CN116178510A

CN116178510A - Triple inactivated vaccine for newcastle disease, avian influenza and infectious rhinitis of chickens as well as preparation and application thereof

Info

Publication number: CN116178510A
Application number: CN202211554357.5A
Authority: CN
Inventors: 王楠; 崔平; 张飞; 王慧; 王成举; 乔培壮
Original assignee: Shandong Binzhou Wohua Biotech Engineering Co ltd
Current assignee: Shandong Binzhou Wohua Biotech Engineering Co ltd
Priority date: 2022-12-06
Filing date: 2022-12-06
Publication date: 2023-05-30

Abstract

The invention discloses a triple inactivated vaccine for newcastle disease, avian influenza and infectious rhinitis of chickens, and preparation and application thereof, and relates to the technical field of bioengineering. A preparation method and application of a subunit protein triple inactivated vaccine for newcastle disease, avian influenza and infectious rhinitis (A, B, C three serotypes) of chickens; comprises construction and expression of a subunit protein expression strain of the avian bacillus paragallinarum of the serum type B; preparing triple inactivated vaccine by subunit proteins of newcastle disease, H9 subtype avian influenza and chicken infectious rhinitis (A, B, C three serotypes) according to a certain proportion; the triple inactivated vaccine has good safety, can be used for preventing three serotypes of parachicken avian bacillosis of newcastle disease, H9 subtype avian influenza and A, B, C of laying hen groups, does not cause reduction of laying rate by immunization, can promote antibody levels of newcastle disease and H9 avian influenza by subunit proteins of parachicken avian bacillosis, and can protect A, B, C three serotypes of parachicken avian bacillosis by the vaccine to reach 100 percent.

Description

Triple inactivated vaccine for newcastle disease, avian influenza and infectious rhinitis of chickens as well as preparation and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a triple inactivated vaccine for newcastle disease, avian influenza and infectious rhinitis of chickens, and preparation and application thereof.

Background

Newcastle disease, a major infectious disease in poultry, has exploded in various areas of the world, including the united states, korea, china, etc., with serious economic losses to the poultry industry being secondary to avian influenza. Conventional vaccines mainly comprise inactivated vaccines and live vaccines. H9 subtype avian influenza is low pathogenic avian influenza, and infection of poultry can cause clinical symptoms such as dyspnea, reduced feeding rate, reduced laying rate and the like, and is easy to be secondary to other diseases, and has great harm. Currently, in addition to the H5N1 and H7N9 subtypes which are already popular, H9N2 is still one of three main avian influenza subtypes which harm the poultry farming industry, and the prevention and control of the H9 subtype avian influenza can be better protected when the antibody level is more than 7log2 by mainly depending on the H9 subtype total inactivated vaccine.

Infectious rhinitis of chicken is an upper respiratory disease of chicken caused by avian bacillus paragallinarum, and infected chicken has the symptoms of face swelling, tearing, nasal discharge and egg drop. After 2015, A, B, C three serotypes of avibacterium paragallinarum are popular in China. The inactivated vaccine for the disease can effectively prevent the occurrence of infectious rhinitis of chickens, and the three serotypes lack effective cross protection.

All chicken infectious vaccines in the current market are whole-cell inactivated vaccines, oil-divided emulsion inactivated vaccines and aluminum gel adjuvant inactivated vaccines. The aluminum gel adjuvant can adsorb endotoxin, has good safety, but can only stimulate Th2 humoral immune response, has a protective effect which is inferior to that of an oil emulsion vaccine and has a short duration; the oil emulsion vaccine can generate higher antibody level and stimulate certain Th1 cell immune response, has good immune effect and long duration, and is easy to cause the reduction of the laying rate of the laying hen group after immunization due to the lack of slow release of bacterial endotoxin, the reduction rate can reach 5-20%, and the oil emulsion vaccine is not suitable for immunization of the chicken group during egg laying.

The subunit vaccine can control the endotoxin content of the semi-finished product in the production process, and has better safety than the whole-thallus vaccine. The outer membrane protein HMTp210 of the A-type and C-type avian paragallinarum has been identified to have better immunogenicity, and the E.coli expression is inclusion body. The HMTp210 reported and identified by the prior art has poor immunogenicity of the B-type avian paragallinarum, and can only provide 30-60% protection.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides the triple inactivated vaccine for newcastle disease, avian influenza and chicken infectious rhinitis and the preparation and application thereof, and realizes the effects of simultaneously preventing diseases caused by infection of three serotypes of newcastle disease virus, H9 subtype avian influenza virus and A, B, C by avian bacillus paragallinarum, reducing the number of immunization times, reducing the immunization cost and really playing the role of reducing the immunization burden. The invention is realized by the following technical scheme: the invention provides a triple inactivated vaccine for newcastle disease, avian influenza and infectious rhinitis of chicken, and preparation and application thereof, comprising,

a protective antigen protein of a type B avian paragallinarum of serum has an amino acid sequence shown as SEQ ID NO. 1.

