CN111454977A

CN111454977A - Novel goose astrovirus composite vaccine and yolk antibody preparation method

Info

Publication number: CN111454977A
Application number: CN202010172345.0A
Authority: CN
Inventors: 王秀云; 王宏华; 焦绪娜; 张勇; 王辉; 刘磊; 李佳礼; 辛瑞祥
Original assignee: Weifang Huaying Biotechnology Co Ltd
Current assignee: Weifang Huaying Biotechnology Co Ltd
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-07-28
Anticipated expiration: 2040-03-12
Also published as: CN111454977B

Abstract

The invention aims to provide a novel goose astrovirus compound vaccine and a preparation method of a yolk antibody, namely, a recombinant PVAX1-Cap plasmid containing a novel goose astrovirus Cap protein gene and a novel goose astrovirus mCAP protein are mixed according to a certain proportion and then emulsified with a white oil adjuvant to prepare the compound vaccine, the average antibody titer of eggs collected 7-150 days after three-time immunization reaches more than 1:64, and the highest antibody titer can reach 1: 1024. The yolk antibody product prepared by extracting and purifying the hyperimmune egg can completely protect the goose group infected with the novel goose astrovirus. The novel goose astrovirus egg yolk antibody prepared by the method has the advantages of reliable effect, low cost and obvious economic and social benefits.

Description

Novel goose astrovirus composite vaccine and yolk antibody preparation method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel goose astrovirus compound vaccine and a preparation method of a yolk antibody.

Background

Astrovirus (AstV) is an unencapsulated, single-stranded, positive-sense RNA virus with a diameter of about 35 nm. The astrovirus comprises three Open Reading Frames (ORFs), wherein ORF1 encodes a nonstructural protein comprising a protease (ORF1a) and an RNA-dependent RNA polymerase (ORF1 b). ORF2 encodes the viral capsid protein (Cap protein, a hypervariable region in the genome, particularly the Splike region at the 3' end of ORF2, contains multiple neutralizing antibody epitopes of astrovirus, and is often used as a target protein for vaccine development.

Since 2017 in 2 months, an acute infectious disease mainly characterized by gout is outbreak in the main goose-raising areas of Shandong, Jiangsu, Anhui, Henan, Liaoning, Guangdong and the like in China. The disease mainly attacks goslings within 2 weeks of age, a large amount of urate is deposited on the surfaces of internal organs and in joint cavities of the goslings, the death rate can reach 50 percent at most, and huge economic loss is caused to the goose breeding industry in China. And then virus separation, identification and animal regression tests confirm that the goose astrovirus is infected with the novel goose astrovirus. As a new epidemic disease, the goose industry needs legal vaccine and special therapeutic medicine.

The yolk antibody product is often used as a specific medicine for the emergency prevention and treatment of epidemic diseases. In the preparation process of the yolk antibody, the immune antigen with outstanding immunogenicity is preferably selected, so that the cellular immunity and humoral immunity level of the laying hens are stimulated to the maximum extent, the high-immunity eggs with high antibody titer and long duration are obtained, and the preparation method has obvious economic value for reducing the production cost of the yolk antibody. Methods for preparing yolk antibodies of novel goose astrovirus are reported by Tang dynasty et al (patent application No. CN201810494058.4), Heng-Tai et al (patent application No. CN201810941620.3), Song dynasty et al (patent application No. CN201811315086.1), but the methods all adopt the novel goose astrovirus cultured by goose embryos as antigens for immunization, the virus production titer is low, other goose-origin pathogens are easily introduced, and the titer of the prepared high-immunity eggs is relatively low, thereby affecting the production efficiency and the product quality of the yolk antibodies.

Disclosure of Invention

The invention aims to provide a novel goose astrovirus compound vaccine and a preparation method of a yolk antibody. The novel goose astrovirus compound vaccine optimized by the preparation method can remarkably improve the titer of the novel goose astrovirus antibody in egg yolk after immunizing laying hens, the extracted and purified egg yolk antibody has stable property and high purity, the production cost of the novel goose astrovirus egg yolk antibody can be greatly reduced, and the novel goose astrovirus compound vaccine has great application value in preventing and treating novel goose astrovirus diseases.

