A type c avian infectious coryza subunit vaccine and its preparation method and application

Abstract

The present application relates to the technical field of veterinary biological products, and particularly relates to a C-type chicken infectious rhinitis subunit vaccine and a preparation method and application thereof. The present application uses Region 2 (abbreviated as TP5-BP5-BS-C-R2) of the outer membrane protein HMTp210 gene of the C-type chicken infectious rhinitis containing an immune enhancement sequence fusion of TP5-BP5-BS as an immunogen to prepare a subunit vaccine. The C-type chicken infectious rhinitis subunit vaccine prepared by the method of the present application has high safety, overcomes the side reactions such as injection site swelling caused by the whole bacteria inactivated vaccine, and can quickly induce the immune animals to produce high levels of neutralizing antibodies, realizes the efficient immune protection of the C-type chicken infectious rhinitis, and the immune effect is better than that of the C-type rhinitis subunit vaccine and the whole bacteria inactivated vaccine without the immune enhancement sequence. Moreover, the preparation process of the subunit vaccine is simple, the cost is low, and the subunit vaccine is easy to mass-produce.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and in particular to a subunit vaccine for type C infectious coryza in chickens, its preparation method and application. Background Technology

[0002] Avian infectious coryza is an acute upper respiratory tract infection in chickens caused by *Avibacterium paragallinarum* (Apg). Currently, this disease is widespread in various poultry farming areas across China, particularly impacting the production performance of laying hens. It primarily causes poor growth in pullets and a significant decrease in egg production in laying hens, and can also significantly increase the culling rate of broilers, resulting in substantial economic losses.

[0003] *Avianobacterium paragallinarum* is a short, Gram-negative bacillus belonging to the Pasteuraceae family. Page et al. classified *Avianobacterium paragallinarum* into three serotypes: A, B, and C. Studies have shown that all three serotypes have varying degrees of pathogenicity in chickens. In recent years, with the increasing incidence of bacterial diseases such as infectious coryza in chickens, and the widespread use of drugs in clinical practice, drug resistance in infectious coryza strains has generally increased, and drug sensitivity has decreased, significantly impacting treatment effectiveness. Since sulfonamides are prohibited during the laying period in laying hens, vaccine prevention has gradually become the preferred method for controlling infectious coryza in chickens, leading to a growing market demand for infectious coryza vaccines, especially high-quality ones. Currently, there are various vaccine products on the market for controlling coryza, including bivalent and trivalent vaccines. In particular, the trivalent whole-cell A, B, and C vaccine can provide protection against all coryza serotypes, thus having a large market potential. Because *Avianobacterium paragallinarum* is a causative bacterium, its cultivation cost is high. Furthermore, whole-cell inactivated vaccines contain large amounts of bacterial protein, endotoxins, other toxic substances, and byproducts, which can easily cause severe side effects and lead to focal necrotic patches at the injection site. In addition, currently available commercially available inactivated chicken contagious coryza vaccines use bacterial antigens, which are particulate antigens, resulting in uneven emulsification and easy demulsification and separation during vaccine transportation and storage. To develop safer and more effective vaccines, some researchers have investigated the feasibility of preparing recombinant vaccines using genetic recombination technology. For example, Ryuichi Sakamoto et al. cloned and expressed HMTp210-R2 of *Avianobacterium paragallinarum* types A and C, achieving good protective effects after immunizing chicken flocks.

[0004] The bursa of Fabricius is a central lymphoid organ unique to birds and the earliest site of B cell development. Cystoxin tripeptide, the first bioactive peptide extracted from the bursa of Fabricius, is an important active component of the bursa, enhancing the body's immune system function and also possessing significant immunological and physiological activities in mammals. Previous research has primarily focused on cystin. In recent years, with the widespread application of mass spectrometry in protein separation and identification, numerous bioactive peptides have been isolated and identified from the bursa of Fabricius, representing significant breakthroughs in their immunological activity and mechanisms of action. Some bioactive peptides from the bursa of Fabricius possess immunomodulatory functions, and some also exhibit antitumor potential. To date, there are no reports of type C infectious coryza subunit vaccines containing TP5-BP5-BS immunomodulatory sequences. Currently, the immunoprotective rate of commercially available inactivated infectious coryza vaccines is generally around 70%, and in clinical practice, cases of non-protection and disease onset after immunization occur frequently. Summary of the Invention

