Newcastle disease, avian influenza and infectious coryza triple inactivated vaccine and its preparation and application

Abstract

The application discloses a chicken Newcastle disease, avian influenza and chicken infectious rhinitis triple inactivated vaccine and preparation and application thereof, and relates to the technical field of bioengineering. The application discloses a preparation method and application of a chicken Newcastle disease, avian influenza and chicken infectious rhinitis (A, B and C three serotypes) subunit protein triple inactivated vaccine. The preparation method comprises the following steps: constructing and expressing a serum B paracolon avian bacillus subunit protein expression strain; and preparing the chicken Newcastle disease, H9 subtype avian influenza and chicken infectious rhinitis (A, B and C three serotypes) subunit protein triple inactivated vaccine at a certain ratio. The triple inactivated vaccine has good safety, can be used for the prevention of chicken Newcastle disease, H9 subtype avian influenza and A, B and C three serotypes paracolon avian bacillus diseases in a laying hen group, and does not cause the decrease of the laying rate after immunization. Meanwhile, the paracolon avian bacillus subunit protein can improve the antibody level of the Newcastle disease and the H9 avian influenza, and the protection of the vaccine on the A, B and C three serotypes paracolon avian bacillus can reach 100%.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a trivalent inactivated vaccine against Newcastle disease, avian influenza and infectious coryza in chickens, as well as its preparation and application. Background Technology

[0002] Newcastle disease, a major infectious disease in poultry, has broken out in many parts of the world, including the United States, South Korea, and China, causing severe economic losses to the poultry industry, second only to avian influenza. Conventional vaccines mainly include inactivated and live vaccines. H9 subtype avian influenza is a low-pathogenic avian influenza; infection in poultry causes clinical symptoms such as difficulty breathing, decreased feed intake, and decreased egg production, and it easily leads to secondary diseases, posing a significant threat. Currently, in addition to the already prevalent H5N1 and H7N9 subtypes, H9N2 remains one of the three major avian influenza subtypes threatening poultry farming. Control of H9 subtype avian influenza still mainly relies on the H9 subtype fully inactivated vaccine; antibody levels >7 log2 provide good protection.

[0003] Infectious coryza in chickens is an upper respiratory tract disease caused by *Avianella paragallinarum*. Infected chickens exhibit facial swelling, tearing, runny nose, and decreased egg production. Since 2015, serotypes A, B, and C of *Avianella paragallinarum* have been prevalent in China. Inactivated vaccines are effective in preventing infectious coryza in chickens, but there is no effective cross-protection among the three serotypes.

[0004] Currently, all infectious disease vaccines for chickens on the market are whole-cell inactivated vaccines, divided into oil-emulsion inactivated vaccines and aluminum-adjuvant inactivated vaccines. Aluminum-adjuvant vaccines can adsorb endotoxins and have good safety, but they only stimulate a Th2 humoral immune response, resulting in less protection than oil-emulsion vaccines and a shorter duration of protection. Oil-emulsion vaccines can produce higher antibody levels and stimulate a certain Th1 cell immune response, resulting in better immunity and a longer duration of protection. However, because oil-emulsion vaccines lack sustained release of bacterial endotoxins, they can easily cause a decrease in egg production in laying hens after immunization, with a reduction of 5%-20%, making them unsuitable for immunization of laying hens.

[0005] Subunit vaccines allow for control of endotoxin content in the semi-finished product during production, resulting in better safety compared to whole-cell vaccines. The outer membrane protein HMTp210 of sera-type A and C avian paraguinea has been identified as having good immunogenicity, and its expression in Escherichia coli is consistently in inclusion bodies. However, existing reports and identifications of HMTp210 in sera-type B avian paraguinea show poor immunogenicity, providing only 30-60% protection. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a trivalent inactivated vaccine against Newcastle disease, avian influenza, and infectious coryza in chickens, along with its preparation and application. This vaccine simultaneously prevents diseases caused by Newcastle disease virus, H9 subtype avian influenza virus, and three serotypes of Avianella paragallinarum (A, B, and C), reducing the number of immunizations, lowering immunization costs, and truly achieving the effect of reducing the burden of immunization. This invention is achieved through the following technical solution: This invention provides a trivalent inactivated vaccine against Newcastle disease, avian influenza, and infectious coryza in chickens, along with its preparation and application, including...

