Recombinant protein s-trimer of infectious bronchitis virus antigen and subunit vaccine therefor

Abstract

The present application belongs to the field of veterinary drugs, and provides a recombinant protein S-Trimer of an infectious bronchitis virus antigen and a subunit vaccine therefor. Provided in the present application is a recombinant protein S-Trimer of an infectious bronchitis virus antigen, wherein the recombinant protein S-Trimer of the antigen has an amino acid sequence as set forth in SEQ ID No. 4. Also provided in the present application is an infectious bronchitis virus antigen. The infectious bronchitis virus antigen is of a trimer structure of the recombinant protein S-Trimer described above. The present application provides a subunit vaccine for an infectious bronchitis virus, wherein the subunit vaccine comprises a pharmaceutically acceptable carrier and an immunogenic amount of the infectious bronchitis virus antigen.

Description

Recombinant protein S-Trimer of antigen of infectious bronchitis virus of chicken and subunit vaccine thereof Cross-reference to related applications This application claims priority to the Chinese patent application No. 2024108296854, filed on June 25, 2024 to the Chinese Patent Office, entitled "Recombinant protein S-Trimer of antigen of infectious bronchitis virus of chicken and subunit vaccine thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0001] The present application relates to the field of veterinary medicine, in particular to a recombinant protein S-Trimer of antigen of infectious bronchitis virus of chicken and subunit vaccine thereof. BACKGROUND

[0002] Infectious bronchitis (IB) is a deformity, highly contagious, viral respiratory infectious disease caused by infectious bronchitis virus (IBV). It first occurred in North Dakota in 1930, was first reported in 1931, and the first isolated IBV strain was reported in 1937. Currently, the disease is almost universal in all countries of the world's poultry industry, and is one of the important infectious diseases that endanger the poultry industry, causing serious economic losses.

[0003] Infectious bronchitis of chicken is mainly infected through the respiratory tract, and the virus is discharged through the respiratory tract, which is transmitted through the air, and can also be transmitted through contaminated eggs, feed, water, utensils, etc. It occurs all year round, mostly in winter and spring, and chickens of all ages are susceptible, with the most cases in chicks, with a morbidity of 70% to 100% and a mortality of 10% to 40%. Infectious bronchitis spreads rapidly, and crowded chicken flocks, dirty air, damp ground, temperature fluctuations, and lack of vitamins and minerals in feed can induce the disease.

[0004] Prevention is the main method for infectious bronchitis of chicken, and the most important thing is to do a good job of vaccine immunization. The existing IBV vaccine products are all whole virus inactivated vaccines or live vaccines produced by chicken embryos, which have the risk of virus dissemination, and the chicken embryos are difficult to handle and easy to contaminate. Relatively speaking, genetic engineering subunit vaccine is a better choice. Therefore, it is of great practical significance to develop a chicken infectious bronchitis subunit vaccine with low production cost, high production efficiency and good vaccine immunization effect. SUMMARY

[0005] The first object of the present application is to provide a recombinant protein S-Trimer of antigen of infectious bronchitis virus of chicken.

[0006] The second object of the present application is to provide a recombinant protein S-Trimer of antigen of infectious bronchitis virus of chicken.

[0007] The third object of the present application is to provide an application of the antigen recombinant protein S-Trimer.

[0008] The fourth object of the present application is to provide a subunit vaccine of the infectious bronchitis virus of chicken.

[0009] In order to achieve the above objects, the present application adopts the following technical solutions:

[0010] An antigen recombinant protein S-Trimer of the infectious bronchitis virus of chicken, the amino acid sequence of the antigen recombinant protein S-Trimer is shown in SEQ ID No. 4.

[0011] An S protein fragment of the infectious bronchitis virus of chicken, the amino acid sequence of the S protein fragment is shown in SEQ ID No. 2.

[0012] An antigen of the infectious bronchitis virus of chicken, the antigen is a trimeric structure of the above-mentioned recombinant protein S-Trimer.

[0013] A biological material related to the antigen recombinant protein S-Trimer or the S protein fragment, the biological material includes:

[0014] (1) a nucleic acid encoding the above-mentioned antigen recombinant protein S-Trimer or S protein fragment;

[0015] (2) an expression cassette containing the nucleic acid in (1);

[0016] (3) a vector containing the nucleic acid in (1) or the expression cassette in (2);

[0017] (4) a recombinant cell containing the nucleic acid in (1), the expression cassette in (2) or the vector in (3).

