CN111494617A - Quadruple inactivated vaccine and preparation method thereof - Google Patents

Quadruple inactivated vaccine and preparation method thereof Download PDF

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CN111494617A
CN111494617A CN202010267860.7A CN202010267860A CN111494617A CN 111494617 A CN111494617 A CN 111494617A CN 202010267860 A CN202010267860 A CN 202010267860A CN 111494617 A CN111494617 A CN 111494617A
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hexon
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virus
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杨振
王建国
李琛
钱钟
马玉峰
李甜甜
杨玉倩
徐萍
魏荣荣
江媛
范娟
潘杰
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Yangzhou Uni Bio Pharmaceutical Co ltd
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Abstract

The invention provides a method for preparing a quadruple inactivated vaccine by adopting VP2 protein of a bursa of Fabricius virus variant strain FJ19 (with the preservation number of CGMCC NO: 19381) and a corresponding vaccine.A vaccine antigen is a Newcastle disease L a Sota virus strain, an avian influenza T L strain (with the preservation number of CGMCC NO: 19382), VP2 protein of the bursa of Fabricius virus variant strain FJ19 and I group 8b type avian adenovirus Hexon protein.

Description

Quadruple inactivated vaccine and preparation method thereof
Technical Field
The invention belongs to the technical field of biological veterinary drugs, and particularly relates to a novel inactivated quadruple vaccine for newcastle disease, infectious bursal disease, avian influenza and adenovirus and a preparation method thereof.
Background
Newcastle Disease (ND) is an acute, febrile, septicemic and highly contagious infectious disease of birds caused by Newcastle disease virus, characterized by high fever, dyspnea, diarrhea, neurological disorder, mucosal and serosal bleeding, with high morbidity and mortality, is one of the most serious virulent infectious diseases that endanger the global poultry industry.
Infectious Bursal Disease (IBD) is a virulent, highly contagious and viral Infectious Disease that harms young chickens, mainly affecting bursa of Fabricius, caused by IBDV. Infectious Bursal Disease (IBD) is one of the most important immunosuppressive diseases, leading to a higher incidence and mortality of infected chickens. In the past decades, the bursa of Fabricius, which is clinically prevalent in China, is mainly ultra-virulent virus (vvIBDV) and moderately virulent bursal virus, and infectious bursal virus (IBDV) variants never attract attention. The currently used IBD vaccines include attenuated vaccines with moderate virulence, inactivated whole virus vaccines and genetic engineering vaccines, but IBD still occurs in chicken flocks immunized with IBD vaccines in some areas, which suggests that IBDV infection and spread still pose a threat to the prevention and control of IBD in China. The disease mostly occurs in white feather broilers and 817 broilers; can occur all the year round, and is increased in 4-7 months; the chickens are susceptible after 3-7 weeks of age, the disease is found in 16 days at minimum, the infection routes comprise digestive tracts, respiratory tracts, eye conjunctiva and the like, and can also be transmitted by contact, and other infections can be indirectly transmitted by drinking water appliances, food troughs, dust, personnel, insects and transport vehicles, and the infected and diseased chickens are main infection sources. The traditional vaccine can not provide protection for epidemic variant virus, and meanwhile, the live vaccine of the infectious bursal disease causes continuous pollution to a breeding field, so that biosafety risk is brought to the breeding field. The development of the subunit inactivated vaccine aiming at the IBDV variant strain FJ19, which has low production cost, high production efficiency and good vaccine immune effect, has important practical significance for the clinical prevention and control of IBD at present.
Group I avian adenovirus serum 8b is a highly pathogenic pathogen, which has been prevalent in many provinces of China since 2013, resulting in death of a large number of poultry, with the main lesion being liver bleeding. Epidemiological investigation on the disease discovers that the disease has high morbidity in chicken flocks in China, particularly has serious disease to chicks, and mainly occurs to the chicks, and the sick chicks are hindered in growth, disordered in feather and crouched. Death or gradual recovery occurred within more than 48 h. After the chicken group is sick, the death peak of 3-5 days can be highlighted. The mortality rate is low, possibly 10%, and individually can reach 30%. Sometimes, the course of disease may last for 2-3 days. Because of the current clinical non-approved avian adenovirus vaccine, the avian adenovirus epidemic causes huge losses to the breeding industry. In order to reduce the cost input and avoid the injection stress to chicken flocks, a safe and efficient multi-connected vaccine convenient to put in is urgently needed by a farm to deal with the continuously changing epidemic disease prevention and control situation.
Disclosure of Invention
The antigen is a VP2 protein of a Newcastle disease L a Sota virus strain, an H9 subtype avian influenza T L18 strain, a bursal disease virus variant strain FJ19 and a group I8 b type avian adenovirus Hexon protein.
The T L18 strain is classified and named as avian influenza A virus H9 subtype, the preservation number of the strain is CGMCC No.19382, the strain is preserved in China general microbiological culture Collection center at 03.12.2020, the preservation address is No.3 Siro-1 of the south China district facing Yang in Beijing, the variant strain of the bursal disease virus FJ19 is separated from a bursa of Fabricius tissue sample of a white feather broiler, the virus is a new domestic separated virus which is different from the current super virulent virus and classical infectious bursal disease virus, the strain has No agglutination characteristic to chicken erythrocytes, the strain FJ19 strain is classified and named as avian double strand RNA virus, the preservation number of the strain is CGMCC No. 19381, the strain is preserved in China general microbiological culture Collection center at 12.03.12.2020, and the preservation address is No.3 of the Siro-3 of the south China microbiological culture Collection of the south China general microbiological culture Collection center at 03.12.s.
Preferably, the nucleotide of the variant bursa of Fabricius virus VP2 protein is shown in SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2; the nucleotide of the group I8 b avian adenovirus Hexon protein is shown in SEQ ID NO. 3; the amino acid sequence is shown as SEQ ID NO. 4.
More preferably, the content of the Newcastle disease L a Sota virus strain is more than or equal to 108.5EID500.1m L, and the content of the H9 subtype avian influenza T L18 strain is more than or equal to 107.5EID500.1m L, meets the production specification of the vaccine, the agar expansion titer of the content of VP2 protein of the variant bursal disease virus is not less than 1:128, and the agar expansion titer of the content of the I group 8b type avian adenovirus Hexon protein is not less than 1: 32.
In another aspect of the present invention, a method for preparing a quadruple inactivated vaccine is provided, which comprises the following steps: s1, preparing variant bursal disease virus VP2 protein and I group 8b type avian adenovirus Hexon protein; s2, preparing an oil phase; s3, preparing a water phase; s4, emulsification.
Preferably, step S1 includes:
s101, preparation of pMD-IBDV-VP2 recombinant plasmid and pMD-Hexon:
respectively amplifying to obtain VP2 gene of FJ19, and Hexon gene of I group 8b avian adenovirus, connecting with pMD to obtain pMD-IBDV-VP2 and pMD-Hexon recombinant plasmid;
s102, preparation of a pFastBac I-IBDV-VP2 transfer plasmid and a pFastBac I-Hexon transfer plasmid:
carrying out double enzyme digestion on the pFastBac I plasmid and the pMD-IBDV-VP2 and pMD-Hexon recombinant plasmids obtained in S101, and then connecting to obtain pFastBac I-IBDV-VP2 and pFastBac I-Hexon transit plasmids;
s103, construction of recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon:
respectively transferring the transfer plasmids pFastBac I-IBDV-VP2 and pFastBac I-Hexon obtained in S102 into escherichia coli DH10Bac to obtain recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon;
s104, transfecting sf9 cells by using recombinant bacmid:
transfecting the recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon obtained in S103 with insect cell sf9, and culturing to obtain F0 generations of recombinant baculovirus rBac-IBDV-VP2 strain and F0 generations of recombinant bacmid rBac-Hexon strain;
s105, amplification of the recombinant baculovirus:
respectively inoculating the F0 generation of the recombinant baculovirus rBac-IBDV-VP2 strain and the F0 generation of the recombinant baculovirus rBac-Hexon strain obtained in S104 to insect cells sf9, and culturing to obtain F1 generation recombinant baculovirus;
s106, obtaining variant bursal disease virus VP2 protein and group I8 b type avian adenovirus Hexon protein:
respectively inoculating the two F1-generation recombinant baculoviruses obtained in the S105 into insect cells sf9, culturing, collecting cell cultures, centrifuging and taking supernate to obtain variant bursa of Fabricius virus VP2 protein and I group 8b type avian adenovirus Hexon protein;
preferably, the oil phase preparation of S2 comprises: mixing white oil for injection and aluminum stearate, heating, adding span 80, continuing to heat, and naturally cooling to room temperature to obtain an oil phase;
preferably, the aqueous phase preparation of step S3 comprises: mixing the inactivated newcastle disease virus concentrated solution, the inactivated avian influenza virus concentrated solution, the inactivated variant bursal disease virus VP2 protein, the inactivated I group 8b type avian adenovirus Hexon protein and the like to obtain a mixed antigen solution, adding Tween 80 into the mixed antigen solution, and uniformly mixing to obtain a water phase.
Preferably, the emulsifying of step S4 includes: and (3) putting the oil phase obtained in the step (S2) into an emulsifying tank, adding the water phase obtained in the step (S3) while stirring to obtain an emulsion, and shearing to obtain the tetrad inactivated vaccine.
