CN107266538B - Chicken infectious rhinitis subunit vaccine and preparation method thereof - Google Patents

Abstract

The invention relates to the field of epidemic prevention of poultry infectious diseases, in particular to a chicken infectious rhinitis subunit vaccine and a preparation method thereof. Provides an avibacterium paragallinarum immunoprotection antigen protein which is characterized by comprising 16 antigen epitopes or epitope domains with amino acid sequences shown as Seq ID No. 1-16; preferably, the amino acid sequence is selected from one of Seq ID No.17, 18, 19 and 20. Also provides a chicken infectious rhinitis subunit vaccine, and the immune active component of the chicken infectious rhinitis subunit vaccine comprises the avibacterium paragallinarum immune protective antigen protein. The vaccine can obviously enhance the resistance of the chicken to the A, B and C serotype avibacterium paragallinarum, has better protection effect than that of a whole-bacterium inactivated vaccine, and can be used for preventing infectious coryza of the chicken caused by different serotype avibacterium paragallinarum.

Description

Chicken infectious rhinitis subunit vaccine and preparation method thereof

This application is a divisional application of the patent application with the application number 201610606316.4 and the invention titled "Chicken Infectious Rhinitis Subunit Vaccine and Its Preparation Method" submitted on July 28, 2016.

technical field

The invention relates to the field of poultry infectious disease prevention, in particular to a chicken infectious rhinitis subunit vaccine and a preparation method thereof.

Background technique

Avian infectious coryza (Avian infectious coryza) is one of the most important respiratory diseases caused by Avibacterium paragallinarum (Apg) infection. Chickens suffering from chicken infectious rhinitis mainly present clinical symptoms such as runny nose, swollen eyelids and epiphora. Infectious rhinitis in chickens can lead to a delay in egg production and a decline in egg production, resulting in a lot of economic losses.

Apg is a short gram-negative bacillus belonging to the family Pasteuraceae, with basic features of no motility, pleomorphism, and virulent strains with capsules. Page et al. divided Apg into three serotypes, A, B, and C. The study showed that Apg of the three serotypes A, B, and C had different degrees of pathogenicity, but the inactivated bacteria of the three did not exist. cross-immunity. In order to prevent chicken infectious rhinitis, inactivated vaccines have been widely used at present, and these vaccines are obtained by inactivating the cells of Avian Bacillus paragallinarum with formalin, thimerosal and the like. At present, the vast majority of inactivated vaccines commonly used in the world contain type A and type C Apg. Trivalent inactivated vaccines of three serotypes A, B, and C. Since Avian bacillus paragalliformis is a harsh bacteria, the high cost of cultivation leads to high cost of conventional inactivated bacteriocin. In addition, Avian bacillus paragalliformis contains toxins such as LPS, and the toxic and side effects caused by a large number of inoculations will also affect the production of chickens. Eggs and growth, focal necrotic plaques also formed in vaccinated chickens. The preventive effect of inactivated chicken infectious rhinitis vaccine on the infection of wild chickens is about 70-80%, and there may be different results in its efficacy test due to differences in environment and evaluation methods.

In order to develop safer and more effective vaccines, some scholars have studied the feasibility of preparing recombinant vaccines through genetic recombination technology. For example, Ryuichi Sakamoto et al. cloned and expressed the outer membrane proteins of type A and type C Avian paragallinarum, and the obtained recombinant proteins can be used as protective antigens against infection of type A and type C chicken infectious rhinitis, respectively. However, when designing recombinant antigens for vaccine preparation, it is very difficult to screen for antigenic epitopes with neutralizing activity shared by the three serotypes of Apg A, B, and C. So far, there is no evidence that can resist A, B simultaneously. , C A report on the protective antigens of three serotypes of A. paragallinarum infection.

SUMMARY OF THE INVENTION

After in-depth research by the inventors, the present invention provides a novel immunoprotective antigen protein of Avian paragallinarum, with strong immunogenicity, and the subunit vaccine prepared by using the antigen protein as an active ingredient can effectively prevent A, B and Infection with Avianbacterium paragallinarum type C.

The technical solution claimed in the present invention is as follows:

An immunoprotective antigenic protein of Avian Bacillus paragallinarum is characterized by comprising 16 antigenic epitopes or epitope domains whose amino acid sequences are shown in Seq ID No. 1-16.