[0007] A protective antigen protein of sera-associated avian bacillus, having the amino acid sequence shown in SEQ ID NO.1.

[0008] The encoding gene for the protective antigen protein of avian bacterium paragallinarum as described above has the nucleotide sequence of SEQ ID NO.2.

[0009] The trivalent inactivated vaccine against Newcastle disease, avian influenza, and infectious coryza in chickens includes Newcastle disease strain LaSota, H9 subtype avian influenza WD strain, infectious coryza subunit protein A-HMTp210, infectious coryza subunit protein B-HA-C, and infectious coryza subunit protein C-HMTp210.

[0010] The method for preparing the chicken Newcastle disease, avian influenza, and chicken infectious coryza subunit protein triple inactivated vaccine according to claim 3 is characterized by comprising the following steps:

[0011] (1) Preparation of Newcastle disease antigen and H9 subtype avian influenza antigen in chickens,

[0012] Newcastle disease virus strain La Sota and H9 subtype avian influenza virus strain WD were cultured and propagated using the chicken embryo method. Allantoic fluid antigen was collected, and 0.2%-0.3% formaldehyde solution was added. The mixture was inactivated at 37°C for 24 hours, and the mixture was shaken every 6 hours.

[0013] (2) Preparation of subunit proteins of three serotypes of Avian bacillus paragallinarum (A, B, and C),

[0014] Engineered Escherichia coli strains BL21-A-HMTp210, BL21-B-HA-C, and BL21-C-HMTp210 were inoculated into LB medium and induced with IPTG for 5-6 h. The bacterial sludge was collected by centrifugation, resuspended in 50 mM Tris buffer (1 / 10 of the original volume), lysed, purified by ammonium sulfate precipitation, and detoxified with Triton X-114. Endotoxin levels were controlled to <2500 EU / ml. Protein concentration was determined using a BCA kit (thermo). Formaldehyde solution (0.2%-0.3% total concentration) was added, and the cultures were inactivated at 2-8℃ for 120 h. The resulting target protein solutions A-HMTp210, B-HA-C, and C-HMTp210 were obtained.

[0015] (3) Preparation of the trivalent inactivated vaccine,

[0016] Prepare the aqueous phase by adding 0.05 ml of Newcastle disease virus antigen, 0.05 ml of H9 avian influenza virus antigen, 50 μg of A-HMTp210, 50 μg of B-HA-C, and 50 μg of C-HMTp210 per milliliter of vaccine. Add 2%-4% Tween-80 to the aqueous phase, and make up the aqueous phase with PBS. Mix thoroughly to obtain the aqueous phase. Add 6%-8% Span 80 to Marcol 52 white oil and dissolve thoroughly to obtain the oil phase. Autoclave the oil phase and set aside. Add the oil to the oil phase at an oil-water ratio of 2:1-3:1 and shear at a linear velocity of 18-24 m / s for 5-40 minutes to prepare a homogeneous emulsion. This is the trivalent inactivated vaccine for Newcastle disease, H9 subtype avian influenza, and infectious coryza subunit proteins (serotypes A, B, and C). Store at 2-8℃.

[0017] The above-mentioned trivalent inactivated vaccine is used to prevent Newcastle disease, H9 subtype avian influenza, and avian paraguinea (serotypes A, B, and C).

[0018] The beneficial effects of this invention are:

[0019] The combined vaccine developed in this invention, containing subunit protein of Avianella paragallinarum, Newcastle disease virus, and avian influenza virus, provides single-dose immunization, effectively reduces vaccine endotoxins, has good safety, and will not cause a decrease in egg production when applied to laying hen flocks. Simultaneously, it prevents three diseases with a single dose, reducing the stress caused by multiple immunizations, lowering immunization costs, and truly achieving the effect of reducing the burden of immunization. It effectively prevents diseases caused by Newcastle disease virus, H9 subtype avian influenza virus, and three serotypes (A, B, and C) of Avianella paragallinarum in laying hen flocks.