According to the amino acid sequence of the goose astrovirus Cap protein in GenBank, a novel goose astrovirus Cap protein is designed, and the amino acid sequence is SEQ ID NO 1. Further designing and obtaining a novel goose astrovirus Cap protein gene according to codon usage preference, wherein one nucleotide sequence is SEQ ID NO. 2.

The recombinant PVAX1-Cap plasmid is prepared by carrying out enzyme digestion and connection on the obtained novel goose astrovirus Cap protein gene and a eukaryotic expression vector PVAX1, and the recombinant PVAX1-Cap plasmid is transformed into escherichia coli DH5 α and then is subjected to fermentation, DNA extraction and purification and other steps.

Selecting a Spike functional region of the 3' end of the Cap protein of the novel goose astrovirus, which is rich in neutralizing antibody epitopes, and naming the Spike functional region as the mCAP protein of the novel goose astrovirus, wherein the amino acid sequence of the protein is SEQ ID NO 3. Carrying out escherichia coli rare codon optimization on the novel goose star virus mCAP protein to obtain a novel goose star virus mCAP protein nucleotide sequence SEQ ID NO. 4.

Connecting the novel goose star virus mCAP protein gene with an expression vector pET28a, transforming an escherichia coli expression strain B L21 (DE3), and performing high-density fermentation, thallus crushing, protein purification and other steps to prepare the novel goose star virus mCAP protein in large scale.

The recombinant PVAX1-Cap plasmid was mixed with the novel goose-star virus mCAP protein to prepare an aqueous phase.

The optimal final concentration of the recombinant plasmid in the water phase is 20-100 mu g/ml, and the final concentration of the recombinant mCAP protein is 0.2-0.8 mg/ml.

More preferably, the final concentration of the recombinant plasmid in the aqueous phase is 60. mu.g/ml, and the final concentration of the recombinant mCAP protein is 0.5 mg/ml.

Emulsifying the water phase and the white oil adjuvant to prepare the novel goose astrovirus compound vaccine.

The high-immunity egg is subjected to the steps of yolk separation, inactivation, extraction, filtration, subpackaging and the like to prepare a finished yolk antibody product, wherein the final product has the antibody agar-agar titer of 1: 16.

The novel goose-star virus compound vaccine prepared by the invention is used for immunizing laying hens for three times, the antibody titer of eggs laid by 7 days after three-time immunization reaches 1:128, the highest antibody titer of eggs in the immunization period can reach 1:1024, the antibody titer of eggs still can reach 1:64 after 5 months of three-time immunization, and the high-immunity egg requirement is met. Compared with the conventional preparation method, the method can improve the titer of the high immunity egg antibody by more than 4 times, thereby greatly reducing the preparation cost of the novel goose astrovirus egg yolk antibody and having great popularization and application values. The prepared novel goose astrovirus egg yolk antibody is used for preventing or treating the novel goose astrovirus disease, has excellent effect, and completely protects the goose group injected with the antibody.

Detailed Description

The present invention is further described below with reference to specific embodiments, but it will be understood by those skilled in the art that modifications or substitutions in details and forms of the technical solution of the present invention may be made without departing from the technical solution of the present invention, and these modifications and substitutions fall within the scope of the present invention.

Example 1 construction and preparation of recombinant PVAX1-Cap plasmid

1 vector construction of recombinant PVAX1-Cap plasmid

1.1 carrying out homologous comparison on the amino acid sequences of the Cap proteins of the goose astrovirus in GenBank, introducing online MOSAIC vaccine design software to screen the Cap proteins covered by more potential 9-mer epitopes, and finally obtaining the novel Cap protein of the goose astrovirus, wherein the amino acid sequence is SEQ ID NO: 1.

1.2 according to the codon usage preference of chicken, a novel goose astrovirus Cap protein gene is designed and obtained, wherein one nucleotide sequence is SEQ ID NO. 2.

1.3 adding Kpn I and Xho I enzyme cutting sites to the two ends of the obtained novel goose astrovirus Cap protein gene respectively and then carrying out whole gene synthesis.