The coding gene of the protective antigen protein of the serogroup B avian paragallinarum has the nucleotide sequence of SEQ ID NO. 2.

The triple inactivated vaccine for newcastle disease, avian influenza and chicken infectious rhinitis comprises a newcastle disease LaSota strain, an H9 subtype avian influenza WD strain, a chicken infectious rhinitis subunit protein A-HMTp210, a chicken infectious rhinitis subunit protein B-HA-C and a chicken infectious rhinitis subunit protein C-HMTp210.

A method for preparing the triple inactivated vaccine of chicken newcastle disease, avian influenza and chicken infectious rhinitis subunit proteins according to claim 3, which is characterized by comprising the following steps:

(1) Preparing newcastle disease antigen and H9 subtype avian influenza antigen,

respectively carrying out proliferation culture on chicken newcastle disease virus La Sota strain and H9 subtype avian influenza virus WD strain by adopting a chick embryo method, collecting allantoic fluid antigen, adding formaldehyde solution with the total amount of 0.2% -0.3%, inactivating at 37 ℃ for 24 hours, and shaking once every 6 hours;

(2) A, B, C preparation of subunit proteins of three serotypes of avian paragallinarum,

inoculating the engineering bacteria BL21-A-HMTp210, BL21-B-HA-C and BL21-C-HMTp210 into LB culture medium, inducing with IPTG for 5-6h, centrifuging to obtain bacterial sludge, re-suspending with 50mM Tris buffer solution with the volume of 1/10, crushing, ammonium sulfate salting-out and purifying, triton X-114 detoxification, controlling endotoxin to be less than 2500EU/ml, measuring protein concentration with BCA kit (thermo), adding formaldehyde solution with the total amount of 0.2% -0.3%, and inactivating at 2-8 ℃ for 120h; preparing target protein solutions A-HMTp210 and B-HA-C, C-HMTp210;

(3) The preparation of the triple inactivated vaccine,

adding chicken newcastle disease virus antigen, H9 avian influenza virus antigen, A-HMTp210, B-HA-C, C-HMTp210 in an amount of 0.05ml, 50 mug and 50 mug into each milliliter of vaccine, preparing a water phase, adding tween-80 with the proportion of 2% -4% of the water phase, and adding PBS into the water phase, and fully mixing the water phase to obtain a water phase; adding 6% -8% span 80 into Marcol 52 white oil, dissolving fully to obtain oil phase, and sterilizing under high pressure; adding into oil phase at an oil-water ratio of 2:1-3:1, shearing at 18-24m/S linear speed for 5min-40min, and making into uniform emulsion, which is a triple inactivated vaccine of newcastle disease, H9 subtype avian influenza, and chicken infectious rhinitis subunit proteins (A, B, C serotypes), and preserving at 2-8deg.C.

The triple inactivated vaccine is applied to preventing chicken newcastle disease, H9 subtype avian influenza and parachicken bacillus (A, B, C three serotypes) diseases.

The beneficial effects of the invention are as follows:

the subunit protein of the parachicken avian bacillus, newcastle disease virus and avian influenza virus combined vaccine developed by the invention can play a role in immunization by one needle, can effectively reduce vaccine endotoxin, has good safety, can not cause the reduction of laying rate when being applied to laying hen groups, can prevent three diseases by one needle of inoculation, reduces stress caused by immunization times, reduces immunization cost, and truly plays a role in immunization burden reduction; effectively prevent diseases caused by newcastle disease virus, H9 subtype avian influenza virus and three (A, B, C) serotypes of parachicken bacillus in the laying hen group.

The chicken newcastle disease virus, H9 subtype avian influenza virus and A, B, C serotype parachicken avian bacterial infection can be effectively protected, and the subunit protein of the parachicken avian bacterial infection can also promote the antibody level of the chicken newcastle disease virus and H9 subtype avian influenza virus.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1B-HA-C gene PCR amplification diagram, in which M is molecular Marker, lanes 1-3 are PCR products, and lane 4 is negative control;

FIG. 2B-HA-C expression evaluation chart, in which M is a molecular Marker, lanes 1-3 are whole cell, broken supernatant, broken pellet, respectively;

FIG. 3 shows graphs of A-HMTp210 and C-HMTp210 expression, wherein M is a molecular Marker, lane 1 is an A-HMTp210 cell disruption supernatant, and lane 2 is a C-HMTp210 cell disruption supernatant.

Detailed Description

In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below.

