CN111905100A - Goose astrovirus bivalent inactivated vaccine and yolk antibody and preparation method thereof - Google Patents
Goose astrovirus bivalent inactivated vaccine and yolk antibody and preparation method thereof Download PDFInfo
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- CN111905100A CN111905100A CN202010827091.1A CN202010827091A CN111905100A CN 111905100 A CN111905100 A CN 111905100A CN 202010827091 A CN202010827091 A CN 202010827091A CN 111905100 A CN111905100 A CN 111905100A
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Abstract
The invention discloses a goose astrovirus bivalent inactivated vaccine, a yolk antibody and a preparation method thereof, wherein the goose astrovirus bivalent inactivated vaccine is obtained by respectively inoculating goose astrovirus type 1 and type 2 strains to domesticated LMH cells for suspension culture to obtain a virus liquid which is high in virus content and does not need concentration, inactivating the virus liquid, mixing and emulsifying the virus liquid with a vaccine adjuvant to obtain the virus liquid, and separating yolk from high-immunity eggs obtained by immunizing laying hens by using the inactivated vaccine to obtain the goose astrovirus bivalent yolk antibody. The goose astrovirus bivalent inactivated vaccine and the egg yolk antibody prepared by the invention can provide effective immune protection for goslings, can effectively prevent goose gout, and have good commercial development prospect.
Description
Technical Field
The invention belongs to the field of preparation of vaccine antibodies, and particularly relates to a goose astrovirus bivalent inactivated vaccine, a yolk antibody and a preparation method thereof.
Background
Since 2016, infectious diseases mainly characterized by visceral and articular gout are developed in goose farms in more than ten provinces such as Henan, Jiangsu, Shandong and the like, and serious economic loss is caused to the goose farms. The disease mainly attacks goslings with the age of less than 3 weeks, the disease attack of 1-day-old goslings can be seen at the earliest, the mortality rate is 20-70%, diseased geese usually show depression, diet reduction, lying-down difficulty, unwilling to walk, white loose stool discharge, and necropsy show that kidney, heart, liver, lung, ureter, joint and the like have a large amount of urate deposition. The disease occurs in different varieties of goose groups using different feeds and different medicines, and the reduction of the protein content in the feed and the reduction of the feeding amount are both ineffective.
Literature reports and laboratory pathogen detection show that the gosling gout is mainly caused by 2 goose astrovirus types with different genotypes, namely goose 1 astrovirus and goose 2 astrovirus types. Sequence alignment analysis shows that the sequence homology between the type 1 and type 2 goose astrovirus strains is low (< 70%); seroneutralization studies have shown that type 1 and type 2 strains lack effective cross-protection from each other. At present, most goose embryos or goose embryonic kidney cells are adopted for culturing the goose astrovirus, but the goose embryos have the defects of high cost, limited sources, low virus content, no SPF (specific pathogen free) goose embryos and easy pollution of exogenous factors; the goose embryonic kidney cells are complex to prepare, small in amount and easy to pollute, so that the goose embryonic or goose embryonic kidney cells are not beneficial to large-scale production. The development of vaccines and related products is seriously restricted due to a plurality of difficulties in the culture of the goose astrovirus, and no commercial bivalent inactivated vaccine and antibody for effectively preventing and treating the goose astrovirus at home and abroad currently exists, so that the development of the bivalent inactivated vaccine and antibody containing the goose astrovirus type 1 and 2 is an inevitable requirement for preventing and controlling the goose astrovirus.
Disclosure of Invention
In order to solve the above problems, a bivalent inactivated vaccine of goose astrovirus and a method for preparing a yolk antibody have been proposed. In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of a goose astrovirus bivalent inactivated vaccine comprises the following steps:
(1) selecting two different goose astrovirus strains, and proliferating the two different goose astrovirus strains in an LMH cell culture mode, wherein the LMH cell culture method comprises the steps of culturing and proliferating the goose astrovirus strains in LMH cells, then harvesting virus liquid, continuously carrying out virus passage on the harvested virus liquid through the LMH cells until the virus content of the goose astrovirus in the harvested virus liquid is more than or equal to 106.0TCID500.1ml, inoculating the virus solution of the next generation into the domesticated LMH cells for suspension culture and proliferation, and harvesting the virus solution;
(2) respectively inactivating virus liquid finally obtained by culturing and proliferating two different strains of goose astrovirus by adopting LMH cells, and then mixing and emulsifying the two virus liquids respectively inactivated and a vaccine adjuvant to obtain the goose astrovirus bivalent inactivated vaccine.
Preferably, the two different goose astrovirus strains are goose type 1 astrovirus and goose type 2 astrovirus respectively, and the two different goose astrovirus strains have no serological relationship with each other.
Preferably, the two different goose astrovirus strains are a novel goose astrovirus WF strain with a preservation number of CCTCC No. V202047 and a novel goose astrovirus GD0122 strain with a preservation number of CCTCC No. V201820, and the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain have no serological relationship.
Preferably, the goose type 1 astrovirus and the goose type 2 astrovirus used for inoculation into LMH cells in step (1) are obtained by the following method:
(A) inoculating virus liquid of goose type 1 astrovirus and goose type 2 astrovirus into 12-13 day old goose embryos, harvesting dead goose embryo allantoic fluid for 24-168 hours and alive goose embryo allantoic fluid for 168 hours, and continuously carrying out passage for 3 generations in the way;
(B) and (3) performing virus identification on the virus liquid obtained after the continuous passage for 3 generations, inoculating the virus liquid meeting the requirements into goose embryos aged 12-13 days, obtaining the allantoic fluid of the dead goose embryos for 24-168 hours and the allantoic fluid of the alive goose embryos for 168 hours, and continuing the passage for 3 generations in the goose embryos aged 12-13 days.
Preferably, the purified goose-star virus is inoculated into LMH cells at 37 deg.C and 5% CO in the LMH cell culture method of step (1)2Culturing under the condition, harvesting virus liquid after culturing and proliferating, continuously inoculating virus to LMH cells for 3-6 generations until 80% of LMH cells are diseased and the diseased time is controlled within 72-96 hours, harvesting virus liquid, determining as F1 generation, continuously propagating virus in LMH cells for 5-10 generations by the harvested F1 generation virus liquid according to the same method, respectively measuring the virus content of each generation, and taking the goose astrovirus with the virus content more than or equal to 106.0TCID500.1ml of virus solution of generation is inoculated in the domesticated LMH cells for suspension culture and proliferation, and the virus solution is obtained.