1.4 the synthesized novel goose-star virus Cap protein gene is double digested with Kpn I and Xho I and then ligated into the corresponding restriction site of PVAX1 vector, CaCl₂The method is used for transforming escherichia coli DH5 α, extracting plasmids for Kpn I and Xho I double enzyme digestion identification, sending to Shanghai worker for sequencing after the plasmids are correct, and a sequencing result shows that the novel goose astrovirus Cap protein gene is connected with the corresponding enzyme digestion site of the PVAX1 vector, so that the recombinant PVAX1-Cap plasmid is successfully constructed.

2 in vitro expression and identification of recombinant PVAX1-Cap plasmid

2.1 selecting PK-15 cells with good growth to inoculate a 6-hole cell culture plate, culturing the cells in a 5% carbon dioxide incubator at 37 ℃ until the cells have 85% -90% confluency, and carrying out recombinant PVAX1-Cap plasmid transfection.

2.2 transfection of cells Using L ipofectamine^TM2000 kit instructions, the steps are as follows:

2.2.1 Take 4. mu.g of recombinant PVAX1-Cap plasmid DNA, dilute to 250. mu.l with serum-free cell culture medium, mix gently.

2.2.2 mu.l of L ipo2000 was diluted to 250. mu.l with serum-free cell culture medium, gently mixed, and allowed to stand at room temperature for 5 min.

2.2.3 mixing the DNA suspension and L ipo2000 suspension, gently mixing, and standing at room temperature for 20 min.

2.2.4 discarding the culture medium in the 6-well plate with the well-grown PK-15 cells, washing the serum-free cell culture medium for 2 times, sucking the culture medium, adding the mixed solution, slightly mixing uniformly, supplementing 1ml of the serum-free cell culture medium, and culturing in a 5% CO2 incubator at 37 ℃.

2.2.5 after 6h of transfection, 6 well plates of cell fluid were discarded and 2ml of DMEM medium containing 10% newborn calf serum was added to each well.

2.2.6 PK-15 cells transfected with empty vector pVAX1 were used as negative controls.

2.3 after transfection for 48 hours, washing the cell plate after inoculation with PBS (phosphate buffer solution) with pH7.2 for 1 time, taking care of gentle movement to prevent cell shedding, fixing the cells with precooled methanol at 4 ℃ for 15-20 min, washing with PBS for 3 times, adding 100 mul of rabbit anti-goose-star virus positive serum, incubating for 1 hour at 37 ℃, washing with PBS for 3 times, adding 100 mul of FITC-labeled goat anti-rabbit secondary antibody diluted by 100 times, incubating for 45min in a dark place at 37 ℃, washing with PBS for 3 times, and observing the result by a fluorescence microscope.

As a result, an obvious fluorescent signal can be observed after the recombinant PVAX1-Cap plasmid is transfected into PK-15 cells, and no obvious fluorescent signal exists in a control group, so that the constructed recombinant PVAX1-Cap plasmid can correctly express the novel goose astrovirus Cap protein.

Large-scale preparation of 3 recombinant PVAX1-Cap plasmid

3.1 high-density fermentation of recombinant bacteria and thalli lysis of Escherichia coli DH5 α containing recombinant PVAX1-Cap plasmid DNA are subjected to high-density fermentation culture in a 50L fermentation tank, centrifuged at 5000r/min for 10min to collect thalli, added with Solution I in a proportion of 5ml per gram of wet thalli, added with Solution II and Solution III in a proportion of 1:2:1.5 respectively, incubated at room temperature for 15min, centrifuged at 10000r/min for 10min at room temperature, collected supernatant, added with 0.7 times volume of isopropanol, precipitated at-20 ℃ for 30min, centrifuged at 10000r/min for 10min at room temperature, discarded, and the precipitate is dissolved in 10 mmol/L TE buffer Solution.

3.2 removal of endotoxin purified plasmid DNA solution was added to 10% TritonX-114 to a final concentration of 1%, mixed and ice-cooled for 10min, then incubated at 42 ℃ for 10min, centrifuged at 10000r/min at room temperature for 10min, carefully pipetted supernatant into a pyrogen-free container, and the procedure was repeated 1 time if necessary.

3.3, quantitatively subpackaging, diluting to 480 mu g/ml by using 10 mmol/L TE buffer solution, and quantitatively subpackaging to obtain the recombinant PVAX1-Cap plasmid.

