CN111388662B - Composite vaccine of gosling plague virus and preparation method of egg yolk antibody - Google Patents
Composite vaccine of gosling plague virus and preparation method of egg yolk antibody Download PDFInfo
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Abstract
The invention aims to provide a gosling plague virus composite vaccine and a preparation method of a yolk antibody, namely, the recombinant PVAX1-VP2 plasmid containing the VP2 gene of gosling plague virus and the VP3 protein of gosling plague virus are mixed according to a certain proportion and then emulsified with a white oil adjuvant to prepare the composite vaccine, the average antibody titer of eggs collected 7-120 days after three-immunization reaches more than 64, the highest antibody titer can reach 1 512, and the immunization duration can reach more than 4 months. The yolk antibody product prepared by extracting and purifying the hyperimmune eggs can completely protect goose groups infected with gosling plague virus. The gosling plague virus egg yolk antibody prepared by the method has the advantages of definite effect, low cost and obvious economic and social benefits.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a composite vaccine of gosling plague virus and a preparation method of a yolk antibody.
Background
Gosling plague is one of several common diseases causing death of goslings and young muscovy ducks, and has great threat to the development of the waterfowl breeding industry. The fatality rate of the disease to susceptible groups reaches up to 70%, and the common morbidity rate and mortality rate can reach up to more than 90%, even 100%. Therefore, the development of targeted gosling plague virus vaccines and antibody therapeutic preparations has urgent market needs. So far, gene sequence comparison shows that GPV viruses separated from all over the world have little difference, the gene sequences are almost the same and only have one serotype, and the possibility is provided for developing universal gosling plague virus vaccines and antibody preparations.
The gosling plague virus genome is a single strand DNA, mainly comprising two Open Reading Frames (ORFs), the right ORF encodes a structural protein VP, which is divided into VP1, VP2 and VP3, and the three proteins have different initiation codon sites, but share the same termination codon TAA. The VP2 protein is one of the surface antigens of gosling plague virus, can spontaneously form virus-like particles (VLPS), has good antigenicity, can stimulate the organism to generate neutralizing antibodies, and is an important candidate protein for preparing genetic engineering vaccines. The VP3 protein is a main capsid protein, accounts for about 80% of the whole virus capsid protein, is also a main component of an antigenic determinant, is a main protective antigen, can stimulate an organism to generate a neutralizing antibody, plays an important role in the diagnosis and prevention of gosling plague virus, and is a primary protective antigen for preparing genetic engineering vaccines. At present, a plurality of documents report in vitro expression of goose pestivirus VP2 protein and VP3 protein, such as Zhao Wen and the like (prokaryotic expression of goose pestivirus VP3 gene, chinese veterinary magazine, 2012, 48 (3): 29-31) realize expression of VP3 protein in escherichia coli, chengxing and the like (establishment of VP3-ELISA diagnostic method of goose parvovirus VP3 protein expressed based on insect cells, chinese veterinary science 2019, 49 (9): 1112-1118) realize expression of VP3 protein in baculovirus, and Kokungjust and the like (a method for preparing goose pestivirus-like particles by using an escherichia coli system, patent application number: CN 201710049003.8) realize escherichia coli soluble expression of VP2 protein and spontaneous assembly of virus-like particles. Zhu Dekang (comparison of goose pestivirus VP3 eukaryotic expression plasmid and attenuated vaccine induced goose immune response, chinese agricultural science 2011, 44 (3): 595-603) connects VP3 protein with eukaryotic expression vector pcDNA3.1 and then serves as gene vaccine to immunize white geese of 30 days old, and the result shows that the immunity response capability of goose groups is higher compared with that of gosling pestivirus vaccine.
The gosling plague yolk antibody can be used as a specific medicine for the emergency prevention and treatment of 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.
Disclosure of Invention
The invention aims to provide a composite vaccine of gosling plague virus and a preparation method of a yolk antibody. The gosling plague virus compound vaccine optimized by the preparation method can obviously improve the titer of the gosling plague virus antibody in egg yolk after immunizing laying hens, the extracted and purified egg yolk antibody has stable property and high purity, can greatly reduce the production cost of the gosling plague virus egg yolk antibody, and has great application value for preventing and treating gosling plague virus.
