Avian infectious bronchitis subunit vaccine based on nano-skeleton technology

Abstract

The invention relates to an infectious bronchitis subunit vaccine based on a nano-skeleton technology. Specifically, the invention provides a fusion protein which sequentially comprises an infectious bronchitis virus S protein fragment or a mutant thereof, a connecting peptide and helicobacter pylori ferritin from an N terminal to a C terminal, wherein the infectious bronchitis virus S protein fragment is S1 protein, and the amino acid sequence of the S protein fragment is as shown in SEQ ID NO. 2; the mutant of the chicken infectious bronchitis virus S protein fragment is S1-2 protein or S1-3 protein, the amino acid sequence of the S1-2 protein is shown as SEQ ID NO.4, and the amino acid sequence of the S1-3 protein is shown as SEQ ID NO.5. The invention further discloses a preparation method of the chicken infectious bronchitis virus S protein fragment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a recombinant protein subunit vaccine of chicken infectious bronchitis virus (IBV) and its preparation method. Background Technology

[0002] Avian infectious bronchitis (IB) is an acute, highly contagious disease of chickens caused by the Avian infectious bronchitis virus (IBV). The World Organisation for Animal Health (OIE) has listed IB as a notifiable animal disease, and my country has classified it as a Class II animal disease.

[0003] This disease is characterized by respiratory symptoms, nephritis, and decreased production performance. In particular, nephropathy-associated infectious bronchitis (IBV) in broilers, characterized by kidney swelling, causes high mortality rates, exceeding 30% in white-feathered broilers and around 15% in high-quality broilers. Affected broilers experience reduced weight gain and feed conversion ratios. Mixed infections with E. coli and mycoplasma can lead to air sacculitis, further degrading broiler quality. In laying hens, IBV infection, besides causing death, can cause permanent, irreversible damage to the reproductive system, manifesting as underdeveloped oviducts and ovaries, or extensive hydrocele and cysts in the oviducts and uterus, resulting in pseudo-hens. This manifests as no peak egg production, decreased egg quantity and quality, and the production of white-shelled, soft-shelled, rough-shelled, and deformed eggs. Some IBV strains can also cause lesions in the intestines, proventriculus, and muscles. IBV can harm chickens of all ages and breeds, causing enormous economic losses to the global poultry industry and is a major infectious disease seriously affecting the world's poultry sector. Clinical signs of IB include sneezing, tracheal rales, nasal discharge, and wheezing. Broiler poultry experience reduced body weight, while laying hens produce fewer and lower-quality eggs. Respiratory infections make chickens susceptible to secondary bacterial infections, which can be fatal. The virus can also cause permanent damage to the oviducts, particularly in chickens, leading to reduced egg production and quality; and permanent damage to the kidneys, sometimes resulting in fatal kidney disease.

[0004] IBV has been reported to cause more economic losses to the poultry industry than any other infectious disease. To date, vaccination remains the most common and effective measure for controlling IB in production. Currently, IBV vaccines submitted for approval in China are mainly divided into: 1) Whole-virus inactivated vaccines: Whole-virus inactivated vaccines induce a shorter duration of immune response and only induce antibody production, without mediating T-cell immune responses. Their immunization effect is determined by both the dosage and the antigen content in the vaccine. Immunization often requires doses many times higher than live vaccines, and adjuvants must be added, which may increase vaccine production costs and thus limit their widespread application. 2) Attenuated live vaccines: The preparation process is time-consuming and labor-intensive; there are risks of in vivo mutation, recombination, and virulence reversion; maternal antibody neutralization reduces vaccine response; some recombinant DNA vaccines require effective vector delivery; the preparation technology is stringent; post-translational protein modifications may alter protein immunogenicity.

[0005] Subunit vaccines contain the natural components of a complete organism, consisting only of the major viral surface proteins. This avoids the production of antibodies induced by many irrelevant antigens, reducing vaccine side effects, vaccine-related diseases, and virulence reversion. The advantages of subunit vaccines include good safety, reducing or eliminating pyrogens, allergens, immunosuppressants, or other harmful reactants that are difficult to avoid with conventional live or inactivated vaccines; good vaccine stability, facilitating storage and transportation; and the immune response produced can be distinguished from that produced by wild-type virus infection, which is beneficial for disease control and eradication. They can also be mass-produced. However, subunit vaccines also have significant drawbacks: high production costs, lower immunogenicity compared to attenuated and inactivated vaccines, and limited application. As a third-dose booster vaccine, subunit vaccines have a large market potential and are of great significance.

[0006] IBV belongs to the Coronaviridae family and is a representative strain of coronaviruses. The IBV genome primarily encodes four structural proteins: S, N, M, and E proteins, with S, M, and N being more abundant in mature viral particles. The S protein, encoded by the IBV S gene, is a highly glycosylated transmembrane protein, a major component of the outermost spikes of the coronavirus, located on the viral surface. It contains antigenic sites related to viral neutralization, induction of host hemagglutination and inhibition of antibody production, cell adhesion, tissue tropism, and serotype, and is also the most variable structural protein in IBV. It contains the main IBV antigenic neutralizing epitopes, which can stimulate the body to produce specific neutralizing antibodies. After translation, the S gene is cleaved by host cell proteases into two parts, S1 and S2 proteins, linked by disulfide bonds. Both are glycoproteins that mediate viral infection of susceptible cells. The S1 protein, located at the amino terminus, is one of the most important immunogenic components of the virus, mainly forming the outer functional region of the S protein and its external globular structure, playing a crucial role in mediating viral infection of cells. It contains antigenic epitopes that induce the production of neutralizing antibodies and binds to receptors through virus-cell interactions. Furthermore, the S1 protein is associated with the tissue affinity and virulence of the strain and is a major protein determining the IBV serum-specific antigenic determinant.

[0007] IBV M41 is a classic strain of infectious bronchitis virus (IBV) in chickens. This strain is widely used in China, exhibits good immunogenicity and stable biological characteristics, and is currently the main vaccine strain used in my country for producing inactivated vaccines containing IBV antigen components. This strain has strong pathogenicity in chickens. Therefore, cloning and expressing the S1 protein of IBV type M41 provides guidance for clinical vaccine use.

[0008] Existing infectious bronchitis (IBB) vaccine technologies for chickens all have significant shortcomings: while whole-virus inactivated vaccines are relatively safe, their immune response is limited, their protection period is short, and their production costs are high; while attenuated live vaccines have good immunization effects, they pose safety risks such as virulence reversion and genetic recombination, and are susceptible to interference from maternal antibodies; traditional subunit vaccines have the highest safety profile, but their core bottleneck lies in their weak immunogenicity, making it difficult to effectively induce full immunization. Furthermore, all technologies face a common challenge: the critical S1 antigen protein of the virus mutates rapidly, and the correct three-dimensional conformation upon which effective immunization depends is difficult to maintain and reproduce in traditional production systems, resulting in often less than ideal vaccine protective effects. Ultimately, existing technologies have failed to achieve an ideal balance between "absolute safety" and "highly effective protection" in vaccines. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a fusion protein that, as a subunit vaccine against IBV, can increase antibody expression levels and neutralizing antibody titers, thereby enhancing immunogenicity.

[0010] In one aspect of the invention, a fusion protein is provided, comprising, from N-terminus to C-terminus, a fragment of chicken infectious bronchitis virus S protein or a mutant thereof, a linker peptide, and Helicobacter pylori ferritin; wherein...