3.4 detection of recombinant PVAX1-Cap plasmid

3.4.1 concentration determination of recombinant PVAX1-Cap plasmid the plasmid concentration should not be less than 480. mu.g/ml, as determined by ultramicro nucleic acid analyzer.

3.4.2 restriction identification the purified recombinant PVAX1-Cap plasmid was double digested with Kpn I and Xho I and analyzed by agarose gel electrophoresis. As a result, two bands of about 3000bp and 2115bp in size were observed.

3.4.3 host protein assay Standard curves were prepared with known concentrations of BSA according to the BCA protein assay kit instructions. The purified recombinant PVAX1-Cap plasmid is diluted with sterile water in a gradient way, and the mycoprotein in the purified plasmid DNA is quantitatively detected under the same condition, and the mycoprotein content of the recombinant plasmid is lower than 10 mu g/mg.

3.4.4 endotoxin test was performed by limulus reagent method, and the endotoxin content should be lower than 1000 EU/mg.

Example 2 preparation of recombinant novel goose-star virus mCap protein

1 production strains

1.1 analyzing the amino acid sequence of the novel goose astrovirus Cap protein obtained in 1.1 of example 1 by using biological software DNAStar, selecting a Spike functional region (416-621aa) with the 3' end rich in neutralizing antibody epitope, and naming the protein as the novel goose astrovirus mCAP protein, wherein the amino acid sequence is SEQ ID NO: 3.

1.2 using online biological software DNAworks to optimize the rare codon of the novel goose astrovirus mCAP protein to obtain a nucleotide sequence SEQ ID NO. 4.

1.3 Nde I enzyme cutting sites and HindIII enzyme cutting sites are respectively added at two ends of a nucleotide sequence of the newly obtained novel goose star virus mCAP protein for full-gene synthesis.

1.4 the novel goose-star virus mCAP protein gene synthesized by the whole gene is double-digested by Nde I and Hind III and then connected with the corresponding restriction enzyme site of pET28a vector to construct expression vector.

1.5 with CaCl₂The expression vector is transformed into Escherichia coli B L21 (DE3), spread on an agar plate containing 50 mug/ml kanamycin and cultured overnight at 37 ℃, 10 single colonies are selected to extract plasmids, and colonies which are positive by Nde I and Hind III double enzyme digestion are selected for further sequencing and identificationAnd (3) fermenting and culturing the clone in an L B culture medium to 0.6-0.8, adding 0.2-0.5 mM IPTG (isopropyl-beta-thiogalactoside) for induction for 4-9 hours, centrifugally collecting thalli, ultrasonically crushing, centrifugally taking supernatant, detecting protein expression by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis, and simultaneously setting uninduced thalli as a control, wherein after the induction, a protein band is added at a position of 23kD compared with a control bacterium, the protein band is consistent with the theoretical molecular weight of recombinant protein, and the expression amount is about more than 20%.

1.6 the expression strain is broken, supernatant fluid is run for electrophoresis, and Western-blot identification is carried out by using goose astrovirus positive serum, and the result shows positive reaction.

The results prove that the obtained positive clone is a novel goose astrovirus mCAP protein engineering bacterium and is named as GS strain.

2 preparation and test of recombinant novel goose star virus mCAP protein

2.1 seed preparation for production

2.1.1 first-stage seed propagation the freeze-dried strain is respectively inoculated in L B liquid culture medium containing kanamycin, and is subjected to shaking culture at 37 ℃ for 8-10 hours, and then is subjected to streak inoculation on L B solid culture medium containing kanamycin and is subjected to culture at 37 ℃ for 16-18 hours to serve as first-stage seeds.

2.1.2 propagation of Secondary seeds 10 representative colonies were selected from the primary seeds, mixed in a small amount of L B medium, inoculated in L B medium containing kanamycin, cultured at 37 ℃ for 8-10 hours, and subjected to a pure test.

2.2 the fermentation medium is modified L B medium, each 1000ml of the medium contains tryptone 10g, yeast extract 5g, sodium chloride 10g, glucose 5g, MgSO 5₄·7H₂O 5g。

2.3 preparation of recombinant novel goose-star virus mCAP protein

2.3.1 aerobic culture in a culture tank for bacterial liquid culture, charging a proper amount of culture medium (about 70%) and antifoaming agent according to the volume of the culture tank, sterilizing, inoculating secondary seed bacterial liquid according to 1-10% of the culture medium, aerobic culture at 37 deg.C, and waiting for OD of the bacterial liquid₆₀₀When the value reaches 7.0, 2-10 g/L of α -lactose is added for induction, then the culture is continued for 6-8 hours, the pH is adjusted by using 20% NaOH, the dissolved oxygen is controlled by the association of the rotating speed, and when the dissolved oxygen rapidly rises, the feeding is carried out.

