are recorded. The results are shown in Table 1.
Table 1:
| group of | Toxin counteracting toxic strain | Preventive protection |
| Test group | Novel goose astrovirus | 10/10 |
| Control group | |
5/10 |
| Blank control group | |
1/10 |
Note: the prevention and protection are the number of healthy survival goslings/total number of goslings after the challenge.
As can be seen from Table 1, the yolk antibody of the invention has excellent preventive protection efficacy, can provide 100% preventive protection for the novel goose astrovirus, and has better protective efficacy than the yolk antibody prepared by the existing inactivated vaccine.
2. Application in treatment of novel goose astrovirus
In a novel goose astrovirus infection disease area, symptoms are nephritis and gout, 30 goslings with similar disease courses are taken as test objects and randomly divided into 3 groups, and each group comprises 10 goslings which are all separately fed, wherein:
test groups: injecting the yolk antibody prepared in the example 3 into the neck part subcutaneously or intramuscularly, wherein each amount of the yolk antibody is 1.0 ml;
control group: immunizing laying hens with the currently marketed avian astrovirus inactivated vaccine, preparing egg yolk antibodies by the method of example 3, injecting the egg yolk antibodies into the neck subcutaneously or intramuscularly, wherein each egg yolk antibody is 1.0 ml;
blank control group: physiological saline was injected subcutaneously or intramuscularly, 1.0ml each.
Observing for 10 days, and recording the illness state and death condition of each group of goslings.
The results were: after the test group is injected with the egg yolk antibody for 2 days, the feed intake of the sick gosling begins to increase, the mental state is obviously improved, and no gosling dies within 10 days; the gosling of 1/2 was pounded and dissected after 10 days, and as a result, it was found that the body cavity and intra-articular urate deposition of the affected gosling had substantially disappeared. After the egg yolk antibody is injected into the control group, the feed intake and the mental state of the sick gosling are not improved remarkably, the sick gosling die from the 5 th day of the injection of the egg yolk antibody, and the death rate of the sick gosling is 60% within 10 days; 1/2 of the surviving gosling was pounded and dissected after 10 days, and as a result, it was found that urate deposition in the body cavity and joints of the affected gosling remained small. The mortality rate of the diseased gosling in the blank control group within 10 days reaches 90 percent.
The test results show that the yolk antibody has good safety, good prevention effect and high cure rate, can be used for preventing and treating the novel goose astrovirus infection, and has great economic and social benefits.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> egg yolk antibody for preventing and treating novel goose astrovirus and preparation method thereof
