CN107287218B - Avian infectious bronchitis virulent strain S1 gene and virulent strain and application thereof - Google Patents

Abstract

The invention relates to a virulent strain S1 gene of infectious bronchitis and a virulent strain containing the virulent strain S1 gene. The virulent strain S1 gene is homologous with the chicken infectious bronchitis attenuated strain S1 gene, and can be used for the efficacy test of an anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition. The invention also relates to a chicken infectious bronchitis low virulent strain S1 gene and a low virulent strain containing the low virulent strain S1 gene. The polypeptide coded by the low virulent strain S1 gene can stimulate an organism to generate a neutralizing antibody, and the infectious bronchitis YB160 strain containing the low virulent strain S1 gene can be used for preparing chicken infectious bronchitis vaccine compositions or subunit vaccines, can effectively prevent IB epidemic strain attack, and has better protection on glandular stomach type infectious bronchitis. The invention also relates to an infectious bronchitis diagnostic agent prepared from the infectious bronchitis attenuated strain.

Description

Avian infectious bronchitis virulent strain S1 gene and virulent strain and application thereof

The application is a divisional application of Chinese invention patent application with the application number of 201210519405.7, which is filed 12 months and 06 days 2012 and is named as 'avian infectious bronchitis attenuated strain S1 gene and attenuated strain and application thereof'.

Technical Field

The invention belongs to the technical field of poultry biopharmaceuticals, and relates to a virulent strain S1 gene of infectious bronchitis, a virulent strain S1 gene of infectious bronchitis containing chicken and application of the virulent strain in vaccine efficacy test.

Background

Infectious Bronchitis (IB) of chickens is an acute, highly contagious viral disease of chickens. In China, the respiratory tract disease variant IB is reported from 1953, the renal disease variant IB is found in Guangdong for the first time in 1982, and in recent years, IB is popular all over the country, wherein the renal disease variant is mainly used. Since 1995, Jiangsu, Shandong, Shanxi, Anhui provinces in China developed a glandular stomach pathologic change IB which mainly has the symptoms of growth retardation, extreme emaciation and diarrhea and is mainly characterized by glandular stomach swelling. The disease incidence rate is 30-50%, and the disease death rate is more than 30%. The weight of the sick chicken is increased negatively, the feed reward is obviously reduced, and the serious loss is caused to the poultry industry.

The reports on adenogastric infectious bronchitis in 1997 in Wangyondong, et al, are as follows: 25-70 days old chickens bred in chicken farms in Qingdao and nearby areas since 9 months in 1996 continuously generate an infectious disease characterized by lacrimation, eye swelling, respiratory symptoms accompanied with extreme emaciation, diarrhea and death, and the main caesarean examination symptoms are as follows: the glandular stomach is swollen like a ball, the wall of the glandular stomach is thickened, the mucosa of the glandular stomach is bleeding and ulcerated, and the pancreas is swollen and bleeds. The morbidity can reach 100 percent, and the mortality is different from 3 to 95 percent. Different breeders suffer from diseases. Research shows that the pathogeny is coronavirus, and the disease is tentatively named as avian adenogastric infectious bronchitis.

Immunization is the most effective means for the prevention and treatment of infectious bronchitis viruses. At present, some attenuated vaccines are applied clinically in the market, and some new strains are separated. Because of numerous IBV serotypes and the fact that the epidemic strains have larger variation on the gene level compared with vaccine strains, the existing vaccine strains cannot effectively prevent the attack of the IB epidemic strains, and the IB prevention effect is not ideal. For example, chinese patent CN 101514334a discloses an avian infectious bronchitis virus attenuated vaccine strain and its application; chinese patent CN 102220287A discloses a chicken infectious bronchitis cold-adapted attenuated vaccine strain and application thereof; chinese patent CN 101100656A discloses a chicken kidney type infectious bronchitis HN99 strain virus strain. The above patents all relate to some new chicken infectious bronchitis attenuated vaccine strains which can be used for preparing live vaccines, but none of the strains shows that the strains can have good protective effect on glandular stomach type chicken infectious bronchitis viruses.

Therefore, the problem is that a vaccine which can effectively prevent the attack of the IB epidemic strain and has better protection to glandular gastric type infectious bronchitis needs to be researched and developed.

Disclosure of Invention

Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide the attenuated strain S1 gene of the infectious bronchitis, and the polypeptide coded by the attenuated strain S1 gene can stimulate an organism to generate a neutralizing antibody.

The invention further provides a low virulent strain containing the S1 gene, and the low virulent strain can be used for preparing a vaccine composition for resisting the avian infectious bronchitis, can effectively prevent the attack of the adeno-gastric infectious bronchitis virus and has better protection on the adeno-gastric infectious bronchitis.

The invention also provides a subunit vaccine prepared from the chicken infectious bronchitis attenuated strain.

In particular, the present invention provides an infectious bronchitis diagnostic agent prepared from the attenuated strain of infectious bronchitis of chicken of the present invention.

The invention further provides a virulent strain S1 gene of the avian infectious bronchitis which is homologous with the attenuated strain S1 gene of the avian infectious bronchitis and a virulent strain of the avian infectious bronchitis which contains the virulent strain S1 gene, and the virulent strain can be used for the efficacy test of an anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition.

Therefore, the invention provides an infectious bronchitis low virulent strain S1 gene, the nucleotide sequence of which is shown as SEQ ID NO.1 in a sequence table.

The infectious bronchitis low virulent strain S1 gene is the gene which is most easy to be mutated in the evolution process of the virus, the encoded polypeptide can stimulate an organism to generate a neutralizing antibody, and the hypervariable region of the gene is mainly positioned in the S1 gene. Through analyzing the gene sequence of the chicken infectious bronchitis low virulent strain S1, the total sequence length of the chicken infectious bronchitis low virulent strain S1 gene is 1562 bp.

In a specific embodiment of the invention, the gene of the chicken infectious bronchitis attenuated strain S1 is isolated and purified. Firstly, extracting nucleic acid from the virus liquid of the chicken infectious bronchitis low-virulent strain, taking the nucleic acid as a template, carrying out reverse transcription, carrying out PCR amplification by using a specific primer, and then recovering a target fragment by using a PCR product purification kit to obtain the purified chicken infectious bronchitis low-virulent strain S1 gene.

IBV H52, H120 and W93 vaccine strain S1 gene sequences are downloaded from GenBank, DNASAR software is used for carrying out multiple comparison with the chicken infectious bronchitis attenuated strain S1 gene sequence, and the comparison method comprises the following steps: DNAStar software is opened, MegAlign function is selected, the Sequence to be aligned is loaded in the ' File ', By ClustalV method ' in the ' Align ' is selected for alignment, and the nucleotide genetic distance of the aligned Sequence can be seen By selecting Sequence Distances under the ' view ' (see figure 1). The homology of the chicken infectious bronchitis attenuated strain S1 gene and the common S1 gene of the chicken infectious bronchitis virus vaccine strain H52, H120 and W93 is respectively 71.4%, 71.6% and 71.3% through comparison.