Preferably, the amino acid sequence of the immunoprotective antigen protein of F. paragallinarum is selected from one of Seq ID No. 17, 18, 19 and 20.

A gene encoding the immunoprotective antigen protein of Avian bacillus paragallinarum.

Preferably, the nucleotide sequence of the gene is selected from one of Seq ID Nos. 22, 23, 24 and 25.

A construct comprising the gene.

A recombinant cell, characterized in that it is obtained by transforming a recipient cell with the construct; optionally, the recipient cell is selected from bacteria, yeast, animal cells and plant cells.

A chicken infectious rhinitis subunit vaccine, characterized in that its immunologically active components include the immunoprotective antigen protein of Avian paragallinarum.

Preferably, the final concentration of the immunoprotective antigen protein of Avian paragallinarum is 5-100 μg/ml.

A kind of preparation method of chicken infectious rhinitis subunit vaccine, is characterized in that, comprises the steps:

(1) construct the expression vector of described Avian bacillus paragallinarum immunoprotective antigenic protein;

(2) transforming the matched protein expression system with the expression vector to obtain recombinant cells;

(3) culturing the recombinant cell, inducing expression to obtain the immunoprotective antigen protein of Avianbacterium paragallinarum and purifying the protein;

(4) mixing the purified Avian bacillus paragallinarum immunoprotective antigen protein with auxiliary reagents, and adjusting the final concentration of the antigen protein to 5-100 μg/ml to obtain a chicken infectious rhinitis subunit vaccine.

Preferably, the expression vector is selected from pET, pQE and pGEX series vectors; the protein expression system is selected from Escherichia coli BL21, DH5ɑ, Top10 and JM109 strains; the auxiliary reagents include immune enhancers, stabilizers, preservatives , saline solution and/or distilled water.

The present invention utilizes the bioinformatics software geneious (Biomatters.Ltd, website http://www.geneious.com/), through the gene (GenBank: KJ867498.1, full length 6255bp) and The encoded protein sequence (2083 amino acid residues) was subjected to epitope analysis. According to the prediction results and experimental experience, several peptides with strong immunogenicity were selected. After multiple sequence optimizations, 16 core antigenic epitopes/epitope domains were determined, and their amino acid sequences were as shown in Seq ID No.1～16 Show.

In some embodiments, several recombinant antigenic proteins are obtained by combining the above-mentioned 16 antigenic epitopes/epitope domains.

In some embodiments, polypeptides comprising the above 16 antigenic epitopes are obtained, and other selected amino acid sequences such as linking peptides or other antigenic epitopes or epitope domains are obtained, and several recombinant antigenic proteins are obtained by sequence splicing.

The example lists the experimental results of five recombinant antigen proteins (amino acid sequences shown in Seq ID No. 17, 18, 19, 20 and 21, named p1, p2, p3, p4, p5 respectively). The data for the remaining recombinant antigen proteins were similar.

The antigenic characteristics of the obtained recombinant antigenic protein are experimentally analyzed, and Western Blotting experiments have proved that the recombinant antigenic protein obtained by the present invention has strong immunogenicity; Bacillus infection.

The immune experiment data shows that the recombinant antigen protein obtained by the present invention can produce immune protection against multiple strains of different serotypes of Avian Bacillus paragallina, such as types A and B such as Hp8, 221, 0083, BJ, 222, 668, Modesto, etc. and C-type Apg strain, and the protective effect is better than that of whole-bacteria inactivated vaccine. In one embodiment of the present invention, the challenge experiment shows that the chicken flocks immunized with the vaccine prepared by using the recombinant antigenic proteins obtained by the present invention (taking p1, p4, p5 as an example) are susceptible to A, B or C type Avian bacillus After challenge, all chickens had no symptoms of rhinitis, and the protection rate was 100%; the chickens immunized with the vaccine prepared by p2 and p3 had no symptoms of rhinitis after challenge with type A Hp8 strain, and the protection rate was 100%. %, only 10% of the chickens developed rhinitis symptoms after challenge with BJ strains B and C Modesto strains, and the protection rate was 90%; flocks immunized with the whole inactivated vaccine had rhinitis within 7 days after challenge. 2-3 chickens showed clinical symptoms, and the protection rate was 70-80%; while the chickens in the control group that were not immunized all developed rhinitis symptoms after challenge, and the protection rate was 0. There was a very significant difference in the protection rate between the immunized group and the non-immunized group.