[0020] It provides effective protection against Newcastle disease virus, H9 subtype avian influenza virus, and three serotypes of Avian bacillus paragallinarum (A, B, and C). The Avian bacillus paragallinarum subunit protein can also enhance the antibody levels of Newcastle disease virus and H9 subtype avian influenza virus. Attached Figure Description

[0021] The invention will now be further described with reference to the accompanying drawings.

[0022] Figure 1 PCR amplification diagram of B-HA-C gene. In the diagram, M is the molecular marker, lanes 1-3 are PCR products, and lane 4 is the negative control.

[0023] Figure 2 B-HA-C expression identification diagram. In the diagram, M is the molecular marker, and lanes 1-3 are whole cells, lysed supernatant, and lysed precipitate, respectively.

[0024] Figure 3 The expression and identification diagrams of A-HMTp210 and C-HMTp210 are shown. In the diagram, M is the molecular marker, lane 1 is the supernatant of A-HMTp210 cell lysis, and lane 2 is the supernatant of C-HMTp210 cell lysis. Detailed Implementation

[0025] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0026] A protective antigen protein of sera *Avianobacter pylori* type B, having the amino acid sequence shown in SEQ ID NO.1; wherein the sequence of SEQ ID NO.1 is:

[0027] QDTIHDAINNVLTKLISLSATEEEVVSGEPVYEPLKGAKPTVSAEANKDITGLVDVVKKANSPITVEPSTDNNKKKTFTVGLMKDIEGVNSITFDKSGQDPNQVTGRMSSAGLTFKKGDTTNGSTTTFAEDGLTIDSTTNSAQTNLVKVSRDGFSVKNGSDESKLAPTKLSIGAENAEHVEVTKSGIALKADNTSDKSRITLAQDAITLAGNATGTAIKLTGVADGNITANSKDAVNGGQLRTLLGVDSGAKIGGTEKTTISEAISDVKQALTDATLVYKADNNKGTVKLTDGLNFTSTTNIGASVEDSGVKFTLKDRTLGLKTIVTESLNASQNIIAGGTVTVGGETEGIVLTKSGSGNDRTLSLSGAGNATDGIKVSGVKAGTADTDAVNKGQLDKLFKAINDALGTTDLAVTKNPNQTSIFNPINGTAPTTFKDAVDKLTTAVNTGWGSKVGI LATGIDGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDEVSPTKTQTTAPTASSTQGGATTANTAGGVAPAGNVAMGDIAPTQPALPEMKTALVDDHLAVPLGGSLKIHGDHNVKTTISAGNQVGISLQPNISIENNLVIGSNKPEKAKLAAQEGNALVITNKDDGNAAMVFNNEKNMLVFSDKAKKPRAVLDGQNGALTLVGNDDSQVTLSSKKGKDI DGNDLSRLSVTTERNADGQLEKVETSFATMDDGLKFKADGDKVINKKLNETVEIVGDENVTTSITDDNKVKVSLNKKIAIDEVKIPNTDPDAQKGDSIVINNGGIHAGNVITGVKASDDPTSAVNRGQLNTVIDNVQNNFNQVNQRIGDLTRESRAGIAGAMATASLQNVVLPGKTTISVGTATFKGENAVAIGMSRLSDNGKVGIRLSGMSTSNGDKGAAMSVGFTF

[0028] The gene encoding the protective antigen protein of avian bacillus type B in serum as described above has a nucleotide sequence with SEQ ID NO.2; wherein SEQ ID NO.2 is:

[0029]

[0030] This invention also relates to constructing recombinant vectors containing the protective antigen protein gene of Avianella paragallinarum type B. The recombinant vectors include, but are not limited to, pET32a, pET28a, pGEX-6P-1, and pCold vectors. The above recombinant vectors were transformed into Escherichia coli BL21(DE3), and clones identified as positive by PCR were named BL21-B-HA-C as research strains.

[0031] The trivalent inactivated vaccine against Newcastle disease, avian influenza, and infectious coryza in chickens includes Newcastle disease strain LaSota, H9 subtype avian influenza WD strain, infectious coryza subunit protein A-HMTp210, infectious coryza subunit protein B-HA-C, and infectious coryza subunit protein C-HMTp210.