[0011] The S protein fragment of the chicken infectious bronchitis virus is the S1 protein, and its amino acid sequence is shown in SEQ ID NO.2;

[0012] The mutant of the S protein fragment of the chicken infectious bronchitis virus is either the S1-2 protein or the S1-3 protein, wherein the amino acid sequence of the S1-2 protein is shown in SEQ ID NO.4, and the amino acid sequence of the S1-3 protein is shown in SEQ ID NO.5.

[0013] Specifically, the inventors analyzed the amino acid sequence of the natural S protein of IBV and determined the amino acid fragment of the S1 protein. Starting from the S1 protein sequence, S1-1, S1-2, and S1-3 proteins were obtained through amino acid mutations at different sites, and SS1, SS2, and SS3 proteins were obtained through different truncations. The obtained S1 proteins and their mutants or truncated forms were used as vaccine components.

[0014] Furthermore, to enhance the immunogenicity of antigen fragments, this invention fuses the S1 protein and its mutants or truncated forms with Helicobacter pylori ferritin. Helicobacter pylori ferritin is used for the self-assembly of antigen proteins. The Helicobacter pylori ferritin polymerization platform has been used to display antigens such as influenza, HIV-1, and EB virus. The principle is that Helicobacter pylori ferritin self-assembles to form 24 subunit particles with eight triplet symmetry axes. By fusing a single protozoan of a viral glycoprotein to the N-terminal region of the Helicobacter pylori ferritin subunit, it facilitates the assembly of protein nanoparticles displaying eight copies of the trimer antigen at three-fold symmetry axes on the surface. Displaying antigens on ferritin generally elicits a stronger neutralizing antibody response against the target pathogen compared to immunization with antigens alone. Importantly, two influenza functionalized ferritin vaccines have been demonstrated to be safe and immunogenic in clinical trials (NCT03186781 and NCT03814720), and a stable foundation for the large-scale manufacture of ferritin vaccines has been established based on this. In this invention, a modified Helicobacter pylori ferritin polymerization platform was established by exemplarily using the modified Helicobacter pylori ferritin, whose amino acid sequence is shown in SEQ ID NO: 17 (see CN112552413A).

[0015] In this invention, the S1 protein and its mutants or truncated forms are combined with the above-mentioned modified Helicobacter pylori ferritin polymerization platform to obtain a fusion protein of the S1 protein and its mutants or truncated forms with Helicobacter pylori ferritin, and the fusion protein is further expressed through the CHO expression system.

[0016] The nucleotide sequence of the S1 protein is shown in SEQ ID NO.10, the nucleotide sequence of the S1-2 protein is shown in SEQ ID NO.12, the nucleotide sequence of the S1-3 protein is shown in SEQ ID NO.13, and the nucleotide sequence of the Helicobacter pylori ferritin is shown in SEQ ID NO.18.

[0017] In a specific embodiment of the present invention, the linker peptide is selected from: [A(EAAAK)nA], (GGGGS)n, (G)n, (XP)n, and preferably its amino acid sequence is shown in SEQ ID NO.19.

[0018] In a specific embodiment of the present invention, the fusion protein further includes a purification tag, such as His, Fc, HA, GST, Flag, MBP, or FLAG tag.

[0019] For convenience, the term "S protein" is sometimes used below to refer to the S proteins and fragments (S, S1, S1-1, S1-2, S1-3, SS1, SS2, SS3 proteins) of the present invention.

[0020] This invention generates functionalized nanoparticles by transfecting a single plasmid encoding a fusion protein of the S protein and ferritin subunit into mammalian cells, which differs from nanoparticle platforms that require binding antigens to vectors or scaffolds after purification.

[0021] In this invention, the mammalian cells used are CHO cells. In addition to CHO cells, host cells such as 293 and Vero can also be used.

[0022] Furthermore, this invention demonstrated that Balb / c mice immunized with the S protein-Ferritin fusion protein exhibited antibodies capable of strongly blocking IBV virus invasion of target cells. This verified that the fusion protein of this invention can produce high titers of neutralizing antibodies in vivo. Moreover, it was also verified that, compared to control vaccines or other mutants or truncated variants, the S1 protein-Ferritin fusion protein, S1-2 protein-Ferritin fusion protein, and S1-3 protein-Ferritin fusion protein exhibit superior antibody expression levels and neutralizing antibody titers.

[0023] Furthermore, the present invention provides a method for efficiently expressing an optimized chicken infectious bronchitis virus antigen and ferritin fusion protein, which includes introducing the nucleotide coding sequence of the aforementioned S protein (S1 protein, S1-2 protein, S1-3 protein)-ferritin into a recombinant plasmid, transfecting the host CHO cell line with the recombinant plasmid by electroporation, and cloning and screening the transfected host CHO cells.

[0024] In another aspect of the invention, a polynucleotide molecule is provided that encodes the fusion protein.

[0025] In another aspect of the invention, a carrier is provided that contains the aforementioned polynucleotide molecule.

[0026] In another aspect of the invention, a host cell is provided that expresses the fusion protein and / or contains the polynucleotide molecule and / or contains the vector.

[0027] In another aspect of the invention, a vaccine composition is provided comprising the fusion protein, and optionally further comprising an immunologically and pharmaceutically acceptable carrier or adjuvant.

[0028] In another aspect of the invention, the use of the fusion protein, polynucleotide molecule, vector, host cell, and vaccine composition described herein is provided in the preparation of a medicament for treating and / or preventing infectious bronchitis virus (IBV) infection in chickens, or infectious bronchitis virus disease in chickens.

[0029] In another aspect of the present invention, a method for preparing a chicken infectious bronchitis virus (IBV) vaccine is provided, characterized by comprising the following steps:

[0030] (1) Construct the aforementioned polynucleotide molecule;

[0031] (2) The fusion protein is expressed in a host cell; preferably, the host cell is a CHO cell;

[0032] (3) Purify the expressed fusion protein and prepare the vaccine.

[0033] Advantages of the present invention

[0034] Compared to the original length of the natural chicken infectious bronchitis virus (IBBV) S protein, the IBV S1 fragment and its mutants (S1-2 and S1-3 proteins) increased antibody expression and neutralizing antibody titers. Furthermore, compared to the full-length S protein, the truncated IBV S1 fragment reduced the risk of non-specific immunity caused by the non-receptor binding region of the entire sequence. The optimized IBV S protein (S1-2 and S1-3 proteins) and their fusion proteins not only retain the receptor domain of the S protein but are also more easily expressed in CHO cells. The resulting products are easy to purify; the fusion proteins appear as nanoparticles, making them very easy to separate from single antigen molecules, appearing as the first single peak on an SEC column, indicating higher purity and better separation. Moreover, the yield was significantly increased while maintaining antibody neutralizing activity.

[0035] Nanostructures obtained by expressing Helicobacter pylori ferritin enabled the repeating arrangement of antigens (antigens arranged on the surface of spherical structures formed by ferritin). This repeating arrangement of antigens drives a stronger humoral immune response than a single antigen, and can induce stronger B cell activation through antigen-driven B cell receptor (BCR) cross-linking. It may also potentially influence antigen transport and localization.