Therefore, the recombinant antigen protein of the present invention has good immunoprotective activity, and its protective effect is better than that of the whole-bacteria inactivated vaccine, and can be used as a candidate antigen for the subunit vaccine of Avian bacillus paragallina.

The construct can be a cloning vector or an expression vector. The expression vector can be a variety of commercially available expression vectors with trp promoter, T7 promoter, cspA promoter and the like. Such expression vectors include pET series of vectors, such as pET-28a; pQE series of vectors, such as pQE30; pGEX series of vectors, such as pGEX-6-1.

The recombinant cells can be cells used for cloning and preservation of plasmids, or cells used for protein expression. The expression system can be a prokaryotic expression system or a eukaryotic expression system. The prokaryotic expression system can be selected from Escherichia coli BL21, DH5ɑ, Top10, JM109 and other strains, preferably Escherichia coli BL21 (DE3); the eukaryotic expression system can be yeast, animal cells or plant cells. Correspondingly, the matched expression vector, transformation condition, expression condition and protein extraction method are selected to prepare the antigen protein.

The subunit vaccine prepared by using the recombinant antigen protein of the present invention can obviously enhance the resistance of chickens to three serotypes of A, B and C A. paragallinarum, and can effectively prevent the infection of Avian. In some embodiments, the subunit vaccine comprises 1, 2, 3, 4, 5 or more amino acid sequences of the recombinant antigenic proteins of F. paragallinarum of the present invention. The subunit vaccine of the present invention can be used alone, or can be combined with one or more antigens of other infectious disease pathogens to prepare a combined vaccine for preventing multiple diseases in one shot. Other antigens include various infectious pathogens that cause chicken infection, such as chicken infectious bursal disease virus, chicken Newcastle disease virus, chicken infectious bronchitis virus and other viruses; Insect disease.

In the subunit vaccine, the final concentration of the immunoprotective antigen protein of F. paragallinarum is preferably 5-100 μg/ml, more preferably 10-50 μg/ml, and most preferably 20 μg/ml.

The present invention also provides a preparation method of chicken infectious rhinitis subunit vaccine, in some embodiments, comprising the following steps:

Construction of expression vector : total gene synthesis of DNA encoding recombinant antigen protein, the DNA encoding fusion peptide shown in SEQ ID No. 17-21 or fusion peptide mutant in which one or several amino acids are deleted, added or replaced; design The primers of PCR amplify the DNA in a large amount. In order to facilitate the connection with the expression vector, appropriate restriction sites are added to the 5' ends of the upstream and downstream primers. The obtained DNA fragment encoding the recombinant antigen protein is ligated into a suitable expression vector, and the expression vector can be a variety of commercially available expression vectors with trp promoter, T7 promoter, cspA promoter and the like. Such expression vectors include pET series of vectors, such as pET-28a; pQE series of vectors, such as pQE30; pGEX series of vectors, such as pGEX-6-1.

Introduction into host cells : The expression vector carrying the gene encoding the recombinant antigen protein is introduced into the host to express the recombinant antigen protein. The host used for expression can be bacteria, insect cells, animal cells, yeast, plant cells, etc. For example, Escherichia coli BL21, DH5ɑ, Top10, JM109 strains can be selected for mass expression of proteins, which is easy to operate and has low production costs. . An appropriate transformation method is selected according to the host cell used, and the expression vector can be introduced into the host cell using conventional methods in the art, such as electroporation, heat shock, lipofection, and the like.

Induce protein expression : Cultivate host cells carrying recombinant antigen protein expression vectors, add inducers to induce protein expression, collect the induced expression host cells by centrifugation, suspend them in a fixed volume of distilled water or PBS, and break them by ultrasonic lysis , and centrifuged to collect the pellet and supernatant. The recovered fixed amount of supernatant and the precipitate were subjected to SDS-PAGE gel electrophoresis, and after staining with Coomassie brilliant blue, whether the target protein was expressed was confirmed by the position and size of the target band relative to the protein marker. It can also be verified by in vitro antigen-antibody reaction tests such as ELISA and Western-blotting.