[0005] This invention provides a subunit vaccine against type C infectious coryza in chickens, its preparation method, and its application, addressing the low immunoprotective rate of commercially available inactivated infectious coryza vaccines in the prior art. This invention fuses multiple immune-enhancing sequences with the Region 2 expression of the HMTp210 gene of type C infectious coryza in chickens, significantly improving the protective efficiency of the subunit vaccine against type C infectious coryza in chickens.

[0006] This invention provides a protective antigen, the amino acid sequence of which is shown in SEQ IN NO.1. The protective antigen of this invention is Region 2 of the HMTp210 gene of type C infectious coryza in chickens, containing a TP5-BP5-BS immunostimulating sequence fusion. Subunit vaccines prepared using this protective antigen have the advantages of high safety and high immunogenicity. After immunization of animals, they can rapidly induce the production of high levels of neutralizing antibodies, achieving highly efficient immunoprotection against type C infectious coryza in chickens. The immunogenicity is superior to that of whole-cell inactivated vaccines and subunit vaccines without immunostimulating sequences.

[0007] The present invention also provides a nucleic acid for encoding the protective antigen, wherein the nucleotide sequence of the nucleic acid is any one of the following:

[0008] (1) The nucleotide sequence shown in SEQ ID NO.2;

[0009] (2) A nucleotide sequence encoding the same function protein obtained by deleting, substituting or inserting one or more bases of the nucleotide sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a biomaterial comprising the nucleic acid, wherein the biomaterial includes an expression cassette, a vector, or a host cell.

[0011] The present invention also provides a recombinant expression vector comprising the nucleotide sequence of the nucleic acid.

[0012] The present invention also provides a recombinant engineered bacterium, which is Escherichia coli containing the recombinant expression vector.

[0013] The present invention provides a recombinant Escherichia coli genetically engineered bacterium containing a nucleotide fragment encoding TP5-BP5-BS-C-R2, the amino acid sequence of which is shown in SEQ IN NO.1.

[0014] In this invention, the vector and host cell can be understood as various vectors and host cells used by those skilled in the art when performing gene cloning or expression. As technology advances, the selection of the vector and host cell may change, but as long as they contain the TP5-BP5-BS-C-R2 encoding gene of this invention, they are all within the scope of protection of this invention.

[0015] The present invention also provides a subunit vaccine for type C infectious coryza in chickens, the antigen component of which is protein TP5-BP5-BS-C-R2, the amino acid sequence of which is shown in SEQ ID NO.1.

[0016] This invention also provides a method for preparing the C-type chicken infectious coryza subunit vaccine, comprising expressing the TP5-BP5-BS-C-R2 protein, wherein the TP5-BP5-BS-C-R2 protein has the amino acid sequence shown in SEQ ID NO.1. This invention provides a method for preparing a C-type chicken infectious coryza subunit vaccine containing a TP5-BP5-BS immune-enhancing sequence fusion, solving the problems of high endotoxin content, high content of bacterial extraneous proteins, and insufficient protection rate (approximately 70%) in existing coryza vaccines.

[0017] This invention provides a simple method for preparing a novel subunit vaccine for infectious coryza type C in chickens containing the TP5-BP5-BS immune-enhancing sequence. The subunit vaccine for infectious coryza type C in chickens prepared according to this invention has high safety, overcomes the side effects such as injection site swelling that are common with whole-bacterial inactivated vaccines, and can rapidly induce high levels of neutralizing antibodies in immunized animals, achieving highly effective immune protection against infectious coryza type C in chickens. Its immunization effect is superior to subunit vaccines for chickens without the immune-enhancing sequence and whole-bacterial inactivated vaccines. Furthermore, the preparation process of this subunit vaccine is simple, low-cost, and easy for large-scale production.