A goose astrovirus bivalent inactivated vaccine is prepared by the preparation method of the goose astrovirus bivalent inactivated vaccine.
Preference is given toThe virus content of the goose type 1 astrovirus and the goose type 2 astrovirus is not less than 107.0TCID50/0.1ml。
A goose astrovirus divalent yolk antibody is obtained by immunizing laying hens with the goose astrovirus divalent inactivated vaccine.
A preparation method of a goose astrovirus bivalent yolk antibody comprises the following steps:
(a) immunizing laying hens by using the goose astrovirus bivalent inactivated vaccine;
(b) separating yolk from the high immune eggs obtained in step (a) and extracting antibodies.
Preferably, the specific method of step (a) is:
basic immunity: injecting 0.5ml of goose astrovirus bivalent inactivated vaccine into breast muscles of each chicken;
and (3) secondary immunization: performing vaccination for the 2 nd time on 14 th day after the basic immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
three times of immunization: performing 3 rd inoculation on 14 th day after the second immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
and (3) maintaining immunity: when the titer of the agar-agar positive antibody of the goose astrovirus type 1 and the goose astrovirus type 2 in the yolk reaches 1:96, maintaining inoculation for 1 time, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
egg collection: after 10 days of three times of intensified immunization, sampling and taking eggs, separating yolk, extracting antibodies, and collecting high-immunity eggs when the titer of the agaropectin antibody of the goose type 1 astrovirus and the goose type 2 astrovirus is greater than or equal to 1: 64;
the specific method of the step (b) is as follows:
adding the yolk liquid of the high-immunity eggs obtained in the step (a) into a pasteurization tank, adding water for injection, stirring and uniformly mixing, heating to 65 ℃, preserving heat and inactivating for 15 minutes, and cooling to below 30 ℃ in water bath after inactivation is finished;
adding injection water with the volume 4 times that of the original yolk into an acidification reaction tank, adjusting the pH value to 4.2 by using 1mol/L HCl solution, cooling the water to 2-4 ℃, then adding the inactivated yolk liquid while stirring, and keeping the temperature at 4-8 ℃ and standing for 4 hours;
freezing and centrifuging the acidified extract, removing precipitate, and taking supernatant for later use;
adding octanoic acid into the supernatant to a final concentration of 0.02%, stirring, mixing, standing at room temperature for 4 hours, filtering with K-type multi-layer plate frame for clarification, adding formaldehyde solution according to a final concentration of 0.05%, stirring, mixing, sealing for 60 minutes, and filtering with ultrafiltration membrane with cut-off molecular weight of 1000KD to remove viruses;
adding Tween-80 to a final concentration of 0.02%, adjusting pH to 6.8 with 1mol/L sodium hydroxide solution, filtering and sterilizing the qualified antibody with 0.22 μm microporous membrane to obtain refined yolk antibody of gosling gout.
Has the advantages that:
(1) according to the invention, the goose astrovirus type 1 and 2 strains are inoculated to the domesticated LMH cells for suspension culture, the obtained virus content is high, concentration is not required, the vaccine can be directly used for vaccine preparation, the method has the advantages of no exogenous factor pollution, easiness for large-scale production, small occupied space of a production line, high production efficiency, low cost, capability of well maintaining the stability of virus antigens and the like, and the defects of culture by using goose embryos, goose embryonic kidney cells and cell bottles are overcome.
(2) The goose astrovirus type 1 and goose astrovirus type 2 bivalent inactivated vaccines screened by the invention are good in safety, free of any local and systemic adverse reaction caused by the vaccines, and stable and effective in all indexes through analysis of characters, safety tests and efficacy test data; the immune effect of the vaccine is evaluated by a serological method and an immune challenge method, and the result shows that the goose astrovirus type 1 and type 2 bivalent inactivated vaccine prepared by the invention can provide effective immune protection for goslings, can effectively prevent the occurrence of gosling gout, and has good commercial development prospect.
(3) The bivalent egg yolk antibody provided by the invention can be used for preparing a medicine for preventing and treating gosling gout, the safety is good, and any local and systemic adverse reaction caused by antibody injection is avoided after gosling inoculation. The prepared antibody is evaluated by adopting antibody titer determination and toxicity attacking protection tests, and the result shows that the titer of the antibody amplified by the method is not lower than 1:16, the gosling can resist the attack of the virus after the antibody is injected, which shows that the antibody prepared by the invention can provide effective immune protection for goose groups, has better protection effect on goose astrovirus type 1 and goose astrovirus type 2, and has good commercial development prospect.
Drawings
FIG. 1 shows the virus identification electrophoretograms of novel goose astrovirus WF strain and novel goose astrovirus GD0122 strain.
Detailed Description
In order to make the technical solutions of the present invention better understood, the following description of the technical solutions of the present invention with reference to the accompanying drawings of the present invention is made clearly and completely, and other similar embodiments obtained by a person of ordinary skill in the art without any creative effort based on the embodiments in the present application shall fall within the protection scope of the present application.