Purified protein : Centrifuge the recombinant E. coli after induction to collect bacterial pellets. The collected E. coli is disrupted by chemical substances, surfactants, lysozyme or ultrasonic lysis, so as to release the recombinant protein outside the cell. The supernatant of the bacteria after ultrasonic lysis is processed by the principle of nickel agarose affinity chromatography. If the target protein exists in the form of inclusion bodies, the solution containing the inclusion bodies is recovered as a precipitate by centrifugation and concentration. After purification, it was purified by affinity chromatography. When the inclusion body protein is purified, the recovered inclusion bodies are dissolved in a solution containing a denaturant. The denaturant involved can be 4M-8M urea, or guanidine hydrochloride. The buffer for dissolving the denaturant or reducing agent can be pH 7-8 phosphate buffer, Tris buffer, glycine buffer and the like. The recombinant protein was renatured by dialysis to restore the normal spatial structure. The dialysis solution can use the same type, temperature and pH buffer used to dissolve the inclusion bodies. The purity of the protein was determined by SDS-PAGE, and the content or concentration of the obtained protein was determined by a spectrophotometer or a special kit.

Obtaining the vaccine : mixing the purified antigen protein with auxiliary reagents, and adjusting the final concentration of the antigen protein to 20 μg/ml. The auxiliary agents include immune enhancers, such as aluminum hydroxide, mineral oil or non-mineral oil; stabilizers, such as polysorbate 80, lactose and sucrose, etc.; and preservatives, such as formalin, thimerosal, benzyl alcohol, etc. . In some embodiments, the recombinant antigen protein is mixed and emulsified in equal volume with Freund's complete adjuvant or incomplete adjuvant to prepare the Avianbacillus paragallina subunit vaccine. The immunization route of the subunit vaccine of the present invention is not particularly limited, and can be subcutaneously, intradermally or intramuscularly injected.

Immune effect verification: use the subunit vaccine to immunize chickens, collect the serum of the immunized chickens, and measure the antibody titer of Avian paragallinarum in the serum; or challenge the immunized chickens with Apg virulent strains and observe the survival and clinical symptoms of the chickens.

Description of drawings

Figure 1. Map of the pET28a plasmid used in an exemplary embodiment of the present invention.

Fig. 2. SDS-PAGE analysis chart of recombinant protein of the present invention,

Among them, M is the protein molecular weight standard; 1. Whole bacteria after induction by pET28a-p1; 2. Whole bacteria after induction by pET28a-p2; 3. Whole bacteria after induction by pET28a-p3; 4. Whole bacteria after induction by pET28a-p4; 5. Whole bacteria after induction of pET28a-p5.

Fig. 3. SDS-PAGE analysis chart of recombinant protein of the present invention,

Among them, M is the protein molecular weight standard; 1. pET28a-p1 induces cleavage and precipitates; 2. pET28a-p2 induces cleavage and then precipitates; 3. pET28a-p3 induces cleavage and then precipitates; 4. pET28a-p4 induces cleavage and precipitates; 5. PET28a-p5 induced precipitation after lysis.

Fig. 4. SDS-PAGE analysis chart of recombinant protein of the present invention,

Among them, M is the protein molecular weight standard; 1. pET28a-p1 induced cleavage supernatant; 2. pET28a-p2 induced cleavage supernatant; 3. pET28a-p3 induced cleavage supernatant; 4. pET28a-p4 induced cleavage supernatant; 5. pET28a-p5 induced lysis supernatant.

Figure 5. SDS-PAGE analysis of purified recombinant protein,

Among them, M is the protein molecular weight standard; 1. p1 protein; 2. P2 protein; 3. P3 protein; 4. P4 protein; 5. P5 protein.

Fig. 6. Western-blotting detection result of recombinant antigen protein of the present invention,

Wherein, M is the protein molecular weight standard; 1. p1 protein; 2. P2 protein; 3. P4 protein; 4. P5 protein; 5. P3 protein.

Figure 7. Antigen analysis results of outer membrane proteins of Avianbacterium paragallinarum 221 strain.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments. It should be stated that the following embodiments are only for explanation and description, and do not limit the scope of the present invention in any way.