A protective antigen protein of a type B avian paragallinarum of serum has an amino acid sequence shown as SEQ ID NO. 1; wherein the sequence of SEQ ID NO.1 is:

QDTIHDAINNVLTKLISLSATEEEVVSGEPVYEPLKGAKPTVSAEANKDITGLVDVVKKANSPITVEPSTDNNKKKTFTVGLMKDIEGVNSITFDKSGQDPNQVTGRMSSAGLTFKKGDTTNGSTTTFAEDGLTIDSTTNSAQTNLVKVSRDGFSVKNGSDESKLAPTKLSIGAENAEHVEVTKSGIALKADNTSDKSRITLAQDAITLAGNATGTAIKLTGVADGNITANSKDAVNGGQLRTLLGVDSGAKIGGTEKTTISEAISDVKQALTDATLVYKADNKNGKTVKLTDGLNFTSTTNIGASVEDSGVVKFTLKDRLTGLKTIVTESLNASQNIIAGGTVTVGGETEGIVLTKSGSGNDRTLSLSGAGNAATDGIKVSGVKAGTADTDAVNKGQLDKLFKAINDALGTTDLAVTKNPNQTSIFNPINGTAPTTFKDAVDKLTTAVNTGWGSKVGILATGIDGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDEVSPTKTQTTAPTASSTQGGATTANTAGGVAPAGNVAMGDIAPTQPALPEMKTALVDDHLAVPLGGSLKIHGDHNVKTTISAGNQVGISLQPNISIENNLVIGSNKPEKAKLAAQEGNALVITNKDDGNAAMVFNNEKNMLVFSDKKAKPRAVLDGQNGALTLVGNDDSQVTLSSKKGKDIDGNDLSRLSVTTERTNADGQLEKVETSFATMDDGLKFKADGDKVINKKLNETVEIVGDENVTTSITDDNKVKVSLNKKIAIDEVKIPNTDPDAQKGDSIVINNGGIHAGNKVITGVKASDDPTSAVNRGQLNTVIDNVQNNFNQVNQRIGDLTRESRAGIAGAMATASLQNVVLPGKTTISVGTATFKGENAVAIGMSRLSDNGKVGIRLSGMSTSNGDKGAAMSVGFTF

the nucleotide sequence of the coding gene of the protective antigen protein of the serogroup B avian paragallinarum has the sequence of SEQ ID NO. 2; wherein the sequence of SEQ ID NO.2 is:

CAAGATACAATCCACGATGCGATTAATAATGTTCTCACCAAATTGATCTCGCTTTCGGCAACAGAAGAAGAAGTGGTGTCAGGGGAACCTGTCTATGAACCACTTAAAGGTGCAAAACCAACGGTTTCAGCAGAAGCCAACAAAGACATTACTGGCTTGGTGGATGTGGTGAAAAAAGCAAATTCACCGATCACAGTTGAGCCTTCTACCGATAACAACAAGAAAAAAACCTTCACTGTCGGCTTAATGAAAGACATTGAAGGGGTAAACAGCATTACCTTTGATAAGTCAGGGCAAGATCCAAATCAAGTTACGGGCAGAATGAGCAGTGCGGGTTTAACCTTCAAAAAAGGCGACACAACAAATGGTTCAACCACCACTTTTGCAGAAGATGGCTTAACCATTGATAGCACAACAAATTCTGCTCAAACAAACTTAGTGAAAGTAAGTCGTGATGGCTTCTCGGTGAAAAATGGCAGCGATGAAAGCAAATTAGCCCCGACAAAATTATCTATCGGTGCGGAAAATGCAGAACACGTTGAAGTAACTAAATCGGGCATAGCCTTAAAAGCGGATAACACCTCCGATAAATCTCGCATCACCTTAGCCCAAGATGCGATTACTCTTGCGGGGAACGCAACCGGAACGGCGATTAAATTGACTGGTGTTGCAGATGGCAACATTACGGCAAATTCAAAAGATGCGGTAAATGGGGGGCAGTTGCGTACGTTATTAGGGGTTGATAGCGGGGCTAAAATTGGCGGTACTGAGAAAACAACGATCAGTGAAGCCATTTCTGATGTGAAGCAAGCTCTTACCGATGCGACATTGGTATATAAAGCGGACAATAAAAACGGTAAAACAGTTAAATTGACTGACGGATTGAATTTTACTAGCACGACCAATATTGGCGCCTCAGTAGAAGATAGTGGTGTGGTGAAATTCACCTTAAAAGATCGATTAACAGGCTTAAAAACTATCGTAACTGAGTCTTTGAATGCTTCTCAAAACATTATTGCTGGCGGCACAGTAACCGTGGGCGGCGAGACAGAGGGCATTGTGCTAACAAAATCTGGCTCAGGAAATGACCGCACTTTATCTTTATCTGGTGCAGGCAATGCAGCAACAGATGGCATTAAAGTCTCTGGCGTGAAAGCAGGGACGGCAGACACCGATGCGGTGAATAAAGGTCAGTTAGATAAACTTTTTAAAGCGATCAATGACGCATTAGGCACAACAGATTTAGCGGTAACCAAAAATCCAAATCAAACCTCTATCTTTAATCCGATAAACGGCACGGCTCCAACCACCTTTAAAGACGCGGTGGATAAATTAACCACCGCTGTGAATACAGGTTGGGGATCAAAGGTAGGTATTTTGGCAACAGGTATTGATGGTATTGATGCTGGGAATAAGAAAATTAGTAATGTCGCCGATGGGGATATTTCTCCAACCAGTGGTGATGTAGTGACAGGTCGTCAGCTCTACGCCTTAATGCAGAAAGGTATTCGCGTGTATGGTGATGAAGTTAGTCCAACGAAGACTCAAACAACAGCACCTACAGCATCTAGCACTCAAGGTGGGGCGACAACGGCGAATACGGCGGGTGGTGTAGCACCAGCAGGTAATGTAGCAATGGGGGATATTGCGCCGACACAGCCAGCATTGCCAGAGATGAAAACGGCATTGGTTGATGATCACTTGGCTGTGCCGTTAGGTGGAAGCCTCAAGATTCACGGAGATCATAATGTGAAAACAACGATTTCTGCGGGTAATCAAGTGGGGATTTCATTACAGCCAAATATTTCTATTGAGAATAACTTGGTAATTGGTTCAAATAAGCCTGAGAAGGCAAAATTAGCCGCACAAGAAGGTAATGCTTTGGTTATCACTAACAAAGATGACGGGAATGCAGCGATGGTCTTTAATAACGAGAAAAATATGCTTGTTTTCAGTGATAAAAAGGCGAAACCAAGAGCGGTTCTTGATGGACAAAATGGGGCATTAACTTTAGTCGGCAATGATGATTCTCAAGTCACCCTTTCCTCTAAGAAAGGTAAAGATATTGATGGAAATGATTTGAGCCGTCTCTCTGTGACGACTGAAAGAACAAATGCTGATGGGCAACTTGAAAAAGTGGAAACCTCATTTGCTACAATGGATGATGGCTTGAAGTTCAAAGCCGACGGGGATAAAGTGATTAATAAGAAACTTAATGAAACCGTTGAAATTGTTGGTGATGAGAATGTGACAACATCTATTACTGATGATAATAAGGTGAAAGTTTCACTGAATAAGAAAATCGCGATTGATGAGGTTAAGATTCCAAATACAGATCCTGATGCTCAAAAGGGAGATAGCATTGTAATCAACAATGGTGGAATCCACGCAGGTAATAAAGTGATTACTGGCGTTAAAGCTAGTGATGACCCAACCAGTGCGGTGAATCGAGGTCAATTAAATACCGTGATTGATAATGTTCAAAATAATTTCAATCAAGTTAATCAACGTATTGGCGATTTAACACGGGAGTCGCGTGCAGGTATTGCAGGTGCAATGGCGACGGCAAGCCTACAAAATGTTGTTTTACCAGGGAAAACAACGATTTCCGTAGGTACAGCAACGTTCAAAGGGGAGAATGCTGTTGCAATAGGGATGTCTAGACTCTCTGATAATGGAAAAGTAGGTATCCGTTTATCTGGTATGAGTACGAGTAACGGAGATAAAGGGGCAGCAATGAGCGTTGGATTTACCTTTTAG

the invention also relates to a recombinant vector which comprises the avian bacillus paragallinarum type B protective antigen protein gene. Such recombinant vectors include, but are not limited to, pET32a, pET28a, pGEX-6P-1, pCold vectors. The recombinant vector is transferred into escherichia coli BL21 (DE 3), and the cloned strain which is positive is identified by PCR and is named BL21-B-HA-C as a strain for research.

A protective antigen protein of avian bacillus paragallinarum type a, which has an amino acid sequence shown as SEQ ID NO.3, wherein the sequence of SEQ ID NO.3 is:

MDGTITFTNIGGTGQATIHDAINNVLTKGIYLKADQNDPTGNQGQKVELGNAITLSATNQWANNGVNYKTNNLTTYNSQNGTILFGMREDPSVKQITAGTYNTTGDANNKNQLNNTLQQTTLEATGITSSVGSTNYAGFSLGADSVTFSKGGAGTVKLSGVSDATADTDAATLKQVKEYRTTLVGDNDITAADRSGGTSNGITYNLSLNKGTVSATEEKVVSGKTVYEAIRNAITGNIFTIGLDDTTLNKINNPADQDLSNLSESGKNAITGLVDVVKKTNSPITVEPSTDSNKKKTFTVGVDFTDTITEGDATDDKKLTTSKSVESYVTNKLANFSTDILLSDGRSGNATTANDGVGKRRLSDGFTIKSENFTLGSKQYNGSDSLGVMYDDQNGVFKLSLNMTALTTSLANTFAKLDASNLTDDSNKEKWRTALNVYSKTEVDAEIQKSKVTLTPDSGLIFATKQAGSGNNAGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDEVSPTKTQTTAPTNANPTATTAPTASSTQ

a protective antigen protein of avian bacillus paragallinarum C, which has an amino acid sequence shown as SEQ ID NO.4, wherein the sequence of SEQ ID NO.4 is:

MDGTITFTNIGGTGQDTIHDAINNVLTKLISLSATEEEVVSGEAVYDALKGAKPTVSAEANKGITGLVDVVKKANSPITVEPSTDNNKKKTFTVGLMKDIEGVNSITFDKSGQDLNQVTGRMSSAGLTFKKGDTTNGSTTTFAEDGLTIDSTTNSAQTNLVKVSRDGFSVKNGSDESKLASTKLSIGAENAEHVEVTKSGIALKADNTSDKSSITLAQDAITLAGNATGTAIKLTGVADGNITVNSKDAVNGGQLRTLLGVDSGAKIGGTEKTTISEAISDVKQALTDATLAYKADNKNGKTVKLTDGLNFTSTTNIDASVEDNGVVKFTLKDKLTGLKTIATESLNASQNIIAGGTVTVGGETEGIVLTKSGSGNDRTLSLSGAGNAATDGIKVSGVKAGTADTDAVNKGQLDKLFKAINDALGTTDLAVTKNPNQTSIFNPINGTAPTTFKDAVDKLTTAVNTGWGSKVGILATGIDGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDKVSPTKTQTTAPTASSTQG

respectively constructing type A and type C strains for research by using a mode of constructing type B strains for research: BL21-A-HMTp210 and BL21-C-HMTp210.

The method for preparing the triple inactivated vaccine of the subunit proteins of the newcastle disease, the avian influenza and the infectious rhinitis of the chickens is characterized by comprising the following steps:

(1) Preparing newcastle disease antigen and H9 subtype avian influenza antigen,

inoculating the engineering bacteria BL21-A-HMTp210, BL21-B-HA-C and BL21-C-HMTp210 into LB culture medium, inducing with IPTG for 5h-6h, centrifuging to obtain bacterial sludge, re-suspending with 50mM Tris buffer solution with the volume of 1/10, crushing, purifying by ammonium sulfate chromatography, and detoxication with Triton X-114 (Shanghai) to obtain endotoxin with the concentration less than 2500EU/ml, measuring protein concentration with BCA kit (thermo), adding formaldehyde solution with the total amount of 0.2% -0.3%, and inactivating at 2-8deg.C for 120h; preparing target protein solutions A-HMTp210 and B-HA-C, C-HMTp210 with higher purity, and hereinafter referred to as p-A, p-B, p-C;

(3) The preparation of the triple inactivated vaccine,

adding chicken newcastle disease virus antigen, H9 avian influenza virus antigen, A-HMTp210, B-HA-C, C-HMTp210 in an amount of 0.05ml, 50 mug and 50 mug into each milliliter of vaccine, preparing a water phase, adding tween-80 with the proportion of 2% -4% of the water phase, and adding PBS into the water phase, and fully mixing the water phase to obtain a water phase; adding 6% -8% span 80 into Marcol 52 white oil, dissolving fully to obtain oil phase, and sterilizing under high pressure; adding into oil phase at an oil-water ratio of 2:1-3:1, shearing at 18-24m/S linear speed for 5-40min, and making into uniform emulsion, which is a triple inactivated vaccine of newcastle disease, H9 subtype avian influenza, and chicken infectious rhinitis subunit proteins (A, B, C serotypes), and preserving at 2-8deg.C.

Preparing a bivalent vaccine:

preparing a water phase according to the addition amount of newcastle disease virus antigen and H9 avian influenza virus antigen in each milliliter of vaccine, namely 0.05ml and 0.05ml, adding tween-80 with the proportion of 2-4% of the water phase, and fully and uniformly mixing to prepare the water phase; the oil phase preparation and emulsification process which are the same as the preparation of the triple inactivated vaccine are adopted, and the uniform emulsion is prepared into the double inactivated vaccine of newcastle disease and avian influenza (H9 subtype), and the double inactivated vaccine is split-packed and placed at 2-8 ℃ for standby, and is hereinafter referred to as 'new flow double inactivated vaccine'.

Embodiment one:

1. construction of avian bacillus paragallinarum type B protein strain

(1) Primer design

The primer sequences shown in the following Table 1 were designed to express the Haemagglutinin (HA) hypervariable region of the B-type Spross strain of avibacterium paragallinarum and the final codon 1591-2037, designated as B-HA-C, were synthesized by Shanghai Bioengineering Co., ltd.