In order to realize the purpose, the invention adopts the following technical scheme:
synthesizing VP2 gene of gosling plague virus ZJ strain, and preparing a recombinant PVAX1-VP2 plasmid by enzyme digestion and connection of the synthesized gene and a eukaryotic expression vector PVAX 1. After the recombinant plasmid is transformed into Escherichia coli DH5 alpha, the recombinant plasmid can be prepared in large scale through steps of fermentation, extraction, purification and the like.
The nucleotide sequence of the VP3 gene of the gosling plague virus is shown in SEQ ID NO. 1.
The synthesized gosling plague virus VP3 gene is connected with an expression vector pET28a, and is transformed into an escherichia coli expression strain BL21 (DE 3) to be subjected to high-density fermentation, thallus crushing, protein purification and the like, so as to prepare the recombinant gosling plague virus VP3 protein.
The recombinant PVAX1-VP2 plasmid and the recombinant gosling plague virus VP3 protein are mixed to prepare a water phase, and the final concentration of the recombinant plasmid in the water phase is preferably 10 to 100 mu g/ml, and the final concentration of the recombinant gosling plague virus VP3 protein is preferably 0.2 to 0.8mg/ml.
More preferably, the final concentration of the recombinant PVAX1-VP2 plasmid in the aqueous phase is 50. Mu.g/ml, and the final concentration of the recombinant gosling plague virus VP3 protein is 0.4mg/ml.
Emulsifying the water phase and the white oil adjuvant to prepare the gosling plague virus compound vaccine. The compound vaccine is used for immunizing laying hens for three times, the antibody titer of eggs laid 7 days after three-time immunization reaches 1. The production process of separating, inactivating, extracting, filtering, subpackaging and the like is carried out on the high-immunity eggs to prepare finished egg yolk antibody products, wherein the antibody titer of the final products reaches 1.
The invention firstly prepares a recombinant PVAX1-VP2 plasmid which is formed by connecting a goose pestivirus VP2 gene with a eukaryotic expression vector PVAX1 through enzyme digestion. The recombinant PVAX1-VP2 plasmid can be prepared in large scale by methods such as fermentation, extraction and purification after being transformed into Escherichia coli DH5 alpha. The recombinant gosling plague virus VP3 protein production strain is prepared by methods of high-density fermentation, thallus crushing, protein purification and the like. The recombinant PVAX1-VP2 plasmid and the recombinant gosling plague virus VP3 protein are mixed to prepare a water phase. Emulsifying the water phase and the white oil adjuvant to prepare the gosling plague virus compound vaccine.
After the prepared gosling plague virus compound vaccine is used for immunizing laying hens, the egg-receiving requirement (not less than 64) can be reached 7 days after the three-immunization, the maximum value can reach 1. The gosling of the gosling plague virus prevention and treatment group has 100% protection on strong virus attack, and 80% of goslings of the normal saline control group have diseases, so that the egg yolk antibody prepared by the invention has good clinical protection effect on goose groups infected with gosling plague virus, and can be clinically popularized and applied.
Compared with the conventional preparation method, the preparation method of the gosling plague virus yolk antibody of the invention has the advantages that the gosling plague virus compound vaccine is obviously superior to a DNA vaccine only containing VP2 gene, a subunit vaccine only containing VP3 protein and a gosling plague complete virus vaccine in the aspects of yolk antibody generation time, antibody generation height and antibody duration. The implementation of the invention can reduce the preparation cost of the egg yolk antibody on one hand, and can reduce the immune stress of the laying hens on the other hand, thereby having great popularization and application values.
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-VP2 plasmid
Vector construction of 1 recombinant PVAX1-VP2 plasmid
1.1 according to the VP2 gene sequence of gosling plague virus ZJ strain (GenBank accession: KR 265071), adding Kpn I and Xho I enzyme cutting sites on both ends for complete gene synthesis.
1.2 the synthesized gosling plague virus VP2 gene is double digested with Kpn I and Xho I and then ligated into the corresponding restriction sites of PVAX1 vector, caCl 2 The method transforms Escherichia coli DH5 alpha, extracts plasmids to carry out Kpn I and Xho I double enzyme digestion identification, sends the plasmids to Shanghai worker for sequencing after the plasmids are correct, and a sequencing result shows that the goose pestivirus VP2 gene is successfully connected with the corresponding enzyme digestion site of the PVAX1 vector, and the recombinant PVAX1-VP2 plasmid is successfully constructed.
2 in vitro expression and identification of recombinant PVAX1-VP2 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-VP2 plasmid transfection.