[0018] In some embodiments, the nucleotide sequence of the nucleic acid of the antigen recombinant protein S-Trimer is shown in SEQ ID No. 3.

[0019] The application of the above-mentioned antigen recombinant protein S-Trimer in the preparation of a medicine for preventing the infection of the infectious bronchitis virus of chicken.

[0020] In some embodiments, the medicine is a subunit vaccine of the infectious bronchitis virus of chicken.

[0021] A subunit vaccine of the infectious bronchitis virus of chicken, the subunit vaccine includes a pharmaceutically acceptable carrier and an immune amount of the above-mentioned antigen of the infectious bronchitis virus of chicken.

[0022] In some embodiments, the content of the antigen is ≥20 μg / ml, preferably 20-60 μg / ml.

[0023] In some embodiments, the pharmaceutically acceptable carrier comprises at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

[0024] Preferably, the adjuvant comprises one or more of an aluminum hydrogel adjuvant, a saponin, Afutidine, DDA, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic or methacrylic acid, a copolymer of maleic anhydride and an alkenyl derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, Monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, and a Gel adjuvant.

[0025] In some embodiments, the concentration of the adjuvant ranges from 5% V / V to 70% V / V, preferably 30% to 70% V / V, and more preferably 66% V / V.

[0026] In some embodiments, the lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

[0027] Preferably, the immunostimulant comprises an alpha-interferon, a beta-interferon, a gamma-interferon, a granulocyte macrophage colony-stimulating factor, a macrophage colony-stimulating factor, or an interleukin 2.

[0028] Compared with the prior art, the technical effects of the present application are:

[0029] By using bioinformatics software to analyze the structural domains and functional domains of the S protein of the chicken infectious bronchitis virus, two proline mutation sites that can stabilize the pre-fusion conformation of the S protein were obtained. Based on this, a trimeric S protein was constructed by fusing a trimeric tag to the C-terminal of the protective antigen of the S protein truncated form containing two proline mutations (P). After expression and purification, the immunogenicity was evaluated by animal evaluation, and it was verified that the vaccine has high safety, good immunogenicity, batch stability, and can provide complete protection against chicken infectious bronchitis virus challenge. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the liquid chromatography detection result of S and S-Trimer protein. DETAILED DESCRIPTION In the following, the specific embodiments of the present application are described in detail with reference to the accompanying drawings.

[0031] The related terms in the present application are explained as follows:

[0032] The term "chicken infectious bronchitis virus (IBV)" belongs to Nido virus order (Nidovirales), Coronaviridae family, Coronavirus genus gamma coronavirus, and the virus genome is a non-segmented single-stranded RNA with an envelope. The virion mainly includes two parts of envelope and nucleocapsid, contains four structural proteins: Spike (S) glycoprotein, Membrane (M) glycoprotein, Nucleocapsid (N) protein and Small Envelope (E) glycoprotein. It often causes the decline of growth performance, the increase of dead and discarded rate, the decline of egg production and eggshell quality of laying hens and breeding hens, causes serious economic losses, and is one of the important diseases in the poultry industry.

[0033] The term "antigen" refers to a substance that can induce an immune response in the body, i.e., can be specifically recognized and combined by the antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, make them proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically combine with the corresponding products in vivo and in vitro.

[0034] The term "S protein" is a type I transmembrane glycoprotein and a receptor binding protein of IBV, which contains an extracellular domain, a transmembrane region and an intracellular domain, and plays an important role in virus entry, virus-host interaction and immunogenicity evaluation. The S protein is connected by 2-3 monomers into a polymer non-covalently, and the molecular weight is 180 kda. The S protein can be cleaved into two subunits of N-terminal S1 and C-terminal S2 after translation in the host cell. The important biological functions of S protein include: binding to glycoprotein receptors on the host cell membrane, which is a prerequisite for IBV adsorption to cells. After the virus is adsorbed to the host cell membrane, the virus envelope fuses with the host cell membrane, and it is through this fusion that the virus is transmitted. S1 gene is the main protein gene for IBV to produce infectivity, and is also the main immunogenic protein, which can induce the body to produce virus neutralizing antibodies, hemagglutination inhibition antibodies, cell-mediated immune response, and can induce the protective effect against pathogenic virus challenge.