2.3.2 after the completion of the disruption culture, the cells were collected by centrifugation. The collected bacteria are cleaned and made into 1-10% suspension by PBS, and bacteria are crushed by a high-pressure homogenizer. The crushed bacterial liquid is centrifuged for 15 minutes at 8000r/min, and the supernatant is collected.

2.3.3 purifying the conventional nickel column by using a nickel column chromatography, putting the collected recombinant protein eluent into a dialysis bag, taking PBS (phosphate buffer solution) as dialysis external liquid, and dialyzing and desalting to obtain the recombinant protein liquid.

2.3.4 inactivation A10% formaldehyde solution was added to the purified supernatant in proportion to a final concentration of 0.2% and was inactivated at 37 ℃ for 12 hours.

2.3.5 quantitatively subpackaging, diluting the protein solution to a final concentration of 2.0mg/ml with sterile normal saline, and performing sterile filtration for later use.

2.4 testing

2.4.1 protein content assay the protein concentration of the supernatant was determined by BCA method and should not be less than 2.0 mg/ml.

2.4.2 sterility test the sterility test is carried out according to the current Chinese veterinary pharmacopoeia, and the sterility growth is required.

2.4.3 detection of endotoxin the endotoxin content should not be higher than 1000EU/ml, as determined by limulus reagent method.

2.4.4 the residual quantity of the formaldehyde and mercury preservatives is determined according to the current Chinese animal pharmacopoeia, and the determination is in accordance with the regulations.

Example 3 preparation and immunization of a novel goose-astrovirus composite vaccine

1 preparation of novel goose astrovirus compound vaccine

1.1 preparation of semi-finished product the recombinant PVAX1-Cap plasmid DNA prepared in 3.3 of example 1 and the recombinant new goose star virus mCAP protein prepared in 2.3.5 of example 2 were diluted appropriately to make the final content of plasmid DNA 60 μ g/ml and the final content of mCAP protein 0.5mg/ml, and fully mixed to obtain a semi-finished product.

1.2 vaccine preparation

1.2.1 preparing oil phase 94 parts of high-quality white oil for injection and 2 parts of aluminum stearate. And (2) uniformly mixing in an oil phase tank, heating to melt the mixture to be semitransparent, adding 806 parts of span, keeping the temperature for 30 minutes when the temperature reaches 125-130 ℃, and cooling to room temperature for later use.

1.2.2 preparing water phase by adding 96 parts of qualified semi-finished product into 804 parts of sterilized Tween-80, and stirring until Tween-80 is completely dissolved.

1.2.3 emulsifying, taking 2 parts of oil phase, placing in a high-speed shearing machine, starting a motor to stir at a low speed, slowly adding 1 part of water phase, emulsifying at 3600r/min for 40 minutes, adding 1% thimerosal solution before stopping stirring, and enabling the final concentration to reach 0.01%. After emulsification, 10ml of the sample is added into a centrifuge tube and centrifuged at 3000r/min for 15 minutes, and an anhydrous phase is separated out at the bottom of the tube.

1.2.4 subpackaging and quantitatively subpackaging, and sealing the bottle mouth.

2 inspection of novel goose astrovirus composite vaccine

2.1 physical Properties

The appearance was a milky white emulsion.

The dosage form is water-in-oil type. A clean pipette is taken, a small amount of vaccine is sucked and dropped into cold water, and the vaccine is not diffused except for the 1 st drop.

Adding 10ml of the stable suction vaccine into a centrifuge tube, centrifuging for 15 minutes at 3000r/min, and separating out an anhydrous phase at the bottom of the tube.

The viscosity is carried out according to the current Chinese animal pharmacopoeia and conforms to the regulations.

The inspection of the loading amount is carried out according to the current Chinese animal pharmacopoeia, which conforms to the regulations.