2.2 transfection of cells by Lipofectamine TM 2000 kit instructions, the steps are as follows:
2.2.1 mu.g of recombinant PVAX1-VP2 plasmid DNA was diluted to 250. Mu.l with serum-free cell culture medium and gently mixed.
2.2.2 mu.l of Lipo2000 was diluted to 250. Mu.l with serum-free cell culture medium, gently mixed, and allowed to stand at room temperature for 5min.
2.2.3 mixing the DNA suspension and the Lipo2000 suspension, gently mixing, and standing at room temperature for 20min.
2.2.4 discard the culture medium from the 6-well plate with well grown PK-15 cells, wash the serum-free cell culture medium 2 times, aspirate the culture medium, add the above mixture, mix gently, add 1ml of serum-free cell culture medium, and culture 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 pVAX1 empty vector were used as negative controls.
2.3 transfection for 48 hours, washing the cell plate after virus inoculation with PBS (pH7.2) for 1 time, taking care of gentle movement to prevent cell shedding, fixing the cells with precooled methanol at 4 ℃ for 15-20min, washing with PBS for 3 times, adding 100 mul of rabbit anti-gosling plague positive serum, incubating for 1h 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-VP2 plasmid is transfected into PK-15 cells, and no obvious fluorescent signal exists in a control group, so that the constructed recombinant PVAX1-VP2 plasmid can be used for correctly expressing the gosling plague VP2 protein.
Large-scale preparation of 3 recombinant PVAX1-VP2 plasmid
3.1 high-density fermentation of recombinant bacteria and thalli lysis of Escherichia coli DH5 alpha containing recombinant PVAX1-VP2 plasmid DNA, after high-density fermentation culture in a 50L fermentation tank, centrifuging at 5000r/min for 10min to collect thalli, adding Solution I according to the proportion of 5ml per gram of wet bacteria, adding Solution II and Solution III according to the proportion of 1.
3.2 removal of endotoxin the 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, the supernatant carefully aspirated into a pyrogen-free container, and the procedure was repeated 1 time as necessary.
3.3 quantitative subpackaging, diluting to 500 mu g/ml with 10mmol/L TE buffer solution, and quantitatively subpackaging to obtain the recombinant PVAX1-VP2 plasmid.
3.4 detection of recombinant PVAX1-VP2 plasmid
3.4.1 concentration determination of recombinant PVAX1-VP2 plasmid the plasmid concentration should not be less than 500. Mu.g/ml, as determined by a picornascope.
8978 enzyme digestion with zxft 8978 identification the purified recombinant PVAX1-VP2 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 1764bp 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-VP2 plasmid is subjected to gradient dilution by using sterile water, and the mycoprotein in the purified plasmid DNA is quantitatively detected under the same condition, wherein 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 1000EU/mg.
Example 2
Preparation of recombinant gosling plague virus VP3 protein
1 production strains
1.1A nucleotide sequence SEQ ID NO 1 is obtained by optimizing the rare codon of the goose pestivirus VP3 protein by using online biological software DNAworks according to the VP3 protein amino acid sequence of goose pestivirus ZJ strain (GenBank accession number: KR 265071).
1.2 will obtainThe gosling plague virus VP3GeneNde I and Hind III enzyme cutting sites are added at two ends of the sequence respectively for full gene synthesis.
1.3 the goose pestivirus VP3 gene synthesized by whole gene is double digested by Nde I and Hind III, then linked with the corresponding restriction enzyme cutting site of pET28a vector, to construct expression vector.
1.4 with CaCl 2 The expression vector was transformed into E.coli BL21 (DE 3), spread on an agar plate containing 50. Mu.g/ml kanamycin, and cultured overnight at 37 ℃. 10 single colonies are selected to extract plasmids, and Nde I and Hind III colonies which are double-enzyme-cut and verified to be positive are further sequenced and identified. Fermenting and culturing the positive clone after sequencing verification in an LB culture medium to 0.6-0.8, adding 0.2-0.5 mM IPTG to induce for 4-9 hours, centrifugally collecting thalli, then ultrasonically crushing, centrifugally taking supernatant, detecting protein expression by SDS-PAGE electrophoresis, and simultaneously setting uninduced thalli as a control. After the induction, a protein band is added to the positive clone at the position of 60kD compared with the control bacterium, the molecular weight is consistent with the theoretical molecular weight of the recombinant protein, and the expression quantity 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 gosling plague virus positive serum, and the result shows positive reaction.