[0036] Building upon existing work, this invention utilizes ferritin nanoparticles to design and successfully demonstrate nanoparticles of the spike protein of chicken infectious bronchitis virus (IBV). Importantly, the inventors designed a ferritin-based antigen (S protein - Ferritin). Since ferritin is a 24-sided sphere composed of eight trimers, and the IBV coronavirus's S protein forms a distinctive corolla structure on the viral surface as a trimer, its expression in mammalian cells is similar to that of the spike trimer (S). This ensures that the fusion of the spike protein, or the truncated fragment used in this invention, with ferritin will not negatively impact protein production.

[0037] Furthermore, glycosylation can affect the correct folding of viral antigens and participate in the translational regulation of proteins. This invention achieves expression in mammalian cells (CHO), exhibiting accurate post-translational modification capabilities, high transfection efficiency, and long-term stable passage to stably express functionally active antibodies. The expressed protein is closest to the natural protein molecule in terms of molecular structure, physicochemical properties, and biological function. It can produce antigenic fragments with similar antigenicity to the spike protein of chicken infectious bronchitis virus (IBV), which is more conducive to inducing an immune response in poultry such as chickens to produce neutralizing antibodies against it. Attached Figure Description

[0038] Figure 1 The image shows the partial Western blot results during the screening of stable CHO cell lines expressing S1-2-Ferritin. From left to right, the bands represent 1E8, the marker (a pre-stained protein molecular weight standard marker in kDa), 2F10, 1C4, 3F2, 1F7, 3F5, 5C3, 8E7, and 9F6. All lanes used a 10 μL sample loading volume. As shown in the figure, the CHO cell line 1C4 expressing S1-2-Ferritin exhibits a distinct band at approximately 75 kDa, consistent with the expected molecular weight of the recombinant protein S1-2-Ferritin, indicating it is the selected S1-2-Ferritin cell line. Detailed Implementation

[0039] Example 1: Construction and screening of expression vector for S protein-Ferritin fusion gene

[0040] 1.1 Obtaining and optimizing the S protein gene sequence of chicken infectious bronchitis virus

[0041] The amino acid sequence of the IBV S protein was derived from the NCBI strain M41. Analysis of its S protein structure yielded the S1 protein nucleotide sequence. Through different amino acid mutations, the S1-1, S1-2, and S1-3 amino acid sequences were obtained. The nucleotide sequences of these S proteins were then optimized using the CHO expression system codons, and the S sequence was synthesized. This work was commissioned to Nanjing Genscript Biotech Co., Ltd., which ligated the sequence into the PUC57 universal vector (obtained from Nanjing Genscript Biotech Co., Ltd.) to obtain the corresponding PUC57-S plasmid. Primers were designed using a truncated approach based on the S1 protein, and the SS1, SS2, and SS3 protein sequences were designed using molecular cloning techniques. The amino acid sequences of the S, S1, S1-1, S1-2, S1-3, SS1, SS2, and SS3 proteins are shown in SEQ ID Nos. 1-8, and their corresponding CHO codon-optimized nucleotide sequences are shown in SEQ ID Nos. 9-16.

[0042] The original ferritin amino acid sequence was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / protein / WP_000949190.1?). The first four amino acids were removed from the original ferritin amino acid sequence, and the N amino acid at position 19 was mutated to the Q amino acid, resulting in the Helicobacter pylori ferritin amino acid sequence shown in SEQ ID No. 17. Further codon optimization was performed according to the CHO expression system to obtain the corresponding ferritin nucleotide sequence (as shown in SEQ ID No. 18). This sequence was synthesized by Nanjing GenScript Biotech Co., Ltd., and inserted into the pCDNA3.0 vector (pCDNA3.0 was purchased from Addgene) to obtain the pCDNA3.0-Ferritin vector.

[0043] Subsequently, the nucleotide sequences corresponding to the fusion protein were obtained by linking the encoding nucleotide sequences of S, S1, S1-1, S1-2, S1-3, SS1, SS2, and SS3 with the nucleotide sequence of ferritin using linker peptide sequences.

[0044] 1.2 Construction of S protein recombinant plasmid

[0045] Based on the nucleotide sequence S designed in Example 1.1, iron protein plasmids were introduced through enzyme digestion, and then recombinant plasmids of the corresponding eight pCDNA3.0-S-Ferritin proteins were constructed using the pCDNA3.0 vector (purchased from Addgene). Taking S1-1 as an example, the specific steps are described in detail below.

[0046] 1.2.1 Enzyme digestion of PUC57-S plasmid

[0047] (1) Enzymes Xhol and NotⅠ were purchased from NEB. The samples were added to 1.5 mL EP tubes according to the table below, and mixed thoroughly. The double enzyme digestion reaction system was 50 μL, and the sample addition was as shown in Table 1 below:

[0048] Table 1. PUC57-S protein glucoprotein digestion system

[0049]

[0050] (2) The above 1.5mL EP tubes were placed in a 37℃ constant temperature water bath and digested for 3 hours.

[0051] 1.2.2 Enzyme digestion of pCDNA3.0-Ferritin vector

[0052] (1) Enzymes Xhol and NotⅠ were purchased from NEB. The samples were added to 1.5 mL EP tubes according to the table below, and mixed thoroughly. The double enzyme digestion reaction system was 50 μL, and the sample addition was as shown in Table 2 below:

[0053] Table 2 pCDNA3.0-Ferritin vector digestion system

[0054]

[0055] (2) The above 1.5mL EP tubes were placed in a 37℃ constant temperature water bath and digested for 3 hours.

[0056] 1.2.3 Double enzyme digestion gel recovery

[0057] The double digestion products of 1.2.1 and 1.2.2 were taken out and subjected to agarose gel electrophoresis to recover the DNA fragments. The universal DNA purification and recovery kit was purchased from Tiangen Company.

[0058] (1) Column equilibration steps: Add 500 μL of equilibration solution to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0059] (2) Cut a single target DNA band from the agarose gel and place it in a clean centrifuge tube. Weigh the tube and record the value.

[0060] (3) Add an equal volume of PC buffer solution to the 1.5 mL centrifuge tube in step (2), and place it in a 50°C water bath for about 10 minutes. During this time, gently rotate the centrifuge tube up and down continuously to ensure that the gel is fully dissolved.

[0061] (4) Add the solution obtained in step (3) into the adsorption column CB2, let it stand for 2 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

[0062] (5) Add 600 μL of PW buffer to the adsorption column CB2, let stand for 3 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

[0063] (6) Repeat step (5).

[0064] (7) Place the adsorption column CB2 into the collection tube and centrifuge at 12,000 rpm for 2 min to remove as much of the washing liquid as possible. Place the adsorption column at room temperature for 10 min to dry completely.

[0065] (8) Place the adsorption column CB2 into a clean centrifuge tube, add 50 μL ddH2O dropwise to the middle of the adsorption membrane, let stand for 10 min, centrifuge at 12,000 rpm for 2 min, and collect the DNA solution. The double digestion product of PUC57-S and pCDNA3.0-Ferritin is obtained.

[0066] 1.2.4 Connection Reaction

[0067] The DNA fragments recovered in 1.2.3 were ligated and inserted into the pCDNA3.0-Ferritin vector.

[0068] (1) Mark the 200μL centrifuge tubes to be used.

[0069] (2) Add the sample to the labeled 200 μL centrifuge tube according to the 20 μL reaction system in Table 3 below:

[0070] Table 3 Connection Reaction System

[0071]

[0072] (3) After adding the sample, gently blow the liquid several times with a pipette to mix the components.