TABLE 1 primer sequences

B-HA-C/F	CGGGATCCAAAATTAGTTATGTCGCCG
		B-HA-C/R	CCCAAGCTTCTATAAGGTAAATCCAACACT
A-HMTp210/F	CGCGGATCCGATGGCACAATTACATTTACA
		A-HMTp210/R	CCCAAGCTTACCTTGAGTGCTAGATGCTGTAGGTGC
C-HMTp210/F	CCCAAGCTTGATGGCACAATTACATTTACA
		C-HMTp210/R	CCGCTCGAGCTAACCTTGAGTGCTAGATGCTGTAGGTGC

(2) Gene amplification

The B-HA-C nucleotide fragment is amplified by taking the genome DNA of the B-type strain Spross of the avian bacterium paragallinarum as a template, as shown in figure 1, the A-HMTp210 nucleotide fragment is amplified by taking the A-type strain Hpg-8 of the avian bacterium paragallinarum as a template, and the C-HMTp210 nucleotide fragment is amplified by taking the C-type strain Hpg-668 of the avian bacterium paragallinarum as a template. The 50. Mu.L reaction system was as follows: 2 Xbuffer 25. Mu.L, high fidelity enzyme 1. Mu. L, dNTP 1. Mu.L, 1. Mu.L each of the upstream primer/downstream primer (20. Mu.M), 2. Mu.L of the template genome, and ultrapure water were fed to 50. Mu.L. The PCR reaction procedure was: 95 ℃ for 5min;95 ℃ for 30s,55 ℃ for 30s,72 ℃ for 3min,30 cycles; extending at 72℃for 10min. And (3) cutting off a target fragment after electrophoresis of the PCR product, and recovering the target fragment by using a radices tenacissima recovery kit.

(3) Construction of the clone strains

(1) Double enzyme cutting

The gel was taken and recovered and digested with BamHI and HindIII with pET-28a and pET-28a-sumo plasmids. The 40. Mu.L cleavage system was as follows: 33. Mu.L of the gene fragment and vector, 1.5. Mu.L of BamH1 and HindIII, and 4. Mu.L of 10 XK buffer. And (3) enzyme cutting for 3 hours at 37 ℃, and after electrophoresis of enzyme cutting products, recovering enzyme cutting products from the gel.

(2) Ligation transformation

The double-enzyme-cut target gene fragment is connected with the gel recovery product of the plasmid by using T4 ligase, and the target gene fragment is prepared according to the molar mass ratio: plasmid was ligated at 16℃overnight at a 3:1 ratio.

Adding 5 μl of overnight connection product into 50 μl of DH5 α competent cells, flicking, mixing, standing on ice for 30 min, heat-shock at 42deg.C for 60s, and ice-bathing for 2min; after 500. Mu.L of LB medium was added and shaking culture was performed at 37℃for 1 hour, 100. Mu.L of the bacterial liquid was added to LB agar medium containing 50 ug/mL kanamycin, and the culture was performed at 37℃overnight.

(3) Identification of Positive clone strains

2-3 bacterial colonies growing on the resistance plate are picked up, transferred to a culture medium containing corresponding antibiotics, and after bacterial liquid becomes turbid, bacterial liquid PCR identification is carried out by using a primer for amplifying a target gene, positive bacterial liquid is identified, and 0.5mL is taken for sequencing by a worker.

(4) Construction of expression strains

(1) Transformation

Plasmids in the bacterial liquid with correct sequencing are extracted and transformed into BL21 (DE 3) strain.

(2) Identification of Positive expression Strain

And (3) PCR identification: bacterial colonies growing on the resistance plate are taken and identified by bacterial liquid PCR.

Induction expression identification: and transferring the bacterial liquid with positive PCR identification into a corresponding resistant culture medium, adding IPTG with final concentration of 0.1mM to induce for 4-6h when the bacterial liquid grows to OD600nm (approximately equal to 0.4-0.6), and carrying out expression identification, wherein the engineering bacteria BL21-A-HMTp210 and BL21-B-HA-C, BL21-C-HMTp210 can express target strips, as shown in the accompanying

drawings

2 and 3.

2. Immunogenicity identification

(1) Preparation of A-HMTp210, B-HA-C, C-HMTp210 protein

The constructed engineering bacteria BL21-A-HMTp210 and BL21-B-HA-C, BL21-C-HMTp210 are respectively inoculated with LB culture medium, after the induction expression is carried out for 5-6 hours by using 0.1mM IPTG with the final concentration of 0.6 to 08, bacterial sludge is centrifugally taken out, purified water with the volume of 1/10 is used for resuspension, and after high-pressure homogenizing and crushing treatment, salting-out purification and Triton X-114 detoxification treatment are carried out by using ammonium sulfate, thus obtaining the target protein solution with higher purity.