More preferably, the preparation of S1, variant bursa of Fabricius virus VP2 protein, group I8 b avian adenovirus Hexon protein comprises:
s101, preparation of pMD-IBDV-VP2 and pMD-Hexon recombinant plasmids:
primer pairs suitable for expressing a VP2 protein nucleic acid sequence and a Hexon protein nucleic acid sequence in a baculovirus expression system are respectively designed and named as VP2-P1, VP2-P2 and FAdV-F, FAdV-R. The nucleotide sequence of the VP2-P1 is shown as SEQ ID NO. 5; the nucleotide sequence of the VP2-P2 is shown as SEQ ID NO. 6; the nucleotide sequence of the FAdV-F is shown as SEQID NO. 7; the nucleotide sequence of the FAdV-R is shown as SEQ ID NO. 8;
carrying out PCR amplification by using a reverse transcription product cDNA of an extracted variant IBDV FJ19 strain total RNA and VP2-P1 and VP2-P2 as primers to obtain a VP2 target fragment, carrying out PCR amplification by using an extracted I group 8B avian adenovirus DNA as a template and FAdV-F and FAdV-R as primers to obtain a Hexon target fragment, detecting by agarose gel electrophoresis, respectively recovering the VP2 target fragment and the Hexon fragment, connecting the Hexon fragment to a pMD vector by using T4DNA ligase, then transforming DH5 α -T1 competent cells under aseptic conditions to obtain recombinant plasmids a and B, culturing the recombinant plasmids a and B on a L B solid culture medium containing ampicillin with the concentration of 50 mug/M L for 12 h-14 h, selecting a single colony, carrying out PCR identification by using an M13-F primer and an M13-R primer, carrying out sequencing by using a PCR primer named as a colony, verifying the correct sequencing of the SEQ ID primer, and detecting the nucleotide sequence of the recombinant plasmid SEQ ID of SEQ 13-F and SEQ ID 2, wherein the nucleotide sequence of the recombinant plasmid is shown by using the SEQ ID primer of the SEQ ID, the SEQ ID NO. SEQ ID of the SEQ ID NO. 3-P-M3-P-K plasmid, and the SEQ ID of the recombinant plasmid DNA of the SEQ ID NO. 3-SEQ ID, and SEQ ID NO.4 plasmid shown in SEQ ID NO.3 plasmid shown by using the SEQ ID NO.4 plasmid of the SEQ ID NO.4 plasmid shown in SEQ ID NO. 4;
preparing transfer plasmids of the transfer plasmids of S102, pFastBac I-IBDV-VP2 and pFastBac I-Hexon:
carrying out double digestion on a pFastBac I plasmid and a pMD-IBDV-VP2 recombinant plasmid obtained from S101 by using Sal II restriction endonuclease and Not I restriction endonuclease respectively to obtain a pFastBac I digestion fragment and a pMD-IBDV-VP2 digestion fragment, carrying out double digestion on the pFastBac I plasmid and a pMD-Hexon recombinant plasmid obtained from S101 by using BamH I restriction endonuclease and EcoR I restriction endonuclease respectively to obtain a pFastBac I digestion fragment and a pMD-Hexon digestion fragment, respectively recovering the pFastBac I digestion fragment, the pMD-IB865-VP 2 digestion fragment and the pMD-Hexon digestion fragment after agarose gel electrophoresis detection, respectively, connecting 12 h-14 h by using T4DNA ligase at the temperature of 4 ℃, then transforming into competent cells of a plasmid 5 α -T5, obtaining plasmid pdc and dd, culturing a recombinant plasmid containing a recombinant plasmid DNA sequence shown by the concentrations of Sal II restriction endonuclease and Not I restriction endonuclease on a pMD-VP 2 recombinant plasmid, obtaining a pFastBac I digestion fragment and a pMD-VP 2 digestion fragment, respectively, carrying out double digestion on a pFastBac-Hexon recombinant plasmid DNA ligase, and a pFastBac DNA ligase, respectively, and a colony containing a plasmid 12 h-12-Hexon digestion sequence shown by using T-DNA ligase for PCR, which is used for verifying the PCR primer and a recombinant plasmid DNA ligase concentration, and a recombinant plasmid DNA ligase for verifying a plasmid DNA sequence shown by using a plasmid DNA ligase for detecting a plasmid containing;
s103, construction of recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon:
respectively transferring the pFastBac I-IBDV-VP2 transfer plasmid and the pFastBac I-Hexon transfer plasmid obtained in S102 into an escherichia coli DH10Bac competent cell to obtain a recombinant escherichia coli DH10Bac, culturing for 12-14 h on a L B solid culture medium containing ampicillin with the concentration of 50 mug/M L, selecting a positive single colony, carrying out colony PCR (polymerase chain reaction) identification by using an M13-F primer and an M13-R primer, recovering after detecting the colony to be consistent with the expected size through agarose gel electrophoresis, and respectively naming the obtained recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon as the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.9 and the nucleotide sequence of the M13-R primer is shown as SEQ ID NO. 10;
s104, transfecting sf9 cells by using recombinant bacmid:
transfecting the recombinant bacmid rBac-IBDV-VP2 and the rBac-Hexon obtained in S103 with insect cell sf9 by using a liposome transfection method respectively, culturing for 96-144 h at the temperature of 27 ℃, collecting cell culture, centrifuging and taking supernate to obtain a recombinant baculovirus rBac-IBDV-VP2 strain F0 generation and a recombinant baculovirus rBac-Hexon strain F0 generation;
s105, amplification of the recombinant baculovirus:
respectively inoculating the F0 generation of the recombinant baculovirus rBac-IBDV-VP2 strain and the F0 generation of the recombinant baculovirus rBac-Hexon strain obtained in S104 to insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell cultures, centrifuging, and taking supernate to obtain F1 generation recombinant baculovirus;
s106, obtaining variant bursal disease virus VP2 protein and group I8 b type avian adenovirus Hexon protein:
respectively inoculating two F1-generation recombinant baculoviruses obtained in S105 to insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell cultures, centrifuging and taking supernate to obtain variant bursa of Fabricius virus VP2 protein and I group 8b type avian adenovirus Hexon protein;
more preferably, step S2 oil phase preparation comprises:
placing white oil for injection and aluminum stearate in an oil phase preparation tank, heating to 80 deg.C, adding span 80, maintaining for 30min until the temperature reaches 116 deg.C, and naturally cooling to room temperature to obtain oil phase; the mass ratio of the white oil for injection, the aluminum stearate and the span 80 is 94:1: 6;
more preferably, the step S3 aqueous phase preparation comprises:
culturing a L a Sota virus strain of newcastle disease to obtain a newcastle disease virus solution, culturing a T L18 strain of H9 subtype avian influenza to obtain an avian influenza virus solution, respectively filtering, concentrating and inactivating the obtained newcastle disease virus solution and the avian influenza virus solution with formaldehyde to obtain an inactivated newcastle disease virus concentrated solution and an inactivated avian influenza virus concentrated solution, inactivating the variant bursa virus VP2 protein and the I8 b type avian adenovirus Hexon protein obtained in S106 with binary ethyleneimine to obtain the inactivated variant bursa virus VP2 protein and the inactivated I8 b type avian adenovirus Hexon protein, mixing the inactivated newcastle disease virus concentrated solution, the inactivated avian influenza virus concentrated solution, the inactivated variant bursa virus VP2 protein, the inactivated I8 b type avian adenovirus Hexon protein and the like to obtain a mixed antigen solution, adding 80 tween into the mixed antigen solution, and uniformly mixing the tween 80 mass ratio of the aqueous phase and the mixed antigen solution to obtain 96: 96;
more preferably, the emulsifying of step S4 includes:
and (3) putting the oil phase obtained in the step (S2) into an emulsifying tank, adding the water phase obtained in the step (S3) while stirring at the stirring speed of 150r/min, continuing stirring for 30min after the water phase is added to obtain an emulsion, and shearing the emulsion for 2 times at the shearing speed of 4000r/min at the temperature of 20-25 ℃ to obtain the tetrad inactivated vaccine.
More preferably, the rotation speed of the centrifugation in S104, S105 and S106 is 4000 rpm-10000 rpm, and the centrifugation time is 30 min-60 min.
More preferably, the infection complex number of the recombinant baculovirus rBac-IBDV-VP2 strain F0 generation and the recombinant baculovirus rBac-Hexon strain F0 generation inoculated insect cell sf9 in S105 is 0.1-3.0; the multiplicity of infection of the F1 generation recombinant baculovirus inoculated insect cells sf9 in S106 is 0.1-3.0.
More preferably, the concentration times of the newcastle disease virus liquid and the avian influenza virus liquid in S3 are both 3-4 times.
More preferably, the storage condition of the tetrad inactivated vaccine in S4 is sealed storage at a temperature of 2-8 ℃.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a vaccine aiming at infectious bursal disease virus variant FJ19 and H9 subtype avian influenza T L18 for the first time, the inactivated VP2 protein of FJ19 and the inactivated I group 8b type avian adenovirus Hexon protein are used as antigens, then the antigens are mixed with the inactivated Newcastle disease virus concentrated solution and the avian influenza virus concentrated solution to obtain a mixed antigen, and the mixed antigen is emulsified to obtain a quadruple inactivated vaccine, the vaccine obtained has high content of variant bursal disease virus VP2 protein and inactivated I group 8b type avian adenovirus Hexon protein, strong immunogenicity, low formaldehyde content and endotoxin content, has better protection effect on the currently epidemic infectious bursal disease virus variant FJ19 and avian influenza variant T L18, and has no injection stress on chicken flocks.
2. The tetrad inactivated vaccine prepared by the invention has good safety performance, no local and systemic adverse reactions caused by the vaccine appear after the experiment chicken is immunized, the analysis of an efficacy test and an immunity duration test shows that the vaccine has a definite immune effect, the vaccine can resist the infection of newcastle disease virus, avian influenza virus, infectious bursal disease virus variant strains and I group 8b type avian adenovirus to clinical chicken flocks, the immune dose is small, the duration is long, the immunity duration can reach five months under the condition that the tetrad inactivated vaccine is used for only one time of 0.3m L vaccine, and at least 70 percent of protection rate is provided for the chicken flocks.
Drawings
FIG. 1 genetic evolutionary Tree of FJ19 Strain and reference Strain sequences
FIG. 2 PCR amplification of the complete open reading frame sequence of strain FJ19 VP2, wherein M is D L2000 DNAmarker, and 1 is PCR product.
FIG. 3 VP2 transfer vector construction PCR identification, in which M: D L5000 DNAmarker and 1-8:8 different colony PCR.
FIG. 4 VP2 recombinant bacmid PCR identification, in which M: D L5000 DNAmarker, PCR of 1-4:4 different colonies.
Figure 5 SDS-PAGE detects VP2 recombinant baculovirus expression product, where M: pre-dyeing a protein Marker; 1, recombinant baculovirus; 2-6 Sf9 cells infected with the hollow-rod virus.
Figure 6 Western Blot identifies VP2 recombinant baculovirus expression products, where M: pre-dyeing a protein Marker; 1, recombinant baculovirus; 2 Sf9 cells infected with the empty baculovirus.
Detailed Description
The invention uses VP2 protein of variant bursal disease virus FJ19 and I group 8b type avian adenovirus Hexon protein, which are respectively a recombinant baculovirus rBac-IBDV-VP2 strain F0 generation expressing VP2 protein of infectious bursal disease virus (variant FJ19 strain) and a recombinant baculovirus rBac-Hexon expressing I group 8b type avian adenovirus Hexon protein to inoculate Sf9 cells, cell culture is obtained, supernatant is obtained after centrifugation, and the inactivated newcastle disease virus concentrated solution and inactivated avian influenza virus concentrated solution are mixed and emulsified to obtain the quadruple inactivated vaccine. The technical solution of the present invention will be described in detail with reference to the following examples. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1 antigenic composition of a quadruple inactivated vaccine
The antigen of the tetrad inactivated vaccine is inactivated newcastle disease L a Sota virus strain, inactivated H9 subtype avian influenza T L18 strain, inactivated variant bursa of fabricius virus VP2 protein and inactivated I group 8b type avian adenovirus Hexon protein, the nucleotide sequence of the variant bursa of fabricius virus VP2 protein is shown as SEQ ID NO.1, the amino acid is shown as SEQ ID NO.2, the nucleotide sequence of the I group 8b type avian adenovirus Hexon protein is shown as SEQ ID NO.3, the amino acid is shown as SEQ ID NO.4, the content of the newcastle disease L a Sota virus strain is more than or equal to 108.5EID500.1m L, and the content of the H9 subtype avian influenza T L18 strain is more than or equal to 107.5EID500.1m L, wherein the agar expansion titer of the content of VP2 protein of the variant bursal disease virus is more than or equal to 1:128, and the agar expansion titer of the content of Hexon protein of the I group 8b type avian adenovirus is more than or equal to 1: 32;
the Newcastle disease L a Sota virus strain is derived from the China institute of veterinary medicine;
the H9 subtype avian influenza T L18 strain belongs to influenza virus, is preserved in China general microbiological culture Collection center (CGMCC) at 03-12.2020, and has a strain preservation number of CGMCC No.19382, and the H9 subtype avian influenza T L18 strain has a blood coagulation valence of 211With a poison value of 107.0EID500.1m L, is suitable for vaccine production, has high cross hemagglutination with a clinical isolate strain in recent years in homology, has the homology of 97.3-98.0% with an epidemic strain clinically isolated in recent years and has the homology of 90.7-91.5% with a vaccine strain used at present, and shows that the H9 subtype avian influenza T L18 strain is matched with the epidemic strain and has good protection effect on clinical H9 subtype avian influenza.