[0073] (4) Place a 200 μL centrifuge tube in a PCR instrument at 16 °C for 3 h to obtain the ligation reaction product containing the nucleotide sequences of each fusion protein, and name them as pCDNA3.0-S-Ferritin plasmid, pCDNA3.0-S1-Ferritin plasmid, pCDNA3.0-S1-1-Ferritin plasmid, pCDNA3.0-S1-2-Ferritin plasmid, pCDNA3.0-S1-3-Ferritin plasmid, pCDNA3.0-SS1-Ferritin plasmid, pCDNA3.0-SS2-Ferritin plasmid, and pCDNA3.0-SS3-Ferritin plasmid, respectively.

[0074] (5) The product of the linkage reaction in step (4) can be directly converted into a product, or it can be stored at -20°C and thawed for conversion when needed.

[0075] 1.2.5 Transformation Reaction

[0076] (1) Quickly add 10 μL of the obtained ligation reaction product into a sample tube containing 100 μL of competent cells DH5α (purchased from Kangti Life), mix by blowing and aspiration, and incubate on ice for 30 min.

[0077] (2) After step (1) is completed, take out the sample tube, place it in a 42°C water bath for 45s, and then immediately place it in an ice bath for 90s to carry out the conversion.

[0078] (3) After step (2) is completed, take out the sample tube, add 200 μL of LB liquid culture medium to the sample tube in the ultra-clean workbench, and then place the sample tube in a constant temperature shaker at 37℃ and 220 rpm for 45 min.

[0079] (4) Prepare transformation plates. Prepare LB ampicillin resistance plates for transformation based on the resistance of pCDNA3.0-S-iron protein particles.

[0080] (5) Spreading: Take out the transformed competent cells and add them to LB ampicillin resistance plates and spread them evenly.

[0081] (6) Invert the plate from step (5) in a biochemical constant temperature incubator and incubate overnight at 37°C.

[0082] (7) Observe and record the transformation results to obtain a transformation plate with single clones growing.

[0083] 1.2.6 Plasmid extraction and enzyme digestion identification

[0084] 1.2.6.1 Plasmid Extraction

[0085] The plasmid extraction kit was purchased from Tiangen Biotech Co., Ltd. The specific procedures are as follows:

[0086] (1) Use a 10 μL pipette tip to pick up a single colony from the above transformation plate and put it into 5 ml of LB liquid medium containing ampicillin resistance. Shake overnight at 37°C and 220 rpm.

[0087] (2) Column equilibration step: Add 500 μL of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0088] (3) Take 5 mL of overnight cultured bacterial solution, add it to a centrifuge tube, and centrifuge at 12,000 rpm for 1 min using a standard benchtop centrifuge. Try to aspirate the supernatant.

[0089] (4) Add 250 μL of plasmid extraction reagent P1 buffer to each centrifuge tube in step (3) to completely suspend the bacterial cells.

[0090] (5) Add 250 μL of P2 buffer to the solution in step (4), and immediately gently invert the centrifuge tube 6-8 times to mix. Let stand at room temperature for 2-4 min.

[0091] (6) Add 350 μL of P3 buffer to the solution from step (5), and immediately gently invert the centrifuge tube 6-8 times to mix thoroughly. A white flocculent precipitate will appear at this point. Centrifuge at 12,000 rpm for 10 min.

[0092] (7) Transfer the supernatant solution from step (6) to the center of the adsorption column CP3, centrifuge at 12,000 rpm at room temperature for 1 min, discard the liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

[0093] (8) Add 600 μL of washing solution PW to the center of the adsorption column, centrifuge at 12,000 rpm at room temperature for 1 min, discard the liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

[0094] (9) Repeat step (8).

[0095] (10) Place the adsorption column CP3 into the collection tube and centrifuge at 12,000 rpm for 2 min at room temperature.

[0096] (11) Place the CP3 adsorption column into a clean 1.5 ml centrifuge tube, add 50 μL ddH2O to the center of the adsorption membrane, let stand at room temperature for 10 min, centrifuge at 12,000 rpm for 2 min, and store the plasmid solution in the tube at 4 °C to obtain the purified pCDNA3.0-S-Ferritin plasmid, pCDNA3.0-S1-Ferritin plasmid, pCDNA3.0-S1-1-Ferritin plasmid, pCDNA3.0-S1-2-Ferritin plasmid, pCDNA3.0-S1-3-Ferritin plasmid, pCDNA3.0-SS1-Ferritin plasmid, pCDNA3.0-SS2-Ferritin plasmid, and pCDNA3.0-SS3-Ferritin plasmid.

[0097] 1.2.6.2 Each plasmid obtained in Example 1.2.6.1 was subjected to enzyme digestion for identification.

[0098] The universal DNA purification and recovery kit was purchased from Tiangen Biotech Co., Ltd. The specific operating procedure is as follows:

[0099] (1) Label the 1.5mL EP tubes to be used, and add the sample into the 1.5mL EP tubes according to Table 4 below and mix well: The reaction system is 20μL:

[0100] Table 4 Enzyme digestion identification system

[0101]

[0102] (2) Place the 1.5 mL EP tube from step (1) in a 37°C constant temperature water bath and digest overnight.

[0103] (3) Electrophoresis verification. The above double enzyme digestion products were taken out and verified by agarose gel electrophoresis. The sequencing results were returned by Beijing Liuhe BGI Genomics Co., Ltd., which identified and confirmed the enzyme digestion products of pCDNA3.0-S-Ferritin plasmid, pCDNA3.0-S1-Ferritin plasmid, pCDNA3.0-S1-1-Ferritin plasmid, pCDNA3.0-S1-2-Ferritin, pCDNA3.0-S1-3-Ferritin, pCDNA3.0-SS1-Ferritin, pCDNA3.0-SS2-Ferritin, and pCDNA3.0-SS3-Ferritin plasmid.

[0104] Example 2: pCDNA3.0-S protein particles were transduced into CHO cells.

[0105] 2.1 Large-scale extraction of pCDNA3.0-S protein Ferritin plasmid

[0106] The eight pCDNA3.0-S protein Ferritin plasmids finally identified in Example 1 were subjected to plasmid extraction. The plasmid extraction kit was purchased from Tiangen Biotech Co., Ltd.

[0107] (1) Column equilibration step: Add 2.5 ml of equilibration solution BL to the adsorption column CP6 (the adsorption column is placed in a 50 ml collection tube), centrifuge at 8,000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0108] (2) Take 100 ml of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 8,000 rpm for 3 min at room temperature to collect bacteria and remove the supernatant as much as possible.

[0109] (3) Try to remove the supernatant and use clean absorbent paper to absorb the water droplets on the bottle wall.

[0110] (4) Add 8 ml of solution P1 to the centrifuge tube containing bacterial precipitate, and use a pipette or vortex mixer to completely suspend the bacterial cell precipitate.

[0111] (5) Add 8 ml of solution P2 to the centrifuge tube, and immediately gently invert it 6-8 times to fully lyse the bacteria. Let it stand at room temperature for 5 min.