(2) Protein immunogenicity identification

Three vaccines are prepared according to the protein content of 25, 50 and 100 mug/ml in the vaccine, SPF chickens with 5-8 weeks of age are immunized, 0.5 ml/feather is immunized, and the chicken bacillus paragallinarum virulent strain Spross strain is used for attacking 28 days after immunization. When the three proteins p-A, p-B, p-C were 50 μg in the vaccine, 100% protection was provided against virulent challenge with the three serotypes A, B, C, and the results are shown in Table 2.

TABLE 2 protection number of immunized chickens with different proteins

3. New flow nose triple inactivated vaccine and preparation method of new flow double inactivated vaccine

(1) Antigen preparation

(1) Preparation of newcastle disease antigen

Diluting La Sota strain for production with sterilized normal saline 10 ⁴ -10 ⁵ Double, 9-11 days old SPF chick embryo is inoculated, each embryo is inoculated with 0.1ml, and the incubation is continued at 37 ℃. Discarding dead embryo 24h after inoculation, timely placing dead embryo 24h-120h at 4deg.C, collecting mixed sample for 120h, and determining HA and EID of toxigenic ₅₀ Greater than or equal to 1:256 and greater than or equal to 10, respectively ^8.0 EID50/0.1ml. And (3) introducing the newcastle disease virus liquid with the measured titer into a deactivation pot, metering 0.2% -0.3% formaldehyde solution, starting a stirrer for stirring, fully mixing, and inactivating at 37 ℃ for 24 hours.

(2) Preparation of avian influenza antigen

Diluting WD strain for production with sterilized physiological saline solution 10 ³ -10 ⁴ Doubling, inoculating 9-11 day-old SPF chick embryo, inoculating 0.1ml per embryo, and incubating at 37deg.C. Discarding dead embryo 48h after inoculation, timely placing the dead embryo 48 h-120h at 4deg.C, collecting mixed sample 120h, and determining HA and EID of seedling toxin ₅₀ Greater than or equal to 1:256 and greater than or equal to 10, respectively ^8.0 EID ₅₀ 0.1ml. And (3) introducing the newcastle disease virus liquid with the measured titer into a deactivation pot, metering 0.2% -0.3% formaldehyde solution, starting a stirrer for stirring, fully mixing, and inactivating at 37 ℃ for 24 hours.

(3) Preparation of chicken infectious rhinitis subunit protein

The protein solutions prepared in this example were each subjected to protein concentration measurement using BCA protein concentration kit for vaccine preparation.

(2) Vaccine preparation

Preparing water phase according to antigen content of table 2, adding tween 80 accounting for 2% -4% of water phase volume, stirring thoroughly to dissolve, emulsifying with oil phase according to oil-water ratio of 3:1 volume, shearing at 18-24m/s linear speed for 5-40min, making into uniform emulsion, quantitatively packaging, and preserving at 2-8deg.C. Three batches of new flow nasal triple inactivated vaccine were prepared, and were respectively designated 01, 02 and 03, and 1 batch of new flow nasal triple inactivated vaccine was prepared as shown in table 3.

TABLE 3 Table of vaccine formulation information (per milliliter of vaccine addition)

Antigen information	New nose-flowing triple inactivated vaccine	New stream bivalent inactivated vaccine
			Newcastle disease	0.05ml	0.05ml
H9 avian influenza	0.05ml	0.05ml
			p-A	50μg	/
p-B	50μg	/
			p-C	50μg	/

4. Vaccine testing

(1) Safety inspection

10 SPF chickens with the age of 4-5 weeks are injected with 1.0ml (2 feather parts) of vaccine subcutaneously or intramuscularly, and the local and systemic adverse reactions caused by the vaccine do not occur after 14 days of observation.

(2) Validity check

Newcastle disease part:

10 SPF chickens of 4-5 weeks old are used, 20 μl of vaccine is injected subcutaneously or intramuscularly, and after 21 days of inoculation, 5 chickens are used together with control chickens, each chicken is respectively sampled, serum is separated, and HI antibody titer is measured according to the current Chinese animal pharmacopoeia. The antibody level of newcastle disease can reach 1:181-1:223 after the three batches of newstream nose triple inactivated vaccines are immunized for 21 days, the newstream double antibody level is 1:111, the antibody level of the three batches of newstream nose triple inactivated vaccine groups is higher than that of the newstream double inactivated vaccine groups, and the results are shown in table 4.

TABLE 4 Newcastle disease antibody levels (geometric mean)

Avian influenza part:

10 SPF chickens of 4 to 5 weeks old were used, each neck was subcutaneously or intramuscularly vaccinated for 0.5m1, and 21 days after vaccination, along with 5 control chickens, each chicken was bled separately, serum was isolated, and HI antibodies were assayed using avian influenza virus H9 subtype antigen. The H9 subtype avian influenza antibodies had a geometric mean of 1:2896-1:3327 and a new stream diabody level of 1:1552 after 21 days of immunization with three batches of new stream nasal vaccine, and the results are shown in table 5.