The infectious bursal disease virus variant strain FJ19 is separated from a bursa of Fabricius tissue sample of a diseased white feather broiler group, is a virus newly separated in China, is different from the current ultra-virulent virus and classical strain infectious bursal disease virus, has no agglutination characteristic on chicken red blood cells, is preserved in China general microbiological culture Collection center (CGMCC) in 03-12 days of 2020, and has the preservation number: CGMCC No: 19381. in terms of homology, the nucleotide homology with a foreign variant strain (Var E) is 95.0 to 96.1 percent, and the amino acid homology is 97.6 to 97.8 percent; the homology with the current ultra-strong toxic nucleotide is 92.4 to 94.0 percent, and the amino acid homology is 95.8 to 96.2 percent; the homology with medium virulent nucleotide is 92.8-94.4%, and the amino acid homology is 95.4-96.2%. Figure 1 shows a genetic evolutionary tree of the sequence of strain FJ19 with a reference strain.
Example 2 preparation of a quadruple inactivated vaccine
S1 separation and identification of variant bursal disease virus
Collecting diseased chicken bursa tissue, grinding, adding PBS containing double antibodies to dilute into suspension, adding chloroform with the same volume into the suspension, placing the suspension in a low-temperature environment at 20rpm for processing for 24 hours, centrifuging at 3000 rpm, taking supernatant, subpackaging in an ampoule, inoculating 10-day-old chicken embryos in a serosa pathway (CAM) for processing, placing at 37 ℃ for incubation, discarding the chicken embryos dead within 24 hours, placing the chicken embryos dead after 24 hours at 4 ℃, collecting allantoic fluid for hemagglutination test, simultaneously collecting embryo bodies and a serosa, aseptically grinding, subpackaging in the ampoule for later use, extracting total RNA of a culture by using a TRIzol Reagent kit, and reversely transcribing the RNA into cDNA by using M-M L V reverse transcriptase.
S2, preparing variant bursa of Fabricius virus VP2 protein and I group 8b type avian adenovirus Hexon protein:
s201, pMD-IBDV-VP2 and pMD-Hexon recombinant plasmid preparation:
primer pairs suitable for expressing a VP2 protein nucleic acid sequence and a Hexon protein nucleic acid sequence by a baculovirus expression system are respectively designed and named as VP2-P1, VP2-P2 and FAdV-F, FAdV-R. The nucleotide sequence of the VP2-P1 is shown as SEQ ID NO. 5; the nucleotide sequence of the VP2-P2 is shown as SEQ ID NO. 6; the nucleotide sequence of the FAdV-F is shown as SEQID NO. 7; the nucleotide sequence of the FAdV-R is shown as SEQ ID NO. 8;
PCR amplification is carried out by taking cDNA of a reverse transcription product of total RNA of a variant IBDV FJ strain extracted and VP-P as primers to obtain a VP target fragment, PCR amplification is carried out by taking I group 8B avian adenovirus DNA as a template and FAdV-F and FAdV-R as primers to obtain a Hexon target fragment, agarose gel electrophoresis detection is carried out, the VP target fragment and the Hexon fragment are respectively recovered, electrophoresis results are shown in a figure 2a and a figure 2B, wherein M is D2000 DNAmarker, the size of the recovered VP fragment is 1356bp in a figure 2a, the size of the Hexon fragment is 855 in a figure 2B and is connected to a pMD vector by T4DNA ligase, DH-T competent cells are transformed under an aseptic condition, recombinant plasmids a and B are obtained, the recombinant plasmids a and B are subjected to PCR amplification by taking a DNA-T competent cell under the aseptic conditions, the PCR amplification system is carried out by taking a DNA of a variant IBDV FJ strain SEQ ID, PCR amplification system of 5 mu, a DNA, PCR amplification system, PCR amplification is carried out by taking a PCR amplification of a PCR amplification 5-P, PCR amplification is carried out by taking a PCR amplification method of a PCR amplification medium of a DNA of a strain of a DNA of a group 8B, a DNA of a group I, a group 8B, a FAdV-F, a FAdV-P as a DNA of a group 8B, a group, a DNA of a group 8B, a group, a FAdXgroup, a DNA of a FAdXgroup, a FAdXE, a DNA of a group, a DNA of a group I, a DNA of a group I, a group, a DNA of a group I, a group, a DNA of a group, a DNA of a group I, a group I, a group, a DNA of a group, a group I of a group, a group I, a group, a DNA of a group I of a DNA of a group I, a DNA of a group, a group I, a group, a DNA of a.
Preparation of S202, pFastBac I-IBDV-VP2, pFastBac I-Hexon transfer plasmids:
carrying out double digestion on a pFastBac I plasmid and a pMD-IBDV-VP2 recombinant plasmid obtained in S201 by using Sal II restriction endonuclease and Not I restriction endonuclease respectively to obtain a pFastBac I digested fragment and a pMD-IBDV-VP2 digested fragment, carrying out double digestion on the pFastBac I plasmid and the pMD-Hexon recombinant plasmid obtained in S101 by using BamH I restriction endonuclease and EcoR I restriction endonuclease respectively to obtain a pFastBac I digested fragment and a pMD-Hexon digested fragment, respectively recovering the pFastBac I digested fragment, the pMD-IBDV-VP2 digested fragment and the pMD-Hexon digested fragment after agarose gel electrophoresis detection, respectively, connecting for 12 h-14 h by using T4DNA ligase at the temperature of 4 ℃, then converting for 12 h-1-T plasmid competent cells under aseptic conditions to obtain a pFastBac plasmid DNA, obtaining a recombinant plasmid DNA, adding a recombinant DNA sequence containing DNA for PCR, verifying that the recombinant DNA is a recombinant plasmid containing DNA sequence of pFastBac 5-26 min, 5 mu.26 min, carrying out a PCR extension reaction under the conditions of PCR extension, verifying that the concentration of a PCR, the PCR is equal to 0.5 mu.10 mu.26 mu.7 min, the PCR, the concentration of a PCR, the PCR extension of a colony concentration of a PCR extension of a PCR amplification medium DNA sequence of PCR amplification medium, and a PCR amplification medium DNA of PCR amplification medium DNA of PCR, and a PCR amplification medium of PCR amplification medium of PCR 5-10, and a PCR amplification medium of PCR 5-10 mu DNA of PCR 5-10 mu DNA of PCR 5-10, and a PCR of PCR 5-PCR 7, and DNA of PCR 7, and DNA of PCR 5, and a colony of PCR 5, and.
S203, construction of recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon:
respectively transferring the pFastBac I-IBDV-VP2 transfer plasmid and the pFastBac I-Hexon transfer plasmid obtained in S202 into a competent cell of escherichia coli DH10Bac to obtain recombinant escherichia coli DH10Bac, culturing for 12-14 h on a L B solid culture medium containing 50 mu g/M L ampicillin, selecting a positive single colony, carrying out colony PCR identification by using an M13-F primer and an M13-R primer, recovering after detecting the consistency with the expected size through agarose gel electrophoresis, respectively naming the recombinant bacillus rBac-IBV-VP 2 and rBac-Hexon as the positive single colony, wherein the nucleotide sequence of the M13-F primer is shown as SEQ ID NO.9, the nucleotide sequence of the M13-R primer is shown as SEQ ID NO.10, the reaction system for colony PCR identification is colony DNA 1.0 mu L, the nucleotide sequence of M13-F primer is shown as SEQ ID NO.9, the nucleotide sequence of the M13-R primer is shown as SEQ ID NO.10, the colony PCR identification system is shown as colony DNA 1.0 mu.8525, the M37-890.5-F primer with the concentration of 10 mmol/36, the concentration of the PCR is shown as 5834. mu.5 min, the result of the pre-PCR extension reaction system is shown as the result, the PCR extension result is shown as 3695-95 ℃ and the result is shown as the denaturation of the PCR extension of the PCR 2-95 ℃ and the PCR extension of the PCR extension.
S204, transfecting sf9 cells by the recombinant bacmid:
transfecting the recombinant bacmid rBac-IBDV-VP2 and the rBac-Hexon obtained in S203 with insect cell sf9 by using a liposome transfection method, culturing for 96-144 h at the temperature of 27 ℃, collecting cell culture, centrifuging and taking supernate to obtain a recombinant baculovirus rBac-IBDV-VP2 strain F0 generation and a recombinant baculovirus rBac-Hexon strain F0 generation;
s205, amplification of the recombinant baculovirus:
respectively inoculating the F0 generation of the recombinant baculovirus rBac-IBDV-VP2 strain and the F0 generation of the recombinant baculovirus rBac-Hexon strain obtained in S204 to insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell cultures, centrifuging, and taking supernate to obtain F1 generation recombinant baculovirus; the infection complex number of the recombinant baculovirus rBac-IBDV-VP2 strain F0 generation and the recombinant baculovirus rBac-Hexon strain F0 generation inoculated insect cell sf9 is 0.1-3.0;
s206, obtaining variant bursal disease virus VP2 protein and I group 8b type avian adenovirus Hexon protein:
respectively inoculating two F1-generation recombinant baculoviruses obtained in S205 to insect cells sf9, culturing for 96-144 h at the temperature of 27 ℃, collecting cell cultures, centrifuging and taking supernate to obtain variant bursa of Fabricius virus VP2 protein and I group 8b type avian adenovirus Hexon protein; the multiplicity of infection of the F1 generation recombinant baculovirus inoculated insect cell sf9 is 0.1-3.0;
(1) identifying the expression proteins of the variant bursal disease virus VP2 protein and the I group 8b type avian adenovirus Hexon protein obtained in S206:
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) characterization: performing SDS-PAGE electrophoresis on the variant bursal disease virus VP2 protein and the I group 8b type avian adenovirus Hexon protein obtained in the step S206 respectively; after electrophoresis is finished, the molecular weights of the proteins are respectively found at the positions of 48kDa and 30kDa after dyeing and decoloring, and the molecular weights are consistent with the theoretical sizes, which indicates that the variant bursa fabricius virus VP2 protein and the group I8 b type avian adenovirus Hexon protein are successfully expressed (the SDS-PAGE identification result is shown in figures 5a and 5 b);
western Blot assay, the gel after SDS-PAGE electrophoresis is taken and directly transferred onto an NC membrane (nitrocellulose membrane) by a BIO-L AB transfer device, Western Blot assay is carried out according to a conventional method after transfer, a chicken anti-variant infectious bursal disease virus multi-anti-positive serum reference product IgG (1:200) is respectively used as a primary antibody (prepared by Youbo Youba biological medicine limited of Yangzhou), a chicken anti-avian adenovirus (I group 8b type) multi-anti-positive serum reference product IgG (1:200) is used as a primary antibody, a goat anti-chicken secondary antibody IgG labeled by horseradish peroxidase (purchased from Beijing Bailey Boke technology limited) (1:2000) is used as an enzyme-labeled secondary antibody, TMB (3,3',5,5' -tetramethylbenzidine) is finally used for developing (purchased from Biyunyan biotechnology research institute), a blank reaction results of the chicken anti-variant infectious bursal disease virus multi-positive serum reference product IgG and the anti-positive serum reference product IgG (VP 1, VP 6 b) are shown by a binding protein of the chicken anti-variant virus, a binding protein of the chicken anti-variant virus and a monoclonal antibody of the anti-avian adenovirus negative antibody are added in about a monoclonal antibody, and a monoclonal antibody of the chicken anti-avian adenovirus antigen binding protein antigen of the chicken anti-avian adenovirus antigen of the chicken anti-variant virus antigen of the chicken infectious bursal disease virus antigen of the chicken monoclonal antibody is shown by a, the antibody of the chicken monoclonal antibody of the chicken anti-avian adenovirus positive antibody of the chicken monoclonal antibody of avian adenovirus of the chicken monoclonal antibody of the fowl infectious bursal disease antigen of the fowl infectious bursal disease virus is shown by the fowl adenovirus of avian adenovirus of the fowl adenovirus of the fowl infectious bursal disease virus of fowl adenovirus of.