[0035] The term "T4 trimer tag" is also called T4 phage fibritin foldon C-terminal or T4 phage fibritin foldon C-terminal trimerization sequence, each subunit is composed of 27 amino acids, and the domain is composed of 3 identical subunits, each subunit contains a β-hairpin structure.

[0036] The present application provides a chicken infectious bronchitis virus S protein fragment, and the amino acid sequence is shown in SEQ ID NO. 2.

[0037] The present application selects the 19th to 1095th amino acid fragment of the S protein of the chicken infectious bronchitis virus as an antigen target, and replaces the 859th-860th amino acid of S with proline (A859P, I860P). Verification has confirmed that it can effectively prevent chicken infectious bronchitis.

[0038] The present application further studies and finds that the antigen recombinant protein can form a trimer form and has a good immune effect by adding a trimer tag to the above-mentioned S protein fragment.

[0039] In some embodiments, the trimer tag can be a T4 trimer tag, and the amino acid sequence is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO. 5), obtaining the chicken infectious bronchitis virus antigen recombinant protein S-Trimer, and the amino acid sequence is shown in SEQ ID NO. 4.

[0040] The present application also provides a chicken infectious bronchitis virus antigen, which is a trimer structure of the recombinant protein S-Trimer. The trimer structure can present the original structure state of the protein as much as possible, which is beneficial to stimulating the body to produce a stronger immune response.

[0041] The present application provides biological materials related to the above-mentioned S protein fragment or recombinant protein S-Trimer, such as a nucleic acid fragment encoding the above-mentioned protein, an expression cassette containing the nucleic acid fragment, a vector (such as a cloning plasmid and an expression plasmid, etc.) of the expression cassette, and a recombinant cell containing the nucleic acid fragment, the expression cassette or the vector. These biological materials can be directly used for the production of the protein of the present application as a biological module, which has the advantages of being fast and efficient. The nucleic acid fragment of the present application can be obtained by primer amplification or artificial synthesis, and the nucleotide sequence encoding the recombinant protein S-Trimer is shown in SEQ ID NO. 3.

[0042] The recombinant protein S-Trimer or the chicken infectious bronchitis virus antigen provided by the present application is used for preparing related products for preventing chicken infectious bronchitis virus infection, such as vaccines, antibody detection reagents, etc.

[0043] The present application provides a chicken infectious bronchitis virus subunit vaccine, which can contain a pharmaceutically acceptable carrier in addition to the effective component mainly being the recombinant protein S-Trimer or the chicken infectious bronchitis virus antigen provided by the present application, in order to meet different needs in actual application, such as production, transportation, dosage form, administration method, etc.

[0044] The term "vaccine" refers to a pharmaceutical composition containing chicken infectious bronchitis virus protein antigens, which can induce, stimulate or enhance the immune response of a pig to chicken infectious bronchitis virus.

[0045] The term "immunizing amount" is understood to mean an "immunologically effective amount", also referred to as an immunoprotective amount or an effective amount to generate an immune response, which is an amount of antigen effective to induce an immune response in the recipient sufficient to prevent or ameliorate signs or symptoms of disease, including adverse health effects or complications thereof. The immune response can be sufficient for diagnostic purposes or other assays, or can be suitable for preventing signs or symptoms of disease, including adverse health effects or complications thereof resulting from infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. An animal's immune response to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms following challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, a reduction in clinical signs such as mortality, morbidity, temperature values, overall physiological condition and general health and performance of the subject. The immune response can include, but is not limited to, the induction of cellular and / or humoral immunity.

[0046] In some embodiments, the subunit vaccine contains an immunizing amount of chicken infectious bronchitis virus antigens, the content of chicken infectious bronchitis virus antigens is ≥ 20 μg / ml, preferably 20-60 μg / ml. For example, but not limited to, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 55 μg / ml or 60 μg / ml.

[0047] In the present application, the chicken infectious bronchitis virus antigen recombinant protein S-Trimer or chicken infectious bronchitis virus antigen can be prepared by eukaryotic expression system, or can be prepared by prokaryotic expression system, cell expression system or chemical synthesis method.