[0018] In some preferred embodiments of the present invention, the type C chicken infectious coryza subunit vaccine includes an antigen and a vaccine adjuvant, wherein the antigen used is the TP5-BP5-BS-C-R2 protein prepared in this invention.

[0019] In some embodiments of the present invention, the vaccine adjuvant includes mineral oil, aluminum stearate, and Span.

[0020] According to the preparation method of the present invention, the expression is achieved by ligating the nucleotide sequence shown in SEQ ID NO.2 to the expression vector.

[0021] According to the preparation method described in this invention, the expression vector is one of the pET28, pET30, and pET32 series vectors.

[0022] According to the preparation method described in this invention, the host for expression is Enterobacter spp. bacteria.

[0023] The preparation method according to the present invention includes the following steps:

[0024] (1) Construct an expression vector containing the nucleotide sequence shown in SEQ ID NO.2;

[0025] (2) The expression vector was transformed into Enterobacteriaceae to obtain recombinant bacteria expressing TP5-BP5-BS-C-R2 protein;

[0026] (3) Culturing the recombinant bacteria to express the TP5-BP5-BS-C-R2 protein;

[0027] (4) Extract the TP5-BP5-BS-C-R2 protein and prepare a subunit vaccine for type C chicken infectious coryza.

[0028] In some embodiments of the present invention, the preparation method includes codon optimization of the encoding gene of TP5-BP5-BS-C-R2 to obtain the nucleotide sequence shown in SEQ ID NO.2. The present invention uses Region 2 (abbreviated as TP5-BP5-BS-C-R2) of the HMTp210 gene, a type C chicken infectious coryza outer membrane protein fused with the TP5-BP5-BS immunostimulating sequence, as an immunogen to prepare a subunit vaccine. After optimization using a specific codon optimization method, it is cloned into the PET-30a expression vector. After transformation into BL21(DE3) host bacteria, the soluble expression level of TP5-BP5-BS-C-R2 is significantly increased.

[0029] The application of the protective antigen, the nucleic acid, the biological material, or the type C chicken infectious coryza subunit vaccine in chicken infectious coryza immunization.

[0030] Preferably, its use in the preparation of drugs for the treatment or prevention of type C infectious coryza in chickens.

[0031] The beneficial effects of this invention are:

[0032] The subunit vaccine prepared using the obtained TP5-BP5-BS-C-R2 protein as an antigen has the advantages of high safety and high immunogenicity. After immunizing animals, it can rapidly induce the production of high levels of neutralizing antibodies, achieving highly efficient immune protection against type C infectious coryza in chickens. The immunization effect is superior to whole-cell inactivated vaccines and subunit vaccines without immune-enhancing sequences. Moreover, the vaccine preparation process is simple, low-cost, and easy to mass-produce. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 The codon optimization scheme of Embodiment 1C-HMTp210-R2 of the present invention is shown, wherein the optimized codons are uppercase letters.

[0035] Figure 2 This is a diagram illustrating the construction and expression of the TP5-BP5-BS-C-R2 genetically engineered bacteria in Example 2 of this invention.

[0036] From left to right: 1 mg / mL BSA, 0.5 mg / mL BSA, Marker, pET30a-C-HMTP210-R2 supernatant, and pET30a-TP5-BP5-BS-C-R2 supernatant.

[0037] Figure 3 This is the expression and quantification diagram of pET30a-TP5-BP5-BS-C-R2 protein in Example 3 of the present invention, i.e., the result of SDS-PAGE electrophoresis detection;

[0038] From left to right, these are lanes 1-7; lane 1 is the marker, lanes 2-4 are 0.5, 1, and 2 mg / ml BSA protein standards respectively, and lanes 5-7 are 5 μL, 7.5 μL, and 10 μL pET30a-TP5-BP5-BS-C-R2 expression supernatant respectively. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] Example 1: Codon Optimization of C-HMTp210-R2

[0043] To improve the expression level and soluble expression of the target protein, codon optimization was first performed on C-HMTp210-R2 (as shown in SEQ ID NO. 3). The C-HMTp210-R2 codon optimization scheme is as follows: Figure 1 As shown, the optimized codons are uppercase letters, specifically as shown in SEQ ID NO.2 nucleotide sequence 166-1923 (1-165 are TP5-BP5-BS nucleotide sequences).