(2) And (3) identifying the content of the variant bursal disease virus VP2 protein and the group I8 b type avian adenovirus Hexon protein obtained in the S206:
the contents of expressed variant bursal disease virus VP2 protein and I group 8b type avian adenovirus Hexon protein were detected by agarose gel electrophoresis, and the positive serum was variant infectious bursal disease virus polyclonal antiserum (manufactured by Youbang, Yoghou, Youngi Co., Ltd.), avian adenovirus (I group 8b type) polyclonal antiserum (commercially available) and the negative control was physiological saline. The variant infectious bursal disease virus polyclonal antiserum contains an antibody aiming at the variant bursal disease virus, on an agar amplification detection plate, antigen-antibody reaction is carried out between a variant bursal disease virus polyclonal antiserum hole and a variant bursal disease virus VP2 protein hole to generate a precipitation line, the precipitation line is judged to be positive, and the highest dilution multiple of a sample with the clear precipitation line is used as the measured antigen agar amplification titer; the avian adenovirus (I group 8b type) polyclonal antiserum contains antibodies aiming at the avian adenovirus (I group 8b type), on an agar amplification detection plate, an antigen-antibody reaction is carried out between an avian adenovirus (I group 8b type) polyclonal antiserum hole and an I group 8b type avian adenovirus Hexon protein hole, a precipitation line appears, the result is judged to be positive, and the highest dilution multiple of a sample with a clear precipitation line is used as the antigen agar amplification titer to be detected. The positive serum hole and the normal saline hole do not have antigen-antibody reaction, do not have precipitation line, and are negative. Carrying out 2-time series gradient dilution on VP2 protein of the variant bursal disease virus and I group 8b type avian adenovirus Hexon protein in advance, carrying out antigen-antibody reaction on positive serum and proteins with different concentrations, reacting the positive serum after the VP2 protein is diluted to 128 times, and identifying the positive serum after continuous dilution, thereby indicating that the agar amplification titer of the VP2 protein of the variant bursal disease virus is 1: 128; when the Hexon protein is diluted to 32 times, the Hexon protein can still be reacted by positive serum, and can not be identified by the positive serum after being continuously diluted, which shows that the agar amplification titer of the group I8 b type avian adenovirus Hexon protein is 1: 32;
s3, preparing an oil phase:
placing white oil for injection and aluminum stearate in an oil phase preparation tank, heating to 80 deg.C, adding span 80, maintaining for 30min until the temperature reaches 116 deg.C, and naturally cooling to room temperature to obtain oil phase; the mass ratio of the white oil for injection, the aluminum stearate and the span 80 is 94:1: 6;
s4, preparing a water phase:
culturing L a Sota virus strain of newcastle disease to obtain newcastle disease virus liquid, culturing H9 subtype avian influenza T L18 strain to obtain avian influenza virus liquid, respectively filtering the obtained newcastle disease virus liquid and the avian influenza virus liquid to remove particle impurities to obtain transparent or semitransparent solution, then concentrating by using an ultrafiltration concentration system, concentrating by 3-4 times, inactivating by using formaldehyde to respectively obtain inactivated newcastle disease virus concentrated solution and inactivated avian influenza virus concentrated solution, inactivating the variant bursa virus VP2 protein and the I group 8b type avian adenovirus Hexon protein obtained in S206 by using binary imine to obtain inactivated variant bursa virus VP2 protein and inactivated I group 8b type avian adenovirus Hexon protein, and mixing the inactivated newcastle disease virus concentrated solution, the inactivated influenza virus concentrated solution, the inactivated variant bursa virus VP2 protein and the I group 8b type avian adenovirus Hexon protein in an aqueous phase to obtain a mixed antigen solution, and uniformly mixing the inactivated Tween 80 and the antigen solution to obtain a mixed antigen solution, wherein the mixed antigen solution is Tween 80;
s4 the method for culturing the Newcastle disease virus liquid includes such steps as taking the virus seed of L a Sota strain of Newcastle disease, diluting it by sterilized physiological saline solution 10%4Or 105Inoculating healthy susceptible chick embryos of 10-11 days old, inoculating 0.1-0.2 m L into each chick through an allantoic cavity, sealing a pinhole, continuously incubating at 36-37 ℃, removing the chick embryos dying within 48 hours, taking out the dead chick embryos 2-3 times to 96 hours every day, taking out the dead chick embryos at any time, taking out all the chick embryos not dying after 96 hours, standing the air chamber upwards, and placing at 2-8 DEG CCooling for 4-24 hours; taking out the cooled chick embryo, separating the live chick embryo from the dead chick embryo, and aseptically harvesting chick embryo liquid; mixing several chick embryos into a group, sucking allantoic fluid, placing in the same sterilization container, sampling each group, performing aseptic and 1% erythrocyte agglutination test, discarding those with hemagglutination value lower than 1: 512 (micro method), harvesting to obtain Newcastle disease virus liquid, storing at-20 deg.C for no more than 6 months;
the culture method of the avian influenza virus liquid in S4 comprises collecting H9 subtype avian influenza T L18 strain virus seed for production, and diluting with sterilized normal saline 104Or 105Inoculating healthy susceptible chick embryos of 10-11 days old, inoculating 0.1-0.2 m L into each chick embryo through an allantoic cavity, sealing a pinhole, continuously incubating at 36-37 ℃, removing the chick embryos dying within 48 hours, irradiating eggs for 2-3 times to 96 hours every day, taking out the dead chick embryos at any time, taking out all the chick embryos not dying after 96 hours, standing an air chamber upwards, cooling at 2-8 ℃ for 4-24 hours, taking out the cooled chick embryos, separating the living embryos from the dead embryos, and aseptically harvesting chick embryo liquid, mixing a plurality of chick embryos into a group, sucking allantoic liquid, placing the allantoic liquid into the same sterilization container, sampling each group, carrying out asepsis and 1% erythrocyte agglutination test, discarding the chick embryos with the blood coagulation price lower than 1: 256 (micro-dose method), harvesting to obtain avian influenza virus liquid, and storing at the temperature of-20 ℃ for no more than 6 months;
the content of the Newcastle disease L a Sota virus strain obtained by measuring the content of the L a Sota virus strain of the Newcastle disease virus liquid which is not filtered, concentrated and inactivated in S4 is more than or equal to 107.0EID500.1m L, and determining the content of the H9 subtype avian influenza T L18 strain in the avian influenza virus liquid in S4 to obtain the content of the H9 subtype avian influenza T L18 strain which is more than or equal to 108.0EID50/0.1mL；
The sterilization method of the filtered and concentrated Newcastle disease virus liquid and the filtered and concentrated avian influenza virus liquid in the S4 comprises the following steps: respectively putting the filtered and concentrated Newcastle disease virus liquid and the filtered and concentrated avian influenza virus liquid into an inactivation tank, respectively adding 10% formaldehyde solution while stirring until the final concentration of the formaldehyde solution is 0.1%, stirring and mixing for 5min after the addition is finished, heating to 37 ℃ for inactivation for 24 hours (timing is started when the temperature of the Newcastle disease virus liquid or the avian influenza virus liquid in the tank reaches 37 ℃), respectively sampling for inactivation inspection and sterile inspection after the inactivation is finished, and storing the obtained inactivated Newcastle disease virus concentrated liquid and the inactivated avian influenza virus concentrated liquid at the temperature of 2-8 ℃ for no more than 1 month;
taking the inactivated newcastle disease virus concentrated solution or the inactivated avian influenza virus concentrated solution, inoculating 10 SPF (specific pathogen free) chick embryos in 10 days old in an allantoic cavity, incubating each embryo at 0.2m L for 120 hours at 37 ℃, removing dead chick embryos within 24 hours, determining the hemagglutination price per embryo, and avoiding hemagglutination, wherein the number of the nonspecific dead chick embryos is not more than 1, the hemagglutination price is determined one by one, the hemagglutination does not occur, the chick embryo solutions are mixed in equal amount, and then the blind transmission is carried out for 1 generation according to the method, the hemagglutination price is determined, the hemagglutination does not occur, and the inactivation is complete;
the method for inactivating the variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein in S4 comprises the following steps:
(1) cyclizing 2-bromoethylamine hydrobromide, namely mixing 2-bromoethylamine hydrobromide with the concentration of 2 mol/L and NaOH solution with the concentration of 2 mol/L in equal volume, then placing the mixture in a water bath at the temperature of 37 ℃, shaking the mixture for 1 time every 10 minutes, and terminating the cyclization after 1 hour to obtain a diethylene imine solution with the concentration of 1 mol/L;
(2) inactivating and blocking, namely introducing variant bursa of Fabricius virus VP2 protein and I group 8b type avian adenovirus Hexon protein into an inactivation tank, adding 1 mol/L of diethylene imine solution while stirring until the final concentration of the diethylene imine solution is 0.005 mol/L, stirring and mixing for 10min after the addition is finished, then inactivating for 20 hours when the temperature is raised to 32 ℃ (starting timing when the temperature of the variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein reaches 32 ℃), and immediately adding 2 mol/L of Na for filtration and sterilization into the variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein2S2O3Solution to Na2S2O3Inactivating the solution at final concentration of 0.005 mol/L, stirring, mixing, and inactivatingThe variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein are stored at the temperature of 2-8 ℃ and should not exceed 3 months;
taking the inactivated variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein, inoculating the inactivated variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein to Sf9 cells which form a good monolayer according to 5% of the total mass of a culture medium, carrying out culture and observation at 27 ℃ for 3 days, then carrying out etching for 1 generation, continuously culturing for 3 days, then sucking culture solution in the culture medium, inoculating the inactivated variant bursa of Fabricius virus VP2 protein and the I group 8b type avian adenovirus Hexon protein of Sf9 cells into 4 holes in a 6-hole plate as samples, simultaneously setting 1 hole of each negative control and positive control and 1 hole of each positive control, and 1m L hole, wherein the negative control is normal Sf9 cells, the positive control is a positive control which expresses the variant bursa virus VP2 protein and the I group 8b type avian adenovirus Hexon protein, carrying out experimental judgment on the negative control, carrying out plaque inactivation for 7-10 days, and completely carrying out plaque inactivation on the positive control, wherein the control shows that no plaque appears in the positive control;
measuring the content of the newcastle disease L a Sota strain of the inactivated newcastle disease virus concentrated solution in S4 to obtain the newcastle disease L a Sota strain with the content more than or equal to 108.5EID500.1m L, and determining the content of the H9 subtype avian influenza T L18 strain in the inactivated avian influenza virus concentrated solution in S4 to obtain the content of the H9 subtype avian influenza T L18 strain which is more than or equal to 107.5EID500.1m L, measuring the protein content of the inactivated variant bursal disease virus VP2 protein in S4, wherein the agar amplification titer is more than or equal to 1:128, and measuring the protein content of the inactivated I group 8b type avian adenovirus Hexon protein in S4, wherein the agar amplification titer is more than or equal to 1: 32;
s5, emulsification:
putting the oil phase obtained in the step S3 into an emulsifying tank, adding the water phase obtained in the step S4 while stirring at a stirring speed of 150r/min, continuing stirring for 30min after the water phase is added to obtain an emulsion, and shearing the emulsion for 2 times at a shearing speed of 4000r/min at a temperature of 20-25 ℃ to obtain the tetrad inactivated vaccine; the storage condition of the tetrad inactivated vaccine is sealed storage at the temperature of 2-8 ℃.