[0048] The term "pharmaceutically acceptable carrier" refers to all components other than chicken infectious bronchitis virus subunit protein antigens in the subunit vaccine of the present application, which do not stimulate the body and do not hinder the biological activity and characteristics of the compound used as a carrier or diluent, preferably an adjuvant.

[0049] The term "adjuvant" can include aluminium hydrogel adjuvants; saponins such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic or methacrylic acid; co-polymers of maleic anhydride and alkenyl derivatives selected from the group consisting of compounds.

[0050] The term "emulsion" can be based on, inter alia, light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from the oligomerisation of isoolefins, such as squalane or squalene oil, in particular iso-butene or eucalyptene; linear alkyl esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / euric)ate), glycerol tri-(octanoate / euric)ate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearate. The oil is used in combination with an emulsifying agent in order to form the emulsion. The emulsifying agent is preferably a non-ionic surfactant, in particular an ester of sorbitan, an ester of mannide (such as anhydrous mannide oleate), an ester of a fatty glycol, an ester of polyglycerol, an ester of propylene glycol and an ester of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See "The theory and practical application of adjuvants" by Hunter et al. (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and "Vaccine" by Todd et al. (1997, 15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of "Vaccine design, the Subunit and adiuvant approach" by Powell M and Newman M (Plenum Press, 1995) can be used.

[0051] The term "polymers of acrylic or methacrylic acid" are preferably cross-linked polymers of acrylic or methacrylic acid, in particular cross-linked with polyalkenyl ethers of sugars or polyalcohols, which compounds are known under the name Carbomer (Carbopol, trade name) (Pharm. Europa, 1996, 8(2)). The skilled person can also refer to US patent 2 909 462, which describes such acrylic acid polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, in which at least 3 of the hydroxyl groups have their hydrogen atoms replaced by an unsaturated aliphatic radical having at least 2 carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves can contain further substituents, such as methyl groups. These products are sold under the name Carbopol (BF Goodrich, Ohio, USA) and are particularly suitable. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P can be mentioned, the most preferred being Carbopol 971P.

[0052] The term "co-polymers of maleic anhydride and alkenyl derivatives" also contemplates co-polymers of maleic anhydride with ethylene, EMA (Monsanto), which polymers dissolve in water to produce an acidic solution, which is neutralized, preferably to physiological pH, in order to produce an adjuvant solution into which the immunogenic, immunizing or vaccinal composition itself can be incorporated.

[0053] The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, etc.

[0054] In preferred embodiments, the adjuvant comprises one or more of mineral oil, aluminum hydrogel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 201 or Gel adjuvant.

[0055] Preferably, the adjuvant is a mineral oil adjuvant, which is used to prepare a water-in-oil emulsion.

[0056] In some embodiments, the concentration of the adjuvant ranges from 5% V / V to 70% V / V, preferably 30% to 70% V / V, more preferably 66% V / V. The concentration of the adjuvant can range, but is not limited to, 5% V / V, 10% V / V, 15% V / V, 20% V / V, 25% V / V, 30% V / V, 35% V / V, 40% V / V, 45% V / V, 50% V / V, 55% V / V, 60% V / V, 65% V / V or 70% V / V.

[0057] Mineral oil, also known as paraffin oil, white oil, or white mineral oil, is a widely used adjuvant in inactivated vaccines. It has the ability to delay the retention time of the immunogen in the body, allowing for a sustained slow release, and enhances the phagocytic and bactericidal capacity of macrophages.

[0058] The term "lyoprotectant" refers to an ingredient other than an excipient that protects the pharmaceutical active during the process of freeze-drying and during the storage phase after lyophilization. Lyoprotectants can be selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids.

[0059] In some embodiments, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin 2.

[0060] The term "preventing" in relation to an infection with IBV refers to inhibiting the replication of IBV, inhibiting the spread of IBV or preventing IBV from establishing itself in its host, as well as alleviating the symptoms of the disease or condition associated with IBV infection.

[0061] The application will be further described in conjunction with specific embodiments. The advantages and features of the application will become more apparent with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the application without departing from the spirit and scope of the application, and such modifications and substitutions all fall within the protection scope of the application.