[0112] (6) Add 8 ml of solution P4 to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Then let it stand at room temperature for about 10 minutes. Centrifuge at 8,000 rpm for 10 minutes to allow the white precipitate to settle to the bottom of the tube. Carefully pour the entire solution into filter CS1, and slowly push the push handle to filter. Collect the filtrate in a clean 50 ml tube.

[0113] (7) Add 0.3 times the volume of isopropanol to the filtrate, mix by inverting, and then transfer to the adsorption column CP6 (place the adsorption column in a 50 ml collection tube).

[0114] (8) Centrifuge at 8,000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube. Pass the solution obtained in step 7 through the column twice, each time under the above conditions.

[0115] (9) Add 10 ml of washing solution PW to the adsorption column CP6, centrifuge at 8,000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0116] (10) Repeat step (9)

[0117] (11) Add 3 ml of anhydrous ethanol to the adsorption column CP6, centrifuge at 8,000 rpm for 2 min at room temperature, and discard the waste liquid.

[0118] (12) Put the adsorption column CP6 back into the collection tube and centrifuge at 8,000 rpm for 5 min to remove the residual washing solution in the adsorption column.

[0119] (13) Place the adsorption column CP6 in a clean 50 ml collection tube, add 1-2 ml of elution buffer TB dropwise to the middle of the adsorption membrane, incubate at room temperature for 5 min, and then centrifuge at 8,000 rpm for 2 min at room temperature. Transfer all the elution buffer from the 50 ml centrifuge tube into a clean 1.5 ml centrifuge tube. Store at -20℃.

[0120] 2.2 Plasmid transfection based on electroporation

[0121] A. Culture medium preparation: Dialyze the FBS (purchased from Gibco, USA) using a 3500 dialysis bag, then prepare 1L of CSC-03 culture medium containing 10% dFBS. After preparation, place the medium in an incubator for preheating, and set the incubator temperature to 37℃.

[0122] B. Host cell preparation: The initial cell concentration after inoculation is 0.5 × 10⁻⁶. 6 CHO cell line (imported from ATCC by Beijing Dingchi Biotechnology Co., Ltd. on May 1, 2018, ATCC number: CCL61. This cell line was expanded and cultured at Beijing Dingchi Biotechnology Co., Ltd. to establish a cell bank, cell bank number: BJDC-201800010) in 125mL Erlenmeyer flasks and cultured in suspension for 3 days; the host cell CHO viable cell density and cell viability were recorded using a Countstar automated cell counter at a ratio of 1.0 × 10⁻⁶ cells / ml. 7 The total number of cells was taken, and the cells were centrifuged at 800 r / m for 5 min to remove the supernatant. The cells were then washed twice with 5 mL of CD-Pro medium to remove the supernatant. After the second wash, the cells were resuspended in 600 μL of CD-Pro medium and set aside for use.

[0123] C. Take 200 μg of each pCDNA3.0-S protein Ferritin plasmid (dissolved in electroporation medium CD-Pro (purchased from Yishengke (Shenzhen) Co., Ltd.)) and add it to the CHO cell suspension resuspended in CD-Pro medium, and incubate at room temperature for 5 minutes.

[0124] D. Set the electroporation program of the electroporator to 320V, 900uF, ∞, 4mm;

[0125] E. Resuspend the cell solution in CD-Pro medium containing plasmids and transfer it into an electroporation cuvette. Let it stand for 2 minutes before starting electroporation. Record the duration and voltage of electroporation.

[0126] F. After electroporation, add the cell solution in the electroporation cup to the prepared CSC-03 medium containing 10% dFBS and mix by pipetting and aspiration.

[0127] G. Mix the electroporation cell solution by pipetting and aspiration, and spread the cell solution into 96-well plates at a volume ratio of 100 μL / well. Place the 96-well plates in an incubator at 37°C with 5% CO2.

[0128] 2.3 Cell line screening

[0129] 24 hours after electroporation, the CHO cell lysate in 96-well plates was treated with MSX (methionine imino sulfone) at a concentration of 30-50 µM. After culturing for about 20 days, the cell line was transferred to 24-well plates for further culture in a single-clone culture medium CSC-03 containing 5% dFBS (purchased from Yishengke (Shenzhen) Co., Ltd.).

[0130] After 3 days of culture, cells were expanded to obtain a 24-well plate. After standing for 7 days at 37℃ and 5% CO2, the supernatant was collected for testing. Positive cell lines, i.e., successfully transfected cell lines, were screened using Western blotting. The screened cell lines were then transferred to 6-well plates for culture in dFBS-free monoclonal medium. After 3 days of culture, the cells were transferred to shake flasks for culture in dFBS-free CHO-K1 + 15uM MSX medium for 3 days. The CHO-K1 medium was purchased from Yishengke (Shenzhen) Co., Ltd. This culture process simultaneously completed the screening of cell lines and the adaptation of cell lines to serum-free medium, as shown in Table 5.

[0131] Table 5. Serum-free adaptation process schedule

[0132]

[0133] The selected cell lines were amplified. NF604 medium (purchased from Shenzhen Yishengke) was added starting on day 4. When cell viability reached approximately 60%, the cells were centrifuged at 12,000 rpm for 15 minutes. The cell supernatant was collected, and 1 mL of the supernatant was used for detection. Western blotting was used (primary antibody: IBV chicken infectious bronchitis positive hyperimmune serum, purchased from China Veterinary Drug Information Network; secondary antibody: goat anti-chicken IgG-HRP, purchased from Abbkine) to screen for high-yielding positive cell lines, i.e., successfully transfected cell lines (see [link to relevant documentation]). Figure 1Cell lines with high expression levels underwent two more rounds of monoclonal screening. This resulted in several CHO cell lines stably expressing S-Ferritin, S1-Ferritin, S1-1-Ferritin, S1-2-Ferritin, S1-3-Ferritin, SS1-Ferritin, SS2-Ferritin, and SS3-Ferritin.

[0134] The highest yields of S-Ferritin, S1-Ferritin, S1-1-Ferritin, S1-2-Ferritin, S1-3-Ferritin, SS1-Ferritin, SS2-Ferritin, and SS3-Ferritin were determined in clones, with yields of 36.24 mg / L, 148.68 mg / L, 57.86 mg / L, 368.96 mg / L, 263.56 mg / L, 15.28 mg / L, 22.36 mg / L, and 6.28 mg / L, respectively. Correspondingly, the cell-based yields were 6.02 × 10⁻⁶. -9 mg / cell, 24.68×10 -9 mg / cell, 9.60×10 -9 mg / cell, 61.24×10 -9 mg / cell, 43.77×10 -9 mg / cell, 2.54×10 -9 mg / cell, 3.71×10 -9 mg / cell, 1.04×10 -9 mg / cell was used to expand and preserve these clones for subsequent experiments.

[0135] Example 3: Protein Purification

[0136] Chromatographic purification of fusion proteins

[0137] (1) The cell supernatants containing eight optimized S protein-Ferritin groups obtained in Example 2 were purified by chromatography using GE Core 400. Specifically, the eight corresponding cell supernatants obtained in Example 2 were centrifuged at 10000×g for 30 min, the supernatants were collected, filtered at 0.22 μM, and the filtered supernatants were concentrated 10-20 times by 30 kDa membrane ultrafiltration and used as the loading solution.

[0138] (2) Wash the medium with 5 column volumes of distilled water.

[0139] (3) Wash the medium with 5 column volumes of equilibration buffer (PBS, pH 7.4).