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7252
<212> DNA
<213> Goose astrovirus (Goose astrovirus)
<400> 1
tgtagcctct gtgttttcgc ttcctgcgca tcaaggcacg ccttctcacc caaattcata 60
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ccatacctcc cattgtcaac cccttttcct cacttgacac tcgtgaggaa aggaagactg 180
atgagattca gtcaggcctt gagaaggtct tccacttcaa tggtgttaat gaattataca 240
ccaagatgaa aggcttttat ggtcgcactc cagcctggag tgctctcatg aagtgtaatg 300
ccatctatct taaagattgg aaaacagcta taggggttga caatgggagg tatggtctct 360
ttttcgccga tgatgtcacc aaaccaacat ggtcacccga tattggtgca aaccttatga 420
atttgggtga gaaggcgtgc cttgatgcgc aggaagcgaa aacacagagg ctacaatgct 480
cgctgaaacg atgtggagct cttgtccaac aagttcttga gaagagcaaa caagcaaagg 540
aacaaatgca aaaagccgag gaattacaga agaagattga tcaaattaat gaaagcaaca 600
aggttctaca tcgcaaaatg caggagagaa atgttgagta ttctcagaag gtcataactc 660
gtattcatga gctgcaaaaa gacagagacc tgtggatgtc gcgtgctgct cattacgagc 720
aggagaacgt gcggctacgt attcaactgg ggacatatga tccgaacagg tctaagctga 780
tgactttcct agcatggatg tgtgttgcga tcattgcttt cagcttgatt caattggcgg 840
atgctagtga gattaaggat aacagaacag aaaattgttt cccaacatct ggctgtgttc 900
tctcagacgt tccttggact aagtcgtaca cctttatgga gctaatggac aagtgtgtga 960
ccggtactgc catcatacct aaaaacacat ttaatggtga agggcttgag ctggagtgta 1020
ttaagaattt tggcaagaaa tggtgcaaag gaagggttga gaagctcata ccgcattatt 1080
gtgatagaga taatactttt gatgccctct atcagtatta tgttgaaaca gtagcaattg 1140
ctgttgactt ctttaaagtt gcggtgcagt atcggattga caattggatt gtggctctct 1200
ttagtctagt attgactggg aagatggaga agtttcttaa gacgctaccc tttgtaattt 1260
tggcatggtg gtttaagttg cccatctttg ttatgagcat tgccacgaca atctttcctg 1320
tgcatgctct gccattcttg ttcttccagg tgtgctttcc aaactttgtc atcatcactg 1380
gttttctctt gtggatgacc tctatattag tggcttttta ttggtttgat gggactgcaa 1440
ttttggttga ggtttcttat gcggtattct atactattat cttctttgtg tggtctacag 1500
cattgacaat tatttcaact ttgtcattga cagtccctgt acagatttta ttattctgtg 1560
tttcattaac catttattgt ggcacaaagt ttgcgtgttc cactattaca gtcactaatc 1620
ctgacggtac tgtgtcgaaa tactcaagag ttgccaaaat gaaggatact gttgccatac 1680
agtgcaaaaa gatggtgagc tttgggcagg cccgtggtgt gataccagca actccagtga 1740
aggtgaattc cattgtcgtt gttgaaggaa aaaaaggttc aggggttggc ttccggttca 1800
tgaattatat aattacagca ggacacgtca cattaggctc tgactgggta acagtaaaac 1860
atgctggcac agtagccaaa acaaaagtcc tgaagcagat tgaggtgttt gagtgcgtcg 1920
atacaatagc ggttataaag ctgccccccg aattacagaa tttgaaaccc ataaaactcg 1980
caaaacgtgt tgagtccaca tacttaacat tgacagcttt tgatcctaat tttcaaaatg 2040
ttgtgtcata cactggctgg tgtattattg atggaaattg gatcagtaat gcatttaata 2100
ctcaatttgg aaacagtggt ggcccatacg ttgatgcaga gggtaaattg gttgggattc 2160
atcttggaac tcagggagtt atgtcccaag gtattgttgt ggtggatgcc cttaaaacgc 2220
atttcacgct cgcagaccag tgcaaggatt ttgattatga tggcttcctg gagaaagtta 2280
ttgcaggaac aaagacatca catgaagcaa ttctcaaagg tattgaggaa ttgcgagagg 2340
acgtcgacac gctcaagaac aagtgcaaga attatgatga gtactggctt gttcaaacta 2400