The invention also provides a chicken infectious bronchitis low virulent strain containing the low virulent strain S1 gene. The low virulent strain can be used for preparing a vaccine composition for resisting avian infectious bronchitis, can effectively prevent attack of the adenovirus-associated infectious bronchitis virus, and has good protection effect on the adenovirus-associated infectious bronchitis.

The term "viral vaccine strain" as used herein refers to a viral strain used for the preparation of a vaccine.

The term "S1 protein" as used herein refers to the expression product of the S1 gene.

By "expression of a gene" is meant the transformation of genetic information stored in a DNA sequence into a biologically active protein molecule by transcription and translation of the information during the life of the cell.

In the present invention, the term "adenogastric infectious bronchitis virus, abbreviated as adenogastric infectious bronchitis virus" (IB) means that the infectious bronchitis virus of the present invention is an adenovirus infectious bronchitis attenuated strain, which has the following characteristics:

(1) the diseased chicken has swollen glandular stomach like a sphere, thickened glandular stomach wall and bleeding ulcer of glandular stomach mucosa papilla;

(2) the virus isolate interferes with the propagation of the NDV LaSota strain virus;

(3) the virus isolate does not agglutinate 1% of chicken red blood cells;

(4) the results of the specific seroneutralization tests show that virus isolates of sick chickens react positively with YB160 positive sera and negatively with positive sera of NDV LaSota strain, IBDV B87 strain, AIV H9N2 strain and EDS 76.

In the present invention, the "chicken infectious bronchitis attenuated strain" should be understood in a broad sense, and includes a chicken infectious bronchitis attenuated strain containing the S1 gene of the attenuated strain having the nucleotide sequence of SEQ ID No.1 or the S1 gene having a nucleotide sequence having homology of 80% or more with the S1 gene of the attenuated strain having the nucleotide sequence of SEQ ID No.1, preferably a chicken infectious bronchitis attenuated strain containing the S1 gene having a nucleotide sequence of homology of 90% or more with the S1 gene of the attenuated strain having the nucleotide sequence of SEQ ID No.1, more preferably a chicken infectious bronchitis attenuated strain containing the S1 gene having homology of 95% to 99% or more with the S1 gene of the attenuated strain having the nucleotide sequence of SEQ ID No. 1.

The term "homology" as used in the present invention refers to the degree of similarity of two amino acid sequences or two nucleotide sequences. The homology of amino acid sequences or nucleotide sequences can be calculated by any suitable method known in the art, for example, by aligning the target amino acid (or nucleotide) sequence with a reference amino acid (or nucleotide) sequence, introducing a gap if necessary, optimizing the number of identical amino acids (or nucleotides) between the two aligned sequences, and calculating the percentage of identical amino acids (or nucleotides) between the two amino acid (or nucleotide) sequences on the basis thereof. Alignment of amino acid (or nucleotide) sequences and calculation of homology can be achieved by software known in the art, such as, but not limited to, BLAST software (available at the website of the National Center for Biotechnology Information (NCBI): http:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi, or see, e.g., Altschul S.F.et al, J.mol. biol., 215: 403-.

In a specific embodiment, the low virulent strain is Avian infectious bronchitis virus strain YB160 (Avian infectious bronchitis virus strain YB160), and the preservation number is CCTCC NO: v201235, date of deposit: and 08/29/2012, collected in China center for type culture Collection (CCTCC; address: Wuhan university, No. 16, Lojia mountain Loe, Wuchang, Wuhan City, Hubei province).

The invention also provides an anti-infectious bronchitis vaccine composition containing the low virulent strain for infectious bronchitis.

In a preferred embodiment, the vaccine composition comprises the infectious bronchitis virus strain YB 160.

According to the invention, the vaccine composition further comprises a lyoprotectant. The lyoprotectant includes, but is not limited to, gelatin and sucrose.

In a particular embodiment of the invention, the vaccine composition consists of the infectious bronchitis virus strain YB160 and a lyoprotectant: taking a production virus seed of the infectious bronchitis virus YB160 strain, diluting the production virus seed by 10 ten thousand times by using sterilized normal saline, inoculating the production virus seed into allantoic cavities of 9-10 day-old SPF (specific pathogen free) chicken embryos, inoculating 0.1ml of the production virus seed into each embryo, sealing a pinhole after inoculation, and incubating at the temperature of 37 ℃. Discarding the dead embryos for 24h, observing the chick embryos for 72h, and introducing the chick embryos into a gas chamber for 30-72 hAnd upward, cooling for 4-24 h at 4 ℃. Collecting chick embryo allantoic fluid, mixing 6-8 chick embryo allantoic fluids into a group, placing in a sterilization bottle, storing at 2-8 deg.C, performing sterility test, and determining virus content to be 10^7.0EID₅₀0.1 ml. Mixing the chick embryo allantoic fluid qualified by aseptic inspection and virus content determination, then preparing the chick embryo allantoic fluid with a sucrose gelatin protective agent according to a ratio of 1:1(v/v), sterilizing the chick embryo allantoic fluid at 115 ℃ for 40min by using 8% (w/v) gelatin and 40% (w/v) sucrose protective agent in a freeze-drying protective agent, storing the chick embryo allantoic fluid at 4 ℃ and using up the chick embryo allantoic fluid within 72 h. The virus liquid is shaken continuously in the adding process and is fully and uniformly mixed to obtain the vaccine composition stock solution. And (4) carrying out sterile quantitative subpackage on the vaccine composition stock solution, quickly freezing, vacuum-drying, covering and sealing. The IBV of each feather of the avian infectious bronchitis vaccine composition prepared by the invention is 10^4.5EID₅₀。

In a preferred embodiment of the invention, the vaccine composition further comprises other attenuated vaccine strains. Wherein, the other attenuated vaccine strains include but are not limited to chicken Newcastle disease virus attenuated strains and/or bursa of Fabricius attenuated strains and the like. Preferably, the other attenuated vaccine strains are Newcastle disease attenuated strains.

The invention also provides application of the avian infectious bronchitis attenuated strain in preventing and treating avian proventriculic bronchitis. The use is understood to be the use in the preparation of vaccines and compositions thereof for the prevention and treatment of avian proventriculic bronchitis.

The invention provides a subunit vaccine for avian infectious bronchitis, which is characterized in that: the subunit vaccine essentially comprises the amino acid sequence of SEQ ID NO.3 of the sequence Listing.

In one embodiment of the present invention, the subunit vaccine is prepared by a method comprising the steps of:

(1) cloning the coding gene of the YB160 strain S1 protein by using a reverse transcription-polymerase chain reaction (RT-PCR) method;

(2) constructing a recombinant transfer plasmid pFASTYB160S1 containing an S1 gene;

(3) constructing a recombinant baculovirus rAcYB160S 1;

(4) infecting insect cells by the recombinant baculovirus rAcYB160S1 and culturing the insect cells;

(5) infectious bronchitis virus recombinant S1 gene expression infectious chicken bronchitis of recombinant baculovirus

The recombinant S1 protein of the tracheitis virus, and the recombinant S1 protein of the infectious bronchitis of chicken is harvested and purified;

(6) prepared by emulsifying chicken infectious bronchitis virus recombinant S1 protein and adjuvant

Avian infectious bronchitis virus subunit vaccine.