[0062] The chemical reagents used in the embodiments of the application are all of analytical purity and are purchased from the National Pharmaceutical Group. The experimental methods described in the application are all conventional methods unless otherwise specified. The biological materials described in the application are all available through commercial channels unless otherwise specified.

[0063] Example 1 Construction of recombinant eukaryotic expression plasmid

[0064] The IBV S protein is a type I transmembrane protein containing an extracellular domain, a transmembrane domain and an intracellular domain. The signal peptide segment and the transmembrane segment of the protein are analyzed using SignalP 5.0 and TMHMM online tools. The 19th to 1095th amino acid segment of the protein is selected as the target antigen. A Kozak sequence is added before the ATG at the 5' end of the sequence, an HSA protein signal peptide sequence is added after the ATG, the 859th-860th amino acids of S are replaced with proline (A859P, I860P), and a T4 phage trimer tag and a 6×His nucleotide sequence are added before the stop codon at the 3' end of the sequence. The nucleotide sequence is optimized for CHO preferred codons and synthesized by Kingsway. The nucleotide sequence is as shown in SEQ ID NO. 3, and is cloned into the pUC57 vector through Xba I / Hind III enzyme cutting sites (named pUC57-IBV-S-Trimer).

[0065] The synthesized plasmid is recovered by Xba I / Hind III enzyme cutting and is connected to the pcDNA3.1 vector with the same enzyme cutting sites by T4 DNA ligase to construct an expression vector carrying a T4 phage trimer tag. The ligation product is transformed into DH5α E. coli competent cells, and single colony colonies are picked from the transformation plate for bacterial liquid PCR identification. The clone bacterial liquid showing positive in PCR identification is sent to Genscript for sequencing analysis. Finally, the clone with correct sequencing (pcDNA3.1-S-Trimer) is selected for the next step.

[0066] The primer S-F: 5'-CTAGTCTAGAGCCACCATGAAATGGGTGACATTTAT-3' (SEQ ID NO. 6) and S-R: 5'-CCCAAGCTTTCAATGGTGATGGTGGTGATGATACCAGGGCCACTTGA TGT-3' (SEQ ID NO. 7) were designed to amplify the S gene from the pUC57-IBV-S-Trimer plasmid as a template, the nucleotide sequence was as shown in SEQ ID NO. 1, to construct an expression vector without a T4 bacteriophage trimer tag, and finally a correct sequencing clone (pcDNA3.1-S) was selected for the next step.

[0067] The correct sequencing clone broth was taken out, inoculated at 1:1000 into 50 mL, and cultured overnight at 37°C. The cultured broth was used to extract plasmids using an Omega plasmid extraction kit, the plasmids were identified using Xba I / Hind III, and the plasmid concentration was determined using an enzyme-labeled detector. The results showed that the recombinant expression plasmid constructed had the correct band size after enzyme digestion, which was consistent with the expectation.

[0068] Example 2: Expression and purification of recombinant protein

[0069] The recombinant plasmid prepared in Example 1 was used to transfect Expi-CHO cells, the plasmid transfection amount was 0.8 μg / mL, 25 mL was transfected, and the fed-batch culture was performed on the second day after transfection. The sample was collected on the 7th day after transfection for SDS-PAGE identification, and it was found that the protein was expressed in the secreted supernatant. The expression product was harvested on the 9th day after transfection.

[0070] The harvested Expi-CHO cell expression product was centrifuged at 6500 rpm for 20 min, and the culture supernatant was taken and subjected to protein affinity chromatography purification using a protein chromatography purification system. The chromatography medium was Ni Sepharose 6 Fast Flow, and the system flow rate was 90 cm / h. Before loading, the chromatography column was equilibrated with an equilibration buffer (0.02 mol / L Tris (pH 7.0), 0.02 mol / L imidazole, 0.5 mol / L NaCl), and after loading, the impurities were eluted with a buffer solution (0.02 mol / L Tris (pH 7.0), 0.05 mol / L imidazole, 0.5 mol / L NaCl), the target protein was eluted with a buffer solution (0.02 mol / L Tris (pH 7.0), 0.5 mol / L imidazole, 0.5 mol / L NaCl), and the elution product was collected. After the protein was purified by SDS-PAGE electrophoresis, a clear target protein band should be visible after staining. The Expi-CHO expression system can express and secrete soluble IBV S and S-Trimer proteins, and the protein bands are all located between 130-250 KDa, which is significantly greater than the theoretical molecular weight of the amino acid sequence of S and S-Trimer proteins (the theoretical molecular weights of S and S-Trimer are 119 kDa and 123 kDa, respectively), indicating that the expressed recombinant protein has a large amount of glycosylation modification.