Example 3 safety test of vaccines
Two batches of the tetrad inactivated vaccine were prepared according to the preparation method of the tetrad inactivated vaccine of example 1, with the batch numbers of NABFs01 and NABFs02, respectively, and subjected to safety tests:
one-time inoculation safety test of single dose
Taking 10-day-old SPF (specific pathogen free) chickens, wherein the injection dose of the quadruple inactivated vaccine is 0.3m L/SPF, 10 SPF/group, and three groups are provided, wherein the first group is used for injecting the quadruple inactivated vaccine with the batch number of NAIBFs01, the second group is used for injecting the quadruple inactivated vaccine with the batch number of NAIBFs02, and the third group is used for injecting 0.3m L/SPF in the neck;
simultaneously, taking 7-day-old AA white feather broilers (broilers for short), wherein the injection dose of the quadruple inactivated vaccine is 0.3m L/broilers, 10 broilers/group, and three groups are provided, wherein the first group is injected with the quadruple inactivated vaccine with the batch number of NAIBFs01, the second group is injected with the quadruple inactivated vaccine with the batch number of NAIBFs02, and the third group is injected with 0.3m L/broilers at the neck part of 10 broilers;
the chickens of all the groups are raised in an isolator, the diet drinking water, the whole body, the local reaction and other clinical symptoms of the chickens of all the groups are continuously observed for 21 days every day, the injection part of the chickens of all the immune groups is checked by touching 7 days, 14 days and 21 days after inoculation, whether the local injection reaction such as red swelling exists or not is checked, all the chickens are killed after 21 days of immunization, and the change of the injection part, including the histopathological changes of the injection part, is mainly checked. As shown in Table 1, the results show that the two batches of vaccine do not cause obvious adverse reactions to injection parts and the whole body, the test chickens eat and drink water normally in the whole observation period, dissect 21 days after immunization, and absorb the injection parts well, thereby proving that the vaccine is safe to target animals.
TABLE 1 Single dose one-time vaccination safety test for target animals
Figure RE-GDA0002506282460000191
Figure RE-GDA0002506282460000201
(III) safety test of overdose one-time inoculation
Taking 10-day-old SPF (specific pathogen free) chickens, wherein the injection dose of the quadruple inactivated vaccine is 1.0m L/SPF, 10 SPF/group, and three groups are provided, wherein the first group of 10-day-old SPF chickens are injected with the quadruple inactivated vaccine with the batch number of NAIBFs01, and the second group of 10-day-old SPF chickens are injected with the quadruple inactivated vaccine with the batch number of NAIBFs 02;
taking 7-day-old AA white feather broilers (broilers for short), wherein the injection dose of the quadruple inactivated vaccine is 1.0m L/broilers and 10 broilers/group, the injection dose is three groups, the first group is injected with the quadruple inactivated vaccine with the batch number of NAIBFs01, the second group is injected with the quadruple inactivated vaccine with the batch number of NAIBFs02, and the injection dose of the physiological saline injected into the neck of the 7-day-old AA white feather broilers of a negative control group is 1.0m L/broilers;
the chickens of all the groups are fed in an isolator, the mental state of the SPF chickens is not obviously abnormal after vaccine injection, and the AA white feather broilers show slight clinical symptoms of lying, piling and dullness after vaccine injection; after 8 hours, all experimental group chickens began to return to normal feeding;
no chicken died in each immunization group after 21 days of immunization. The inoculated parts of the immunized chickens were observed one by one on the 7 th day and the 14 th day respectively, the inoculated parts of some chickens had no red and swollen symptom, but some chickens on the eye had milky vaccine residues, and the chickens were killed 21 days after immunization, and the statistical results are shown in table 2. The reason why the chicken in each group injected with the vaccine has a small amount of vaccine residues at the injection part but the injection part does not have inflammatory symptoms, and the chicken in the test group injected with the vaccine shows certain clinical symptoms at the initial stage of injection is probably that the chicken is small or the injection dose is too large, the local short-time pain of injection is stimulated, the state of the chicken is abnormal and is not general reaction caused by vaccine toxicity, and the test chicken does not die after 21 days of observation.
TABLE 2 overdose one-shot vaccination safety test for target animals
Figure RE-GDA0002506282460000202
Figure RE-GDA0002506282460000211
Example 4 Immunopotentiality test of quadruple inactivated vaccine
Two batches of the quadruple inactivated vaccines with the respective batches NAIBFs01 and NAIBFs02 of example 3 were used for the immunoefficacy test:
partial efficacy test of newcastle disease
Performing a newcastle disease virus efficacy experiment on the chicken by adopting a serology method and an immune toxicity counteracting method respectively;
(1) the serological method comprises the steps of taking 21-day-old SPF (specific pathogen free) chickens, wherein the immunization dose of subcutaneous inoculation of the quadruple inactivated vaccine is 0.02m L/SPF, 10/group and 3 groups in total, subcutaneously inoculating the quadruple inactivated vaccine with the batch number of NAIBFs01 to the 21-day-old SPF chickens in the first group, subcutaneously inoculating the quadruple inactivated vaccine with the batch number of NAIBFs02 to the 21-day-old SPF chickens in the second group, subcutaneously inoculating the 5-day-old SPF chickens in the third group with PBS (phosphate buffered saline) with the same dose in the first two groups, feeding the chickens in an isolator, collecting blood and separating serum of each chicken 21 days after immunization, and performing HI (hemagglutination inhibition) antibody titer determination, wherein the results are shown in Table 3, and the HI antibody average geometric titer of the serum 21 days after immunization is higher than 1:16 and meets the requirements of vaccine regulations.
(2) The immunization challenge method comprises collecting blood of two groups of immunization groups and negative control group of 21 days after immunization by serological method, simultaneously challenging three groups of chicken with Newcastle disease virulent strain Beijing strain F3 generation wet toxin, and intramuscular injecting 0.2m L/chicken (0.2m L of Newcastle disease virulent strain Beijing strain F3 generation challenge dose is 105.0EID50) And observed for 14 days. The results are shown in table 3, which indicates that the mean geometric titer of serum HI antibodies at 21 days after immunization of the two batches of the quadruple inactivated vaccines is higher than 4log 2; as shown in Table 4, after the challenge of the two groups of immunization groups, the chickens are healthy and alive 14 days, the challenge protection rate on the Newcastle disease virus of the challenge chickens reaches 100%, 5 chickens die within 5 days after the challenge of the negative control group, and the death rate is 100%.
The efficacy experiment of the newcastle disease virus shows that the chickens are healthy and alive 14 days after the vaccine group is attacked, and 5 chickens in the control group die completely. The protection rate of the vaccine to chicken flocks reaches 100 percent, and the vaccine can provide complete protection to newcastle disease in the clinical application.
TABLE 3 Newcastle disease virus antibody detection of two batches of quadruple inactivated vaccines
Figure RE-GDA0002506282460000221
TABLE 4 Newcastle disease immune challenge protection results of the quadruple inactivated vaccine of the two batches
Figure RE-GDA0002506282460000222
(II) efficacy test of H9 subtype avian influenza Virus
Performing efficacy experiments of H9 subtype avian influenza virus by adopting a serology method and an immune toxicity attacking method respectively;
(1) the serological method comprises the steps of taking 21-day-old SPF (specific pathogen free) chickens, enabling the immunization dose of subcutaneous inoculation of the four-combined inactivated vaccine to be 0.3m L/SPF, enabling 10 SPF chickens to be divided into two groups, enabling the first group of 21-day-old SPF chickens to be subcutaneously inoculated with the four-combined inactivated vaccine with the batch number of NAIBFs01, enabling the second group of 21-day-old SPF chickens to be subcutaneously inoculated with the four-combined inactivated vaccine with the batch number of NAIBFs02, meanwhile, setting 5 21-day-old SPF chickens not injected with the four-combined inactivated vaccine as a negative control group, enabling the 21-day-old SPF chickens in the negative control group to be subcutaneously inoculated with PBS (phosphate buffer solution) with equal doses, feeding the four-combined inactivated vaccine in an isolator, separating serum of each chicken after 21 days of immunization, determining the HI antibody titer, enabling the result to be shown in table 5, enabling the geometric mean value of the avian influenza (H9 subtype) HI antibody in the 21-day serum to be higher than 6log2, enabling.
(2) The immune challenge method comprises the steps of collecting blood of three groups of immune groups and negative control groups which are immunized by the serological method for 21 days, diluting the three groups of immune groups and the negative control groups with H9 subtype avian influenza T L strain F3 wet virus at the same time for 1:10, carrying out intravenous injection, 0.2m L/chicken, respectively collecting throat and cloacal cotton swab samples of each chicken 5 days after challenge, freezing and thawing the throat and cloacal cotton swab samples of each chicken, respectively inoculating 5 SPF embryos through allantoic cavities by using double antibodies, respectively inoculating the 10-day-old embryos to each SPF embryo, wherein the dose of the cloacal cotton swab inoculated to each SPF embryo is 0.2m L, incubating and observing for 5 days, determining HA (hemagglutination) titer of all chicken embryo liquids, determining that only 1 of 5 embryos in 5 10-day-old embryos inoculated to each cotton swab sample HAs the allantoic liquid HA titer which is more than or equal to 1:16, judging that the subtype of the avian influenza virus is H9, and determining that the subtype of the negative influenza virus in the chicken after separation, the H6-passage vaccine, and the negative control group after the serological method, the avian influenza virus infection, the negative control group, and the avian influenza virus infection, wherein the subtype of the avian influenza virus infection is 100, and the avian.