2.2 sterile test is carried out according to the current Chinese veterinary pharmacopoeia, and the bacteria-free growth is carried out.

2.3 the measurement of the residual quantity of the formaldehyde and the mercury preservatives is carried out according to the current Chinese animal pharmacopoeia and meets the regulations.

3 research of novel goose-star virus composite vaccine on immunization program of laying hens

80 laying hens of 120 days old are selected and averagely divided into 4 groups, each group comprises 20 DNA vaccine groups, the recombinant PVAX1-Cap plasmid prepared in example 1 is immunized by the DNA vaccine groups, the recombinant PVAX1-Cap plasmid is diluted to 10 mu g/ml by 10 mmol/L TE buffer solution, the subunit vaccine groups are used for immunizing a recombinant novel goose astrovirus subunit vaccine, the recombinant novel goose astrovirus mCAP protein prepared in example 2 is emulsified with white oil adjuvant according to a ratio of 1:2, wherein the final concentration of the Cap protein in an aqueous phase is 0.5mg/ml, the novel goose astrovirus compound vaccine prepared in example 3 is immunized by the compound vaccine groups, physiological saline with the same volume is injected into an immunization control group, the corresponding vaccine immunization and subcutaneous injection are respectively carried out on each group of laying hens for a duration, 0.5 ml/egg, the immunization interval is 14 days, three times of immunization are carried out, eggs are respectively collected before immunization, before three times of immunization (14 days after two times of immunization), 7, 14, 21, 28 days after three times of immunization, the egg yolk antibody amplification is collected every 30 days, and the yolk antibody titer and the yolk antibody and yolk antibody generation period is determined.

As a result: after the novel goose-star virus composite vaccine is used for immunizing laying hens, the 7-day agaropectin antibody titer after three-immunization can reach 1:128, which is higher than the conventional egg-receiving standard (not lower than 1:64) (see table 1) and can reach 1:1024 at most, and the egg yolk antibody titer still meets the requirement when the detection is carried out 5 months after three-immunization (see table 2). The results show that the composite vaccine group is far superior to the DNA vaccine group and the subunit vaccine group no matter the yolk antibody production time, the antibody production height or the antibody duration, and shows good application prospect.

TABLE 1 yolk antibody production period of egg-laying hens immunized by each vaccine group

TABLE 2 egg yolk antibody duration of the hens immunized by each vaccine group

Example 4 preparation and potency assay of novel goose-astrovirus yolk antibody

1 preparation of novel goose astrovirus egg yolk antibody

Preparing hyperimmune eggs by immunizing laying hens with the novel goose-star virus compound vaccine according to the method in the part 3 in the example 3, collecting qualified hyperimmune eggs (the antibody titer is not less than 1:64), sterilizing egg shells, and then carrying out methods such as yolk separation, extraction, inactivation, filtration sterilization, dilution and split charging, wherein the antibody titer of a final product is not less than 1: 16.

2 safety experiment of novel goose astrovirus egg yolk antibody

20 healthy and susceptible goslings of 1 day age are divided into two groups on average, and each group comprises 10 goslings. The first group was injected subcutaneously with 1.0 ml/egg yolk antibody prepared in the present invention, and the second group was injected intramuscularly with the same volume of physiological saline. Goose groups are observed 21 days after immunization, and all goose groups are healthy and alive without any adverse reaction.

3 potency assay for novel goose-star Virus yolk antibodies

The 2-day-old healthy susceptible goslings 60 are divided into three groups of 20 average. The first group is a yolk antibody treatment group, 0.5ml of the novel goose astrovirus virulent GV strain is injected intramuscularly, and 0.5 ml/egg yolk antibody of the novel goose astrovirus prepared by the invention is injected intramuscularly after 24 hours. The second group is a yolk antibody and prevention group, 0.5ml of the novel goose astrovirus yolk antibody prepared by the invention is injected intramuscularly, and 0.5ml of the novel goose astrovirus virulent GV strain is injected intramuscularly after 24 h. The third group is a normal saline control group, 0.5ml of the novel goose astrovirus virulent GV strain is injected intramuscularly, and 0.5ml of sterile normal saline is injected intramuscularly after 24 hours. The treatment effect is counted 14 days after the toxic materials are attacked. Wherein the pathogenesis standard is as follows: death, enlarged kidney and spleen, white spots on the liver, white capsule on the pericardium and cheese-like urate deposition in joints were observed in individual geese.