The invention also provides a chicken infectious bronchitis virulent strain S1 gene which is homologous with the S1 gene of the attenuated strain, and the nucleotide sequence of the gene is shown as SEQ ID NO.2 in the sequence table.

The invention also provides a virulent strain of infectious bronchitis containing the virulent strain S1 gene, which is characterized in that: the virulent strain is an Avian infectious bronchitis virus YBX strain (Avian infectious bronchitis virus strain YBX), and the preservation number of the virulent strain is CCTCC NO: v201236, date of deposit: and 08/29/2012, collected in China center for type culture Collection (CCTCC; address: Wuhan university, No. 16, Lojia mountain Loe, Wuchang, Wuhan City, Hubei province).

The invention further provides the application of the virulent strain in the efficacy test of the anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition. It adopts an infectious bronchitis virus YBX strain as a virulent strain to carry out a virus attack test.

In the present invention, the virulent strain is an important source of the attenuated avian bronchitis strain and the S1 gene of the attenuated avian bronchitis strain of the present invention, in addition to being used for efficacy test of an anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition. The avian infectious bronchitis virulent strain is obtained by passage weakening of an avian infectious bronchitis virulent strain of the virulent strain S1 gene. The infectious bronchitis low virulent strain S1 gene is obtained by separating and purifying the infectious bronchitis low virulent strain.

The invention also particularly provides an infectious bronchitis diagnostic agent prepared from the infectious bronchitis attenuated strain, which can be used for determining the antibody level in the infectious bronchitis vaccine efficacy test.

The gene of the avian infectious bronchitis low virulent strain S1 is the gene which is most easy to be mutated in the evolution process of viruses, the encoded polypeptide can stimulate the organism to generate neutralizing antibodies, and the hypervariable region of the gene is mainly positioned in the S1 gene. The chicken infectious bronchitis vaccine composition or subunit vaccine prepared from the chicken infectious bronchitis attenuated strain containing the attenuated strain S1 gene can effectively prevent and treat the attack of IB epidemic virus, and particularly has better protective capability on glandular stomach type infectious bronchitis.

The infectious bronchitis diagnostic agent prepared from the infectious bronchitis low-virulent strain can be used for determining the antibody level in the efficacy test of the infectious bronchitis vaccine. The infectious bronchitis virulent strain can be used for testing the efficacy of an anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition.

Drawings

FIG. 1 shows the result of comparing the nucleotide sequences of the gene S1 of the avian infectious bronchitis attenuated strain of the invention and the genes S1 of the IBV common vaccine strains H120, H52 and W93 in example 1.

FIG. 2 shows the nucleotide sequence alignment results of the F115, F125, F145, F160 and F170 viral fluid S1 genes of the IBV YB160 strain of the invention in example 2.

FIG. 3 shows the results of PCR amplification product identification of IBV S1 gene in example 5; the reference numerals in fig. 3 have the following meanings: 1IBV S1 gene amplification product; 2DNA Marker 2000.

Strain preservation

The Avian infectious bronchitis virus YB160 strain (Avian infectious virus strain YB160) was isolated and identified by Proecho bioengineering GmbH, and was preserved in China center for type culture Collection (CCTCC; address: Lopa Alkya mountain road No. 16 Wuhan university in Wuchang, Wuhan, Hubei province) at the preservation date: day 29, 08/2012, deposit number: CCTCC NO: and V201235.

An infectious bronchitis virus YBX strain (Avian infectious bronchitis virus strain YBX) is separated and identified by Proecho bioengineering GmbH, and has been preserved in China center for type culture Collection (CCTCC; address: Lopa Ganyhan, Langhua, No. 16 Wuhan university, Wuhan district, Wuhan City, Hubei province) with the preservation date: day 29, 08/2012, deposit number: CCTCC NO: and V201236.

Detailed Description

In order that the present invention may be more readily understood, the following detailed description of the invention is given, with reference to the accompanying drawings and examples, which are given by way of illustration only and are not intended to limit the scope of the invention, and the following examples are given without reference to specific experimental procedures, which are generally conducted in accordance with routine experimentation.

Examples

Example 1: preparation of attenuated vaccine strain (IBV) of infectious bronchitis virus of chicken

The IBV virulent strain is the chicken infectious bronchitis virus YBX strain

The infectious bronchitis virus strain is attenuated by passage, 9-10 day-old SPF (specific pathogen free) chick embryos are used for carrying out continuous passage attenuation on the strain, 3 chick embryos are inoculated through an allantoic cavity in each generation and continuously transmitted to 170 generations for carrying out attenuation evaluation test. The strains of F30, F65, F90, F115, F125, F145, F160 and F170 are respectively inoculated to SPF chickens of 1 day old, each chicken is dripped into the nose, orally and intramuscularly injected with 1ml, wherein the chicks inoculated with the strains of F145, F160 and F170 have no morbidity and mortality, which indicates that the strains are completely attenuated in the strains of F145, F160 and F170 and have better safety to the chickens. Meanwhile, the low virulent strain is used for immunizing a chick of 15 days old, and the homologous virulent strain is used for attacking the chick 15 days after immunization, so that the immunized chick is found to be in a good state and has no disease symptoms. While the control group had significant morbidity, which was about 30% (6-7/20) of the chickens died after challenge. The experiment is repeated for 2 times to obtain consistent results, which indicates that the attenuated strain has good immune protection, and F160 generation virus is selected as a candidate strain for vaccine development.

Example 2: the cultivation of the attenuated vaccine strain avian infectious bronchitis virus YB160 strain and the gene stability thereof

1. Materials and methods

1.1 Strain YBX attenuated by passage

YBX strains are subjected to continuous passage weakening by using SPF chick embryos of 9-10 days old. Inoculating 3 chick embryos to each secondary virus through an allantoic cavity, inoculating 0.1ml of each chick embryo, incubating at 37 ℃ for 72h (discarding dead chick embryos within 24 h), observing the chick embryos twice a day, and preserving at 4 ℃ if the chick embryos die within 24-72 h. And after 72h, taking out all the inoculated embryos, placing at 4 ℃ for 4-24 h, observing and recording the pathological change condition of the chick embryos, and aseptically collecting the chick embryo allantoic fluid for next generation passage. Chick embryos that did not die for 72h but had typical lesions and clear allantoic fluid were selected for passage.

1.2 Virus titre assay

Taking the chicken embryo allantoic fluid which is secondarily diluted by F30, F65, F90, F115, F125, F145, F160 and F170 in turn, and respectively carrying out 10 times serial dilution; 4 dilution times (10) were selected⁴～10⁷Or 10⁵～10⁸) Respectively inoculating 5 SPF (specific pathogen free) chick embryos of 9-10 days old to the virus suspension, inoculating 0.1ml of the virus suspension to each chick embryo, incubating the chick embryos in a 37 ℃ incubator for 1 week, irradiating the chick embryos every day, recording the infection number and survival number of the chick embryos within 1 week, and calculating the EID (EID) according to a Reed-Muench method₅₀。

1.3 sterility and Mycoplasma assays

The YB strains F30, F65, F90, F115, F125, F145, F160 and F170 are subjected to sterility test and mycoplasma test according to the appendix of the animal pharmacopoeia of the people's republic of China.