TABLE 5H 9 subtype avian influenza virus antibody detection of two batches of the quadruple inactivated vaccine
Figure RE-GDA0002506282460000231
TABLE 6 protective results of H9 subtype avian influenza virus challenge of the two-batch inactivated vaccine
Figure RE-GDA0002506282460000232
(III) efficacy test of variant infectious bursal disease Virus
Performing efficacy experiments of the variant infectious bursal disease virus by adopting a serology method and an immune virus counteracting method respectively;
(1) the serological method comprises the steps of taking 50 SPF (specific pathogen free) chickens of 21 days old, performing neck subcutaneous immunization on 2 groups of the SPF chickens, respectively performing neck subcutaneous immunization on 0.3m L/SPF chickens and 10 SPF chickens/SPF chickens, performing subcutaneous inoculation on 10 SPF chickens with the same amount of PBS as a control group, respectively numbering the SPF chickens, feeding the SPF chickens in an isolator, performing blood collection on each SPF chicken after 21 days of immunization, separating serum, and performing E L ISA (infectious bursal disease Virus) and E L ISA (infectious bursal disease Virus) antibody titer determination on IBDV (infectious bursal disease Virus), wherein the serum agar titer of the 10 SPF chickens in the immunization group is not lower than 3log2, the E L ISA antibody.
Table 721 day-old SPF chicken immune serum IBDV agar-amplified antibody/E L ISA antibody titer
Figure RE-GDA0002506282460000241
Note that the antibody value of E L ISA of not less than 396 is positive
(2) Immune challenge method blood is collected 21 days after immunization, an immune group and a control group are challenged by using separated IBDV variant strains at the same time, the total amount of 0.2m L per chicken (containing 100BID) is dripped into anus, the observation is carried out for 96 hours, all chickens are killed after 96 hours, and the symptom of bursa of Fabricius is observed, as shown in Table 8, the chickens are healthy and alive 96 hours after challenge of the vaccine group, the bursa of Fabricius is consistent with a non-immune challenge group, the protection rate of the disease-free symptom (at least one disease such as obvious swelling, bleeding, yellowing and jelly-like secretion in bursa of Fabricius) reaches 100%, and the disease incidence rate reaches 100% within 5 days after challenge of the control group, namely 10/10.
TABLE 821 day SPF chicken passive immunization challenge (variant infectious bursal disease FJ19 strain)
Figure RE-GDA0002506282460000242
Note: the standard of the bursal disease is at least one symptom of obvious swelling, bleeding, yellowing, jelly-like secretion and the like.
(IV) group I8 b avian adenovirus potency assay
Performing a group I8 b avian adenovirus efficacy experiment by adopting a serology method and an immune challenge method respectively;
(1) the serological method comprises the steps of taking 21-day-old SPF (specific pathogen free) chickens, enabling the immunization dose of subcutaneous inoculation of the quadruple inactivated vaccine to be 0.3m L/SPF, enabling 10 SPF chickens in the first group to be subcutaneously inoculated with the quadruple inactivated vaccine with the NAIBFs01, enabling 21-day-old SPF chickens in the second group to be subcutaneously inoculated with the quadruple inactivated vaccine with the NAIBFs02, meanwhile, setting 10 SPF chickens in the 21-day-old without injection of the quadruple inactivated vaccine as a negative control group, enabling the SPF chickens in the 21-day-old of the negative control group to be subcutaneously inoculated with PBS (phosphate buffer solution) in equal doses, feeding the SPF chickens in an isolator for 21 days after immunization, respectively separating serum of each chicken, carrying out AGP (agar diffusion experiment) antibody titer determination, enabling the results to be shown in table 9, enabling the SPF chickens in the second group to be immunized with each chicken in the 21-day-old day of immunization 0.3m L, enabling the positive rate of the antibodies in the first group after immunization to be 90%, enabling the positive rate in the AGP antibodies in the second group.
(2) The immunization challenge method comprises collecting blood of chicken of three groups of immunization groups and negative control group 21 days after immunization by serology method, simultaneously using group I8 b type avian adenovirus effective detection F3 generation wet toxin to challenge the immunization groups and negative control group, injecting neck part subcutaneously, and using 100EID dose of challenge dose of 0.2m L/chicken (0.2m L group I8 b type avian adenovirus effective detection F3 generation50) And after 14 days of observation, the result shows that each chicken in the immunization group is healthy and alive, and the protection rate is 100 percent; the non-immune negative control group continuously attacks 3-4 days after virus attack, 9 chickens attack within 14 days, 2 chickens begin to die within 10 days, and the diseased chickens and the dead chickens have obvious typical liver swelling and bleeding symptoms and kidney swelling after the autopsy. The challenge results in table 13 show that the challenge protection rates of the three immunization groups for group I8 b avian adenovirus are all 100%, 90% of chickens are attacked and 20% of chickens are killed 14 days after challenge of the non-immunized negative control group, and both the killed chickens and the attacked chickens have the symptoms of inclusion body hepatitis.
TABLE 9 detection of group I8 b avian adenovirus antibody for two batches of the quadruple inactivated vaccine
Figure RE-GDA0002506282460000251
Note: "+" indicates positive for agar, "-" indicates negative for agar, and "1" indicates a 1log2 agar titer.
TABLE 10 protective results of group I8 b avian adenovirus challenge with two-batch four-way inactivated vaccine
Figure RE-GDA0002506282460000261
The efficacy test shows that the quadruple inactivated vaccine has a definite effect and can resist the infection of newcastle disease virus, avian influenza virus, variant infectious bursal disease virus and I group 8b avian adenovirus to clinical chicken flocks.
Example 4 duration of immunization test for quadruple inactivated vaccine
The duration of immunization test was carried out with the quadruple inactivated vaccines of example 3, batch nos. NAIBFs01 and NAIBFs02, respectively:
(1) immune duration antibody test results:
in order to research the lasting efficacy of the vaccine, 260 incubated SPF (specific pathogen free) chickens of 21 days old are selected and divided into three groups, wherein 95 chickens in each group are immunized with quadruple inactivated vaccines of batches of NAIBFs01 and NAIBFs02 respectively, the immunization dose of neck subcutaneous injection is 0.3m L/chicken, 70 immune equivalent PBS in the third group is used as a non-immune control group and is raised in 8 isolators, antibody detection is carried out in 2 weeks, 1 month, 1.5 months, 2 months, 3 months and 4 months after immunization respectively, adenovirus is only subjected to challenge virus mode for efficacy test, as shown in table 11, ND HI (AGP HI) in the table represents newcastle disease hemagglutination inhibition antibodies in the sera of the chickens after immunization, H5660 (subtype) represents avian influenza (H9) hemagglutination inhibition antibodies in the sera of the chickens after immunization, IBDV (AGP HI) represents variant bursa virus expansion antibodies in the sera of the chickens after immunization, and the results show that the peak value of the vaccine of 21 days of the 21 days old chickens after immunization in 0.3m L/21 months, the vaccine is reduced by the normal vaccine, the log of the vaccine, the vaccine is reduced by 14 months after 3 months, and the vaccine is reduced by the peak value of the vaccine after the vaccine is increased by 100.7 months, and the vaccine is increased by the vaccine after the vaccine is increased by the vaccine, and the vaccine is increased by the peak value of the vaccine after the vaccine is increased by more than the vaccine after the vaccine is increased by 100 months, and the vaccine is increased by the vaccine after the vaccine is increased by the.
TABLE 12 two-batch detection of antibodies for duration of immunization of the four-way inactivated vaccine
Figure RE-GDA0002506282460000271
(2) Challenge results for duration of immunity:
after 21-day-old SPF chickens are immunized for 1 month and after 3 months, 40 immune group chickens and 30 non-immune control group chickens are selected for each batch, wherein 40 immune group chickens are selected as one group, the chickens are respectively attacked by newcastle disease L a Sota virus strain, H9 subtype avian influenza T L18 strain, variant strain bursa virus and I group 8b type avian adenovirus to form newcastle disease virus, H9 subtype avian influenza virus, variant strain bursa virus and I group 8b type avian adenovirus, 30 non-immune negative control group chickens are attacked by newcastle disease, 5H 9 subtype avian influenza virus, 10 variant strain bursa virus and 10I group 8b type avian adenovirus according to the specification, the attacking result is shown in a table 13, NDV in the table represents viruses for newcastle disease virus, AIV (H9N2) represents viruses for avian influenza virus, IBDV represents viruses for attacking bursa virus, and FAV-8 b represents for avian adenovirus.
TABLE 13 challenge results for duration of immunization for two-batch quadruple inactivated vaccine
Figure RE-GDA0002506282460000272
Figure RE-GDA0002506282460000281
As can be seen from table 13, after 21-day-old SPF chickens are immunized with 0.3m L/chicken dose of the quadruple inactivated vaccines of the batch numbers of NAIBFs01 and NAIBFs02, the HI antibodies of newcastle disease virus and H9 subtype avian influenza virus are 100% above the specification standard, the antibody positive rate of variant bursa virus reaches 100%, the challenge results show that the protection rates of newcastle disease virus, H9 subtype avian influenza virus, variant bursa virus and group I8 b type avian adenovirus are 100%, after immunization for 4 months, the antibody water of each component is above the specification standard on average, the challenge results show that the immune protection rates of each component are 100%, the antibody levels of each component are reduced at 4 months after immunization, but the challenge results show that the protection rates of newcastle disease and H2 avian influenza reach 90%, the protection rates of variant bursa virus and group I8 b type adenovirus of all reach 90%, the challenge results show that the vaccine of newcastle disease and group I8 b type avian influenza virus can provide at least 90% of the vaccine when the vaccine is used as a vaccine for preventing newcastle disease virus, the chicken vaccine when the chicken is used for at least 3m vaccine, and the chicken, and when the chicken is used for at least for 3H vaccine for 3 months.