Example 1
The preparation method of the goose astrovirus bivalent inactivated vaccine provided in the specific embodiment comprises the following steps:
(1) inoculating virus liquid of a novel goose astrovirus WF Strain with the preservation number of CCTCC No. V202047 and a novel goose astrovirus GD0122 Strain (Noval goose astrovirus GD 0122) with the preservation number of CCTCC No. V201820 into goose embryos of 12-13 days old, wherein the novel goose astrovirus WF Strain and the novel goose astrovirus GD0122 Strain have no serological relationship, harvesting goose embryo allantoic fluid dying within 24-168 hours and goose embryo allantoic fluid surviving within 168 hours, and continuously carrying out passage for 3 generations in the way;
(2) performing virus identification on the virus liquid obtained after continuous passage for 3 generations, inoculating the virus liquid meeting the requirements into goose embryos aged 12-13 days, obtaining the allantoic fluid of the dead goose embryos for 24-168 hours and the allantoic fluid of the alive goose embryos for 168 hours, and continuously transmitting for 3 generations in the goose embryos aged 12-13 days in this way;
(3) carrying out continuous 3 generations of the step (2) and purifying the new goose astrovirus WF strain and the new goose astrovirus WF strainThe goose-star virus GD0122 strain is proliferated by LMH cell culture method, which comprises inoculating goose-star virus into LMH cells at 37 deg.C and 5% CO2Culturing under the condition, harvesting virus liquid after culturing and proliferating, continuously inoculating virus to LMH cells for 3-6 generations until 80% of LMH cells are diseased and the diseased time is controlled within 72-96 hours, harvesting virus liquid and naming as F1, continuously propagating virus in LMH cells for 5-10 generations according to the same method for the harvested virus liquid F1, respectively measuring the virus content of each generation, and taking the goose astrovirus with the virus content more than or equal to 106.0TCID500.1ml of virus solution of generation is inoculated in the domesticated LMH cells for suspension culture and proliferation, and the virus solution is obtained;
(4) and (3) respectively inactivating virus liquid finally obtained by culturing and proliferating the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain in the step (3) by adopting LMH cells, and then mixing and emulsifying the two virus liquids respectively inactivated and a vaccine adjuvant to obtain the goose astrovirus bivalent inactivated vaccine. (ii) a
The specific embodiment also provides a goose astrovirus bivalent inactivated vaccine, which is prepared by the preparation method of the goose astrovirus bivalent inactivated vaccine, and the virus content of the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain is not less than 107.0TCID50/0.1ml。
Example 2
1. Isolation and characterization of viruses
In the specific embodiment, 2 strains of goose astrovirus are used, namely a novel goose astrovirus WF Strain and a novel goose astrovirus GD0122 Strain (Noval goose astrovirus Strain GD 0122), the novel goose astrovirus WF Strain is preserved in China center for type culture Collection, Wuhan university, Wuchang district, Wuhan City, North Hubei province, 7-29 days 2020, and the preservation numbers are: CCTCC No. V202047; the novel goose astrovirus GD0122 Strain (Noval goose astrovirus Strain GD 0122) is preserved in China center for type culture Collection (China center for type culture Collection, Wuhan university, Wuchang district, Wuhan City, North Hubei province in 2018, 5 months and 13 days, and the preservation numbers are: CCTCC No. V201820. The novel goose astrovirus WF Strain is goose type 1 astrovirus, and the novel goose astrovirus GD0122 Strain (Noval goose astrovirus Strain GD 0122) is goose type 2 astrovirus.
The novel goose astrovirus WF strain is separated from 6-day-old diseased goose groups in a certain Shandong goose farm, and the novel goose astrovirus GD0122 strain is separated from 10-day-old diseased goose groups in a certain Guangdong goose farm.
1.1 isolation of viruses
Selecting 50-100 g of tissues of livers, spleens, kidneys, pancreas and the like of dead geese with typical disease symptoms, grinding the tissues by using a mortar, adding sterile PBS (0.1mol/L, pH 7.2) containing double antibodies (containing 1000U/ml penicillin and 1000 mu g/ml streptomycin) according to the proportion of 1:5(w/v), homogenizing, repeatedly freezing and thawing for 3 times, centrifuging at 6000r/min for 10 minutes, taking supernate, filtering and sterilizing by using a 0.22 mu m filter, and storing for later use after the qualified aseptic inspection. Inoculating the virus filtrate into 12-13-day-old goose embryos, 0.2 ml/embryo, irradiating the embryos for 2 times per day at 36-37 ℃, discarding the dead goose embryos within 24 hours, harvesting the dead goose embryos within 24-168 hours and live goose embryo allantoic fluid within 168 hours, cooling at 2-8 ℃ for 12-24 hours, harvesting the goose embryo allantoic fluid, and continuously carrying out passage for 3 generations by using the method.
1.2 identification of viruses
1.2.1PCR identification
Respectively taking 200 mu l of frozen and thawed virus liquid, extracting virus RNA by using an RNA extraction kit, extracting virus DNA by using a DNA extraction kit, detecting goose paramyxovirus, H5, H7 and H9 subtype avian influenza, reovirus and astrovirus by PCR after RNA reverse transcription, and detecting circovirus, avian adenovirus, gosling plague and Muscovy duck parvovirus by DNA PCR. And (5) observing the result by electrophoresis on a 1% agarose gel after the PCR is finished.
And (3) PCR reaction system: 25 μ l
To a 0.5m 1PCR tube were added in sequence:
PCR reaction parameters: the pre-denaturation conditions were 98 ℃ for 3 min, the denaturation conditions 98 ℃ for 10 sec, the annealing temperature and time 50 ℃ for 30 sec, the extension temperature 72 ℃ for 20 sec, 34 cycles, and finally 72 ℃ for 3 min.
After amplification, the separated strain only amplifies a target fragment corresponding to the astrovirus, the size of the target fragment is consistent with the size of an expected target fragment, and the electrophoresis result is shown in figure 1, wherein 1 is a novel goose astrovirus GD0122 strain, 2 is a novel goose astrovirus WF strain, 3 is a negative control, and M is a Marker. The detection results of goose paramyxovirus, H5, H7 and H9 subtype avian influenza, reovirus, circovirus, adenovirus, gosling plague, muscovy duck parvovirus and the like are negative. Sequencing the amplified target fragment, comparing the sequences, analyzing and constructing an evolutionary tree, and showing that the WF strain is the astrovirus type 1, and the GD0122 strain is the astrovirus type 2. It was analyzed from the molecular evolutionary tree that WF strain was in the same branch as the astrovirus type 1 strains FLX (ID: KY271027) and AHDY (ID: MH410610), and GD0122 strain was in the same branch as the astrovirus type 2 CXZ strains CXZ (ID: MH807626) and GD strain (ID: MG934571) and other type 2 strains.