1.4 exogenous Virus assay

Carrying out exogenous virus detection on YB160 strains F30, F65, F90, F115, F125, F145, F160 and F170 subspecies.

1.5 evaluation of attenuation of YB160 Strain

180 SPF chickens aged 1 day are randomly divided into 9 groups (20 chickens in each group), 9 groups of chickens are respectively raised in 9 negative pressure isolators, and chicks freely eat drinking water. At 15 days of age, 1-8 groups of chicks were respectively replaced with chick embryos to be detoxified F30 (10)^6.5EID₅₀)、F65(10^7.2EID₅₀)、F90(10^7.5EID₅₀)、F115(10^7.8EID₅₀)、F125(10^8.0EID₅₀)、F145(10^8.2EID₅₀)、F160(10^8.3EID₅₀) And F170 (10)^8.2EID₅₀) Inoculating, and dripping 0.1ml of the solution into each nose; group 9 chicks served as control groups, each dropping 0.1ml of normal allantoic fluid. Observing and recording the morbidity and the mortality of chicken flocks every day from the day of inoculation, performing autopsy on the dead chickens, observing the pathological changes of IBV target organs and tissues, and attacking with homologous virulent YBX 14 days after inoculation, wherein 0.1ml of nasal drops are dripped into each eye; after counteracting toxic materials, observing and recording the disease condition and death condition of the chicken group every day, performing autopsy on the dead chicken, and observing the pathological changes of target organs and tissues.

1.6 evaluation of the immunopotency of strain YB160

1.6.115 day old SPF chicken immunopotency test

40 SPF chickens aged 1 day are randomly divided into 2 groups (20 chickens in each group), and are respectively raised in a negative pressure isolator, and the chicks eat drinking water freely. When the chicken is 15 days old, a group of chicks are inoculated by YB160 strains, and each chick is 0.1ml in nose; the other group of chicks served as a control group, and each chick had 0.1ml of normal allantoic fluid. Observing and recording the morbidity and mortality of the inoculated chicken group every day from the day of inoculation, and detecting specific antibodies of each group of collected serum 15 days after immunization, wherein the specific method is carried out by referring to the specification of the infectious bronchitis virus antibody detection kit; simultaneously, 2 groups of chicks are subjected to nasal drip attack by homologous virulent YBX, and each chick is 0.1 ml; after the challenge, the disease condition and death condition of the chicken flocks are observed and recorded every day.

1.6.23 day old SPF chicken immunopotency test

The method is the same as 1.6.1, and SPF chickens are 3 days old.

1.7 Gene stability test of YB160 Strain

The genes of S1 of strains F115, F125, F145, F160 and F170 were sequenced, and the gene stabilities were compared.

2 results

2.1 attenuation of the YB160 strain by passage

The IBV-YBX virulent strain is continuously passaged on SPF chick embryos for 170 generations, and an IBV attenuated strain with obviously weakened virulence and good immunogenicity is cultured. The experiment proves that the IBV-YB160F160 generation toxicity is obviously reduced by 10^5.5EID₅₀The inoculated SPF chicken 5/5 with the age of 1 day has no adverse reaction. IBV-YB160F160 generation virus continuously passes 5 generations of SPF chickens of 1 day age, and the virulence return is not seen. At 10³EID₅₀SPF chickens 1 day old were immunized and 10/10 protected after challenge, control 10/10 developed disease.

2.2 results of viral titer determination

The YB160 strain can be highly suitable for EID of chick embryo, F30 generation₅₀Is 10^6.50.1ml, the virus titer gradually increases after passage, and F65-F125 generations are more than or equal to 10^7.0EID₅₀0.1ml, the F145 to F160 generations tended to be stable, the titer was about 10^8.2EID₅₀/0.1ml。

TABLE 1 measurement of the chick embryo passage titer of YB160 strain

2.3 sterility and Mycoplasma assays

F30, F65, F90, F115, F125, F145, F160 and F170 generations in the process of chick embryo generation passage are tested to be free from bacterial, mould and mycoplasma pollution.

2.4 exogenous Virus testing

F30, F65, F90, F115, F125, F145, F160 and F170 generation secondary poison in the process of chick embryo generation is tested to have no exogenous virus pollution.

2.5 evaluation of attenuation of different subsynthes of strain YB160

The YB160 strain becomes progressively less pathogenic to SPF chicks after continuous passage on chick embryos. The results are shown in Table 2.

TABLE 2 virulence of different YB160 subsynes on SPF chickens

2.6 primary evaluation of the immunopotency of strain YB160

2 immunization groups of different ages in days (immunization with strain YB160F160 generation, 10)^5.0EID₅₀) And collecting chicken serum samples 15 days after immunization, and detecting by using an indirect ELISA kit to find that all the serum samples inoculated with the YB160 strains are positive to IBV antibodies, while the serum samples of a control group are negative. The immune group and the control group are attacked by homologous virulent YBX and then observed for 5 days, and the immune group chicken is found to be in a good state and has no disease symptoms; the control group had significant morbidity manifested by symptoms of depression, hunch, rough and disordered hair, mouth breathing, and about 30% (6-7/20) death after toxicity attack (see table 3).

TABLE 3 immunopotency evaluation of YB160 strains

2.7 YB160 strain gene stability:

the homology of the nucleotide sequence of the S1 gene of the F115, F125, F145, F160 and F170 strains is more than 97.5 percent (see figure 2), which indicates that the gene sequence is kept stable during the passage.

Example 3: vaccine preparation

2.1 preparation of avian infectious bronchitis live vaccine (YB160 strain)

Taking YB160 strain virus seeds (the preservation number is CCTCC.V201235), diluting the virus seeds by 10 ten thousand times with sterilized normal saline, inoculating the diluted virus seeds into allantoic cavities of SPF (specific pathogen free) chick embryos of 9-10 days old, inoculating 0.1ml of virus seeds into each embryo, sealing pinholes after inoculation, and incubating at 37 ℃. Discarding the dead embryos for 24h, observing the chick embryos for 72h, and placing the chick embryos for 30-72 h with an upward air chamber at 4 ℃ for cooling for 4-24 h. Collecting chick embryo allantoic fluid, mixing a plurality of chick embryo allantoic fluids into a group, placing the group in a sterilization bottle, storing at 2-8 ℃, simultaneously performing sterile inspection, and measuring the virus content to be 10^7.0EID₅₀0.1 ml. Mixing the allantoic fluid of chick embryo qualified by aseptic inspection and virus content determination, then mixing with sucrose gelatin protective agent at a volume ratio of 1:1, sterilizing the mixture at 115 deg.C for 40min with 8% (W/W) gelatin and 40% (W/W) sucrose protective agent, and placingStoring at 4 deg.C, and using up within 72 h. The virus solution is continuously shaken in the adding process and is fully and uniformly mixed to obtain the vaccine stock solution. And (4) packaging the vaccine stock solution in a sterile and quantitative manner, quickly freezing, drying in vacuum, and covering and sealing. The IBV virus content in each feather of the obtained avian infectious bronchitis vaccine is 10^4.5EID₅₀. Active immunoassay with lot number 07012.2

1-3 groups of 3-day-old SPF chicks are respectively inoculated with 3 batches of vaccines, and 1 using dose (namely 10) is inoculated to each nasal drop^4.5EID₅₀). Group 43 day old SPF chicks were inoculated with 1 drop of sterile saline per nasal drip. 14 days after immunization, 4 groups of chickens were inoculated with 1 drop (about 0.03ml) of YBX virulent vaccine (10 ml) by nasal drop and eye drop method^6.0EID₅₀) Simultaneously, 1.5ml is injected intramuscularly. The observation was made for 14 days and morbidity and mortality were recorded.