[0044] Example 2: Soluble expression of C-HMTp210-R2 protein

[0045] The codon-optimized TP5-BP5-BS-C-R2 gene sequence was synthesized. The unoptimized C-HMTp210-R2 and TP5-BP5-BS-C-R2 gene sequences were digested with Nde I / Xho I and cloned into the pET-30a vector. The resulting recombinant expression vectors were named pET30a-C-HMTP210-R2 and pET30a-TP5-BP5-BS-C-R2, respectively, and transformed into *E. coli* BL21(DE3). The resulting engineered bacteria were inoculated into kanamycin-resistant medium and cultured at 37°C with shaking for 4-5 hours until OD500 reached. 600 Once the bacterial count reaches approximately 0.8, IPTG is added, and the cells are induced at 16°C for 15 hours. The cells are harvested, sonicated, centrifuged, and subjected to SDS-PAGE electrophoresis. Expression characteristics are as follows: Figure 2 The results showed that codon optimization increased the soluble expression level of TP5-BP5-BS-C-R2 protein by more than two times.

[0046] Example 3: Preparation of Type C Chicken Infectious Coryza Subunit Vaccine

[0047] 1. Preparation of TP5-BP5-BS-C-R2 antigen

[0048] (1) Activation of genetically engineered bacteria: 10 μL of the engineered bacteria pET30a-TP5-BP5-BS-C-R2 protein glycerol bacteria constructed in Example 2 was inoculated into 5 ml of kanamycin-resistant LB medium and cultured overnight with shaking.

[0049] (2) Inoculate 200 mL of kanamycin-resistant medium at a ratio of 1:200, and incubate at 37°C with shaking for 4-5 hours until OD reaches 1. 600 The value reaches around 0.8;

[0050] (3) Add IPTG at a ratio of 1:2000 and induce at 16℃ for 15h;

[0051] (4) Collect bacterial cells;

[0052] (5) Resuspend the bacteria in 20 mL of a dedicated buffer;

[0053] (6) Ultrasonic disruption of bacteria;

[0054] (7) Centrifuge at 12000 rpm for 10 min, repeat twice;

[0055] (8) Add 40% ammonium sulfate to precipitate the target protein, centrifuge at 12000 rpm for 10 min, and resuspend the precipitate in 20 ml of special buffer.

[0056] (9) Add 1% Triton X114 to remove endotoxin.

[0057] (10) Protein concentration was detected by SDS-PAGE electrophoresis (results are shown below). Figure 3 (As shown).

[0058] The concentration of C-HMTP210-R2 antigen was calculated through grayscale analysis.

[0059] 2. Preparation of Type C Chicken Infectious Coryza Subunit Vaccine

[0060] (1) Oil phase preparation: Take 95 parts of mineral oil and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tube and heat to 80°C. Then add 5 parts of Span 80 and maintain the temperature at 115°C for 30 minutes. After cooling, the oil phase preparation is complete.

[0061] (2) Aqueous phase preparation: Take the TP5-BP5-BS-C-R2 subunit antigen prepared above and prepare type C chicken infectious coryza subunit inactivated vaccine at a dose of 20 μg / bird. Take 95 parts of antigen solution, add 5 parts of sterile Tween 80, and mix thoroughly;

[0062] (3) Emulsification: Take 2 parts of oil phase and 1 part of water phase, put them into an emulsification tank, stir at 3500 r / min for 5 minutes, stir at 8000 r / min for 15 minutes to complete the emulsification preparation and obtain the subunit vaccine.

[0063] 3. Testing of subunit vaccines

[0064] ① Properties

[0065] Appearance: Milky white, uniform emulsion.

[0066] Dosage form: water-in-oil emulsion. Take a clean pipette, draw a small amount of vaccine and drop it onto a clean, cold water surface. Except for the first drop, none of the drops spread.