[0140] (4) Load 1 / 3 CV each time.

[0141] (5) Collect external water volume peaks.

[0142] (6) Wash the chromatography column with 1M NaOH + 30% isopropanol, then equilibrate it to pH neutral with PBS, and store the chromatography column with 20% ethanol.

[0143] Purified S-Ferritin, S1-Ferritin, S1-1-Ferritin, S1-2-Ferritin, S1-3-Ferritin, SS1-Ferritin, SS2-Ferritin, and SS3-Ferritin fusion proteins (as antigens) were obtained, which can be used as candidate antigens for subunit vaccines against chicken infectious bronchitis virus.

[0144] Example 4: Preparation and Immunization of Subunit Vaccines

[0145] The S protein-Ferritin antigen solution obtained in Example 3 was diluted with 0.01 mol / L PBS (pH 7.2) to the required protein concentration for the experiment. The solution was mixed at a volume ratio of 1:1 and then prepared with adjuvant white oil adjuvant (purchased from Henan Tongshang Import & Export Co., Ltd.). After passing the quality inspection, the solution was stored at 4°C under aseptic conditions.

[0146] (1) Animal vaccination

[0147] The experimental chickens were 23-day-old SPF chickens, purchased from Beijing Boehringer Ingelheim Biotechnology Co., Ltd.

[0148] The H120 live vaccine was produced by Qingdao Weilan; the M41 inactivated vaccine was also produced by Qingdao Weilan. The IB S protein subunit vaccine was prepared under this application.

[0149] The grouping details are shown in Table 6, and the vaccine immunization trial arrangements are as follows.

[0150] Table 6 Vaccine Immunization Trial Arrangement

[0151]

[0152] After vaccination, the 13 groups of chicks were isolated and raised in 6 isolators, maintaining the same feeding and management conditions. Chicken coops were cleaned and disinfected regularly, and the health of each flock was observed.

[0153] (2) Immunization procedures and blood collection

[0154] Vaccination. Immunize and label according to the table above. Collect 1 ml of blood from group 13 chickens and separate the serum for later IBHI antibody testing. 21 days after the first immunization / before the second immunization, band chickens in groups 1-13, collect blood again, and administer booster immunizations according to the table above. Collect 1 ml of blood and separate the serum for later IBHI antibody testing. 42 days after the first immunization / 21 days after the second immunization, collect 1 ml of blood from chickens in groups 1-13, separate the serum, and simultaneously test the IBHI antibody using the serum from the first immunization. Compare the inhibition fold of the second immunization serum antibody to the first immunization serum antibody.

[0155] When chicks are under 30 days old, use the cardiac blood collection method. After 30 days old, wing vein blood collection can be performed. Each blood collection should ensure 1 ml of blood to ensure sufficient serum is extracted. Each group of serum should be labeled.

[0156] (3) Preparation and preservation of serum

[0157] Tilt the 1 ml syringe containing blood and place it in a 37°C incubator for 2 hours. Then remove it and transfer the blood clot and serum mixture from the syringe to a 2 ml centrifuge tube. Centrifuge at 2000 r / min for 5 min and store the supernatant at -40°C.

[0158] Example 5: Study on immune response to S-Ferritin subunit vaccine

[0159] Serum samples prepared and stored 21 days and 42 days after the first immunization with the subunit vaccine in Example 4 were used for hemagglutination inhibition titer experiments. Higher hemagglutination inhibition titers indicate better neutralization of viral antigens by the serum and better protection against IBV virus by the vaccine. The hemagglutination inhibition titer experimental method complies with the requirements of the current Chinese Veterinary Pharmacopoeia.

[0160] (1) Preparation of HI antigen

[0161] The IBV M41 strain antigen reference material for the hemagglutination inhibition test, purchased from the China Institute of Veterinary Drug Control, was aliquoted and stored at -40°C. The hemagglutination titer for this antigen was 1:256. The IBV antigen reference material was treated with trypsin (final concentration 5 μg / mL) at 37°C for 15-30 minutes, and then the reaction was immediately terminated by ice bath. This treated antigen was diluted to a working concentration containing 4 hemagglutination units, i.e., the HA titer of the hemagglutination antigen was 256. Therefore, this HA antigen, diluted 256 / 4 = 64 times, yielded 4 units of antigen.

[0162] (3) HI test of IBV

[0163] Add 25 μL of PBS (pH 7.2) to each well of a 96-well "V"-shaped reaction plate. Use a pipette to take 25 μL of the serum to be tested and serially dilute it from well 1 to well 11. Discard the 25 μL of diluent. Well 12 is the hemagglutination antigen control well without serum.

[0164] Add 25 μL of the prepared hemagglutination antigen solution (4 units) to each well, shake well on a micro-shaker, and let stand at room temperature for 40 min. Then add 25 μL of 1% chicken red blood cells (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) to each well and let stand at room temperature for about 40 min to observe the results.

[0165] Table 7. Methods for determining serum hemagglutination inhibition titer against IBV

[0166]

[0167] To ensure the reliability of experimental results, control tests should be set up simultaneously for red blood cells, the tested serum, and hemagglutination antigens in each experiment.

[0168] Table 8. Types of control wells in the IBV hemagglutination inhibition test.

[0169]

[0170] Result determination:

[0171] When making a determination, first look at the control. When both the red blood cell control and the tested serum control are negative, that is, there is no agglutination, and the hemagglutination antigen control shows + or ++ agglutination in one well and 100% agglutination in the other two wells, the inhibition titer of the tested serum can be determined well by well from front to back. It is expressed as the highest dilution factor of the tested serum that can completely inhibit red blood cell agglutination.

[0172] Table 9. Results of HI antibody titer determination after immunization with different types of IB-S protein subunit vaccines

[0173]

[0174] Note: The numbers in the table are expressed as log2 of the serum dilution factor and are geometric mean.

[0175] The results showed that chicken serum from chickens not immunized with live, inactivated, or subunit vaccines (i.e., the blank control) did not show any inhibitory effect against IBV virus, meaning no neutralizing antibodies against IBV virus were produced. In all chickens injected with the H120 live vaccine, a titer of 2 was produced. 1.1 -2 1.9After booster immunization, the neutralizing antibody titers of all chickens significantly increased. Chickens boosted with the IBV S protein subunit vaccine showed significantly higher neutralizing antibody titers than those boosted with the M41 inactivated vaccine. Specifically, the neutralizing antibody titer in chickens boosted with the IBV S protein subunit vaccine was 2. 3.7 -2 5.6 The neutralizing antibody titer in chickens vaccinated with the M41 inactivated vaccine was 2. 2.4 The neutralizing antibody titers in chickens immunized with the IBV S1 protein fusion ferritin protein vaccine were significantly higher than those in the control group containing only IBV S1 protein and the control group containing a mixture of IBV S1 protein and ferritin protein. This indicates that, as a booster immunization, the IBV S subunit vaccine used in this example provides stronger neutralizing protection than the M41 inactivated vaccine. The neutralizing antibody titers (i.e., neutralizing effects) produced by each protein in the IBV S subunit vaccine, in descending order, are: S1-2-Ferritin > S1-3-Ferritin > S1-Ferritin > S1-1-Ferritin > S-Ferritin > SS2-Ferritin = SS1-Ferritin > SS3-Ferritin, verifying that S1-2-Ferritin, S1-3-Ferritin, and S1-Ferritin significantly enhance immunogenicity.