The biochemical reagents not specifically described in the examples of the present invention are all conventional reagents in the art, and can be obtained commercially or prepared by conventional methods in the art, and the specifications are laboratory pure grade.

Embodiment 1. Antigen epitope prediction of F. paragallinarum outer membrane protein

Outer membrane protein: its amino acid sequence is shown in Seq ID No. 27, which is derived from the gene encoding the outer membrane protein of GenBank No. KJ867498.1 Avian Bacillus paragallinarum 221 strain.

Epitope prediction: use the epitope prediction software geneious (Biomatters.Ltd, website http://www.geneious.com/) to predict the epitope of the outer membrane protein, and analyze the secondary structure, hydrophilicity, hydrophobicity, antigenicity and other information (Figure 7).

Sequence analysis and splicing: Comprehensive software analysis, according to the antigenicity of the protein sequence, considering the difficulty of protein expression and purification, avoiding the signal peptide region, and selecting the appropriate epitope sequence or epitope concentration region for sequence splicing; Based on the prediction results and accumulated experimental experience, the selected antigenic epitope sequences are optimized for many times, and part of the antigenic epitope region is intercepted, so that the spliced antigenic protein is highly immunogenic and suitable for protein expression and animal immunization. Finally, 16 core epitopes/epitope domains were determined, and their amino acid sequences are shown in Seq ID No.1-16.

Several recombinant antigenic proteins were obtained by combining the above 16 epitopes/epitope domains.

Several recombinant antigenic proteins were also obtained by sequence splicing with selected other protein sequences such as linker peptides or other epitopes or epitope domains.

Among them, five recombinant antigen proteins of Avian Bacillus paragallinarum were named p1, p2, p3, p4, and p5, and their amino acid sequences were shown in Seq ID No. 17, 18, 19, 20 and 21.

Embodiment 2, the prokaryotic expression of Avian bacillus paragallinarum recombinant antigenic protein

1. Materials

1.1 Plasmids and strains

The pET28a plasmid (product of Pharmacia Company, purchased from Beijing Bailingke Biotechnology Co., Ltd.) used in this example; the pET28a plasmid map is shown in Figure 1 .

Escherichia coli BL21(DE3) competent cells were purchased from Beijing Tiangen Biotechnology Co., Ltd.

The strains of Avian bacillus paragallius used in the present invention are Hp8, 221, 0083, BJ, 222, Modesto, and 668 strains, which are purchased from the China Veterinary Drug Administration in Beijing, China, and belong to commercial strains.

1.2 Avian Bacillus paragallinarum antigenic protein

In Example 1, the five recombinant antigen proteins p1, p2, p3, p4, and p5 obtained by bioinformatics software were predicted, analyzed and spliced.

1.3 Main reagents and consumables

Sodium chloride, EDTA disodium salt, ethanol, methanol, Ponceau S, trichloroacetic acid (TCA) are products of China National Pharmaceutical Company.

Tris base (Tris-HCL), dithiothreitol (DTT), glycerol, sodium dodecyl sulfonate (SDS), acrylamide, ammonium persulfate, tetramethylethylenediamine (TEMED), sodium carbonate, Sodium acetate was purchased from Shanghai Sangon Bioengineering Technology Co., Ltd.

Glycine and Coomassie brilliant blue R-250 were purchased from AMRESCO Company.

Bovine serum albumin (BSA), trypsin, protease inhibitor (PMSF), and formaldehyde are all products of Sigma.

Proteinase K (the concentration of the stock solution is 20 mg/ml, and the concentration of the working solution is 1 mg/ml) was purchased from Shanghai Huashun Bioengineering Co., Ltd.

Kanamycin, fetal bovine serum, and IPTG (isopropyl-β-D-thiogalactoside) are all products of Invitrogen.

The tips and centrifuge tubes are products of AxyGen.

Taq enzyme and 10xTaq enzyme buffer, NcoI, Xho I and other endonucleases and related buffers, T4 ligase and 10x T4 ligase buffer, Rnase, DNA marker (DL-2000, DL-15000) are all Bio-Bio (Dalian) Co., Ltd. product.

Column centrifugal DNA gel recovery kit and horseradish peroxidase (HRP)-labeled rabbit anti-chicken IgG were purchased from Sigma Company.