1
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
SEQ ID NO.1
1356
IBDV-FJ19 strain VP2 protein nucleotide sequence
ATGACAAACCTGCAAGATCAAACCCAACAGATTGTTCCGTTCAT ACGGAGCCTTCTGATGCCAACAACCGGACCGGCGTCCATCCCG GACGACACCCTGGAGAAGCACACTCTCAGGTCAGAGACCTCG ACCTACAATTTGACTGTGGGGGACACAGGGTCAGGGCTAATTG TCTTTTTCCCTGGCTTCCCTGGCTCAATTGTGGGTGCTCACTACATACTGCAGAGCGATGGGAGCTACAAGTTCGATCAGATGCTCCTG ACGGCCCAGAACCTACCTGCCAGCTACAACTACTGCAGGCTAG TGAGTCGGAGTCTCACAGTAAGGTCAAGCACACTCCCTGGTGG CGTTTATGCGCTAAACGGCACCATAAACGCCGTGACCTTCCAAG GGAGCCTGAGTGAACTGACAGATGTTAGCTACAACGGGTTGAT GTCTGCAACAGCCAACATCAACGATAAAATTGGGAACGTCCTA GTAGGGGAAGGGGTAACCGTTCTCAGCTTACCCACATCATATGA TCTCGGGTATGTGAGGCTTGGTGACCCCATACCTGCTGTAGGGC TCGACCCAAAGATGGTAGCAACATGTGACAGCAGTGACAGGCC CAGAGTCTACACCATAACTGCAGCCGACAATTACCAATTCTCAT CACAGTACAAGACAGGTGGGGTAACAATCACACTGTTCTCAGC CAACATTGATGCCATCACTAGTCTCAGCGTTGGGGGGGAGCTTGTGTTCAAAACCAGCATCCAAAACCTTGTACTGGGCGCCACAAT CTACCTTATAGGCTTTGATGGGACTGCGGTAATCACCAGAGCTG TAGCTGCAAACAATGGGCTGACGGCCGGCATCGACAACCTCAT GCCATTCAACCTTGTGATTCCGACCAGCGAGATAACCCAGCCAA TCACATCCATCAAATTGGAGATAGTGACCTCCAAAAGTGATGGC CAGGCAGGGGAACAGATGTCGTGGTCGGCAAGTGGGAGTCTA GCAGTGACGATCCATGGTGGCAACTATCCAGGAGCCCTCCGTCC CGTCACGCTAGTGGCCTACGAACGAGTGGCAAAAGGATCCGTT GTTACGGTCGCCGGGGTGAGCAACTTCGAGCTGATCCCAAATC CTGAACTAGCAAAGAACCTGGTCACAGAATACGGCCGATTCGA CCCAGGAGCCATGAACTACACGAAACTGATACTGAGTGAGAGG GACCGTCTTGGCATTAAGACCGTCTGGCCAACAAGGGAGTACA CCGACTTTCGCGAGTACTTCATGGAGGTGGCCGACCTCAACTCT CCCCTGAAGATTGCAGGAGCATTTGGCTTCAAAGACATAATCCG GGCCATAAGG
SEQ ID NO.2
452
IBDV FJ19 strain VP2 protein amino acid sequence
MTNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNL TVGDTGSGLIVFFPGFPGSIVGAHYILQSDGSYKFDQMLLTAQNLP ASYNYCRLVSRSLTVRSSTLPGGVYALNGTINAVTFQGSLSELTDV SYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIP AVGLDPKMVATCDSSDRPRVYTITAADNYQFSSQYKTGGVTITLFS ANIDAITSLSVGGELVFKTSIQNLVLGATIYLIGFDGTAVITRAVAAN NGLTAGIDNLMPFNLVIPTSEITQPITSIKLEIVTSKSDGQAGEQMS WSASGSLAVTIHGGNYPGALRPVTLVAYERVAKGSVVTVAGVSNF ELIPNPELAKNLVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTR EYTDFREYFMEVADLNSPLKIAGAFGFKDIIRAIR
SEQ ID NO.3
855
ATGAATGTCACTACCGAGAAGGCCCAGCGGCTTCAGATCAGGT TCTATCCCACCCAGACGGACGACACCCCCAACAGTTACCGGGT TCGGTACAGCTTAAACGTGGGGGACAGCTGGGTGTTGGACATG GGAGCGACCTACTTCGACATCAAAGGGGTGCTCGACAGAGGTC CTTCCTTCAAGCCCTACGGCGGCACGGCTTACAACCCCCTGGCCCCTCGCGAAGCCTTCTTTAACAACTGGATCGAGGACGATGAAA ACAACACAACCATCACCGGGCAAATGACCAATCCGTACAAGAA CGAGCAGCAAAACACAGCTACGGCAACAGCTGGGGCAATCGC CAGCGTTTCAGGCTCTTATCCTAACCCTAACGTGGGGCTGGCCA TTAGCGAAATGGGAGCCCTCACCCCGACACAAGCAGCACAGGT CGGCAGCACAGGTCGGTCTGGCCGGTCGGTTTGCCAAGGTGTC GAGCGAGAACACGCGGCTGGCTTATGGAGCGTACGTGAAGCCT CTAAAAGACGACGGCTCTCAGTCACTTGGAACAACGCCTTACT ACGTGTTAGACACCACTGCACAGAAATACTTGGGCGTCATGGG GGTAGAAGACTTTACGCAAAGTCTTACCTACCCAGACAGTCTGT TAATCCCCCCTCCTTCTGAGTACGGAGCGGTTAACAGCGGGGTG ATGAAAGCCAACAGACCCAACTACATCGGGTTCCGTGACAATT TCATCAACCTCCTGTACCACGATACCGGCGTGTGCTCCGGGACC CTCAACTCCGAACGGTCAGGCATGAACGTGGTGGTGGAATTGC AGGACCGAAATACCGAACTCAGTTACCAGTACATGCTCGCCCA GTAA
SEQ ID NO.4
280
Met Asn Val Thr Thr Glu Lys Ala GlnArg Leu Gln Ile Arg Phe Tyr
Pro ThrGlnThr Asp AspThr Pro Asn Ser Tyr Arg Val Arg Tyr Ser
Leu Asn Val Gly Asp Ser Trp Val Leu Asp Met Gly Ala Thr Tyr Phe
Asp Ile Lys Gly Val Leu Asp ArgGly Pro Ser Phe Lys Pro Tyr Gly
GlyThr Ala Tyr Asn Pro Leu Ala Pro Arg Glu Ala PhePheAsnAsn
Trp Ile Glu Asp Asp Glu Asn Asn Thr Thr Ile ThrGlyGln Met Thr
Asn Pro Tyr Lys Asn Glu GlnGlnAsnThr Ala Thr Ala Thr Ala Gly
Ala Ile Ala Ser Val Ser Gly Ser Tyr Pro Asn Pro Asn Val Gly Leu
Ala Ile Ser Glu Met Gly Ala Leu Thr Pro ThrGln Ala AlaGln Val
Gly Leu Ala GlyArgPhe Ala Lys Val Ser Ser Glu AsnThrArg Leu
Ala Tyr Gly Ala Tyr Val Lys Pro Leu Lys Asp AspGly Ser Gln Ser
Leu GlyThrThr Pro Tyr Tyr Val Leu Asp ThrThr Ala Gln Lys Tyr
Leu Gly Val Met Gly Val Glu Asp PheThrGln Ser Leu Thr Tyr Pro
Asp Ser Leu Leu Ile Pro ProPro Ser Glu Tyr Gly Ala Val Asn Ser
Gly Val Met Lys Ala AsnArg Pro Asn Tyr Ile Gly Phe Arg Asp Asn
Phe Ile Asn Leu Leu Tyr His Asp ThrGly Val Cys Ser GlyThr Leu
Asn Ser Glu Arg Ser Gly Met Asn Val ValVal Glu Leu Gln Asp Arg
AsnThr Glu Leu Ser Tyr Gln Tyr Met Leu Ala Gln
MNVTTEKAQRLQIRFYPTQTDDTPNSYRVRYSLNVGDSWVLDMGATYFDIK GVLDRGPSF
KPYGGTAYNPLAPREAFFNNWIEDDENNTTITGQMTNPYKNEQQNTATATA GAIASVSGS
YPNPNVGLAISEMGALTPTQAAQVGLAGRFAKVSSENTRLAYGAYVKPLKD DGSQSLGTT
PYYVLDTTAQKYLGVMGVEDFTQSLTYPDSLLIPPPSEYGAVNSGVMKANR PNYIGFRDN
FINLLYHDTGVCSGTLNSERSGMNVVVELQDRNTELSYQYMLAQ
SEQ ID NO.5
VP2-P1:ACGCGTCGACATGACAAACCTGCAAGATCAAAC
SEQ ID NO.6
VP2-P2:ATAAGAATGCGGCCGCCCTTATGGCCCGGATTATGTCT
SEQ ID NO.7
FAdV-F:ATAGGATCAATGAATGTCACTACCGAGAAG
SEQ ID NO.8
FAdV-R:ATAGAATTCT TACTGGGCGA GCATGTACTG
SEQ ID NO.9
M13-F：TGTAAAACGACGGCCAGT
SEQ ID NO.10
M13-R：CAGGAAACAGCTATGAC
SEQUENCE LISTING
<110> Youbang, Yangzhou biopharmaceutical Co Ltd
<120> quadruple inactivated vaccine and preparation method thereof
<130>2020
<160>10
<170>PatentIn version 3.3
<210>1
<211>1356
<212>DNA
<213>Infectious bursal disease virus
<400>1
atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60
ccaacaaccg gaccggcgtc catcccggac gacaccctgg agaagcacac tctcaggtca 120
gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
cctggcttcc ctggctcaat tgtgggtgct cactacatac tgcagagcga tgggagctac 240
aagttcgatc agatgctcct gacggcccag aacctacctg ccagctacaa ctactgcagg 300
ctagtgagtc ggagtctcac agtaaggtca agcacactcc ctggtggcgt ttatgcgcta 360
aacggcacca taaacgccgt gaccttccaa gggagcctga gtgaactgac agatgttagc 420
tacaacgggt tgatgtctgc aacagccaac atcaacgata aaattgggaa cgtcctagta 480
ggggaagggg taaccgttct cagcttaccc acatcatatg atctcgggta tgtgaggctt 540
ggtgacccca tacctgctgt agggctcgac ccaaagatgg tagcaacatgtgacagcagt 600
gacaggccca gagtctacac cataactgca gccgacaatt accaattctc atcacagtac 660
aagacaggtg gggtaacaat cacactgttc tcagccaaca ttgatgccat cactagtctc 720
agcgttgggg gggagcttgt gttcaaaacc agcatccaaa accttgtact gggcgccaca 780
atctacctta taggctttga tgggactgcg gtaatcacca gagctgtagc tgcaaacaat 840
gggctgacgg ccggcatcga caacctcatg ccattcaacc ttgtgattcc gaccagcgag 900
ataacccagc caatcacatc catcaaattg gagatagtga cctccaaaag tgatggccag 960
gcaggggaac agatgtcgtg gtcggcaagt gggagtctag cagtgacgat ccatggtggc 1020
aactatccag gagccctccg tcccgtcacg ctagtggcct acgaacgagt ggcaaaagga 1080
tccgttgtta cggtcgccgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca 1140
aagaacctgg tcacagaata cggccgattc gacccaggag ccatgaacta cacgaaactg 1200
atactgagtg agagggaccg tcttggcatt aagaccgtct ggccaacaag ggagtacacc 1260
gactttcgcg agtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga 1320
gcatttggct tcaaagacat aatccgggcc ataagg 1356
<210>2
<211>452
<212>PRT
<213>Infectious bursal disease virus
<400>2
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg
1 5 10 15
Ser Leu Leu Met Pro Thr Thr GlyPro Ala Ser Ile Pro Asp Asp Thr
20 25 30
Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr
35 40 45
Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro
50 55 60
Gly Ser Ile Val Gly Ala His Tyr Ile Leu Gln Ser Asp Gly Ser Tyr
65 70 75 80
Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr
85 90 95
Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr
100 105 110
Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr
115 120 125
Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu
130 135 140
Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val
145 150 155 160
Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly
165 170 175
Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Val Gly Leu Asp Pro Lys
180 185 190
Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile
195 200 205
Thr Ala Ala Asp Asn Tyr Gln Phe Ser Ser Gln Tyr Lys Thr Gly Gly
210 215 220
Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu
225 230 235 240
Ser Val Gly Gly Glu Leu Val Phe Lys Thr Ser Ile Gln Asn Leu Val
245 250 255
Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Ala Val Ile
260 265 270
Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr Ala Gly Ile Asp Asn
275 280 285
Leu Met Pro Phe Asn Leu Val Ile Pro Thr Ser Glu Ile Thr Gln Pro
290 295 300
Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Asp Gly Gln
305 310 315 320
Ala Gly Glu Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr
325 330 335
Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val
340 345 350
Ala Tyr Glu Arg Val Ala Lys Gly Ser Val Val Thr Val Ala Gly Val
355 360 365
Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val
370 375 380
Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu
385 390 395 400
Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr
405 410 415
Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu
420 425 430
Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile
435 440 445
Arg Ala Ile Arg
450
<210>3
<211>868
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>3