1.2.2 detection of Virus content
Taking 3 rd generation goose embryo allantoic fluid of WF strain and GD0122 strain, respectively, properly diluting, inoculating 12-13 days old healthy goose embryos, 0.2 ml/embryo, culturing in an incubator with 60% humidity and 37 ℃ for 168 hours, discarding goose embryos dying within 24 hours, immediately taking out goose embryos dying within 24-168 hours, storing at 4 ℃ for 168 hours, taking out all the embryos not dying, storing at 4 ℃ for 12-24 hours, and collecting the allantoic fluid of the goose embryos. The harvested allantoic fluid of the goose embryo is continuously subjected to 3 generations in 12-13-day-old goose embryos by the method, and the generations are respectively marked as D4-D6 generations. After purifying the D5 and D6 virus liquid, half of the infection amount EID was measured50The results are shown in Table 1, and D5 and D6 are EID50Are all at 105.0EID50More than 0.2 ml.
TABLE 1 measurement of Virus content of WF Strain and GD0122 Strain ((10)n EID50/0.2ml)
1.2.3 animal regression test
10 goslings with the age of 2 days are inoculated with 1.0ml of WF strain and GD0122 strain respectively in an intramuscular injection way, the goslings begin to die at the 4 th day after challenge, the death peak is concentrated at 7-11 days after challenge, the death rate of the WF strain is 7/10, and the death rate of the GD0122 strain is 8/10. The diseased gosling is depressed, discharges white thin feces, is emaciated, and dissects the dead gosling, so that visceral urate deposition and ureter swelling can be seen, and cheese-like urate deposition at joints can be seen in severe cases.
1.2.4 Virus neutralization assay
The WF strain and the GD0122 strain are subjected to subculture and purification to prepare inactivated vaccines, and the inactivated vaccines are used for immunizing SPF (specific pathogen free) chickens of 30 days old respectively, wherein the first immunization dose is 0.5ml per chicken, the boosting immunization dose is 1.0ml per chicken, the immunization is performed once every 2 weeks for 3 times, blood sampling is performed 10 days after 3-immunization, serum is separated, and the inactivated vaccines are inactivated for 1 hour at 56 ℃.
And performing cross neutralization titer determination on positive serum of WF strain and GD0122 strain by adopting a fixed virus dilution serum method. Diluting WF strain and GD0122 strain to 200EID respectively50And mixing the diluted positive serum with WF strain and GD0122 strain one by one in equal volume, shaking and mixing uniformly, neutralizing for 1 hour at 37 ℃, inoculating 5 goose embryos with the age of 10 days, 0.2 ml/goose embryo, observing once every 24 hours, discarding the dead goose embryos within 24 hours, and continuously observing for 7 days. The results are shown in Table 2, and show that there is no neutralization between the goose astrovirus WF strain and GD0122 strain serum viruses, and the neutralization titer is uniform<1:4. This shows that there is no cross-neutralization between WF and GD0122, and their antibodies can only neutralize the virus, but not neutralize the virus. From the results of the cross-neutralization test, there was no serological relationship between the goose astrovirus WF strain and the GD0122 strain.
TABLE 2 serum cross-neutralization test results for goose-star virus WF strain and GD0122 strain
2. Preparation of vaccines
2.1 Virus propagation and harvesting
Selecting LMH cells full of monolayer, pouring out cell culture solution in the bottle, washing with PBS for three times, collecting purified 6 th generation of WF strain in 1.2.2, inoculating virus according to the ratio of 2% -5% of culture solution, inoculating virus at 37 deg.C and 5% CO2Culturing under the condition for about 96 hours, harvesting virus liquid, and repeatedly freezing and thawing for 1-2 times. Continuously inoculating the virus liquid to LMH cells for 3-6 generations until 80% of the cells have pathological changes and the pathological change time is controlled within 72-96 hours, harvesting the virus liquid, determining the virus liquid as F1 generation, continuously propagating the virus liquid of the harvested F1 generation for 5-10 generations according to the same method, respectively measuring the virus content of each generation, and taking WF strain with the virus content of more than or equal to 106.0TCID500.1ml of virus liquid of the generation virus is inoculated in the domesticated LMH cells for suspension culture, the virus is collected after 48 to 72 hours of proliferation, and finally the sterile WF strain virus liquid for the detection of the same generation is obtained and mixed and stored at the temperature of minus 20 ℃.
The GD0122 strain virus is cultured and proliferated according to the same method, and finally the sterile GD0122 strain virus solution which is tested in the same generation is mixed and stored at the temperature of minus 20 ℃.
In the scheme, 1.2.2 WF strain and GD0122 strain 6 th generation embryo allantoic fluid are selected because viruses can be detected in 3 generations on embryos generally, the virus content is low at this time, the virus content can be improved only by continuous generation, the virus content in the sixth generation is higher, and the propagation on cells is facilitated, so the sixth generation is selected.
2.2 inactivation of the Virus
Respectively introducing virus liquid of WF strain and GD0122 strain into an inactivation tank, adding formaldehyde solution according to the final concentration of the virus liquid of 0.1%, inactivating for 24 hours at 37 ℃ under the condition of continuous stirring, taking out, and storing at 2-8 ℃.2.3 inspection of the semi-finished product
2.3.1 sterility test
The inactivated virus solution was collected and examined according to 2015 edition of appendix of Chinese veterinary pharmacopoeia, and aseptically grown.
2.3.2 inactivation assay
The inactivated virus solution is inoculated into 4 wells of well-growing LMH cells (24-well plate), each well is 0.2ml, maintenance solution is supplemented to 2.0ml, incubator culture is carried out at 37 ℃, cells are observed for 2 times every day, and 168 hours of observation are carried out. Collecting cell sap, performing blind passage for 2 generations according to the same method, and observing and recording the cytopathic condition. As a result, the cells are free from pathological changes and are completely inactivated.