Substrate solution preparation: Substrate solution A: 0.006% H ₂ O ₂ buffer; Substrate solution B: take Na ₂ HPO ₄ .12H ₂ O 14.2g, citric acid 10.5g, dilute to 500mL with double distilled water into 0.1 phosphate citrate buffer (pH 5.0), and then add benzidinediamine (TMB). When using, mix equal volume of liquid A and liquid B, use within 5 minutes after mixing, and use it now.

Escherichia coli medium: LB liquid medium and solid medium: each liter contains 5g yeast extract, 10g tryptone, 10g NaCl, adjust pH to 7.5 with 10mol/L NaOH, sterilize by autoclaving at 121°C for 20min, and store at 4°C for later use . Add 1.5 g of agar to every 100 ml of LB liquid medium to obtain solid LB medium, sterilize by autoclaving at 121°C for 20 min, and store at 4°C for later use.

TSB, TSA, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma company.

The antigenic protein of Avian bacillus paragallinarum was purified using Sepharose 4B purification column (product of Amershan pharmacia) and purchased from Beijing Bailingke Biotechnology Co., Ltd.

PBS buffer: NaCl 8.0g, KCl 0.2g, KH ₂ PO ₄ 0.24g, Na ₂ HPO ₄ 12H ₂ O 3.628g, dissolved in 800ml of distilled water, adjusted to pH 7.4 with hydrochloric acid, dilute to 1000ml with distilled water, Autoclave at 121°C for 20 min and store at room temperature.

Western-blotting buffer:

Electroporation buffer: 39 mmol/L glycine, 48 mmol/L Tris base, 0.037% SDS (electrophoresis grade), 20% methanol. To prepare 1000 mL of transfer buffer, 2.9 g of glycine, 5.8 g of Tris base, 0.37 g of SDS should be weighed, 200 mL of methanol, and ddH ₂ O should be added to make the total amount of 1000 mL.

TBS (pH8.0) buffer: 10 mmol/L Tris.HCl, 150 mmol/L NaCl.

TBST buffer: add 0.05% Tween-20 to the above TBS.

Ponceau S (10×) stock solution: 2 g Ponceau S, 30 g trichloroacetic acid, 30 g sulfosalicylic acid, add dd _H2O to 100 mL.

1.4 Experimental animals: 6-week-old SPF chickens were purchased from Beijing Merial Weitong SPF Chicken Experimental Animal Center.

2. Sequence Design and Synthesis

The gene coding sequences of antigenic proteins p1, p2, p3, p4, p5 (p1-5) are shown in Seq ID No. 22, 23, 24, 25, 26. Send the gene coding sequences to Huada Technology (Beijing) Co., Ltd. Perform whole gene synthesis.

Design upstream and downstream primers according to their respective base sequences, and introduce NcoI and XhoI restriction sites into the upstream and downstream primers, respectively, and send them to Huada Technology (Beijing) Co., Ltd. for primer synthesis. The primer sequences are such as Seq ID No. 28 to 37.

3. Construction of recombinant expression plasmids

3.1 PCR amplification of the gene encoding antigenic protein p1-5

Taking the synthetic product obtained in step 2 as a template, using the synthetic primers to carry out PCR,

PCR reaction system:

The amount of each component of PCR: (where the primer concentration is 1OD dissolved in 400μl ddH ₂ O)

PCR reaction program: After 1 minute at 98°C, 30 cycles of "denaturation (95°C, 10 seconds), annealing (56°C, 15 seconds) and extension (72°C, 120 seconds)" were performed, followed by repair extension Reaction (72°C, 7 minutes).

3.2 Enzyme digestion and recovery of PCR products

The above PCR product was digested with NcoI and XhoI,

Enzyme cleavage system:

PCR product fragment digestion system (50ul):

The above system was put into a constant temperature water bath at 37°C to react for 2h.

After all the digested products were separated by 0.8% agarose gel electrophoresis, DNA fragments were recovered by using the ordinary DNA gel recovery kit from Beijing Tiangen Biochemical Technology Co., Ltd. according to the steps of the kit instructions. The recovered target DNA fragments can be used immediately or stored at -20°C for later use.