[0032] A protective antigen protein of Avianobacter paragallinarum type A, having the amino acid sequence shown in SEQ ID NO.3, wherein the sequence of SEQ ID NO.3 is:

[0033] MDGTITFTNIGGTGQATIHDAINNVLTKGIYLKADQNDPTGNQGQKVELGNAITLSATNQWANNGVNYKTNNLTTYNSQNGTILFGMREDPSVKQITAGTYNTTGDANNKNQLNNTLQQTTLEATGITSSVGSTNY AGFSLGADSVTFSKGGAGTVKLSGVSDATADTDAATLKQVKEYRTTLVGDNDITAADRSGGTSNGITYNLSLNKGTVSATEEKVVSGKTVYEAIRNAITGNIFTIGLDDTTLNKINNPADQDLSNLSESGKNAITG LVDVVKKTNSPITVEPSTDSNKKKTFTVGVDFTDTITEGDATDDKKLTTSKSVESYVTNKLANFSTDILLSDGRSGNATTANDGVGKRRLSDGFTIKSENFTLGSKQYNGSDSLGVMYDDQNGVFKLSLNMTALTT SLANTFAKLDASNLTDDSNKEKWRTALNVYSKTEVDAEIQKSKVTTLTPDSGLIFATKQAGSGNNAGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDEVSPTKTQTTAPTNANPTATTAPTASSTQ

[0034] A protective antigen protein of Avianella paragallinarum type C, having the amino acid sequence shown in SEQ ID NO.4, wherein the sequence of SEQ ID NO.4 is:

[0035] MDGTITFTNIGGTGQDTIHDAINNVLTKLISLSATEEEVVSGEAVYDALKGAKPTVSAEANKGITGLVDVVKKANSPITVEPSTDNNKKKTFTVGLMKDIEGVNSITFDKSGQDLNQVTGRMSSAGLTFKKGDTT NGSTTTFAEDGLTIDSTTNSAQTNLVKVSRDGFSVKNGSDESKLASTKLSIGAENAEHVEVTKSGIALKADNTSDKSSITLAQDAITLAGNATGTAIKLTGVADGNITVNSKDAVNGGQLRTLLGVDSGAKIGGT EKTTISEAISDVKQALTDATLAYKADNKNGKTVKLTDGLNFTSTTNIDASVEDNGVVKFTLKDKLTGLKTIATESLNASQNIIAGGTVTVGGETEGIVLTKSGSGNDRTLSLSGAGNAATDGIKVSGVKAGTADT DAVNKGQLDKLFKAINDALGTTDLAVTKNPNQTSIFNPINGTAPTTFKDAVDKLTTAVNTGWGSKVGILATGIDGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDKVSPTKTQTTAPTASSTQG

[0036] Using the method for constructing type B research strains, type A and type C research strains were constructed respectively: BL21-A-HMTp210 and BL21-C-HMTp210.

[0037] A method for preparing a triple inactivated vaccine against Newcastle disease, avian influenza, and infectious coryza subunit proteins in chickens, characterized by comprising the following steps:

[0038] (1) Preparation of Newcastle disease antigen and H9 subtype avian influenza antigen in chickens,

[0039] Newcastle disease virus strain La Sota and H9 subtype avian influenza virus strain WD were cultured and propagated using the chicken embryo method. Allantoic fluid antigen was collected, and 0.2%-0.3% formaldehyde solution was added. The mixture was inactivated at 37°C for 24 hours, and the mixture was shaken every 6 hours.

[0040] (2) Preparation of subunit proteins of three serotypes of Avian bacillus paragallinarum (A, B, and C),

[0041] Engineered Escherichia coli strains BL21-A-HMTp210, BL21-B-HA-C, and BL21-C-HMTp210 were inoculated into LB medium and induced with IPTG for 5-6 hours. The bacterial sludge was collected by centrifugation, resuspended in 50 mM Tris buffer (1 / 10 of the original volume), lysed, purified by ammonium sulfate precipitation, and detoxified with Triton X-114 (Shanghai Sangon Biotech). Endotoxin levels were controlled to <2500 EU / ml. Protein concentration was determined using a BCA kit (thermo). Formaldehyde solution (0.2%-0.3% total concentration) was added, and the cultures were inactivated at 2-8℃ for 120 hours. High-purity target protein solutions A-HMTp210, B-HA-C, and C-HMTp210 were obtained, hereinafter referred to as pA, pB, and pC.