[0071] Example 3 Identification of recombinant protein

[0072] I. Western blot (WB) identification of reactivity

[0073] The product after SDS-PAGE electrophoresis in Example 2 was transferred to an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with chicken-derived anti-chicken infectious bronchitis virus specific serum for 2 hours, rinsed, incubated with HRP-labeled rabbit anti-chicken IgY antibody secondary antibody for 2 hours, rinsed, and then developed using a DAB developing kit. The results showed that the Expi-CHO recombinant expressed IBV S and S-Trimer proteins could react with IBV positive serum.

[0074] II. Liquid chromatograph detection analysis

[0075] The purified protein obtained in Example 2 was filtered and then detected using a Shimadzu high-performance liquid chromatograph. The chromatographic column was a Sartorius SRT-C SEC500 chromatographic column, PBS was used as the mobile phase, the column was equilibrated for 30 min, the flow rate was set to 0.5 ml / min, the sample volume was 50 μl, and each protein was detected for 40 min. The results are shown in FIG. 1. The S protein (monomer protein theoretical molecular weight 119 kDa) without the addition of a T4 trimer tag had an elution time of about 18.8 min, and the size was about 120 kDa according to a standard curve, indicating that the recombinant S protein existed in the form of a monomer. The S-Trimer protein (monomer protein theoretical molecular weight 123 kDa) had an elution time of about 16.9 min, and the size was about 370 kDa according to a standard curve, indicating that the recombinant S-Trimer protein mainly existed in the form of a trimer.

[0076] Example 4 Preparation of a subunit vaccine

[0077] The chicken infectious bronchitis virus antigen prepared above (trimer form of the antigen recombinant protein S-Trimer) was slowly added to the adjuvant, and the motor was started to stir at 17,500 r / min for 5 min. The adjuvant suitable for use in the present application can be an adjuvant known to those skilled in the art. In the present application, the adjuvant selected was a mineral oil adjuvant (water-in-oil emulsion). The specific ratio is shown in Table 1.

[0078] Table 1 Subunit vaccine ratio

[0079] Example 5 Evaluation of the immunogenicity of a subunit vaccine

[0080] Forty 21-28 day-old SPF chickens were used, of which 30 were divided into three groups of 10 for the immunization groups, and the remaining 10 were used as the control group. The chickens in the immunization groups were each inoculated intranasally and ocularly with one dose of chicken infectious bronchitis live vaccine (H120 strain) containing 10 3.5 EID 50 ), and the chickens in the control group were inoculated with the same volume of sterile PBS. Twenty-one days after inoculation, blood was collected from all the chickens, and the serum was separated. The chickens in the three immunization groups were each injected intramuscularly in the leg with 0.5 ml of the vaccine prepared in Example 4 (vaccine 1 to vaccine 3), and the control group was inoculated with 0.5 ml of sterile PBS. Twenty-eight days after the second immunization, blood was again collected from all the chickens, and the serum was separated. The serum collected 21 days after the first immunization and 28 days after the second immunization was detected for IB HI antibody titer. The results are shown in Table 2.

[0081] Table 2 Results of the immunogenicity test of a subunit vaccine

[0082] From the results, the geometric mean of the HI antibody titer of the second immune serum of the three groups of immunization test chickens was far higher than 4 times of the geometric mean of the HI antibody titer of the first immune serum, which met the standard that the geometric mean of the HI antibody titer of the second immune serum should not be lower than 4 times of the geometric mean of the HI antibody titer of the first immune serum.