3.3 Enzyme digestion and recovery of expression vector

The expression vector PET28a was subjected to Nco I and XhoI digestion and the recovery of the products, and the methods and steps were the same as those in 3.2. The digestion and recovery of PCR products.

3.4 Linking the target fragment to the vector

The target DNA fragment recovered and purified in step 3.2 and the expression vector PET28a recovered and purified in step 3.3 were connected to obtain a recombinant plasmid.

The ligation system was: target DNA fragment, 8ul; expression vector pET28a, 4ul; 10×T4 DNA ligase buffer, 2ul; T4 DNA ligase, 1ul (5u/ul); ddH ₂ O supplemented to 20ul.

Ligation conditions: The mixture of the above ligation system can be placed in a PCR instrument at 22°C for 1 hour.

3.5 Transform and screen clones

Take 100 μl of E. coli DH5ɑ competent cells into a 1.5 ml EP tube, and add 5-10 μl of the recombinant plasmids pET28a-p1, pET28a-p2, pET28a-p3, pET28a-p4, and pET28a-p5 obtained in step 3.4 to their respective EP tube and mix. After 30 minutes on ice, heat shock at 42°C for 90 seconds and ice bath for 3-5 minutes. 400 μl of LB was added, and the mixture was cultured at 37° C. and 200 rpm for 45 min to recover. The resuscitated recombinant E. coli suspension was centrifuged at 25,000 rpm for 10 min at 4°C, 400 μl of supernatant was discarded, the remaining 100 μl was used to resuspend the pellet and spread on LB agar plates containing 25 μg/ml kanamycin. Proliferate at 37°C for 1 h, then turn the plate over and invert at 37°C for 14h-16h until colonies appear.

3.6 Recombinant plasmid digestion and identification

Pick single colonies grown on the plate in step 3.5, inoculate them in LB liquid medium containing 25 μg/ml kanamycin respectively, and shake them at 37°C at 200 rpm until the OD ₆₀₀ reaches 0.6-1.

The bacterial cells were collected, and the recombinant plasmid was extracted using an ordinary plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the specific operation steps were carried out in accordance with the kit instructions. The recombinant plasmid was identified by NcoI+XhoI digestion, and the exogenous fragment and vector fragment of the expected size after digestion was the correct recombinant plasmid.

4. Construction of recombinant expression strains

Take 100 μl of competent cells BL21(DE ₃ ) and add it to a 1.5 ml EP tube, and add 0.5 μl of the correctly identified recombinant plasmids pET28a-p1, pET28a-p2, pET28a-p3, pET28a-p4, and pET28a-p5 to their respective EPs tube and mix. After 30 minutes on ice, heat shock at 42°C for 90 seconds and ice bath for 3-5 minutes. It was spread on LB agar plates containing 25 μg/ml kanamycin. Incubate at 37°C for 1 hour, then turn the plate over and invert at 37°C for 14h-16h until colonies appear. Pick a single colony, perform colony PCR and sequence comparison according to the reaction system and reaction procedure in step 3.1, and identify positive clones.

5. Expression and purification of the target gene in Escherichia coli

5.1 Induction and expression of target gene: Inoculate positive clones containing recombinant plasmids pET28a-p1, pET28a-p2, pET28a-p3, pET28a-p4, pET28a-p5 in 3mL LB liquid medium containing 25μg/ml kanamycin respectively , cultured with shaking at 37°C. Take 100 μl of the cultured bacterial liquid and inoculate it into 10 mL of fresh LB liquid medium containing 25 μg/ml kanamycin, shake and culture at 37 °C for about 3 hours, when the OD ₆₀₀ reaches 0.6-1.0, add IPTG to the final concentration was 0.8 mmol/L, and the cells were collected after culturing for 3 h.

5.2 SDS-PAGE electrophoresis analysis of expression products

(1) Gel preparation: The preparation method of SDS-PAGE gel is shown in Table 1:

Table 1 SDS-PAGE gel preparation method

12% Separating Gum Preparation: Mix the ingredients quickly after adding them, add them to the rubber plate, and add purified water on top. Then, prepare a 5% stacking gel: add the relevant ingredients in the table and mix quickly, add it to the top of the separating gel on the gel plate (pour out the purified water on the separating gel first), and insert the sampling comb after filling. After the stacking gel has set, remove the comb.