[0067] Stability: Add 10 mL of vaccine to a centrifuge tube and centrifuge at 3000 rpm for 15 minutes. The aqueous phase precipitated at the bottom of the tube should not exceed 0.5 mL.

[0068] ② Sterility test: The test was conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia. The results showed that there was no sterile growth.

[0069] Example 4: Efficacy analysis of type C chicken infectious coryza subunit vaccine

[0070] 1. Safety testing of subunit vaccines

[0071] (1) Safety trials of single-dose administration via different routes of administration at the minimum age of administration.

[0072] Seventy 7-day-old SPF chickens were divided into four groups: groups 1-3 (immunization groups, 20 chickens each) and group 4 (control group, 10 chickens each). Each of the immunization groups 1-3 was further divided into two subgroups of 10 chickens each. The chickens were immunized via different routes (intramuscular injection or subcutaneous injection in the neck) with inactivated subunit C-type infectious coryza vaccine (batch numbers 20220120, 20220421, and 20220518, prepared using the same method as in Example 3), at a dose of 0.5 mL / chick. The control group (10 SPF chickens) received a subcutaneous injection of sterile saline solution in the neck, at a dose of 0.5 mL / chick. All chickens were raised and managed under the same conditions and observed for 14 consecutive days. If any chickens died, they were necropsed to check for lesions in their internal organs. The live chickens were also observed for any adverse reactions. Fourteen days after vaccination, all surviving chickens were euthanized to check for lesions in their internal organs.

[0073] Safety trials of single-dose administration via different routes at the minimum age of administration are shown in Table 1.

[0074] Table 1. Safety trial results of single-dose administration via different routes of administration at the minimum age of administration.

[0075]

[0076] (2) Safety trials of single-dose repeated administration

[0077] Forty 14-day-old SPF chickens were divided into four groups of 10 each. Groups 1, 2, and 3 were administered a subcutaneous injection of type C infectious coryza subunit inactivated vaccine (batch numbers 20220120, 20220421, and 20220518, respectively, prepared using the same method as in Example 3) via neck injection at a dose of 0.5 mL / chick. The control group of 10 SPF chickens received a subcutaneous injection of 0.5 mL / chicken of sterile saline via neck injection. All chickens were raised and managed under the same conditions and observed for 14 consecutive days. Adverse reactions were observed; any deaths were examined by necropsy to check for lesions in the internal organs. A second vaccination was administered 14 days after the first vaccination at the same dose, and observation continued for another 14 days. Adverse reactions were observed; any deaths were examined by necropsy to check for lesions in the internal organs. Local inflammation and tissue lesions were assessed. Fourteen days after the second vaccination, all surviving chickens were euthanized, and lesions in the internal organs were observed. The results of the single-dose repeated vaccination safety trial are shown in Table 2.

[0078] Table 2. Safety trial results of single-dose repeated vaccination

[0079]

[0080] (3) Safety trial of single overdose inoculation

[0081] Forty 14-day-old SPF chickens were divided into four groups of 10 each. Groups 1, 2, and 3 were administered type C infectious coryza subunit inactivated vaccine (batch numbers 20220120, 20220421, and 20220518), respectively, via subcutaneous injection in the neck at a dose of 2.0 mL / chick. The control group (n=10) received 2.0 mL / chick of sterile saline solution via subcutaneous injection in the neck. All chickens were raised and managed under the same conditions and observed for 14 consecutive days. All surviving chickens were then euthanized, and their internal organs were examined for lesions. The safety results of the overdose vaccination are shown in Table 3.