tttttggcca gaagaaaaag ggcaaaacaa agaaaacagc ccgggggaat aaacatcttg 2460
taacaaagag attcctgagt aaaggccact tcatgaaact caaaatgctc accgatgagg 2520
aatataatca tatgattgag caaggtttca ccgctgatga gatacgtgag gcagtcaatg 2580
agctgagaga acaagcatgg ctcaactact gcattgataa tgatattgat gaagaaggtg 2640
cggaagagtg gtatgaagat atgattgaag atgaccaaat taatgaaaag attgatgaag 2700
ccattgagaa agcaatggaa gaaaggggcg agttttatca agtcaaaaaa aggaaaacat 2760
ttgcacaaca agcattatta cacctcatcc gtgtcagtaa agccaggagc caaactgcaa 2820
agcaagaggt tcagaaagaa aacgcagatc agctttacga gatgttcaaa tctgccgtaa 2880
aggatgaaca agttgatgaa ggcacaacct cttacgcact cttcgccaat ggtgatcaga 2940
ttgagatggt cgaaggaaag gaacttgatt ttgaaaaggt aaagcttatc cacgttgatg 3000
gtgaacaaaa aaatgagacc attccgggga cgaaagtcac tgagatttct actggccctg 3060
agaataagaa gaacattttg cgcaagaagg acactcaaat taatccagaa caagctcaac 3120
ctcttgagca gcgcaagagg atctgcaagt ggtgtgggtc gagccaaaag catgactttc 3180
gtgattgcaa gttccagcgt gagaaaaggt tttgtgtctt ctgtgcaaag atgcactcat 3240
tgtttgatgg tcatcaacgg gacgtcttgt gcgacgtttg tgggaagaag ttcaagagtg 3300
tagaggagct tgaacagcac gtaactcgtg aaacgtgtga aaaaaactag ataaggggga 3360
cgatgccatc tgggtccccc gcataccacc tgatgattct cacatttttt cttggcagga 3420
tgatattatt gagtgtgata agaaaatcac tttgcccgac tatatagata taattggttt 3480
tatacctgta gataaattag ttgtcagagc aaagaccatt catgacccct tattaaactt 3540
ggttgagcat tggcaacaag atgaatatgc tcctactaaa tgggactata aagcctatac 3600
caagatgttt gaaaaatttt tctatcatga acctcaggat tttgtaaaca attttccaga 3660
gctggtcatc ttaagtgata gtgttgtgtt agaagaacat tcttatatgg ccaattccaa 3720
tttaacaccc ataatggcaa ctaagaagaa tgtcgatagt acccctggtt atccaaaatt 3780
taagtacttt gacacagaga aggactatct tgaacaatgt ggctggggtg agtacttgaa 3840
ggctgtatca gatattgatg agactgtgca aaataggccc ctctggtggt gttttctcaa 3900
aaatgaggtc ctcaaaaaga agaaaatcat ggataatgat attcgtatga tcttgtgtgc 3960
tgatccagta tatacaagga ttggagccat gtttgagcag gaccagaatg agaaaatgaa 4020
gcaacagaca gaacggcggg ctgcacaagt tggttggaca cccttttttg gtggaataca 4080
tcagcgagta tctaggctca ttggtggtgg tgacaggttt tttgtagaga cggactggac 4140
gcgttatgat ggtacgttgc ccaagcctct cttctggcgg atacgacaga tgcgttactt 4200
tttcctgtcc aaccatcata agacaccaca gcttaagaaa ctctatgatt ggtatgtcaa 4260
aaacttagtt gaaaaaatta tattattacc aactggggag gtttgtacag ttaagaaggg 4320
aaatccaagt ggccaatatt caacaacagt ggacaacaat atgtgtaatg tctggctcac 4380
ccattttgag atagcttatc tttattggaa acagcatggg tcattgccga cgctcagatt 4440
actcagggat aatgtcacca tgatttgcta tggcgatgat aggcttgtgt caataaggaa 4500
gggttttatt aattacgaca cgaatgtcgt catagatatg tataaaaata tttttggtat 4560
gtgggttaag ccagaaaatg tcaaggtctc tgatgatatt gagggtatga ctttctgtgg 4620
tcttacgttg gttaaaagga gtgatggtag atttgttgga gttcccaatg ttgataagat 4680
attgtcaaca cttgaaaatc ctgttagaaa attacccaac ttagaatcac tctggggtaa 4740
attggtttct ttaaggatct tgtgcgaaaa tgcacccaat agtgttagag agtttcttga 4800
taaacaaatc aacacggttg aggagcacgc tgccagtgaa aatattaact tacctgaggt 4860
cgggcccgac ttcttttctc aaatctggtg agtggcggac cgaaataaaa tggcagacag 4920
ggcggtggcc ccgcgcgaga aggtgaccaa gaaggttaca aaagtagtca ccgttaagaa 4980