TABLE 5 H9 subtype avian influenza antibody levels (geometric mean)

Infectious coryza part of chicken:

150 SPF chickens, 01, 02 and 03 batches of new running nose triple inactivated vaccine and control vaccine with the age of 4-5 weeks are immunized with 30 animals, 0.5ml of vaccine is injected subcutaneously, and 30 blank control chickens are additionally arranged for no treatment. On 28 days after immunization, 10 Hpg-8 strains (3.5X10) were injected into the infraorbital sinus at 1 onset, respectively, per group ³ CFU/ml), spross strain (7.0X10) ³ CFU/m）、Hpg-668（6.5×10 ³ CFU/ml) strain virulent bacteria solution 0.2ml, and observed for 7 days. Three batches of new running nose triple inactivated vaccines and control vaccines can provide 10/10 protection against the attack of three serotypes of A, B, C virulent strains, and the result shows that the triple vaccine has better protection against three serotypes of avian bacillus paragallinarum and the result is shown in table 6.

TABLE 6 toxicity counteracting protection results for avian bacteria of paragallinarum

5. Effect of vaccine immunization on the laying rate of a group of laying hens

The three-phase inactivated vaccine and the two-phase inactivated vaccine of the new flowing nose of the 01 batch, the 02 batch and the 03 batch are respectively immunized with 500 layers of laying hens for 200 days, 500 layers of commercially available trivalent inactivated oil emulsion vaccines for rhinitis are respectively immunized, and 500 non-immunized control chicken groups are additionally arranged. 1 day before immunization and 7 days after immunization, counting the number of eggs laid, calculating the egg laying rate, and continuously counting for 7 days. The results show that the three batches of the new flow nasal triple inactivated vaccine and the new flow duplex inactivated vaccine have smaller influence on egg production after immunization, have no significant difference (p is more than 0.05) compared with the non-immune control group, and the three-phase inactivated rhinitis vaccine with the commercial whole thallus can cause the reduction of the short-time egg production rate after immunization, and have extremely significant difference (p is less than 0.01) compared with the non-immune control group, and the results are shown in table 7.

TABLE 7 variation of the laying rate (%) in the days after immunization of different vaccines

Grouping	Day-1	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
									01 batch	90.2	89.6	90.2	91.2	90.8	91.2	91.6	91.4
02 batch	90.8	90.4	90.4	90.6	90.4	90.2	90.8	90.6
									Batch 03	90.6	90	91.6	90.6	91.2	91	90.8	90.8
New stream bivalent inactivated vaccine	90.4	89.6	90.8	91.2	91.6	91.2	90.6	90.6
									Control vaccine	90.2	84.2	80.2	79.2	80.6	85.6	88.8	89.8
Non-immune control	91.2	90.6	91	90.8	90.6	90.4	90.6	91.2

Note that: day 0 of immunization, day 1 before immunization was noted as day-1. Egg yield = number of eggs/number of flocks.

Claims

1. A protective antigen protein of a type B avian paragallinarum of serum is characterized in that the protective antigen protein has an amino acid sequence shown as SEQ ID NO. 1.

2. A coding gene of the protective antigen protein of the serogroup B avian paragallinarum as claimed in claim 1, wherein the nucleotide sequence of the coding gene has the sequence of SEQ ID No. 2.

3. The triple inactivated vaccine for newcastle disease, avian influenza and chicken infectious rhinitis is characterized in that: comprises chicken newcastle disease LaSota strain, H9 subtype avian influenza WD strain, chicken infectious rhinitis subunit protein A-HMTp210, chicken infectious rhinitis subunit protein B-HA-C and chicken infectious rhinitis subunit protein C-HMTp210.

4. A method for preparing the triple inactivated vaccine of chicken newcastle disease, avian influenza and chicken infectious rhinitis subunit proteins according to claim 3, which is characterized by comprising the following steps:

(1) Preparing newcastle disease antigen and H9 subtype avian influenza antigen,

inoculating the engineering bacteria BL21-A-HMTp210, BL21-B-HA-C and BL21-C-HMTp210 into LB culture medium, inducing with IPTG for 5h-6h, centrifuging to obtain bacterial sludge, re-suspending with 50mM Tris buffer solution with the volume of 1/10, crushing, purifying by ammonium sulfate chromatography and carrying out Triton X-114 detoxification treatment, controlling endotoxin to be less than 2500EU/ml, measuring protein concentration with BCA kit (thermo), adding formaldehyde solution with the total amount of 0.2% -0.3%, and inactivating at 2-8 ℃ for 120h; preparing target protein solutions A-HMTp210 and B-HA-C, C-HMTp210;

(3) The preparation of the triple inactivated vaccine,

5. The triple inactivated vaccine of claim 4, wherein the triple inactivated vaccine is used for preventing newcastle disease, H9 subtype avian influenza and parachicken bacillus (A, B, C three serotypes) diseases.