The results prove that the obtained positive clone is a gosling plague virus VP3 protein engineering bacterium which is named as a GX strain.
2 preparation and test of recombinant gosling plague virus VP3 protein
2.1 seed preparation for production
10 typical colonies were selected and mixed in a small amount of LB medium, inoculated in LB medium containing kanamycin, and cultured at 37 ℃ for 8 to 10 hours to give a seed solution.
2.2 the fermentation medium is an improved LB medium, each 1000ml of which contains tryptone 10g, yeast extract 5g, sodium chloride 10g, glucose 5g, mgSO 5 4 ·7H 2 O 5g。
2.3 preparation of recombinant gosling plague virus VP3 protein
2.3.1 aerobic culture in a culture tank, filling a proper amount of culture medium (about 70%) and defoaming agent in the culture tank, sterilizing, and inoculating seed bacteria in an amount of 2% of the culture mediumCulturing at 37 deg.C until OD of bacterial liquid is reached 600 When the value reaches 7.0, 2-10 g/L alpha-lactose is added for induction, and then the culture is continued for 6-8 hours. And controlling dissolved oxygen through rotation speed correlation. When the dissolved oxygen rapidly rises, the feeding material is fed.
2.3.2 after the completion of the disruption culture, the cells were collected by centrifugation. The collected bacteria are washed and then broken by a high-pressure homogenizer. And centrifuging the crushed bacterial liquid at 8000r/min for 15 minutes, and collecting the crushed precipitate.
2.3.3 Inclusion body washing Inclusion bodies were washed with 2M urea solution for 30min, and the precipitate was collected by centrifugation.
2.3.4 denaturation of Inclusion bodies the denaturation liquid was added in a proportion of 1g inclusion bodies to 10ml denaturation liquid (8M urea), stirred at room temperature for 30min, centrifuged and the supernatant collected.
2.3.5 purifying the conventional nickel column by nickel column chromatography, performing on-column renaturation recombinant protein by adopting continuous gradient urea concentration, putting 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.
2.3.6 inactivation the purified supernatant was added proportionally to a 10% formaldehyde solution with a final concentration of 0.2% and inactivated at 37 ℃ for 12 hours.
2.3.7 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 2.0mg/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 gosling plague virus composite vaccine
1 preparation of gosling plague virus composite vaccine
1.1 preparation of semi-finished product the recombinant PVAX1-VP2 plasmid DNA prepared in 3.3 of example 1 and the recombinant CSFV VP3 protein prepared in 2.3.7 of example 2 were diluted appropriately so that the final content of plasmid DNA was 50. Mu.g/ml and the final content of VP3 protein was 0.4mg/ml, and mixed well 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. Mixing the components evenly in an oil phase tank, heating and melting the components to be semitransparent, then adding span-80 parts, keeping the temperature for 30 minutes when the temperature reaches 125-130 ℃, and cooling the mixture to room temperature for later use.
1.2.2 preparing water phase by taking 4 parts of sterilized Tween-80, adding 96 parts of qualified semi-finished product, and fully stirring until the Tween-80 is completely dissolved.
1.2.3 emulsifying, namely putting 2 parts of oil phase into a high-speed shearing machine, starting a motor to stir at a low speed, slowly adding 1 part of water phase, emulsifying for 40 minutes at 3600r/min, and adding 1% thimerosal solution before stopping stirring until the final concentration reaches 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 gosling plague virus 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 residual quantity of the formaldehyde and mercury preservatives is determined according to the current Chinese animal pharmacopoeia and conforms to the regulations.