[0083] At 28 days after the second immunization, the chickens of the three groups of immunization and the control group were challenged with 0.1 ml of the infectious bronchitis virus M41 strain virus liquid (containing 10 5.5 EID 50 ) by the way of nasal drops and eye drops, and were isolated and bred. Five days after the challenge, the tracheal swabs were collected, and 9-11 day-old SPF chicken embryos were inoculated for IBV virus isolation. At the same time, all the chickens were euthanized 5 days after the challenge, and the upper, middle and lower segments of the trachea were taken for microscopic observation of the cilia activity. The scoring standard was as follows: if the cilia of the whole segment of the trachea were active, the score was 0; if 75%-99% of the cilia of the whole segment of the trachea were active, the score was 1; if 50%-74% of the cilia of the whole segment of the trachea were active, the score was 2; if 25%-49% of the cilia of the whole segment of the trachea were active, the score was 3; and if less than 25% of the cilia of the whole segment of the trachea were active or the cilia were completely inactive, the score was 4. The challenge test results are shown in Table 3.

[0084] Table 3 Challenge test results of the subunit vaccine *: In the immunization group of the immunized vaccine 1, the tracheal cilia damage scores of two chickens were 1 respectively, and the total was 2.

[0085] The virus isolation results showed that the tracheal swabs of the challenge control group were 10 / 10 IBV virus isolation positive, while the IBV virus isolation of the three groups of immunization was negative. The tracheal cilia damage results showed that the tracheal cilia of the chickens of each immunization group had only slight damage, and the integrity was 75%-100%; the tracheal cilia of the chickens of the challenge control group was severely shed, and the integrity was lower than 25%.

[0086] It is shown that the chicken infectious bronchitis virus subunit vaccine with a content of not less than 20 μg / ml can make the chicken population produce a higher HI antibody titer, and can realize effective immunization protection for the chicken population.

[0087] The above only describes the preferred embodiments of the present application, and does not make any form of limitation to the present application. Although the preferred embodiments of the present application are disclosed as above, however, the present application is not limited thereto. Any person skilled in the art can make some minor changes or modifications to the above disclosed technical contents to form equivalent embodiments with equivalent changes, as long as the changes or modifications do not depart from the technical solution of the present application. Any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application still belong to the scope of the technical solution of the present application.

Claims

1. A recombinant protein S-Trimer of chicken infectious bronchitis virus antigen, wherein the amino acid sequence of the recombinant protein S-Trimer is shown in SEQ ID No.

4.

2. A fragment of the S protein of chicken infectious bronchitis virus, wherein the amino acid sequence of the S protein fragment is shown in SEQ ID No.

2.

3. A chicken infectious bronchitis virus antigen, wherein the chicken infectious bronchitis virus antigen is a trimer structure of the recombinant protein S-Trimer as described in claim 1.

4. A biomaterial relating to the recombinant antigen protein S-Trimer of claim 1 or the S protein fragment of claim 2, said biomaterial comprising: (1) The nucleic acid encoding the recombinant antigen protein S-Trimer or the S protein fragment; (2) An expression cassette containing the nucleic acid in (1); (3) A vector containing the nucleic acid in (1) or the expression cassette in (2); (4) Recombinant cells containing the nucleic acid in (1), the expression cassette in (2), or the vector in (3).

5. The biomaterial according to claim 4, wherein, The nucleotide sequence of the nucleic acid of the recombinant antigen protein S-Trimer is shown in SEQ ID No.

3.

6. The use of the recombinant antigen protein S-Trimer according to claim 1 in the preparation of a drug for preventing infection with infectious bronchitis virus in chickens.

7. The application according to claim 6, wherein the drug is a chicken infectious bronchitis virus subunit vaccine.

8. A subunit vaccine against infectious bronchitis virus in chickens, said subunit vaccine comprising a pharmaceutically acceptable carrier and an immunizing dose of the infectious bronchitis virus antigen of claim 3.

9. The subunit vaccine according to claim 8, wherein, The antigen content is ≥20 μg / ml, preferably 20–60 μg / ml.

10. The subunit vaccine according to claim 8, wherein, The pharmaceutically acceptable carriers include at least one of the following: adjuvants, lyophilization protectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. Preferably, the adjuvant comprises one or more of the following: aluminum gel adjuvant, saponin, avrididine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, block copolymer, monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-sensitive enterotoxin of Escherichia coli, cholera toxin, muramyl dipeptide, and gel adjuvant; Preferably, the concentration range of the adjuvant is from 5% V / V to 70% V / V, more preferably from 30% V / V to 70% V / V, and even more preferably 66% V / V; Preferably, the freeze-drying protectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids; Preferably, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-2.

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