[0176] To explore the tolerability and safety of the subunit vaccine, animal tolerance was observed at an immunization dose of 20 µg / 0.5 ml / bird. Chickens in all intramuscular injection groups showed normal activity, body temperature, and appetite, with no deaths. This indicates the excellent safety profile of the subunit vaccine of this invention.

[0177] In summary, experimental verification has shown that the vaccine prepared by this invention can be used to prevent infection with infectious bronchitis virus (IBV) or infectious bronchitis virus disease in chickens.

[0178] The above description is merely a preferred embodiment of the present invention and does not limit the scope of the invention. Any simple equivalent changes and modifications made in accordance with the claims and description of the invention are still within the scope of the invention. To avoid unnecessary repetition, the present invention will not describe any possible combinations.

[0179] Industrial applicability

[0180] The fusion protein provided by this invention, comprising S1, S1-2, S1-3 proteins and Helicobacter pylori ferritin, is more easily expressed in CHO cells and can significantly increase the expression level of S protein-ferritin. This protein can be used to prepare vaccines for the prevention of infectious bronchitis virus (IBV) disease in chickens, laying a solid foundation for the production of IBV subunit vaccines. This invention also relates to the IBV S protein-ferritin fusion gene, vector, cells, preparation methods, treatment methods, or pharmaceutical applications. These can be applied in a wide range of fields, including basic research on the mechanism of viral action, differential diagnosis, rapid assay, epidemiological surveys, and animal model preparation.

[0181] sequence list

[0182] SEQ ID NO.1, amino acid sequence of the S protein

[0183]

[0184] SEQ ID NO.2, Amino acid sequence of S1 protein

[0185] AFRPPNGWHLHGGAYAVVNISSESNNAGSSPGCIVGTIHGGRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYKYDGCPITGMLQKNFLRVSAMKNGQLFYNLTVSVAKYPTFKSFQCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSDGFYPFINSSLVKQKFIVYRENSVNTTFTLHNFTFHNETGANPNPSGVQNIQTYQTQTAQSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQSVFSGRATCCYAYSYGGPSLCKGVYSGELALNFECGLLVYVTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGILTSRNETGSQLLENQFY

[0186] SEQ ID NO.3, Amino acid sequence of S1-1 protein

[0187] MLVTPLLLVTLLCVLCSAALYDSSSYVYYYQSAFRPPNGWHLHGGAYAVVNISSESNNAGSSPGCIVGTIHGGRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYKYDGCPITGMLQKNFLRVSAMKNGQLFYNLTVSVAKYPTFKSFQCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSDGFYPFINSSLVKQKFIVYRENSVNTTFTLHNFTFHNETGANPNPSGVQNIQTYQTQTAQSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQSVFSGRATCCYAYSYGGPSLCKGVYSGELDLNFECGLLVYVTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGILTSRNETGSQLLENQFYIKITNGTRR

[0188] SEQ ID NO.4, Amino acid sequence of S1-2 protein

[0189] AFRPPNGWHLHGGAYAVVNISSESNNAGSSPGCIVGTIHGGRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYKYDGCPITGMLQKNFLRVSAMKNGQLFYNLTVSVAKYPTFKSFQCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDKSPKGLLACQYNTGNFSDGFYPFTNTTLVREKFIVYRESSVNTTLALTNFTFTNVSNAQPNSGGVNTFHLYQTQTAQSGYYNFNLSFLSQFVYKASDFMYGSYYPRCSFRPETINNGLWFNSLSVSLTYGPLQGGCKQSVFSGKATCCYAYSYNGPRACKGVYSGELSKTFECGLLVYVTKSDGSRIQTRTEPLVLMQHNYNNITLDKCVNYNIYGRVGQGFITNVTDSAANFSYLADGGLAILDTSGAIDVFVVQGIYGPNYYKVNPCEDVNQQFVVSGGNIVGILTSRNETGSEQVENQFY

[0190] SEQ ID NO.5, Amino acid sequence of S1-3 protein

[0191] AFRPPNGWHLHGGAYAVVNISSESNNAGSSPGCIVGTIHGGRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYSSGTGSCPITGMIARDHIRISAMKNGSLFYNLTVSVSKYSRFKSFQCVNNLTSVYLNGDLVFTSNKTTDVTSAGVYFKAGGPVNYSVMKEFKVLAYFVNGTAQDVILCDKSPKGLLACQYNTGNFSDGFYPFTNTTLVREKFIVYRESSVNTTLALTNFTFTNVSNAQPNSGGVNTFHLYQTQTAQSGYYNFNLSFLSQFVYKASDFMYGSYYPRCSFRPETINNGLWFNSLSVSLTYGPLQGGCKQSVFSGKATCCYAYSYNGPRACKGVYSGELSKTFECGLLVYVTKSDGSRIQTRTEPLVLMQHNYNNITLDKCVNYNIYGRVGQGFITNVTDSAANFSYLADGGLAILDTSGAIDVFVVQGIYGPNYYKVNPCEDVNQQFVVSGGNIVGILTSRNETGSEQVENQFY

[0192] SEQ ID NO.6, Amino acid sequence of SS1 protein

[0193] FSDGFYPFINSSLVKQKFIVYRENSVNTTFTLHNFTFHNETGANPNPSGVQNIQTYQTQTAQSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQSVFSGRATCCYAYSYGGPSLCKGVYSGELALNFECGLLVYVTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGILTSRNETGSQLLENQFY

[0194] SEQ ID NO.7, Amino acid sequence of SS2 protein

[0195] MKNGQLFYNLTVSVAKYPTFKSFQCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSDGFYPFINSSLVKQKFIVYRENSVNTTFTLHNFTFHNETGANPNPSGVQNIQTYQTQTAQSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQSVFSGRATCCYAYSYGGPSLCKGVYSGELALNFECGLLVYVTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGILTSRNETGSQLLENQFY

[0196] SEQ ID NO.8, Amino acid sequence of SS3 protein

[0197] TLHNFTFHNETGANPNPSGVQNIQTYQTQTAQSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQSVFSGRATCCYAYSYGGPSLCKGVYSGELALNFECGLLVYVTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGILTSRNETGSQLLENQFY

[0198] SEQ ID NO.9, Nucleotide sequence of S protein

[0199]

[0200] SEQ ID NO.10, nucleotide sequence of S1 protein

[0201]

[0202] SEQ ID NO.11, nucleotide sequence of S1-1 protein

[0203]

[0204] SEQ ID NO.12, nucleotide sequence of S1-2 protein

[0205]

[0206] SEQ ID NO.13, nucleotide sequence of S1-3 protein

[0207]