[0042] (3) Preparation of the trivalent inactivated vaccine,

[0043] Prepare an aqueous phase by adding 0.05 ml of Newcastle disease virus antigen, 0.05 ml of H9 avian influenza virus antigen, 50 μg of A-HMTp210, 50 μg of B-HA-C, and 50 μg of C-HMTp210 per milliliter of vaccine. Add 2%-4% Tween-80 to the aqueous phase, and then add PBS to make up the aqueous phase. Mix thoroughly to obtain the aqueous phase. Add 6%-8% Span 80 to Marcol 52 white oil and dissolve thoroughly to obtain the oil phase. Autoclave the oil phase and set aside. Add the oil to the oil phase at an oil-water ratio of 2:1-3:1 and shear at a linear velocity of 18-24 m / s for 5-40 min to prepare a homogeneous emulsion. This is the trivalent inactivated vaccine for Newcastle disease, H9 subtype avian influenza, and infectious coryza subunit proteins (serotypes A, B, and C). Store at 2-8℃.

[0044] Preparation of the bivalent vaccine:

[0045] Prepare the aqueous phase by adding 0.05 ml of Newcastle disease virus antigen and 0.05 ml of H9 avian influenza virus antigen per milliliter of vaccine. Add 2-4% Tween-80 to the aqueous phase and mix thoroughly to form the aqueous phase. Use the same oil phase preparation and emulsification process as the above-mentioned trivalent inactivated vaccine to make a uniform emulsion, which is the Newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine. After dispensing, store at 2-8℃ for later use. Hereinafter referred to as "Newcastle disease and avian influenza bivalent inactivated vaccine".

[0046] The above-mentioned trivalent inactivated vaccine is used to prevent Newcastle disease, H9 subtype avian influenza, and avian paraguinea (serotypes A, B, and C).

[0047] Example 1:

[0048] 1. Construction of B-type protein strain of *Avianobacterium paragallinarum*

[0049] (1) Primer design

[0050] The primer sequences shown in Table 1 below were designed to express the amino acid peptide segment from the hypervariable region of the Haemagglutinin (HA) gene of Spross strain B avian bacillus to the stop codon 1591-2037, named B-HA-C. The primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. based on the reported primer sequences A-HMTp210 and C-HMTp210.

[0051]

[0052] (2) Gene amplification

[0053] The B-HA-C nucleotide fragment was amplified using the genomic DNA of *Avianobacter pylori* strain B, Spross, as a template, as shown in the attached figure. Figure 1 As shown, the A-HMTp210 nucleotide fragment was amplified using *Avianobacterium paragallinarum* strain A (Hpg-8) as a template, and the C-HMTp210 nucleotide fragment was amplified using *Avianobacterium paragallinarum* strain C (Hpg-668) as a template. The 50 μL reaction system consisted of: 25 μL 2×buffer, 1 μL high-fidelity enzyme, 1 μL dNTP, 1 μL each of upstream and downstream primers (20 μM), 2 μL template genomic DNA, and ultrapure water to a final volume of 50 μL. The PCR program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 3 min, 30 cycles; extension at 72℃ for 10 min. After electrophoresis of the PCR products, the target fragment was excised and recovered using a Tiangen gel extraction kit.

[0054] (3) Construction of cloned strains

[0055] ① Double enzyme digestion

[0056] The gel-recovered products, along with pET-28a and pET-28a-sumo plasmids, were digested with BamHI and HindIII. The 40 μL digestion system consisted of: 33 μL gene fragment and vector, 1.5 μL each of BamH1 and HindIII, and 4 μL 10×K buffer. Digestion was carried out at 37°C for 3 h. After electrophoresis, the digested products were recovered from the gel.