[0082] Table 3. Safety test results of overdose vaccination

[0083] vaccine batch number Immunization age Number of test chickens Immunization dose Immune pathway Result determination 20220120 21 10 2mL Neck subcutaneous normal 20220421 21 10 2mL Neck subcutaneous normal 20220518 21 10 2mL Neck subcutaneous normal Comparison 21 10 2mL Neck subcutaneous normal

[0084] 2. Immunopotency analysis of subunit inactivated vaccine against type C infectious coryza in chickens

[0085] Seventy 10-day-old SPF chickens were divided into 7 groups of 10 each. Groups 1-3 were immunized with three batches of C-type infectious coryza subunit inactivated vaccine (batch numbers 20220120, 20220421, and 20220518, prepared using the same method as in Example 3), 0.5 mL per chicken. Group 4 received a C-type coryza subunit vaccine (antigen content 20 μg / dose) without the immune-enhancing sequence and without codon optimization. Group 5 was the whole-cell inactivated vaccine control group (immunized with 0.5 mL PBS). Group 6 was the non-immunization challenge control group, immunized with 0.5 mL / chicken of PBS. Group 7 was the healthy control group. 28 days post-immunization, 2 x 10 6.0 Chickens were challenged with a dose of CFU / bird via intraorbital injection. They were observed for 7 consecutive days, and clinical symptoms (including swelling of the infraorbital sinus and surrounding areas on one or both sides of the face, as well as tearing and nasal discharge) were recorded. Results showed that the subunit vaccine prepared using the method in Example 3 had good immunogenicity, rapidly and effectively inducing high levels of neutralizing antibodies. It achieved over 90% immune protection after challenge, superior to the whole-cell vaccine group. 100% of unimmunized chickens developed the disease after challenge and exhibited obvious clinical symptoms of infectious coryza.

[0086] Table 4. Tests on the immunogenicity of the type C chicken infectious coryza subunit inactivated vaccine.

[0087] Group Infectious drug dosage SPF chicken count Protection status Group 1 <![CDATA[2X10 6.0 CFU / per]]> 10 10 / 10 protection 2 groups <![CDATA[2X10 6.0 CFU per animal]]> 10 10 / 10 protection 3 groups <![CDATA[2X10 6.0 CFU / per]]> 10 10 / 10 protection 4 groups <![CDATA[2X10 6.0 CFU / per]]> 10 8 / 10 protection 5 groups <![CDATA[2X10 6.0 CFU / per animal]]> 10 8 / 10 protection 6 groups <![CDATA[2X10 6.0 CFU / per animal]]> 10 0 / 10 protection 7 groups / 10 10 / 10 is normal

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protective antigen, characterized in that, The amino acid sequence of the protective antigen is shown as SEQ ID NO.

1.

2. A nucleic acid, characterized in that, The nucleic acid is used for encoding the protective antigen of claim 1, and the nucleotide sequence of the nucleic acid is shown as SEQ ID NO.

2.

3. A biomaterial, characterized by, The biological material comprises an expression cassette, a vector or a host cell.

4. A subunit vaccine for C. avian Influenza of type A, characterized in that, The antigen component is a protein TP5-BP5-BS-C-R2, and the amino acid sequence of the protein TP5-BP5-BS-C-R2 is shown as SEQ ID NO.

1.

5. A process for the preparation of the C-type avian rhinotracheitis subunit vaccine according to claim 4, characterized in that, The biological material comprises an expression cassette, a vector or a host cell.

6. The preparation method according to claim 5, characterized in that, The expression is achieved by connecting the nucleotide sequence shown as SEQ ID NO. 2 to an expression vector.

7. The preparation method according to claim 6, characterized in that, The expression vector is one of pET28, pET30 and pET32 series vectors.

8. The method of any one of claims 5-7, wherein the method further comprises, The host of the expression is an Enterobacteriaceae bacterium.

9. The method of any one of claims 5-7, wherein the method further comprises, The method comprises the following steps: (1) constructing an expression vector containing the nucleotide sequence shown as SEQ ID NO. 2; (2) transforming the expression vector into an Enterobacteriaceae bacterium to obtain a recombinant bacterium expressing the TP5-BP5-BS-C-R2 protein; (3) culturing the recombinant bacterium to express the TP5-BP5-BS-C-R2 protein; (4) extracting the TP5-BP5-BS-C-R2 protein to prepare a C-type avian infectious coryza subunit vaccine.

10. Use of the protective antigen of claim 1, the nucleic acid of claim 2, the biological material of claim 3 or the C-type avian infectious coryza subunit vaccine of claim 4 in the preparation of a medicament for treating or preventing C-type avian infectious coryza.

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