2.3.3 measurement of Virus content
Diluting virus solution before inactivation by 10 times with maintenance solution respectively, and taking 10 times-5、10-6、10-7、10-8The LMH cells (96-well cell plates) grown in full monolayers were seeded at 4 dilutions, each dilution being seeded at 6 wells, 0.1ml per well. Setting virus positive control hole and cell negative control hole at 37 deg.C and 5% CO2Culturing in an incubator, observing for 1-2 times every day, and continuously observing for 168 hours. Carefully observing the pathological condition of cells, judging the cells with cytopathic effect (CPE) as infection, and calculating TCID according to Reed-Muench method50The virus content should be not less than 107.0TCID50/0.1ml。
2.4 preparation of inactivated vaccine
Two aliquots of two inactivated antigen solutions (i.e., inactivated virus solutions) were mixed with SEPPIC 71R oil adjuvant at 3: 7, emulsifying, starting a motor to stir the 71R adjuvant at a low speed for 2 minutes, slowly adding the antigen liquid at 10000R/min, and emulsifying for 15 minutes to obtain the oil emulsion inactivated vaccine. Quantitatively subpackaging, covering and sealing, sticking a label, and storing at 2-8 ℃.
3. Vaccine product inspection
3.1 Properties
Appearance: milk white emulsion.
The preparation formulation is as follows: water-in-oil type. A clean pipette is taken, a small amount of vaccine is sucked and dropped into cold water, and the vaccine does not spread except the 1 st drop.
Stability: 10ml of the vaccine is sucked and added into a centrifuge tube, and the centrifuge tube is centrifuged for 15 minutes at 3000r/min, and the water phase separated out from the tube bottom is not more than 0.5 ml.
Viscosity: according to the appendix of the existing Chinese animal pharmacopoeia, the result conforms to the regulations.
3.2 filling check
The inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the result accords with the regulation.
3.3 sterility testing
The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the result is aseptic growth.
3.4 safety inspection
10 goslings of 7-day old are immunized subcutaneously, 2.0ml of vaccine is injected subcutaneously in different points at each neck, the growth and development are normal after 21 days of observation, the mental state is good, the vaccine is well absorbed at the injection part of the autopsy, and inflammatory reactions such as red swelling, tissue necrosis and the like do not exist.
4. Vaccine immunization effect detection
4.1 serological methods
The vaccine is injected subcutaneously into 10 breeding geese at the neck part with the dose of 1.0 ml/goose, other 10 breeding geese with the same age on the same day are taken as non-immune control, blood is collected every week after 2 weeks after immunization, serum is separated until 8 weeks after immunization, the serum antibody titer of the goose astrovirus type 1 and 2 is respectively determined by an agar immunodiffusion test method, and the detection result is shown in table 3.
TABLE 3 determination of antibody levels after vaccine immunization
Note: the line of precipitation appeared as positive.
It can be seen from the table that, in the goose star virus bivalent inactivated vaccine provided in this embodiment, the agar titer of both star virus type 1 and star virus type 2 can reach 9/10 or more positive at week 3 after immunization, while the agar titer of the non-immunized control group goose is negative.
4.2 immunological challenge
Injecting the vaccine into neck part of goose subcutaneously at a dose of 1.0 ml/goose, and adding other vaccineAnd immunizing a control group, collecting hatching eggs after 4-8 weeks of immunization, and hatching the goslings. 20 goslings of 1 day old are selected as an immunity group, 20 goslings of 1 day old produced by a non-immunity control group are selected as a control group, 10 goslings of the immunity group and the control group are respectively selected at random, and 0.5 ml/g of WF strain virus liquid is injected into muscles (the virus content is about 5 multiplied by 10)6.5TCID50) The goslings of the rest challenge group and the control group are respectively injected with 0.3ml of GD0122 strain virus solution per mouse (the virus content is about 3 multiplied by 10)6.0TCID50) The disease of the offspring of the gosling in the immune group and the gosling in the control group is observed and recorded in detail, and the result is shown in a table 4.
Table 4 vaccine efficacy test results
The result shows that the breeding geese are subjected to challenge after immunization and observed for 14 days, the maternal antibody of the offspring goslings of the immune group of the breeding geese is higher, and the immune group goslings survive and grow normally after challenge of WF strains and GD0122 strains; the control group had all diseases, wherein 6 deaths occurred in group C, 7 deaths occurred in group D, visceral gout symptoms were observed by autopsy, arthritic gout symptoms appeared in some cases, goose dysplasia and emaciation were tolerated.
In conclusion, after the goose astrovirus type 1 and type 2 bivalent inactivated vaccine prepared by the invention is used for immunizing breeding geese, the maternal antibodies of the progeny gosling astrovirus type 1 and type 2 are higher, and the occurrence of gout of the progeny goslings can be effectively prevented.
Example 3
The specific embodiment provides a goose astrovirus divalent egg yolk antibody, which is obtained by immunizing laying hens with the goose astrovirus divalent inactivated vaccine of the embodiment 1, and the preparation method of the goose astrovirus divalent egg yolk antibody comprises the following specific steps:
(a) the laying hens were immunized with the goose-star virus bivalent inactivated vaccine of example 1:
basic immunity: injecting 0.5ml of goose astrovirus bivalent inactivated vaccine into breast muscles of each chicken;
and (3) secondary immunization: performing vaccination for the 2 nd time on 14 th day after the basic immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
three times of immunization: performing 3 rd inoculation on 14 th day after the second immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
and (3) maintaining immunity: when the titer of the agar-amplified antibodies of the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain in the yolk reaches 1:96, maintaining inoculation for 1 time, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
egg collection: after 10 days of three times of enhanced immunization, eggs are sampled and taken, yolk is separated, antibodies are extracted, the titer of the amplified antibodies of the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain is greater than or equal to 1:64, and high-immunity eggs are collected.
(b) Preparing an antibody:
adding the yolk liquid of the high-immunity eggs obtained in the step (a) into a pasteurization tank, adding water for injection, stirring and uniformly mixing, heating to 65 ℃, preserving heat and inactivating for 15 minutes, and cooling to below 30 ℃ in water bath after inactivation is finished;
adding injection water with the volume 4 times that of the original yolk into an acidification reaction tank, adjusting the pH value to 4.2 by using 1mol/L HCl solution, cooling the water to 2-4 ℃, then adding the inactivated yolk liquid while stirring, and keeping the temperature at 4-8 ℃ and standing for 4 hours;
freezing and centrifuging the acidified extract, removing precipitate, and taking supernatant for later use;
adding octanoic acid into the supernatant to a final concentration of 0.02%, stirring, mixing, standing at room temperature for 4 hours, filtering with K-type multi-layer plate frame for clarification, adding formaldehyde solution according to a final concentration of 0.05%, stirring, mixing, sealing for 60 minutes, and filtering with ultrafiltration membrane with cut-off molecular weight of 1000KD to remove viruses;
adding Tween-80 to a final concentration of 0.02%, adjusting pH to 6.8 with 1mol/L sodium hydroxide solution, filtering and sterilizing the qualified antibody with 0.22 μm microporous membrane to obtain refined yolk antibody of gosling gout.