3 gosling plague virus compound vaccine immune program research for laying hens
100 laying hens aged 180 days are selected and averagely divided into 5 groups, and each group comprises 20 laying hens. DNA vaccine group the recombinant PVAX1-VP2 plasmid prepared in example 1 was immunized and diluted to 10. Mu.g/ml with 10mmol/L TE buffer; the subunit vaccine group immunizes the recombinant gosling plague virus subunit vaccine, which is prepared by emulsifying the recombinant VP3 protein prepared in the example 2 and a white oil adjuvant according to the proportion of 1:2, wherein the final concentration of the VP3 protein in a water phase is 0.4mg/ml; composite vaccine group immunizes the composite vaccine prepared in example 3; the whole virus vaccine group is a first-immunity inactivated vaccine prepared by using a commercial gosling plague live vaccine, and a second-immunity third-immunity inactivated vaccine prepared by inactivating a gosling plague virus GX strain and emulsifying the inactivated vaccine with a white oil adjuvant according to the proportion of 1:2; the immunization control group was injected with the same volume of physiological saline. The corresponding vaccine immunization and subcutaneous injection are respectively carried out on each group of laying hens, 0.5ml is injected per egg, and the immunization interval is 14 days, and three times of immunization are carried out. Eggs are collected before immunization, before three-immunization (14 days after two-immunization) and 7, 14, 21 and 28 days after three-immunization respectively to determine the egg yolk agarose antibody titer, and then high-immunity layer eggs are collected every 30 days to determine the egg yolk antibody titer, so that the egg yolk antibody production period and the egg yolk antibody duration of the layer are determined.
After the prepared gosling plague virus compound vaccine is used for immunizing laying hens, the antibody titer can reach 1. The results show that the composite vaccine group is far superior to a DNA vaccine group, a subunit vaccine group and a whole virus vaccine group no matter the egg 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 efficacy experiment of goose plague virus egg yolk antibody
1 preparation of gosling plague virus egg yolk antibody
Preparing hyperimmune eggs by immunizing laying hens with the gosling plague 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.
2 safety experiment of gosling plague virus yolk antibody
20 healthy susceptible goslings of 2 days old are averagely divided into two groups of 10 goslings. The first group of legs are injected with 1.0ml of the yolk antibody prepared by the invention per egg, and the second group of legs are injected 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 efficacy test of yolk antibody of gosling plague virus
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 gosling plague virulent GX strain is injected intramuscularly, and 0.5 ml/yolk antibody of gosling plague virus prepared by the invention is injected intramuscularly after 24h. The second group is a yolk antibody and a prevention group, 0.5ml of the yolk antibody of gosling plague virus prepared by the invention is injected intramuscularly, and 0.5ml of gosling plague virulent GX strain is injected intramuscularly after 24 hours. The third group is a normal saline control group, 0.5ml of gosling plague virulent GX strain is injected intramuscularly, and 0.5ml of sterile normal saline is injected intramuscularly after 24h. Continuously observing for 10 days after virus attack, and taking death of goslings as a disease onset judgment standard of gosling plague viruses.
The goslings of the egg yolk antibody prevention group and the egg yolk antibody treatment group are protected by 100 percent, and the goslings are not killed. 80% of the goslings in the normal saline control group are attacked and 16 goslings die. The result shows that the egg yolk antibody of gosling plague virus prepared by the invention has good clinical protection effect on goose groups infected with gosling plague virus, and can be used for preventing and treating gosling plague virus.
TABLE 3 efficacy test of yolk antibody against gosling plague virus
Sequence listing
<110> Weifang Huaying Biotech Co., ltd