[0057] ② Connection transformation

[0058] The target gene fragment digested with double enzymes was ligated with the gel recovery product of the plasmid using T4 ligase. The ligation was performed overnight at 16°C according to the molar mass ratio of target gene fragment to plasmid of 3:1.

[0059] Take 5 μL of the overnight ligation product and add it to 50 μL of DH5α competent cells. Gently mix and place on ice for 30 min. Heat shock at 42℃ for 60 s and incubate on ice for 2 min. Add 500 μL of LB medium and incubate at 37℃ in a shaker for 1 h. Then take 100 μL of the bacterial culture and add it to LB agar medium containing 50 ug / mL kanamycin. Incubate overnight at 37℃.

[0060] ③ Identification of positive clone strains

[0061] Pick 2-3 colonies growing on antibiotic plates and transfer them to a culture medium containing the corresponding antibiotic. After the bacterial solution becomes turbid, use primers to amplify the target gene to perform bacterial PCR identification. Take 0.5 mL of the positive bacterial solution and send it to Sangon Biotech for sequencing.

[0062] (4) Construction of expression strains

[0063] ① Transformation

[0064] Plasmids were extracted from the correctly sequenced bacterial culture and transformed into the BL21(DE3) strain.

[0065] ② Identification of positive expression strains

[0066] PCR identification: Colonies grown on the resistant plate were subjected to bacterial PCR identification.

[0067] Induced expression identification: PCR-positive bacterial cultures were transferred to corresponding antibiotic-resistant media. When the bacterial culture reached an OD600nm ≈ 0.4-0.6, 0.1mM IPTG was added for induction for 4-6 hours. Expression identification was then performed. The engineered bacteria BL21-A-HMTp210, BL21-B-HA-C, and BL21-C-HMTp210 all expressed the target band, as shown in the attached diagram. Figure 2 and attached Figure 3 As shown.

[0068] 2. Immunogenicity identification

[0069] (1) Preparation of A-HMTp210, B-HA-C, and C-HMTp210 proteins

[0070] The engineered bacteria BL21-A-HMTp210, BL21-B-HA-C, and BL21-C-HMTp210 were inoculated into LB medium. After inducing expression with 0.1 mM IPTG for 5-6 hours at an OD600nm ≈ 0.6-0.08, the bacterial sludge was collected by centrifugation, resuspended in 1 / 10 of the original volume of pure water, homogenized under high pressure, purified by salting out with ammonium sulfate, and detoxified with Triton X-114 to obtain a high-purity target protein solution.

[0071] (2) Identification of protein immunogenicity

[0072] The expressed proteins were collected, and three vaccines were prepared with protein concentrations of 25, 50, and 100 μg / ml. 0.5 ml / bird was administered to 5-8 week old SPF chickens. Twenty-eight days post-immunization, the chickens were challenged with the virulent Spross strain of *Avianella paragallinarum*. When the concentrations of pA, pB, and pC proteins in the vaccine were all 50 μg, 100% protection was provided against virulent challenges of serotypes A, B, and C. The results are shown in Table 2.

[0073]

[0074] 3. Preparation methods of neonatal influenza trivalent inactivated vaccine and neonatal influenza bivalent inactivated vaccine

[0075] (1) Antigen preparation

[0076] ① Preparation of Newcastle disease antigen in chickens

[0077] Take the La Sota strain seed for production and dilute it with sterile physiological saline at a concentration of 10%. 4 -10 5 Inoculate 9-11 day old SPF chicken embryos with 0.1 ml per embryo and incubate at 37°C. Discard any embryos that die within 24 hours of inoculation. Place any dead embryos between 24 and 120 hours after inoculation at 4°C. Collect a mixed sample at 120 hours and determine the HA and EID of the vaccine virus. 50 ≥1:256 and ≥10 respectively 8.0 EID50 / 0.1ml. Pour the Newcastle disease virus solution with the determined potency into the inactivation vessel, add 0.2%-0.3% formaldehyde solution, turn on the stirrer to mix thoroughly, and inactivate at 37°C for 24 hours.