The goose groups in the egg yolk antibody prevention group and the egg yolk antibody treatment group are 100% protected, and the goose groups in the normal saline control group are 90% attacked. The novel goose astrovirus egg yolk antibody prepared by the invention has good clinical protection effect on goose groups infected with the novel goose astrovirus, and can be used for preventing and treating the novel goose astrovirus.

TABLE 3 potency assay for novel goose-astrovirus egg yolk antibodies

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tctccaactg actctttggg tccagctatc aacatgtgga cttacttgaa gactgttaac 660

gctttgtctg ctagagctca agctcaacaa gttccataca cttctgcttt gttcttggtt 720

gaagctactg ttacttacga attctctaac tacggtccaa agccaggttt gtctttgatg 780

acttctgaaa ctttgtctgc ttctggtaag actgctactt tggttaacac tcaagacggt 840

gctttggctt tgactgtttc tggtgctttg caaagattct tggacgaaaa ggaacaacac 900

agaagagttt ctaacgctca aacttctggt gttggtgaag ttttctgggc tgtttctact 960

gaagttgttg aaactgttgc ttctgctttg ggtggttggg gttggttgtt gaagggtggt 1020

tggttcgtta tcagaaagtt gttcggtgct gcttctaact ctggttctac ttacttgatc 1080

tactcttctg tttctgacgc tcaaatcgac tctagaatct accaaactgt tccaccaaac 1140

actccattgc aattggctgc taacactgtt aagttggttc aattgactca accaaacgtt 1200

aacactactg gtcaaggtac tactgttttg tctagagacg ctgactactt gccattgcca 1260

gttgctccaa tgcaagttac tccatctttg gtttacaact tccaaggtga aagacaatct 1320

actactgaat cttgttcttt cttggttttc ggtatcccac aagctgaatc tagatctaga 1380

tacaacgctg ctatcacttt caacgttggt tacagaggta gaacttctac ttctttcact 1440

ttgggtactc acaactggtg ggctgttatg actttgtctc aaactggtgt tatcttcgct 1500

ccaccagctg ttggtactgg tgtttgtaac actttggcta ctgctatcca acacttgaac 1560

ccagaattgg aaactgctgt tttgagagtt aacacttcta ctacttctac tggtggtttg 1620

atcactgaat tgagaaacag attgaacatc gctgacggtg actacgttat ctctatgggt 1680

gacccacaag gtaacagatc tgctttgtac ttcagaaact ctgaccaaaa gtgggtttgg 1740

ttgtgggctg gtgactctaa cccaggtgaa actttccaaa acttcaagat gccagttttg 1800

atcaactggt ctgtttctga ctctcaagaa caatacaacg ctagagttag aatggttcaa 1860

tacgctaacg ctcaacaaca aatcttgact gacccagaag aagacgacga cccattgtct 1920

gacgttactt ctttgttcga cccaactgct gaagacgaaa ctgacttcca cttggctgtt 1980

tctttgaaga cttctgacta cttgaaggaa gaagctgaat actggaaggc taaggctcaa 2040

gctttgttga tggaaaaggc tttgtctgct ccacaagctg gtgctgttag attcgaaaag 2100

ggtggtcacg aa 2112

<210>3

<211>206

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Tyr Leu Pro Leu Pro Val Ala Pro Met Gln Val Thr Pro Ser Leu Val