<120> preparation method of compound vaccine and yolk antibody of gosling plague virus
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cactggggta tccgtccgaa atctctgaaa ttcaaaatct tcaacgttca ggttaaagaa 360
gttaccaccc aggaccagac caaaaccatc gctaacaacc tgacctctac catccaggtt 420
ttcaccgacg acgaacacca gctgccgtac gttctgggtt ctgctaccga aggtaccatg 480
ccgccgttcc cgtctgacgt ttacgctctg ccgcagtacg gttactgcac catgcacacc 540
aaccagaacg gtgctcgttt caacgaccgt tctgctttct actgcctgga atacttcccg 600
tctcagatgc tgcgtaccgg taacaacttc gaattcacct tcgacttcga agaagttccg 660
ttccactcta tgttcgctca ctctcaggac ctggaccgtc tgatgaaccc gctggttgac 720
cagtacctgt ggaacttcaa cgaagttgac tcttctcgtc acgctcagtt caaaaaagct 780
gttaaaggtg cttacggtac catgggtcgt aactggctgc cgggtccgaa attcctggac 840
cagcgtgttc gtgcttacac cggtggtacc gacaactacg ctaactggaa catctggtct 900
aacggtaaca aagttaacct gaaagaccgt cagtacctgc tgcagccggg tccggtttct 960
gctacccaca ccgaagctga agcttcttct atcccggctc agaacatcct gggtctggct 1020
aaagacccgt accgttctgg ttctaccacc gctggtatct ctgacatcat ggttaccgac 1080
gaacaggaag ttgctccgac caacggtgtt ggttggaaac cgtacggtaa aaccgttacc 1140
aacgaacaga acaccaccac cgctccgacc tcttctgacc tggacgttct gggtgctctg 1200
ccgggtatgg tttggcagaa ccgtgacatc tacctgcagg gtccgatctg ggctaaaatc 1260
ccgaaaaccg acggtaaatt ccacccgtct ccgaacctgg gtggtttcgg tctgcacaac 1320
ccgccgccgc aggttttcat caaaaacacc ccggttccgg ctgacccgcc ggttgaatac 1380
gttcaccaga aatggaactc ttacatcacc cagtactcta ccggtcagtg caccgttgaa 1440
atggtttggg aactgcgtaa agaaaactct aaacgttgga acccggaaat ccagttcacc 1500
tctaacttct ctgaccgtac ctctatcatg ttcgctccga acgaaaccgg tggttacgtt 1560
gaagaccgtc tgatcggtac ccgttacctg acccagaacc tgtaa 1605
Claims (8)
1. The gosling plague virus compound vaccine is prepared by water phase and adjuvant, wherein the antigen in the water phase is formed by mixing recombinant PVAX1-VP2 plasmid and recombinant gosling plague virus VP3 protein, and the recombinant PVAX1-VP2 plasmid is formed by enzyme digestion connection of gosling plague virus VP2 gene and eukaryotic expression vector PVAX 1.
2. The composite vaccine of claim 1, wherein one nucleotide sequence of the encoding protein of CSFV VP3 protein is SEQ ID NO. 1.
3. The composite vaccine of claim 1, wherein the final concentration of the recombinant PVAX1-VP2 plasmid in the aqueous phase is 10-100 μ g/ml, and the final concentration of the recombinant CSFV VP3 protein is 0.2-0.8 mg/ml.
4. The combination vaccine of claim 3, wherein the final concentration of the recombinant PVAX1-VP2 plasmid in the aqueous phase is 50 μ g/ml and the final concentration of the recombinant CSFV VP3 protein is 0.4mg/ml.
5. A method for preparing the composite vaccine of claim 1-4, wherein said recombinant PVAX1-VP2 plasmid containing goose pestivirus VP2 gene is mixed with said recombinant goose pestivirus VP3 protein to obtain aqueous phase, and the aqueous phase is mixed with adjuvant to obtain the final product.
6. The combination vaccine of claim 5, wherein the adjuvant is white oil.
7. The combination vaccine of claim 5, wherein the aqueous phase is emulsified with an adjuvant.
8. A method for preparing a yolk antibody of gosling plague virus, wherein the gosling plague virus compound vaccine prepared by the method of claims 5-7 is used for immunizing laying hens, and the yolk antibody of the gosling plague virus is extracted and purified from the yolk of a hyperimmune egg.
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| CN107551267A (en) * | 2017-08-01 | 2018-01-09 | 扬州优邦生物药品有限公司 | A kind of Goose Parvovirus subunit vaccine and its preparation method and application |
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| CN107551267A (en) * | 2017-08-01 | 2018-01-09 | 扬州优邦生物药品有限公司 | A kind of Goose Parvovirus subunit vaccine and its preparation method and application |
Non-Patent Citations (5)
| Title |
|---|
| 朱德康 ; 黎敏 ; 车茜 ; 程安春 ; 汪铭书 ; 陈孝跃 ; .小鹅瘟病毒VP3真核表达质粒与弱毒疫苗诱导鹅体免疫应答的比较.2011,(第03期),595-603. * |
| 杜秋明 ; 鲁承 ; 王暖成 ; 贾文影 ; 赵文婧 ; 申峰勇 ; .鹅细小病毒三种疫苗对Balb/c小鼠的免疫效果.2011,(第19期),141-143. * |
| 甘军纪 ; 魏平华 ; .小鹅瘟研究进展.2012,(第10期),38-42. * |
| 许洪洁 ; 张鑫 ; 夏铭琦 ; 胡桂学 ; .小鹅瘟病毒VP3基因真核表达质粒在小鼠中的免疫效果.2008,(第01期),5-8. * |
| 高光 ; 张鑫 ; 徐佳 ; 王淳玉 ; 许洪洁 ; 胡桂学 ; .鹅细小病毒VP3基因真核表达载体的构建及其在Vero细胞中表达.2010,(第03期),323-326. * |
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