[0208] SEQ ID NO.14, Nucleotide sequence of SS1 protein

[0209] TTCTCCGATGGATTCTACCCTTTCATTAACAGTTCACTGGTGAAGCAGAAATTCATCGTGTACAGAGAGAACTCCGTGAACACCACCTTCACCCTGCACAACTTCACCTTCCACAACGAAACCGGCGCCAACCCTAACCCAAGCGGCGTGCAGAACATCCAGACATATCAGACCCAGACCGCTCAGTCTGGCTATTACAACTTCAACTTTAGCTTTCTCTCCTCTTTTGTGTATAAAGAGTCCAACTTCATGTACGGCTCATACCACCCCTCCTGTAACTTTCGGCTGGAAACCATCAACAATGGGCTGTGGTTCAATTCCCTGTCTGTGTCTATCGCTTACGGCCCTCTGCAAGGCGGCTGCAAGCAGTCCGTGTTCAGTGGTCGGGCTACCTGTTGCTACGCTTACTCCTACGGCGGACCTTCCCTGTGCAAGGGCGTGTACTCTGGCGAACTGGCCCTGAACTTCGAGTGCGGCCTGTTGGTGTACGTGACAAAATCCGGCGGCTCCCGGATTCAGACCGCTACCGAGCCTCCTGTGATCACCAGACATAACTACAACAACATCACCCTGAATACTTGCGTGGACTACAATATCTACGGCCGGACCGGACAAGGATTTATCACCAACGTGACCGACTCTGCTGTGTCCTACAACTACCTGGCTGATGCCGGCCTGGCCATCCTGGACACCTCCGGCTCCATCGACATCTTCGTTGTCCAGGGCGAGTATGGCCTGACCTACTACAAGGTGAACCCTTGCGAGGACGTGAATCAGCAGTTCGTGGTGTCTGGCGGCAAGCTGGTGGGCATCCTGACCTCTAGAAACGAGACAGGCAGCCAGCTTCTGGAAAACCAGTTCTAC

[0210] SEQ ID NO.15, Nucleotide sequence of SS2 protein

[0211]

[0212] SEQ ID NO.16, Nucleotide sequence of SS3 protein

[0213] ACCCTGCACAACTTCACCTTCCACAACGAAACCGGCGCCAACCCTAACCCAAGCGGCGTGCAGAACATCCAGACATATCAGACCCAGACCGCTCAGTCTGGCTATTACAACTTCAACTTTAGCTTTCTCTCCTCTTTTGTGTATAAAGAGTCCAACTTCATGTACGGCTCATACCACCCCTCCTGTAACTTTCGGCTGGAAACCATCAACAATGGGCTGTGGTTCAATTCCCTGTCTGTGTCTATCGCTTACGGCCCTCTGCAAGGCGGCTGCAAGCAGTCCGTGTTCAGTGGTCGGGCTACCTGTTGCTACGCTTACTCCTACGGCGGACCTTCCCTGTGCAAGGGCGTGTACTCTGGCGAACTGGCCCTGAACTTCGAGTGCGGCCTGTTGGTGTACGTGACAAAATCCGGCGGCTCCCGGATTCAGACCGCTACCGAGCCTCCTGTGATCACCAGACATAACTACAACAACATCACCCTGAATACTTGCGTGGACTACAATATCTACGGCCGGACCGGACAAGGATTTATCACCAACGTGACCGACTCTGCTGTGTCCTACAACTACCTGGCTGATGCCGGCCTGGCCATCCTGGACACCTCCGGCTCCATCGACATCTTCGTTGTCCAGGGCGAGTATGGCCTGACCTACTACAAGGTGAACCCTTGCGAGGACGTGAATCAGCAGTTCGTGGTGTCTGGCGGCAAGCTGGTGGGCATCCTGACCTCTAGAAACGAGACAGGCAGCCAGCTTCTGGAAAACCAGTTCTAC

[0214] SEQ ID NO.17, Amino acid sequence of Helicobacter pylori ferritin

[0215] DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0216] SEQ ID NO.18, Nucleotide sequence of Helicobacter pylori ferritin

[0217] gacataatcaaattgctgaacgaacaagtgaataaagagatgcagtcaagtaacctatacatgtctatgtcaagttggtgctataccccactcactggacggggcagggctattcctgtttga tcatgcagctgaagagtacgaacacgctaaaaagttaataattttcctgaacgaaaataacgtacctgtgcaactaacttctatatccgcacccgagcataagtttgaagggttgacccaaa tcttccagaaggcatacgagcacgagcacataagtgaatcaatcaacaatatcgttgaccatgctataaagtctaaagatcacgcaacatttaacttcttgcaatggtacgtagctgag cagcacgaggaagaggtgctatttaaagacatactggataagattgaattgatagggaacgaaaaccacgggttgtatctagctgaccaatacgtaaaggggatcgcaaagagcagaaaaagt

[0218] SEQ ID NO.19, amino acid sequence of the linker peptide

[0219] GGGSGGGS

[0220] SEQ ID NO.21, nucleotide sequence of S1-Ferritin fusion protein

[0221]

[0222] SEQ ID NO.23, nucleotide sequence of S1-2-Ferritin fusion protein

[0223]

[0224] SEQ ID NO.24, nucleotide sequence of S1-3-Ferritin fusion protein

[0225]

Claims

1. Fusion proteins, which, from the N-terminus to the C-terminus, include: Chicken infectious bronchitis virus S protein fragment or its mutant, linker peptide, and Helicobacter pylori ferritin; among which, The S protein fragment of the chicken infectious bronchitis virus is the S1 protein, and its amino acid sequence is shown in SEQ ID NO.2; The mutant of the S protein fragment of the chicken infectious bronchitis virus is either the S1-2 protein or the S1-3 protein, wherein the amino acid sequence of the S1-2 protein is shown in SEQ ID NO.4, and the amino acid sequence of the S1-3 protein is shown in SEQ ID NO.

5.

2. The fusion protein according to claim 1 or 2, wherein, The amino acid sequence of the Helicobacter pylori ferritin is shown in SEQ ID NO.

17.

3. The fusion protein according to claim 1 or 2, wherein, The nucleotide sequence of the S1 protein is shown in SEQ ID NO. 10, the nucleotide sequence of the S1-2 protein is shown in SEQ ID NO. 12, and the nucleotide sequence of the S1-3 protein is shown in SEQ ID NO.

13. Optionally, the nucleotide sequence of the Helicobacter pylori ferritin is shown in SEQ ID NO.

18.

4. The fusion protein according to claim 1 or 2, wherein, The linker peptide is selected from: [A(EAAAK)nA], (GGGGS)n, (G)n, (XP)n, and preferably its amino acid sequence is shown in SEQ ID NO.

19.

5. The fusion protein according to claim 1 or 2, further comprising a purification tag, such as His, Fc, HA, GST, Flag, MBP, or FLAG tag.

6. A polynucleotide molecule encoding the fusion protein of any one of claims 1-5.

7. A carrier comprising the polynucleotide molecule of claim 6.

8. A host cell expressing the fusion protein of any one of claims 1-5, and / or comprising the polynucleotide molecule of claim 6, and / or comprising the vector of claim 7.

9. A vaccine composition comprising the fusion protein of any one of claims 1-5, and optionally further comprising an immunologically and pharmaceutically acceptable carrier or adjuvant.

10. Use of the fusion protein of any one of claims 1-5, the polynucleotide molecule of claim 6, the vector of claim 7, the host cell of claim 8, and the vaccine composition of claim 9 in the preparation of a medicament for treating and / or preventing infectious bronchitis virus (IBV) infection in chickens, or infectious bronchitis in chickens.

11. A method for preparing a chicken infectious bronchitis virus (IBV) vaccine, characterized in that, Includes the following steps: (1) Construct the polynucleotide molecule of claim 6; (2) Expressing the fusion protein as described in any one of claims 1-5 in a host cell; preferably, the host cell is a CHO cell; (3) Purify the expressed fusion protein and prepare the vaccine.

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