[0078] ② Preparation of avian influenza antigen

[0079] Take the WD strain used in production and dilute it with sterile physiological saline at a concentration of 10%. 3 -10 4 Inoculate 9-11 day old SPF chicken embryos with 0.1 ml per embryo and incubate at 37°C. Discard any embryos that die within 48 hours of inoculation. Remove any dead embryos between 48 and 120 hours after inoculation and place them at 4°C. Collect a mixed sample at 120 hours and determine the HA and EID of the vaccine virus. 50 ≥1:256 and ≥10 respectively 8.0 EID 50 / 0.1ml. Pour the Newcastle disease virus solution with the determined potency into the inactivation vessel, add 0.2%-0.3% formaldehyde solution, turn on the stirrer to mix thoroughly, and inactivate at 37°C for 24 hours.

[0080] ③ Preparation of chicken infectious coryza subunit protein

[0081] The protein solutions prepared in this example were used to determine their protein concentrations using a BCA protein concentration kit for vaccine preparation.

[0082] (2) Vaccine preparation

[0083] According to the antigen content in Table 2, an aqueous phase was prepared, and 2%-4% (by volume) of Tween 80 was added. The mixture was thoroughly stirred to dissolve the antigen, and then emulsified with the oil phase at a 3:1 volume ratio. The emulsion was sheared at a linear velocity of 18-24 m / s for 5-40 min to obtain a homogeneous emulsion. The emulsion was then quantitatively dispensed and stored at 2-8℃. Three batches of neonatal influenza trivalent inactivated vaccine were prepared, designated as batches 01, 02, and 03, respectively. One batch of neonatal influenza bivalent inactivated vaccine was also prepared, as shown in Table 3.

[0084]

[0085] 4. Vaccine testing

[0086] (1) Safety inspection

[0087] Ten 4-5 week old SPF chickens were injected subcutaneously or intramuscularly with 1.0 ml of vaccine (2 doses) each. They were observed for 14 days. No local or systemic adverse reactions caused by the vaccine were observed.

[0088] (2) Validity test

[0089] Newcastle disease section:

[0090] Ten 4-5 week old SPF chickens were each injected subcutaneously or intramuscularly with 20 μl of vaccine. Twenty-one days after vaccination, blood was collected from each chicken, along with five control chickens, and serum was separated. HI antibody titers were determined according to the current Chinese Veterinary Pharmacopoeia. Twenty-one days after immunization with the three batches of Newcastle disease-nasal trivalent inactivated vaccine, the Newcastle disease antibody levels reached 1:181-1:223, while the Newcastle disease-bivalent antibody level was 1:111. The antibody levels in the three batches of Newcastle disease-nasal trivalent inactivated vaccine group were higher than those in the Newcastle disease-bivalent inactivated vaccine group. The results are shown in Table 4.

[0091]

[0092] Avian influenza section:

[0093] Ten 4-5 week old SPF chickens were injected subcutaneously or intramuscularly with 0.5 ml of vaccine into their necks. Twenty-one days after vaccination, blood was collected from each chicken, along with five control chickens. Serum was separated, and H9 subtype avian influenza virus antigen was used to measure HI antibodies. Twenty-one days after immunization with the three batches of NIV vaccine, the geometric mean of H9 subtype avian influenza antibodies ranged from 1:2896 to 1:3327, and the NIV dual antibody level was 1:1552. The results are shown in Table 5.

[0094]

[0095] Infectious coryza in chickens:

[0096] One hundred and fifty 4-5 week old SPF chickens were immunized with three batches of the 01, 02, and 03 new-type rhinotracheitis trivalent inactivated vaccine and a control vaccine, with 0.5 ml of vaccine injected subcutaneously. Thirty control chickens were left untreated. Twenty-eight days post-immunization, ten chickens from each group were injected intraorbitally with one dose of the pathogenic Hpg-8 strain (3.5 × 10⁻⁶). 3 CFU / ml), Spross strain (7.0×10 3 CFU / m), Hpg-668 (6.5×10) 3 0.2 ml of a highly virulent strain (CFU / ml) was administered and observed for 7 days. All three batches of the novel coronavirus trivalent inactivated vaccine and the control vaccine provided 10 / 10 protection against challenges to the three serotypes A, B, and C of the highly virulent strain, indicating that the trivalent vaccine provides good protection against all three serotypes of Avian bacillus paragallinarum. The results are shown in Table 6.