2.3 Passive Immunity assay

Respectively inoculating 1-3 groups of 18-week-old AA breeding hens with 3 batches of vaccines, and inoculating 1 dosage, namely 10, to each nasal drop^4.5EID₅). In the 4 th group of 18-week-old AA breeding hens, 1 drop of sterilized normal saline is inoculated to each nasal drop. After 14 days of immunization, 100 chick embryos are randomly picked up for hatching in each group, 60 hatched chicks in each group are respectively tested for maternal antibodies in 7 days, 9 days and 14 days, 1 drop of YBX virulent strain is inoculated by a nose dropping method and an eye dropping method, and 1.5ml of the virulent strain is injected intramuscularly. The observation was made for 14 days and morbidity and mortality were recorded.

3 results

3.1 active immunoassay

After each vaccine immunization group is immunized for 14 days, the group is attacked by strong toxicity, wherein the physiological saline control group is completely attacked, clinical symptoms such as mental depression, feather disorder and the like appear, and the dead chicken in the observation period is dissected to find specific pathological changes with large glandular and gastric swelling. Batch No. 201113 is live vaccine against infectious bronchitis of chicken (W93 strain) from Liaoning Yikang biological products Ltd, batch No. S111101 is live vaccine against infectious bronchitis of chicken (H52) from Qianyuan Hao, and the detailed results are shown in Table 4.

TABLE 4 active immunization efficacy test data of avian infectious bronchitis live vaccine

3.2 Passive Immunity assay

The YB160 strain infectious bronchitis vaccine can protect chickens hatched from eggs laid by healthy AA chickens within 9 days, and the virus attack protection rate is over 90 percent. The antibody detection result shows that although the detection result still keeps positive, the protection rate of the maternal antibody to virulent attack in the continuous infection process is gradually reduced. This may be related to infectious bronchitis mainly dominated by local immunity of the upper respiratory tract on the one hand, and by cellular immunity and humoral immunity as auxiliaries on the other hand, and the detection results and the toxicity attack protection results are shown in table 5.

TABLE 5 Passive immunization test results for avian infectious bronchitis vaccine (YB160 strain)

The experimental result shows that the active immune protection rate is 80%, and the passive immune can obtain more than 90% of protection within 9 days, which indicates that the strain has good protection effect on the infectious bronchitis of chicken.

Example 4: immunity efficacy experiment of new branch (NDV La Sota strain + IBV YB160 strain) dual-combined vaccine

1 Material

The application and dosage are as follows: the feather is marked according to the bottle label, diluted by normal saline, and the vaccine is absorbed by a dropping bottle, and 1 drop (about 0.03ml) is dropped into the nose of each chicken.

2 method

2.1 preparation of New Branch (NDV La Sota + IBV YB160) bivalent live seedlings

Preparing NDV virus liquid: the La Sota strain seed was diluted with a sterilized normal saline (e.g., 10-4) and inoculated with 0.1m1 per allantoic cavity. After inoculation, the pinholes are sealed, and incubation is continued at 36-37 ℃ without turning eggs. After inoculation of the embryos, eggs were given 1 time per day and embryos that died 60 hours ago were discarded. After 60 hours, the eggs are photographed for 1 time every 4-8 hours, and dead chick embryos are taken out at any time. And (3) taking out all the components until 96 hours, and cooling the components for 4 to 24 hours at the temperature of 2 to 8 ℃ with the air chamber upright whether the components die or not. The chick embryo allantoic fluid is harvested, and a plurality of chick embryo allantoic fluids are mixed into a group and placed in a sterilization bottle. Leave sample to assay HA, HA < 1: 256 should be discarded. At the same time, the bacteria should not grow when the bacteria is tested. The virus content was measured to be 108.0EID50/0.1 ml.

Preparation of IBV virus solution: the virus seeds for production are diluted by 10 ten thousand times by using sterilized normal saline, inoculated into allantoic cavities of SPF (specific pathogen free) chick embryos of 10 days old, 0.1nl is inoculated into each embryo, pinholes are sealed after inoculation, and the embryos are incubated at 37 ℃. Discarding the dead embryos for 24h, observing the chick embryos for 72h, and placing the chick embryos for 30-72 h with an upward air chamber at 4 ℃ for cooling for 4-24 h. Collecting chick embryo allantoic fluid, mixing a plurality of chick embryo allantoic fluids into a group, placing the group in a sterilization bottle, storing at 2-8 ℃, and simultaneously performing sterile inspection to obtain a virus content of 10^7.0EID₅₀/0.1ml。

Preparation of new (NDV La Sota strain + IBV YB160 strain) bivalent seedlings: NDV and IBV chick embryo allantoic fluid qualified by aseptic examination and virus content determination is mixed with sucrose gelatin protective agent according to the volume ratio of 1:1, then the mixture is mixed with the sucrose gelatin protective agent according to the volume ratio of 1:1, the freeze-drying protective agent is 8% gelatin and 40% sucrose protective agent, and the mixture is sterilized at 115 ℃ for 40min under high pressure, stored at 4 ℃ and used up within 72 h. The virus solution is continuously shaken in the adding process and is fully and uniformly mixed to obtain the vaccine stock solution. And (4) packaging the vaccine stock solution in a sterile and quantitative manner, quickly freezing, drying in vacuum, and covering and sealing. In the new branch combined vaccine prepared by the invention, the IBV in each feather of the avian infectious bronchitis vaccine is 10^4.5EID₅₀The virus content of the newcastle disease virus La Sota strain is 10^6.5EID₅₀. Lot number 0901.

The newcastle disease and infectious bronchitis bivalent live vaccine (LaSota strain + H120 strain) with the batch number of 201118 is purchased from Harbin group biological vaccine Co., Ltd, and the newcastle disease and infectious bronchitis bivalent live vaccine (LaSota strain + H52 strain) with the batch number of 110516 is purchased from Guangdong Dahua agricultural animal health product Co., Ltd.