Example 4
1. High-immunity egg production
1.1 the laying hens should have the production performance of commercial laying hens.
1.1.1 No avian leukosis and avian influenza infection
Sampling blood according to 0.5 percent of chicken flocks, and taking all negative samples.
1.1.2 pullorum disease and Mycoplasma gallisepticum
The positive rate of pullorum disease and mycoplasma gallisepticum infection is less than or equal to 0.1 percent by detecting according to NY/T536-2002 'diagnosis technology of typhoid and pullorum disease' and NY/T553-2002 'diagnosis technology of mycoplasma gallisepticum disease'.
1.1.3 feeding management of layers
The construction of the chicken farm must meet the requirements of veterinary health epidemic prevention regulations, the chicken farm should leave over 500 meters from the traffic road, and the entrance and exit roads should be separated. The material and the manure channel in the field are separated. The inlet and outlet of the chicken farm are provided with a disinfection pond. Isolation belts should be arranged in brooding houses and adult chicken houses. In addition, the chicken farm should be provided with a feces treatment facility, the whole-in and whole-out treatment is implemented, drinking water in the chicken farm should reach the sanitary standard, and feeding personnel should be sanitary and healthy.
1.1.4 prevention and treatment of epidemic disease in chickens
According to the actual situation of occurrence of local epidemic diseases, related vaccines are timely inoculated according to a scientific immunization program, and according to the situation requirement, antibacterial drugs are added into the feed to prevent bacterial infection and anticoccidial drugs are added to prevent coccidium infection and the like according to the conventional method.
1.1.5 day-old chickens
100-150 days old.
1.2 immunization with bivalent inactivated vaccine of goose astrovirus type 1 and 2 prepared in example 2
Basic immunity: injecting 0.5ml of goose astrovirus bivalent inactivated vaccine into breast muscles of each chicken;
and (3) secondary immunization: performing vaccination for the 2 nd time on 14 th day after the basic immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
three times of immunization: performing 3 rd inoculation on 14 th day after the second immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
and (3) maintaining immunity: when the titer of the agar-amplified antibodies of the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain in the yolk reaches 1:96, maintaining inoculation for 1 time, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
egg collection: after 10 days of three times of enhanced immunization, sampling eggs, separating egg yolks, extracting antibodies, collecting high-immunity eggs, storing at 4-8 ℃ for no more than 10 days, wherein the titer of the amplified antibodies of the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain is more than 1: 64.
2. Antibody production
2.1 Eggshell Disinfection
Soaking high immunity egg in 0.1% benzalkonium bromide water solution at 42 deg.C for 15 min for disinfection, selecting egg shell with serious pollution, sterilizing separately, washing with clear water, and soaking for disinfection once. And then, soaking the eggs in a water bath with the temperature of more than 95 ℃ for disinfection for 5 seconds, taking out the eggs, and airing or blow-drying the eggs for later use.
2.2 separating yolk
Manually or mechanically beating eggs, removing egg white, blastoderm and frenulum, and collecting egg yolk.
2.3 inactivation of I
Adding the egg yolk liquid into a pasteurization tank, adding appropriate amount of water for injection, stirring, heating to 65 deg.C, inactivating for 15 min, and cooling to below 30 deg.C in water bath.
2.4 acidification extraction
Adding injection water with the volume 4 times that of the original yolk into an acidification reaction tank, adjusting the pH value to 4.2 by using 1mol/L HCl solution, cooling the water to 2-4 ℃, then adding the inactivated I yolk liquid while stirring, and keeping the temperature at 4-8 ℃ for standing for 4 hours.
2.5 centrifugal separation
Freezing and centrifuging the acidified extract, removing precipitate, and taking supernatant for later use.
2.6 inactivation of II
Adding octanoic acid to the final concentration of 0.02%, stirring and mixing, and standing at room temperature for 4 hours.
2.7 deep filtration
Clarifying by filtering with K-type multi-layer plate frame.
2.8 inactivation of III
Adding formaldehyde solution according to the final concentration of 0.05%, fully stirring and uniformly mixing, and sealing for 60 minutes.
2.9 Virus removal by Ultrafiltration
The virus is removed by filtration with an ultrafiltration membrane with a molecular weight cut-off of 1000 kD.
2.10 blending Total mix
Tween-80 was added to a final concentration of 0.02%, and the pH was adjusted to 6.8 with 1mol/L sodium hydroxide solution.
2.11 sterile filtration
Filtering and sterilizing the qualified antibody by using a 0.22 mu m microporous filter membrane, and performing sterile subpackage to obtain 100 ml/bottle of the goose astrovirus bivalent egg yolk antibody.
3. Antibody assay
3.1 purity
The detection is carried out according to the existing pharmacopoeia of the people's republic of China, and the pollution of bacteria, mycoplasma and exogenous viruses is avoided.
3.2 Octanoic acid and Formaldehyde residue
The detection is carried out according to the current pharmacopoeia of the people's republic of China, and the result accords with the regulation.
3.3 safety testing
10 healthy goslings of 7 days old are injected with 2.0ml per one time, and the health is kept for 14 days after observation.
3.4 efficacy test
3.4.1 detection of the amplification potency of the antibody
And (4) detecting the titer of the agar-agar and agar-agar antibody according to the current pharmacopoeia of the people's republic of China, and recording the result. The results show that the titer of the setron amplified antibodies of the goose astrovirus WF strain and the GD0122 strain is not lower than 1: 16.