aaaacaccca aaaaagaaac caaagcagaa agtatataag ccccaaaaat tacccatgaa 5040
ggccgagagg aagcttgaga aagaagtgaa aggtttgaag aaaagagtag ctggaccacc 5100
cgttaatgac aaaatgacta ccacgataac acttggtcag atcactggga attcaacaga 5160
cacactcgac cggaagcata aatacttcac aaatccactc atgatgaaaa accaggaaaa 5220
tgggcaaaca gcaactcctc taagtataag ggcctcacaa tataacttgt ggaggatcag 5280
aaagctgcat atccgccttg ttccacttgc tggtagggcc aatattcttg gctcggttgt 5340
cttcctagat atagaacagg aagctaacac agctgggcca gagtccatag ataccataaa 5400
agctcggccg catcttgaac tcccgattgg ctcaaaacat ctttggaggg ttcaacccag 5460
gctgatgcag ggaccccggc aaggatggtg gaatgtagac cccggggatt cacctactga 5520
ctcacttggt ccagcaatca atatgtggac atatttgaaa acagtaaatg cactttcaac 5580
acgggcgcag gcacaacaag ttccctatac ttctgccctc ttcctggttg aagcaacggt 5640
cacttatgag ttctcgaact atggtccaaa gccagggctc tcactcatga ccagtgaaac 5700
actttcagca tcagggaaaa cggcaaccct tgtaaatacc caggatggtg cacttgcttt 5760
aacagttagt ggcgcattgc agagattcct cgatgagaag gagcaacaca ggcgcgtttc 5820
aaatgcgcag acctctggtg tcggtgaggt gttttgggct gtttccacag aggtggtcga 5880
gaccgtagcg agtgcacttg gaggctgggg ttggctcttg aaaggaggat ggttcgtgat 5940
cagaaaattg tttggtgctg cttcaaattc tggttccact tatctgatct attcctcagt 6000
ttcagacgcc cagatagaca gcaggattta tcagacagtt ccaccgaaca cgccactaca 6060
gctggctgca aatacagtta agcttgtaca gctaacacag ccaaatgtga atacaactgg 6120
acaaggtacc actgtccttt cacgggatgc cgattacctg cctctgccag tggcaccaat 6180
gcaggttact ccctcgcttg tgtacaactt ccagggtgaa aggcagagca ctacagaatc 6240
gtgctcattc ctggtgtttg gaataccaca ggcagaatcc aggtcaagat acaatgcaaa 6300
tataaccttc aatgttggct atcgtggaag gacttcaaca tcatttacac ttggaacaca 6360
caattggtgg gctgttatga cactttcaca aacaggagta atttttgcac caccggctgt 6420
gggcacaggg gtctgtaata cattggctac agccatacaa cacttaaacc ctgagcttga 6480
aacagcagtc ctgcgtgtga ataccagtac aacatctact ggtgggctaa taacggaact 6540
caggaatcgg ctcaacatcg ctgatgggga ctatgtgatt tcaatgggtg atccgcaagg 6600
aaataggtca gcactgtact ttaggaattc agaccagaaa tgggtgtggc tctgggctgg 6660
cgactctaac cctggtgaaa ctttccaaag ttttaagatg ccagtcctaa ttaattggtc 6720
agtgtcagat tcccaggaac aatataatgc tagagtcagg atggtacagt atgctaatgc 6780
acaacagcag actttgacag accctgagga agatgatgat cccctctctg atgtcacttc 6840
gctttttgat ccaacagcgg aagatgagac tgacttccac ctagcagtct cactcaagac 6900
ctctgactac ttaaaagaag aggctgagta ctggaaagcg aaggcgcagg ccttgcttat 6960
ggagaaggca ctaagtgcac cacaagcagg gacagtccgc tttgagaagg gcggacatga 7020
gtgatctctt cactagcagc cgcggccacg ccgagtagga tcgagggtac agctgcacct 7080
tctcattagg cttgagggag aatcggccct ctctgcctta aattaacccg gtgacagtca 7140
ggtggtcacc cacacaaatc gcgtgcgggt acccctgacc gggtttttgt ttaaagaatc 7200
aaatgtgatt tttaagcatt taaaatcttt aaaaaaaaaa aaaaaaaaaa aa 7252
Claims (10)
1. The yolk antibody for preventing and treating the novel goose astrovirus is characterized in that the yolk antibody is an inactivated yolk antibody with a preservation number of CCTCC NO: v201808, and extracting and purifying from the yolk of the hyperimmune egg.