2.2 efficacy test

2.2.1 examination with chick embryos

The vaccine is diluted to l feather/0.5 ml with normal saline and is respectively put into two test tubes, each tube lml. The first tube is added with the same amount of serum specific to the Newcastle disease virus, and the second tube is added with the same amount of serum specific to the avian infectious bronchitis virus YB 160. Neutralizing for 1 hour at room temperature (shaking for 1-2 times in the middle), wherein the virus content is 0.1 feather/0.1 ml. Continuously diluting the first tube by 10 times, inoculating 5 SPF (specific pathogen free) chick embryos of 10 days old into allantoic cavities of 3 suitable dilutions, wherein each chick embryo is 0.1ml, observing at 37 ℃ for 6 days, and calculating EID (EID) according to the sum of dead chick embryos after inoculation for 24-144 hours and specific lesion embryos with low water loss, low shrinkage and low development (the weight of the inoculated fetus is more than 2g lower than that of the lightest control fetus) in chick embryos living at 6 days₅₀. And continuously performing 10-fold serial dilution on the second tube, inoculating 5 chick embryos into 3 appropriate dilutions respectively, wherein each chick embryo is 0.1m1, death is not counted before 48 hours, the chick embryos which die after 48-120 hours are taken out at any time, harvesting chick embryo liquid, mixing the chick embryo liquid with the dilution in equal amount, taking out live embryos after 120 hours, harvesting the chick embryo liquid one by one, respectively measuring the blood coagulation price, judging the chick embryo liquid to be infected when the blood coagulation price is more than or equal to 1:128 (a micro method), and calculating EID 50.

2.2.2 Chicken test

(1) Newcastle disease component of chicken

Using 10 SPF chickens of 30-60 days old to inoculate 1/100 dose per nasal drop, and after 10-14 days, injecting 10 doses of the solution into each muscle of 3 nonimmune control chickens with the same conditions^5.0ELD₅₀The strong virus lml of the Newcastle disease Beijing strain is observed for 14 days.

(2) Infectious bronchitis part of chicken

10 SPF chickens of l-3 days old are used, 1 vaccine dosage is dripped into each nose, after 10-14 days, YBX virulent stock solutions are dripped into eyes and 1 drop into the nose respectively together with 10 control chickens, and 1.5ml of virulent stock solutions are simultaneously injected into muscles for observation for 14 days.

3 results

3.1 chick embryo inspection method

3 batches of bigeminy seedlings are respectively diluted to l feather/0.5 ml by using sterile normal saline in 1 bottle of each batch, and the diluted feather is respectively put into two test tubes, each tube lml. The first tube is added with the same amount of anti-newcastle disease virus specific serum, and the second tube is added with the same amount of anti-chicken transmissionSpecific serum of strain YB160 of infectious bronchitis virus. Neutralizing for 1 hour at room temperature (shaking for 1-2 times in the middle), wherein the virus content is 0.1 feather/0.1 ml. Continuously diluting the first tube by 10 times, inoculating 5 SPF (specific pathogen free) chick embryos of 10 days old into allantoic cavities of 3 suitable dilutions, wherein each chick embryo is 0.1ml, observing at 37 ℃ for 6 days, and calculating EID (EID) according to the sum of dead chick embryos after inoculation for 24-144 hours and specific lesion embryos with low water loss, low shrinkage and low development (the weight of the inoculated fetus is more than 2g lower than that of the lightest control fetus) in chick embryos living at 6 days₅₀. Continuously performing 10-fold serial dilution in a second tube, inoculating 5 chick embryos into 3 appropriate dilutions respectively, wherein each chick embryo is 0.1m1, death is not counted before 48 hours, the chick embryos dead after 48-120 hours are taken out at any time, harvesting chick embryo liquid, mixing the chick embryo liquid with the dilution in equal amount, taking out live embryos after 120 hours, harvesting the chick embryo liquid one by one, respectively measuring the blood coagulation price, judging the infection as the blood coagulation price is more than or equal to 1:128 (micro method), calculating EID (EID)₅₀. Checking that the virus content of each feather of the Newcastle disease part of the chicken is more than or equal to 10^6.5EID₅₀The virus content of each feather of the infectious bronchitis part of the chicken is more than or equal to 10^4.0EID₅₀. And (4) testing the efficacy of the new-branch bigeminal seedling (chick embryo method) to be qualified. The results are shown in Table 6.

TABLE 6 results of the two-way vaccine efficacy test (chick embryo method)

3.2 Chicken test method 3 two combined seedlings, each batch was diluted with sterile physiological saline in 1 bottle.

(1) Newcastle disease component of chicken

10 SPF chickens of 30 days old are inoculated with the dosage of l/100 per drop through the nose, after 10-14 days, the non-immune control chickens with the same conditions are injected with Beijing virulent lml strain of Newcastle disease containing 104.0ELD50 through each muscle, and the observation is carried out for 14 days. The control chicken 3/3 in the test chicken died, and the protection rate of the immune chicken reaches 90 percent.

(2) Infectious bronchitis part of chicken

10 SPF chickens of one day age are fed with 1 vaccine dose per nose drop, and the other 15 SPF chickens of the same day age are used as a control and are isolated and raised under the same condition. After 14 days, 10 control chickens were spotted with YBX virulent stock solutions 1 drop by drop through eyes and nose, and were subjected to intramuscular injection of 1.5ml for 14 days. The disease of the control chicken 8/10 which is attacked by the virus in the test chicken is detected, the protection rate of the immunized chicken reaches 80 percent, and the normal control chicken has no any clinical symptoms. The new-branch dual-vaccine efficacy test (chicken inspection method) is qualified, and the results are shown in tables 7 and 8.

TABLE 7 partial results of the two-vaccine efficacy test (Chicken test method) for Newcastle disease

TABLE 8 two-fold vaccine efficacy test (Chicken test) tranexamal partial results

Experimental results show that the protection rate of the new-branch bivalent live vaccine prepared by the avian infectious bronchitis YB160 strain on NDV and IBV (YBX virulent virus) virulent virus attack is over 80 percent, and the new-branch bivalent live vaccine prepared by the strain has good protection effect on newcastle disease and glandular stomach type avian infectious bronchitis.

Example 5: preparation and efficacy test of sub-unit vaccine of transmissible YB160 strain

1. Materials and methods

1.1 sequencing of S1 Gene and construction of pMDYB160S1 recombinant plasmid

Firstly, designing a primer IBV-87 (5'-TAT TGA TTA GAG ATG TTG GG-3') and a primer S1Oligo3 ' (5'-CAT AAC TAA CAT AAG GGC AA-3'), amplifying an IBVS1 gene by RT-PCR by taking the genomic RNA of an IBV-YB160 strain with the preservation number of CCTCC.V201235 as a template, and cloning the amplified S1 gene sequence into a pMD-18T vector to obtain a recombinant plasmid pMDYB160S 1.

1.2 construction of recombinant transposable plasmid

The recombinant plasmid pMDYB160S1 and the transfer vector pFASTBA are respectively enzyme-cut by BamH I and Sal I, S1 gene fragments and vector fragments are recovered, T4DNA Ligase is used for connection, escherichia coli DH5a is transformed, an LB plate containing gentamicin and ampicillin is coated for culture, plasmid DNA is extracted after bacteria selection and enrichment culture, plasmid with higher molecular weight is preliminarily screened out through electrophoresis, then single and double enzyme-cut identification is carried out on the plasmid, the insertion and connection direction of a target gene is determined, and the recombinant transfer plasmid pFASTYB160S1 is obtained.