3.4.2 immunization method for counteracting toxic substance
Taking 3 batches of the egg yolk antibodies prepared above, and injecting 3 days old susceptible goslings (the female source antibodies of the 1 type and 2 type goose astrovirus are less than or equal to 1:4)20 eggs per batch at 1.0 ml/egg yolk antibody; additionally, 20 control groups were injected with 1.0 ml/control group of saline subcutaneously in the neck; all groups are separately fed.
The goslings are respectively attacked after being inoculated for 16 hours, 20 goslings in each immune group and control group are respectively randomly divided into 2 groups, 10 goslings in each group are respectively injected with WF strain and GD0122 strain virus solution through muscle, each group is 0.5ml, the continuous observation is carried out for 14 days, and the death condition of each group of goslings is recorded.
The results are shown in Table 5, the immune group is 9/10 or above for protection, the normal saline control group is 9/10 for disease after challenge, visceral urate deposition can be seen in the dead goose after autopsy, and gout typical symptoms such as white spots, enlarged spleen and swollen kidney of liver can be seen
In conclusion, after a gosling is immunized by the divalent egg yolk antibody prepared by the LMH cell culture novel goose astrovirus, the challenge test of astrovirus type 1 and type 2 strains shows that the divalent egg yolk antibody has better protective effect on both gosling astrovirus type 1 and type 2.
TABLE 5 antibody potency and challenge protection results
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A preparation method of a goose astrovirus bivalent inactivated vaccine is characterized by comprising the following steps:
(1) selecting two different goose astrovirus strains, and proliferating the two different goose astrovirus strains in an LMH cell culture mode, wherein the LMH cell culture method comprises the steps of culturing and proliferating the goose astrovirus strains in LMH cells, then harvesting virus liquid, continuously carrying out virus passage on the harvested virus liquid through the LMH cells until the virus content of the goose astrovirus in the harvested virus liquid is more than or equal to 106.0TCID500.1ml, inoculating the virus solution of the next generation into the domesticated LMH cells for suspension culture and proliferation, and harvesting the virus solution;
(2) respectively inactivating virus liquid finally obtained by culturing and proliferating two different strains of goose astrovirus by adopting LMH cells, and then mixing and emulsifying the two virus liquids respectively inactivated and a vaccine adjuvant to obtain the goose astrovirus bivalent inactivated vaccine.
2. The method for preparing the goose astrovirus bivalent inactivated vaccine as claimed in claim 1, wherein the two different strains of goose astrovirus are goose type 1 astrovirus and goose type 2 astrovirus respectively, and there is no serological relationship between the two different strains of goose astrovirus.
3. The method for preparing the goose astrovirus bivalent inactivated vaccine as claimed in claim 2, wherein the two different goose astrovirus strains are a novel goose astrovirus WF strain with the preservation number of CCTCC No. V202047 and a novel goose astrovirus GD0122 strain with the preservation number of CCTCC No. V201820, and there is no serological relationship between the novel goose astrovirus WF strain and the novel goose astrovirus GD0122 strain.
4. The method for preparing the bivalent inactivated vaccine against goose astrovirus according to claim 2, wherein the goose astrovirus type 1 and the goose astrovirus type 2 used for inoculation into LMH cells in the step (1) are obtained by the following method:
(A) inoculating virus liquid of goose type 1 astrovirus and goose type 2 astrovirus into 12-13 day old goose embryos, harvesting dead goose embryo allantoic fluid for 24-168 hours and alive goose embryo allantoic fluid for 168 hours, and continuously carrying out passage for 3 generations in the way;
(B) and (3) performing virus identification on the virus liquid obtained after the continuous passage for 3 generations, inoculating the virus liquid meeting the requirements into goose embryos aged 12-13 days, obtaining the allantoic fluid of the dead goose embryos for 24-168 hours and the allantoic fluid of the alive goose embryos for 168 hours, and continuing the passage for 3 generations in the goose embryos aged 12-13 days.
5. The method for preparing the inactivated bivalent vaccine against goose star virus according to any one of claims 2 to 4, wherein the purified goose star virus is inoculated into LMH cells at 37 ℃ and 5% CO in the LMH cell culture method of step (1)2Culturing under the condition, harvesting virus liquid after culturing and proliferating, continuously inoculating virus to LMH cells for 3-6 generations until 80% of LMH cells are diseased and the diseased time is controlled within 72-96 hours, harvesting virus liquid, determining as F1 generation, continuously propagating virus in LMH cells for 5-10 generations by the harvested F1 generation virus liquid according to the same method, respectively measuring the virus content of each generation, and taking the goose astrovirus with the virus content more than or equal to 106.0TCID500.1ml of virus solution of generation is inoculated in the domesticated LMH cells for suspension culture and proliferation, and the virus solution is obtained.
6. A bivalent inactivated vaccine of goose astrovirus, which is prepared by the preparation method of the bivalent inactivated vaccine of goose astrovirus according to any one of claims 2-5.
7. The goose astrovirus bivalent inactivated vaccine as claimed in claim 6, wherein the virus content of the goose astrovirus type 1 and the goose astrovirus type 2 is not less than 107.0TCID50/0.1ml。
8. A goose astrovirus bivalent yolk antibody, which is obtained by immunizing laying hens with the goose astrovirus bivalent inactivated vaccine of claim 6.
9. A preparation method of a goose astrovirus bivalent yolk antibody is characterized by comprising the following steps:
(a) immunizing laying hens with the goose-star virus bivalent inactivated vaccine of claim 6;
(b) separating yolk from the high immune eggs obtained in step (a) and extracting antibodies.