[0097]

[0098] 5. The impact of vaccination on egg production rate in laying hens

[0099] Five hundred laying hens each of batches 01, 02, and 03 of the NSFW / MMR trivalent inactivated vaccine and NSFW / MMR bivalent inactivated vaccine were immunized for 200 days. Another 500 hens were immunized with a commercially available trivalent inactivated rhinitis oil emulsion vaccine. A control group of 500 hens was not immunized. Egg production was recorded one day before immunization and seven days after immunization, and the egg production rate was calculated. Results showed that the three batches of NSFW / MMR trivalent inactivated vaccine and NSFW / MMR bivalent inactivated vaccine had little impact on egg production, with no significant difference compared to the unimmunized control group (p > 0.05). However, immunization with the commercially available whole-cell trivalent inactivated rhinitis vaccine caused a short-term decrease in egg production rate, with a highly significant difference compared to the unimmunized control group (p < 0.01). The results are shown in Table 7.

[0100]

[0101] Note: The day of immunization is designated as day 0, and the day before immunization is designated as day -1. Egg production rate = number of eggs produced / number of chickens in the flock.

Claims

1. A trivalent inactivated vaccine against Newcastle disease, avian influenza, and infectious coryza in chickens, characterized by: include, The Newcastle disease strain LaSota, the H9 subtype avian influenza strain WD, the infectious coryza subunit protein A-HMTp210, the infectious coryza subunit protein B-HA-C, and the infectious coryza subunit protein C-HMTp210, wherein the infectious coryza subunit protein B-HA-C is encoded by positions 1591-2037 of the nucleotide sequence shown in SEQ ID NO.

2.

2. A method for preparing the Newcastle disease, avian influenza and infectious coryza triple inactivated vaccine of claim 1, characterized in that, Includes the following steps: (1) Preparation of Newcastle disease antigen and H9 subtype avian influenza antigen in chickens, Newcastle disease virus strain LaSota and H9 subtype avian influenza virus strain WD were cultured and propagated using the chicken embryo method. Allantoic fluid antigen was collected, and 0.2%-0.3% formaldehyde solution was added. The mixture was inactivated at 37°C for 24 hours, and the mixture was shaken every 6 hours. (2) Preparation of subunit proteins of three serotypes of Avian bacillus paragallinarum (A, B, and C), Engineered Escherichia coli strains BL21-A-HMTp210, BL21-B-HA-C, and BL21-C-HMTp210 were inoculated into LB medium and induced with IPTG for 5-6 hours. The bacterial sludge was collected by centrifugation, resuspended in 50 mM Tris buffer (1 / 10 of the original volume), lysed, purified by ammonium sulfate precipitation, and detoxified with Triton X-114. Endotoxin levels were controlled to be <2500 EU / ml. Protein concentration was determined using a BCA kit. Formaldehyde solution (0.2%-0.3% total concentration) was added, and the cultures were inactivated at 2-8℃ for 120 hours. The target protein solutions A-HMTp210, B-HA-C, and C-HMTp210 were thus obtained. (3) Preparation of the trivalent inactivated vaccine, Prepare the aqueous phase by adding 0.05 ml of Newcastle disease virus antigen, 0.05 ml of H9 avian influenza virus antigen, 50 μg of A-HMTp210, 50 μg of B-HA-C, and 50 μg of C-HMTp210 per milliliter of vaccine. Add 2%-4% Tween-80 to the aqueous phase, and make up the aqueous phase with PBS. Mix thoroughly to obtain the aqueous phase. Add 6%-8% Span 80 to Marcol 52 white oil and dissolve thoroughly to obtain the oil phase. Autoclave the oil phase and set aside. Add the oil to the oil phase at an oil-water ratio of 2:1-3:1 and shear at a linear velocity of 18-24 m / s for 5-40 min to prepare a homogeneous emulsion. This is the trivalent inactivated vaccine for Newcastle disease, H9 subtype avian influenza, and infectious coryza subunit protein. Store at 2-8℃.

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