1.3 obtaining of recombinant baculovirus rAcYB160S1

pFASTYB160S1 was transformed into E.coli DH10Bac competent cells (purchased from Invitrogen) containing viral shuttle plasmid Bacmid to obtain recombinant shuttle plasmid rBacmidYB160S 1; PCR screening, extracting positive plasmid, and preparing positive recombinant plasmid rBacmidYB160S1 DNA. By using

The Reagent transfected Sf9 cells (purchased from Invitrogen company), after the cells appeared diseased, recombinant virus plaque purification and virus amplification were performed, and then PCR was performed by using universal primers M13F and M13R to verify the objective recombinant S1 gene. The recombinant baculovirus obtained by purification was named rAcYB160S 1. The amplified recombinant baculovirus rAcYB160S1 is used as seed virus, titer titration is carried out by a plaque method, and then the virus is stored at 4 ℃ for standby.

1.4 expression and identification of recombinant S1 protein

rAcYB160S1 recombinant baculovirus was infected with suspension-cultured insects High FiveTM (purchased from Invitrogen). High FiveTM cells were aseptically suspension cultured in 500ml roller bottles containing 200-350 ml of EXPRESS FIVE SFM medium, and seeded at a cell density of 0.3-0.6X 106 cells/ml. When the cell density reaches 1X 10⁶When each cell/ml is detected, each spinner flask is inoculated with the recombinant virus rAcYB160S1, the recombinant baculovirus inoculated in each spinner flask has different multiplicity of infection (M.O.I), which is 0.01, 0.1 and 1 respectively, after the recombinant baculovirus is inoculated, the cells are cultured for 4 days at the temperature of 26-28 ℃ and the rotating speed of 100rpm。

Each vial was sampled every 12 hours post inoculation, i.e. 48h, 60h, 72h, 84h and 96 h. Each sample was centrifuged at 10000g for 20min and the supernatant and pellet were separated. Filtering the supernatant through a filter membrane of 1um, and performing SDS-PAGE electrophoresis and an ultraviolet spectrophotometer to determine the avian infectious bronchitis virus recombinant S1 protein and the content thereof in the supernatant.

Western blotting is adopted to identify the expressed avian infectious bronchitis virus recombinant S1 protein. High FiveTM cells infected by the recombinant viruses are collected respectively, centrifuged at 3000r/min for 10min, and supernatants are taken for SDS-PAGE electrophoresis.

1.5 harvesting and purification of avian infectious bronchitis Virus recombinant S1 protein

Centrifuging the culture for 20min by 10000g, separating precipitate and supernatant, filtering the supernatant through a filter membrane of 1um, concentrating and purifying by ultrafiltration, wherein the aperture of the ultrafiltration membrane is 10000 (cut molecular weight), inactivating the recombinant baculovirus vector by 7mM of diethyleneimine (BEI) in the purified sample solution, and adding equivalent sodium thiosulfate for neutralization for 36-48 hours.

1.6 preparation of avian infectious bronchitis subunit vaccine

Antigen dilutions were prepared at different concentrations: the recombinant S1 protein was diluted with PBS (pH7.4) to 0.5. mu.g/ml, 1. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml.

The antigen dilutions above were mixed and emulsified with oil adjuvant ISA 206. ISA206 adjuvant is sterilized by moist heat at 121 ℃ and 60Min high pressure, and the ratio of antigen: adjuvant volume ratio 46: 54. Adding adjuvant into adjuvant cylinder of emulsifying machine, slowly dripping antigen water phase into adjuvant under stirring (100r/min), and stirring for 5 min. And (4) circularly emulsifying for 4min by using an emulsifying machine at 5000r/min to obtain the subunit vaccine.

1.7 potency assay for IBV subunit vaccines

a. Immune response and antibody level detection of subunit vaccine in SPF (specific pathogen free) chicken

The test chickens are 15 Specific Pathogen Free (SPF) chickens of 21 days old, 10 immunization groups and 5 control groups, 1 feather of infectious bronchitis live vaccine (H120 strain) is inoculated to the test chickens through nasal drip, blood is collected 21 days after inoculation, and 0.3ml of subunit vaccine is injected subcutaneously or intramuscularly to each test chicken. SPF chickens are raised in isolators. And collecting blood 28 days after injection, separating serum, and measuring HI antibody titer of the two sera respectively. The geometric mean value of the HI antibody titers of the hyperimmune serum should not be less than 4-fold that of the primary serum.

HI antibody detection method:

(1) the chicken infectious bronchitis HI antigen hemagglutination valence determination method comprises the steps of taking 1V-shaped micro reaction plate with 96 holes, adding 25 mu L of PBS (0.01mol/L, pH value 7.0-7.4) into each hole, adding 25 mu L of antigen into the 1 st row, making 2-4 repeated holes, then carrying out 2-time serial dilution on the antigen, adding 25 mu L of PBS (0.01mol/L, pH value 7.0-7.4) into each hole after dilution, finally adding 25 mu L of 1% chicken erythrocyte suspension, uniformly mixing by using a micro oscillator, standing for 40 minutes at 2-8 ℃, and determining a result, wherein the highest dilution multiple of 100% erythrocyte agglutinated antigen is used as a determination end point.

(2)4HA antigen 4HA unit antigen was prepared with PBS (0.01mol/L, pH 7.0-7.4) according to the measured HI antigen HA titer. The prepared 4HA unit antigen is diluted by PBS (0.01mol/L, pH value is 7.0-7.4) to the dilution degree of 1:2, 1:3, 1:4, 1:5, 1:6 and 1: 7. Adding 25 mul PBS (0.01mol/L, pH value 7.0-7.4) into 25 mul antigen of each dilution, adding 25 mul of 1% chicken red blood cell suspension, mixing uniformly, standing for 40 minutes at 2-8 ℃, and judging the result. If the 1:4 dilution is 100% erythrocyte agglutination end point, it indicates that 4HA unit antigen is prepared; if the 100% hemagglutination endpoint is 1:5, 1:6, it indicates that the formulated 4HA unit antigen is actually higher than 4 units; if the 100% hemagglutination endpoint is 1:2, 1:3, it indicates that the 4HA unit antigen formulated is actually less than 4 units. The antigen working solution should be determined to be 4HA units by appropriate adjustment according to the test result.

(3) Hemagglutination inhibition assay (HI)

Firstly, a 96-well V-shaped micro reaction plate is taken, and 25 mu L of PBS (0.01mol/L, pH value is 7.0-7.4) is added into each well.

② respectively sucking 25 mul of serum to be detected, adding into each corresponding hole of the 1 st row of each plate, setting standard positive serum and negative serum contrast on each plate, and then serially diluting by 2 times.

③ respectively adding 25 mu l of 4HA unit antigen into each hole, and standing for 30 minutes at 2-8 ℃.

And fourthly, adding 25 mu l of 1% (V/V) chicken red blood cell suspension into each hole, gently mixing the mixture evenly, and standing the mixture for 40 minutes at the temperature of between 2 and 8 ℃.

The result is judged that the reaction plate is inclined, and the hemagglutination inhibition is judged when the red blood cells in the serum reaction hole and the red blood cell contrast hole flow from the bottom of the hole at the same speed. The assay may be established when the negative serum HI titer is not greater than 1:8 and the error in the positive serum HI titer compared to the specified titer is not greater than 1: 2. The highest dilution of serum that completely inhibited 4HA unit antigen was taken as HI titer.

b. IBV virus challenge test after immunization of SPF (specific pathogen free) chicken with subunit vaccine

The immunized chicken and non-immunized chicken in the above item "a" were subjected to eye drop and nose drop with YBX virulent stock solution 1 drop each 28 days after the second immunization, and were subjected to intramuscular injection of 1.5ml, followed by 14 days of observation.