1 5 10 15

Tyr Asn Phe Gln Gly Glu Arg Gln Ser Thr Thr Glu Ser Cys Ser Phe

20 25 30

Leu Val Phe Gly Ile Pro Gln Ala Glu Ser Arg Ser Arg Tyr Asn Ala

35 40 45

Ala Ile Thr Phe Asn Val Gly Tyr Arg Gly Arg Thr Ser Thr Ser Phe

50 55 60

Thr Leu Gly Thr His Asn Trp Trp Ala Val Met Thr Leu Ser Gln Thr

65 70 75 80

Gly Val Ile Phe Ala Pro Pro Ala Val Gly Thr Gly Val Cys Asn Thr

85 90 95

Leu Ala Thr Ala Ile Gln His Leu Asn Pro Glu Leu Glu Thr Ala Val

100 105 110

Leu Arg Val Asn Thr Ser Thr Thr Ser Thr Gly Gly Leu Ile Thr Glu

115 120 125

Leu Arg Asn Arg Leu Asn Ile Ala Asp Gly Asp Tyr Val Ile Ser Met

130 135 140

Gly Asp Pro Gln Gly Asn Arg Ser Ala Leu Tyr Phe Arg Asn Ser Asp

145 150 155 160

Gln Lys Trp Val Trp Leu Trp Ala Gly Asp Ser Asn Pro Gly Glu Thr

165 170 175

Phe Gln Asn Phe Lys Met Pro Val Leu Ile Asn Trp Ser Val Ser Asp

180 185 190

Ser Gln Glu Gln Tyr Asn Ala Arg Val Arg Met Val Gln Tyr

195 200 205

<210>4

<211>618

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

tacctgccgc tgccagttgc gccgatgcag gttaccccgt ctctggttta caacttccag 60

ggtgagcgtc agtctaccac cgaatcttgc tctttcctgg ttttcggtat ccctcaggcg 120

gaatctcgtt ctcgttacaa tgcggcgatc accttcaatg ttggttaccg tggccgtact 180

tctacttctt tcaccctcgg tacccataat tggtgggcgg ttatgaccct gtctcagacc 240

ggtgttatct tcgcaccgcc tgcggttggc accggcgttt gcaacactct ggcgaccgcg 300

atccagcacc tgaacccgga actggaaacc gctgtcctgc gtgttaatac cagcacgacc 360

tctaccggtg gtctgattac cgaactgcgt aaccgtctga acatcgcgga cggtgattac 420

gttatctcta tgggtgaccc acagggtaat cgttctgcgc tgtacttccg taactctgac 480

cagaaatggg tttggctgtg ggccggtgac tctaacccag gtgaaacctt ccagaacttc 540

aaaatgccgg tactgatcaa ttggtctgtt tctgactctc aggaacaata caacgcacgt 600

gtccgtatgg tacagtac 618

Claims

1. A recombinant PVAX1-Cap plasmid is formed by enzyme digestion connection of a novel goose astrovirus Cap protein gene and a eukaryotic expression vector PVAX1, wherein the amino acid sequence of the coding protein of the novel goose astrovirus Cap protein is SEQ ID NO. 1, and one nucleotide sequence of the coding protein of the novel goose astrovirus Cap protein is SEQ ID NO. 2.

2. The method for mass production of recombinant PVAX1-Cap plasmid according to claim 1, wherein the recombinant PVAX1-Cap plasmid is transformed into Escherichia coli DH5 α, and then the recombinant PVAX 3578-Cap plasmid is prepared by high-density fermentation of Escherichia coli, lysis of bacterial cells, extraction and purification of plasmid DNA.

3. A novel goose astrovirus compound vaccine is prepared by mixing an aqueous phase and an adjuvant, wherein an antigen in the aqueous phase is formed by mixing the recombinant PVAX1-Cap plasmid of claims 1-2 and the recombinant novel goose astrovirus mCAP protein, the amino acid sequence of the protein coded by the recombinant novel goose astrovirus mCAP protein is SEQ ID NO. 3, and one nucleotide sequence of the protein coded by the recombinant novel goose astrovirus mCAP protein is SEQ ID NO. 4.

4. The composite vaccine of claim 3, wherein the final concentration of the recombinant plasmid in the aqueous phase is 20-100 μ g/ml, and the final concentration of the recombinant mCAP protein is 0.2-0.8 mg/ml.

5. The novel goose astrovirus composite vaccine as claimed in claim 4, wherein the final concentration of the recombinant plasmid in the aqueous phase is 60 μ g/ml, and the final concentration of the recombinant mCAP protein is 0.5 mg/ml.

6. The combination vaccine of claim 3, wherein the adjuvant is white oil.

7. The combination vaccine of claim 3, wherein the aqueous phase is emulsified with an adjuvant.

8. A method for preparing a novel goose-star virus yolk antibody, which is obtained by immunizing laying hens with the compound vaccine of claims 3-7 and then extracting and purifying the yolk of a hyperimmune egg.