10. The method for preparing the goose astrovirus bivalent inactivated vaccine as claimed in claim 9, wherein the specific method in the step (a) comprises the following steps:
basic immunity: injecting 0.5ml of goose astrovirus bivalent inactivated vaccine into breast muscles of each chicken;
and (3) secondary immunization: performing vaccination for the 2 nd time on 14 th day after the basic immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
three times of immunization: performing 3 rd inoculation on 14 th day after the second immunization, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
and (3) maintaining immunity: when the titer of the agar-agar positive antibody of the goose astrovirus type 1 and the goose astrovirus type 2 in the yolk reaches 1:96, maintaining inoculation for 1 time, and injecting 1.0ml of goose astrovirus bivalent inactivated vaccine into the breast muscle of each chicken;
egg collection: after 10 days of three times of intensified immunization, sampling and taking eggs, separating yolk, extracting antibodies, and collecting high-immunity eggs when the titer of the agaropectin antibody of the goose type 1 astrovirus and the goose type 2 astrovirus is greater than or equal to 1: 64;
the specific method of the step (b) is as follows:
adding the yolk liquid of the high-immunity eggs obtained in the step (a) into a pasteurization tank, adding water for injection, stirring and uniformly mixing, heating to 65 ℃, preserving heat and inactivating for 15 minutes, and cooling to below 30 ℃ in water bath after inactivation is finished;
adding injection water with the volume 4 times that of the original yolk into an acidification reaction tank, adjusting the pH value to 4.2 by using 1mol/L HCl solution, cooling the water to 2-4 ℃, then adding the inactivated yolk liquid while stirring, and keeping the temperature at 4-8 ℃ and standing for 4 hours;
freezing and centrifuging the acidified extract, removing precipitate, and taking supernatant for later use;
adding octanoic acid into the supernatant to a final concentration of 0.02%, stirring, mixing, standing at room temperature for 4 hours, filtering with K-type multi-layer plate frame for clarification, adding formaldehyde solution according to a final concentration of 0.05%, stirring, mixing, sealing for 60 minutes, and filtering with ultrafiltration membrane with cut-off molecular weight of 1000KD to remove viruses;
adding Tween-80 to a final concentration of 0.02%, adjusting pH to 6.8 with 1mol/L sodium hydroxide solution, filtering and sterilizing the qualified antibody with 0.22 μm microporous membrane to obtain refined yolk antibody of gosling gout.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280750A (en) * | 2020-10-22 | 2021-01-29 | 山东农业大学 | Novel goose astrovirus with cross-species transmission capability and application thereof |
| CN112641936A (en) * | 2021-01-05 | 2021-04-13 | 安徽农业大学 | Goose astrovirus spike protein liposome vaccine and preparation method and application thereof |
| CN112675299A (en) * | 2021-01-06 | 2021-04-20 | 黑龙江省百洲生物工程有限公司 | Preparation method of compound adjuvant inactivated vaccine for preventing and controlling goose astrovirus |
| CN116970574A (en) * | 2023-06-26 | 2023-10-31 | 广东省农业科学院动物卫生研究所 | Application of goose source waveform protein, method for promoting proliferation of goose astrovirus, application and vaccine |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105727277A (en) * | 2014-12-12 | 2016-07-06 | 普莱柯生物工程股份有限公司 | Method for preparation of swine pseudorabies virus vaccine and vaccine product |
| WO2016149089A1 (en) * | 2015-03-13 | 2016-09-22 | The Regents Of The University Of California | Subunit vaccine for prevention of astrovirus disease |
| CN109082415A (en) * | 2018-08-17 | 2018-12-25 | 山东信得科技股份有限公司 | A kind of novel goose astrovirus Strain and its application |
| CN109265540A (en) * | 2018-08-17 | 2019-01-25 | 山东信得科技股份有限公司 | A kind of preparation and its application of novel goose astrovirus Yolk antibody |
| CN110042085A (en) * | 2019-05-15 | 2019-07-23 | 青岛蔚蓝生物制品有限公司 | A kind of cultural method that 4 type aviadenovirus of I group suspends entirely |
| CN111110839A (en) * | 2020-01-06 | 2020-05-08 | 青岛易邦生物工程有限公司 | Goose astrovirus bivalent inactivated vaccine for preventing gosling gout |
-
2020
- 2020-08-17 CN CN202010827091.1A patent/CN111905100B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105727277A (en) * | 2014-12-12 | 2016-07-06 | 普莱柯生物工程股份有限公司 | Method for preparation of swine pseudorabies virus vaccine and vaccine product |
| WO2016149089A1 (en) * | 2015-03-13 | 2016-09-22 | The Regents Of The University Of California | Subunit vaccine for prevention of astrovirus disease |
| CN109082415A (en) * | 2018-08-17 | 2018-12-25 | 山东信得科技股份有限公司 | A kind of novel goose astrovirus Strain and its application |
| CN109265540A (en) * | 2018-08-17 | 2019-01-25 | 山东信得科技股份有限公司 | A kind of preparation and its application of novel goose astrovirus Yolk antibody |
| CN110042085A (en) * | 2019-05-15 | 2019-07-23 | 青岛蔚蓝生物制品有限公司 | A kind of cultural method that 4 type aviadenovirus of I group suspends entirely |
| CN111110839A (en) * | 2020-01-06 | 2020-05-08 | 青岛易邦生物工程有限公司 | Goose astrovirus bivalent inactivated vaccine for preventing gosling gout |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280750A (en) * | 2020-10-22 | 2021-01-29 | 山东农业大学 | Novel goose astrovirus with cross-species transmission capability and application thereof |
| CN112641936A (en) * | 2021-01-05 | 2021-04-13 | 安徽农业大学 | Goose astrovirus spike protein liposome vaccine and preparation method and application thereof |
| CN112641936B (en) * | 2021-01-05 | 2022-03-25 | 安徽农业大学 | Goose astrovirus spike protein liposome vaccine and preparation method and application thereof |
| CN112675299A (en) * | 2021-01-06 | 2021-04-20 | 黑龙江省百洲生物工程有限公司 | Preparation method of compound adjuvant inactivated vaccine for preventing and controlling goose astrovirus |
| CN112675299B (en) * | 2021-01-06 | 2023-10-20 | 黑龙江省百洲生物工程有限公司 | Preparation method of goose astrovirus prevention and control compound adjuvant inactivated vaccine |
| CN116970574A (en) * | 2023-06-26 | 2023-10-31 | 广东省农业科学院动物卫生研究所 | Application of goose source waveform protein, method for promoting proliferation of goose astrovirus, application and vaccine |
| CN116970574B (en) * | 2023-06-26 | 2024-02-09 | 广东省农业科学院动物卫生研究所 | Application of goose source waveform protein, method for promoting proliferation of goose astrovirus, application and vaccine |
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