2 results

2.1 construction and identification of recombinant plasmid PCR product size about 1700bp, the results are shown in figure 3, wherein, the 1 st hole is IBV S1 amplification product; the 2 nd well is a DNA Marker 2000. Sequencing the PCR product, and obtaining the result of electrophoresis and sequencing that the recombinant vector pMDYB160S1 contains the sequence SEQ ID NO.1 in the sequence table.

2.2 the identification of the recombinant transposable plasmid is verified by enzyme digestion, the insertion direction of the S1 gene is correct, and the size of the enzyme digestion fragment is consistent with the expected result.

2.3 transfer vector comprising the infectious bronchitis Virus S1 Gene was transformed into E.coli competent cells containing a shuttle vector to obtain a shuttle vector comprising the infectious bronchitis Virus S1 gene, which was transfected into insect cells (purchased from Invitrogen) and cell death began to occur 24h after transfection, after which the cells tended to continue to proliferate. After 72h, the cells begin to have lesions, the cell nuclei become enlarged, the lesions gradually increase, after 96h, the cells begin to die, and after 1 week, the cells die basically.

2.4 expression and characterization of recombinant S1 protein

The content of the recombinant protein in the supernatant reaches 60 micrograms/ml after the recombinant virus infects the cells for 72 hours. The results show that after the recombinant virus infects cells, the yield of the chicken infectious bronchitis virus recombinant S1 protein can be remarkably improved by recovering culture supernatant after culturing for at least 72 h.

A specific band with the size of about 56KDa is observed in SDS-PAGE protein electrophoresis gel, and the specific band is the recombinant avian infectious bronchitis virus S1 protein. Sequencing the target protein, wherein the protein sequence of the recombinant avian infectious bronchitis virus S1 contains a sequence SEQ ID NO.3 in the sequence table according to electrophoresis and sequencing results.

2.5 recombinant protein harvesting and purification: the content of the purified protein was determined by UV spectrophotometer to reach 68. mu.g/ml.

2.6 testing of subunit vaccines: after the prepared vaccine is subjected to viscosity detection, centrifugal detection, dosage form detection and stability detection, all indexes of the prepared vaccine meet the regulations. The vaccine was stored at 4 ℃.

The protein antigen produced by the method can be mixed with other oily adjuvants, aqueous adjuvants or other adjuvants, such as white oil adjuvants, alumina gel adjuvants, Freund's adjuvants and the like which are common in the field.

2.7 efficacy test results for subunit vaccines

The antibody detection results after subunit vaccine immunization are shown in table 9, and the results in the table show that the geometric mean value of the HI antibody titer of the serum after the second immunization is not lower than 4 times of that of the serum of the first immunization.

TABLE 9 detection of anti-IBVHI antibody production in SPF chickens immunized with the subunit vaccine

In the challenge protection test of the subunit vaccine, the protection rate of the immunized chicken reaches 90 percent when the challenge control chicken 5/5 is tested to be attacked, and the normal control chicken has no clinical symptoms, and the specific results are shown in table 10.

TABLE 10 IBV challenge test results after immunization of SPF chickens with subunit vaccines

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

<110> Puleco bioengineering GmbH

<120> avian infectious bronchitis virulent strain S1 gene and virulent strain and application thereof

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<170> PatentIn version 3.5

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1 5 10 15

Ser Ala Phe Arg Pro Ala Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala

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Val Val Asn Val Ser Leu Glu Thr Asn Asn Ala Gly Thr Ala Ser Gln Cys

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Ile Ala Gly Ala Ile Ser Trp Ser Lys Asn Phe Ser Ala Ser Ala Val Ala

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Met Thr Ala Pro Glu Leu Gly Met Thr Trp Ser Thr Gly Gln Phe Cys Thr

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Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys Phe Lys

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His Gly Asp Gly Leu Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly Phe Ile

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Arg Val Ser Ala Met Arg Lys Gly Ser Asn Ser Leu Phe Tyr Asn Leu Thr

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Val Ser Val Thr Lys Tyr Pro Arg Phe Lys Ser Leu Gln Cys Val Asn Asn

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Tyr Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr Ser Asn Glu Thr

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Lys Pro Val Ser Ala Ala Gly Val Ser Phe Thr Ala Gly Gly Pro Ile Thr

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Tyr Lys Thr Met Ser Glu Val Lys Val Leu Ala Tyr Phe Ala Asn Gly Thr

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Ala Gln Thr Val Ile Pro Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys

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Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Tyr Thr Asn Ser

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Ser Leu Val Lys Glu Arg Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr

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Thr Leu Val Leu Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Pro Pro

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Asn Thr Gly Gly Val His Ser Ile Val Leu His Gln Thr Gln Thr Thr Gln

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Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Arg Tyr Val

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Glu Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Ser Phe Arg Leu

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Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Gly

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Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Asn Met Ala

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Thr Cys Cys Tyr Ala Tyr Ser Tyr Ser Gly Pro Thr Leu Cys Lys Gly Val

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Tyr Ser Gly Glu Leu Gln Lys Thr Phe Glu Cys Gly Leu Leu Val Phe Val

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Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn Glu Pro Leu Val Leu

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Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Lys Cys Val Glu Tyr Asn

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Ile Tyr Gly Arg Val Gly Gln Gly Leu Ile Thr Asn Ile Thr Asp Ser Ala

430 435 440

Ala Asn His Gly Tyr Leu Ala Asp Gly Gly Leu Ala Val Leu Asp Thr Ser

445 450 455

Gly Ala Ile Asp Val Phe Val Val Gln Gly Val Tyr Gly Leu Thr Tyr Tyr

460 465 470 475

Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly

480 485 490

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

495 500 505 510

Glu Asn Arg Phe Tyr Val Lys Phe Pro Asn

515 520

Claims (3)

1. A virulent strain S1 gene of infectious bronchitis, the nucleotide sequence of which is shown as SEQ ID NO.2 in a sequence table, the virulent strain S1 gene is homologous with an attenuated strain S1 gene of infectious bronchitis of chicken shown as SEQ ID NO.1 in the nucleotide sequence table, the virulent strain S1 gene can be used for the efficacy test of an anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition, the anti-infectious bronchitis vaccine or subunit vaccine or vaccine composition can prevent the attack of the adenovirus-associated bronchitis virus, and the adenovirus-associated bronchitis virus has the following characteristics: the diseased chicken has swollen glandular stomach like a sphere, thickened glandular stomach wall, bleeding and ulcer of glandular stomach mucosa papilla.

2. A virulent strain of infectious bronchitis chicken comprising the virulent strain S1 gene of claim 1, wherein: the virulent strain is an infectious bronchitis virus YBX strain, and the preservation number is as follows: CCTCC NO: v201236, deposited in China center for type culture Collection.

3. Use of a virulent strain according to claim 2 for the preparation of a medicament for efficacy testing against an infectious bronchitis vaccine or subunit vaccine or vaccine composition.

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