2. The method for producing a yolk antibody according to claim 1, comprising the steps of:
(1) the preservation number is CCTCC NO: v201808, wherein the goose astrovirus is used as a vaccine production strain to prepare an inactivated vaccine;
(2) injecting the prepared inactivated vaccine into an immunized laying hen to prepare a hyperimmune egg;
(3) the yolk antibody for preventing and treating the novel goose astrovirus is prepared by collecting yolk from the hyperimmune egg and performing primary inactivation, acidification extraction, secondary inactivation, rough filtration, sterilization filtration, concentration and tertiary inactivation.
3. The method according to claim 2, wherein in step (1), the inactivated vaccine is prepared by the following method:
the preservation number is CCTCC NO: v201808, inoculating the goose-star virus into SPF chick embryos, and collecting allantoic fluid of dead chick embryos within 24-120h to obtain virus fluid; concentrating the virus liquid until the virus content is more than or equal to 105.0ELD50Inactivating the concentrated virus solution by formaldehyde, adding Tween-80, mixing to obtain a water phase, mixing white oil, aluminum stearate and Span-80 to obtain an oil phase, mixing the water phase and the oil phase according to the volume ratio of 1:1, and emulsifying to obtain the inactivated vaccine.
4. The process according to claim 2, wherein in the step (2), the layer chicken is immunized with the inactivated vaccine prepared 4 times at intervals of 2 weeks.
5. The preparation method according to claim 2, wherein in the step (3), the primary inactivation specifically comprises: mixing the yolk solution and water according to the volume ratio of 1 (0.5-1.5), uniformly stirring to obtain a yolk diluent, and preserving heat for 25-35 min at the temperature of 60-65 ℃.
6. The preparation method according to claim 2, wherein in the step (3), the acidification extraction is specifically: adding an acetate buffer solution with the volume of 2.5-3.5 times that of the yolk diluent into the yolk diluent, stirring, filtering and collecting filtrate; the pH value of the acetate buffer solution is 4.8-5.2.
7. The preparation method according to claim 2, wherein in the step (3), the secondary inactivation specifically comprises: adding octanoic acid into the filtrate after acidification and extraction until the final concentration is 3.5-4.5%, stirring for 30-120min, and standing for 4-8 hours at 2-8 ℃ after stirring;
in the step (3), the sterilizing filtration specifically comprises: filter sterilized with a 0.22 μm filter.
8. The method according to claim 3, wherein in the step (3), the concentration is specifically: concentrating with PES hollow fiber ultrafiltration membrane of 30-50KD at 2-8 deg.C until the antibody titer is not less than 1: 512.
9. Use of the yolk antibody of claim 1 in the preparation of a preparation for the prevention and/or treatment of a novel goose astrovirus infection; the preservation number of the novel goose astrovirus is CCTCC NO: and V201808.
10. The preservation number is CCTCC NO: v201808, the preparation and treatment of the goose astrovirus are carried out by selecting the goose astrovirus with the preservation number of CCTCC NO: v201808 goose star virus induced gout yolk antibody.
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| CN109336971A (en) * | 2018-11-06 | 2019-02-15 | 哈药集团生物疫苗有限公司 | The preparation method and products thereof of goose astrovirus Yolk antibody |
| CN109456391B (en) * | 2018-11-12 | 2019-09-03 | 扬州优邦生物药品有限公司 | A kind of VLP vaccine and its preparation method and application preventing goose astrovirus infection |
| CN110590945B (en) * | 2019-08-29 | 2021-06-15 | 广东渔跃生物技术有限公司 | Preparation method of duck astrovirus egg yolk antibody |
| CN111471701B (en) * | 2020-04-27 | 2021-12-10 | 浙江鼎持生物制品有限公司 | Method for efficiently expressing ORF2 gene of goose star virus soluble capsid protein and application thereof |
| CN112280750B (en) * | 2020-10-22 | 2022-11-01 | 山东农业大学 | Novel goose astrovirus with cross-species transmission capability and application thereof |
| CN112341539B (en) * | 2020-10-22 | 2022-11-01 | 山东农业大学 | Yolk antibody for preventing and treating novel goose astrovirus with cross-species transmission capability and preparation method thereof |
| CN113214388B (en) * | 2021-05-12 | 2022-06-28 | 南阳师范学院 | Goose star virus egg yolk antibody and preparation method thereof |
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