atgaatgtca ctaccgagaa ggcccagcgg cttcagatca ggttctatcc cacccagacg 60
gacgacaccc ccaacagtta ccgggttcgg tacagcttaa acgtggggga cagctgggtg 120
ttggacatgg gagcgaccta cttcgacatc aaaggggtgc tcgacagagg tccttccttc 180
aagccctacg gcggcacggc ttacaacccc ctggcccctc gcgaagcctt ctttaacaac 240
tggatcgagg acgatgaaaa caacacaacc atcaccgggc aaatgaccaa tccgtacaag 300
aacgagcagc aaaacacagc tacggcaaca gctggggcaa tcgccagcgt ttcaggctct 360
tatcctaacc ctaacgtggg gctggccatt agcgaaatgg gagccctcac cccgacacaa 420
gcagcacagg tcggcagcac aggtcggtct ggccggtcgg tttgccaagg tgtcgagcga 480
gaacacgcgg ctggcttatg gagcgtacgt gaagcctcta aaagacgacg gctctcagtc 540
acttggaaca acgccttact acgtgttaga caccactgca cagaaatact tgggcgtcat 600
gggggtagaa gactttacgc aaagtcttac ctacccagac agtctgttaa tcccccctcc 660
ttctgagtac ggagcggtta acagcggggt gatgaaagcc aacagaccca actacatcgg 720
gttccgtgac aatttcatca acctcctgta ccacgatacc ggcgtgtgct ccgggaccct 780
caactccgaa cggtcaggca tgaacgtggt ggtggaattg caggaccgaa ataccgaact 840
cagttaccag tacatgctcg cccagtaa 868
<210>4
<211>284
<212>PRT
<213> Artificial sequence (Artificial sequence)
<400>4
Met Asn Val Thr Thr Glu Lys Ala Gln Arg Leu Gln Ile Arg Phe Tyr
1 5 10 15
Pro Thr Gln Thr Asp Asp Thr Pro Asn Ser Tyr Arg Val Arg Tyr Ser
2025 30
Leu Asn Val Gly Asp Ser Trp Val Leu Asp Met Gly Ala Thr Tyr Phe
35 40 45
Asp Ile Lys Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Gly
50 55 60
Gly Thr Ala Tyr Asn Pro Leu Ala Pro Arg Glu Ala Phe Phe Asn Asn
65 70 75 80
Trp Ile Glu Asp Asp Glu Asn Asn Thr Thr Ile Thr Gly Gln Met Thr
85 90 95
Asn Pro Tyr Lys Asn Glu Gln Gln Asn Thr Ala Thr Ala Thr Ala Gly
100 105 110
Ala Ile Ala Ser Val Ser Gly Ser Tyr Pro Asn Pro Asn Val Gly Leu
115 120 125
Ala Ile Ser Glu Met Gly Ala Leu Thr Pro Thr Gln Ala Ala Gln Val
130 135 140
Gly Leu Ala Gly Arg Phe Ala Lys Val Ser Ser Glu Asn Thr Arg Leu
145 150 155 160
Ala Tyr Gly Ala Tyr Val Lys Pro Leu Lys Asp Asp Gly Ser Gln Ser
165 170 175
Leu Gly Thr Thr Pro Tyr Tyr Val Leu Asp Thr Thr Ala Gln Lys Tyr
180 185 190
Leu Gly Val Met Gly Val Glu Asp Phe Thr Gln Ser Leu Thr Tyr Pro
195 200 205
Asp Ser Leu Leu Ile Pro Pro Pro Ser Glu Tyr Gly Ala Val Asn Ser
210 215 220
Gly Val Met Lys Ala Asn Arg Pro Asn Tyr Ile Gly Phe Arg Asp Asn
225 230 235 240
Phe Ile Asn Leu Leu Tyr His Asp Thr Gly Val Cys Ser Gly Thr Leu
245 250 255
Asn Ser Glu Arg Ser Gly Met Asn Val Val Val Glu Leu Gln Asp Arg
260 265 270
Asn Thr Glu Leu Ser Tyr Gln Tyr Met Leu Ala Gln
275 280
<210>5
<211>33
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>5
acgcgtcgac atgacaaacc tgcaagatca aac 33
<210>6
<211>38
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>6
ataagaatgc ggccgccctt atggcccgga ttatgtct 38
<210>7
<211>30
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>7
ataggatcaa tgaatgtcac taccgagaag 30
<210>8
<211>30
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>8
atagaattct tactgggcga gcatgtactg 30
<210>9
<211>18
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>9
tgtaaaacga cggccagt 18
<210>10
<211>17
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>10
caggaaacag ctatgac 17

Claims (10)

1. The chicken bursa variant FJ19 is preserved in China general microbiological culture Collection center (CGMCC) at 03 and 12 months 2020, with the preservation number: CGMCC No: 19381.
2. the use of the chicken bursa variant FJ19 of claim 1 in the preparation of a vaccine.
3. The quadruple inactivated vaccine is characterized in that antigens of the quadruple inactivated vaccine are a VP2 protein of newcastle disease virus, avian influenza virus, a variant bursa of fabricius virus FJ19 and a group I8 b type avian adenovirus Hexon protein, wherein the variant bursa of fabricius virus FJ19 is preserved in China general microbiological culture Collection center (CGMCC) in 2020, 03 and 12 days, and the preservation number is as follows: CGMCC No: 19381.
4. the tetrad inactivated vaccine as claimed in claim 3, wherein the strain of Newcastle disease virus is L aSota, the strain of avian influenza is T L18 of H9 subtype, the T L18 strain is deposited at the China general microbiological culture Collection center (CGMCC) on 12 months after 2020, and the strain preservation number is CGMCC No. 19382.
5. The quadruple inactivated vaccine according to claim 4, wherein the content of the Newcastle disease L a Sota virus strain is more than or equal to 108.5EID500.1m L, and the content of the H9 subtype avian influenza T L18 strain is more than or equal to 107.5EID500.1m L, wherein the agar expansion titer of the content of the variant bursal disease virus VP2 protein is more than or equal to 1:128, and the agar expansion titer of the group I8 b type avian adenovirus Hexon protein is more than or equal to 1: 32.
6. The quadruple inactivated vaccine according to any one of claims 3 to 5, wherein the nucleic acid sequence of the VP2 protein is SEQ ID No.1, and the amino acid sequence thereof is SEQ ID No. 2; the nucleic acid sequence of the I group 8b type avian adenovirus Hexon protein is SEQ ID NO.3, and the amino acid sequence thereof is SEQ ID NO. 4.
7. The method for preparing the quadruple inactivated vaccine according to any one of claims 3 to 6, which comprises the following steps:
s1, preparing variant bursal disease virus VP2 protein and I group 8b type avian adenovirus Hexon protein;
s2, preparing an oil phase;
s3, preparing a water phase;
s4, emulsification.
8. The method for preparing the quadruple inactivated vaccine according to claim 7, wherein the step S1 comprises:
s101, preparation of pMD-IBDV-VP2 recombinant plasmid and pMD-Hexon:
respectively amplifying to obtain VP2 gene of FJ19, and Hexon gene of I group 8b avian adenovirus, connecting with pMD to obtain pMD-IBDV-VP2 and pMD-Hexon recombinant plasmid;
s102, preparation of a pFastBac I-IBDV-VP2 transfer plasmid and a pFastBac I-Hexon transfer plasmid:
carrying out double enzyme digestion on the pFastBac I plasmid and the pMD-IBDV-VP2 and pMD-Hexon recombinant plasmids obtained in S101, and then connecting to obtain pFastBac I-IBDV-VP2 and pFastBac I-Hexon transit plasmids;
s103, construction of recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon:
respectively transferring the transfer plasmids pFastBac I-IBDV-VP2 and pFastBac I-Hexon obtained in S102 into escherichia coli DH10Bac to obtain recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon;
s104, transfecting sf9 cells by using recombinant bacmid:
transfecting the recombinant bacmid rBac-IBDV-VP2 and rBac-Hexon obtained in S103 with insect cell sf9, and culturing to obtain F0 generations of recombinant baculovirus rBac-IBDV-VP2 strain and F0 generations of recombinant bacmid rBac-Hexon strain;
s105, amplification of the recombinant baculovirus:
respectively inoculating the F0 generation of the recombinant baculovirus rBac-IBDV-VP2 strain and the F0 generation of the recombinant baculovirus rBac-Hexon strain obtained in S104 to insect cells sf9, and culturing to obtain F1 generation recombinant baculovirus;
s106, obtaining variant bursal disease virus VP2 protein and group I8 b type avian adenovirus Hexon protein:
respectively inoculating the two F1-generation recombinant baculoviruses obtained in the S105 into insect cells sf9, culturing, collecting cell cultures, centrifuging and taking supernate to obtain variant bursa of Fabricius virus VP2 protein and I group 8b type avian adenovirus Hexon protein;
9. the method for preparing the quadruple inactivated vaccine according to claim 7 or 8, wherein the oil phase preparation of S2 comprises: mixing white oil for injection and aluminum stearate, heating, adding span 80, continuing to heat, and naturally cooling to room temperature to obtain an oil phase; the preparation of the aqueous phase of step S3 includes: mixing the inactivated newcastle disease virus concentrated solution, the inactivated avian influenza virus concentrated solution, the inactivated variant bursal disease virus VP2 protein, the inactivated I group 8b type avian adenovirus Hexon protein and the like to obtain a mixed antigen solution, adding Tween 80 into the mixed antigen solution, and uniformly mixing to obtain a water phase.
10. The method for preparing an inactivated vaccine according to any one of claims 7 to 9, wherein the emulsification of step S4 comprises: and (3) putting the oil phase obtained in the step (S2) into an emulsifying tank, adding the water phase obtained in the step (S3) while stirring to obtain an emulsion, and shearing to obtain the tetrad inactivated vaccine.
CN202010267860.7A 2020-04-08 2020-04-08 Quadruple inactivated vaccine and preparation method thereof Pending CN111494617A (en)

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