(2) PAGE: Fix the gel on the electrophoresis device, add a sufficient amount of Tris-glycine electrophoresis buffer, and add each sample to the sample well: electrophoresis voltage 200V, current in the range of 20mA-40mA, electrophoresis for 1h, The electrophoresis was terminated when the bromophenol blue eluted out of the bottom of the gel.

(3) Staining and destaining of polyacrylamide gel: Remove the gel, stain with Coomassie brilliant blue R250 staining solution for 30 min, and then decolorize with destaining solution for 1 min to observe the results.

The results are shown in Figure 2. After the Escherichia coli containing the positive recombinant plasmid was induced with 1 mM IPTG at 37 °C, the expression was detected by SDS-PAGE. The protein target band appeared at the expected position. The expected protein size was the same, and no induction was performed. Bacteria do not have this protein.

5.3 Preparation and purification of recombinant protein

1) Pick the correct positive clones identified in step 4, inoculate in 3 mL of LB medium containing 25 μg/ml kanamycin, activate overnight at 37°C, and dilute 1:100 to LB containing 25 μg/ml kanamycin In the medium, shake at 37°C and 230r/min to cultivate to the logarithmic growth phase (OD ₆₀₀ 0.6～1), add IPTG with a final concentration of 1mmol/L, and shake at 37°C and 230r/min to induce culture for 6-8h. . 1 mL was taken out at different times before and after induction, and the cells were collected by centrifugation at 5,000 r/min for 10 min. After the supernatant was discarded, 2×SDS loading buffer was added, boiled for 10 min, and then washed with 12% sodium dodecyl sulfate-polyethylene glycol. Acrylamide gel electrophoresis (SDS-PAGE) was performed and expression yields were analyzed using Quantity One-4.3.1 (BIO-RAD) software.

2) Collect 500 mL of bacterial liquid after induction and expression for 6 h, centrifuge at 8000 r/min for 10 min, dissolve the precipitate with 5 mL of 10 mmol/L PBS, and after ultrasonication, centrifuge at 12,000 r/min for 10 min, and retain the supernatant. The inclusion body precipitate was dissolved with 8mol/L urea solution, 8M urea was washed in 50mM Tris pH7.0-8.5, 1mM EDTA, 10% Triton X-100, disulfide bond reducing agent. Wash for 4-8h to denature and dissolve the inclusion bodies. The supernatant after the dissolution of the inclusion bodies was purified by nickel affinity chromatography. The operation process was carried out according to the operation manual provided by Amersham Pharmacia Biotech. The specific process is as follows: transfer the resin to the column, after complete precipitation, wash the nickel affinity chromatography column with triple distilled water to remove the ethanol and air in the matrix and prevent the next Ni ²⁺ precipitation; 5 × column volume of Charge with Charge Buffer (50mM NiSO ₄ ); wash with 20×column volume of triple distilled water to remove free Ni ²⁺ ; 10×column volume of Binding Buffer to equilibrate the column; manually load the sample, and determine the sample volume according to the protein concentration ; Wash with 20×column volume of Binding Buffer; Wash with 6×column volume of Wash Buffer until no protein is detected; Elution Buffer, 1ml each time, collect the elution effluent, and elute until no protein is detected; then SDS-PAGE electrophoresis detection.

The results are shown in Figure 3 and Figure 4. The two figures are the SDS-PAGE of the recombinant plasmid constructed in the present invention after induction and expression by ultrasonic cracking. Figure 3 is the precipitation after the cracking. It can be seen that most of p1, p2, p4 and p5 are in the precipitation; Figure 3 4 is the supernatant after lysis, and it can be seen that most of the p3 protein exists in the supernatant after lysis.

If the protein content of the lysed supernatant after induction is higher than that of the precipitate, the lysed supernatant can be directly used for purification, omitting the step of denaturing and renaturing inclusion bodies, and using the principle of nickel agarose affinity chromatography to treat the bacteria after ultrasonic lysis The supernatant, the specific operation is as follows:

①Take 5 mL of Ni-IDA, wash the equilibrated column with Binding buffer of 10 times the column bed volume, and the flow rate is 5 mL/min.

②The sample (lysate supernatant) was loaded on the column, the flow rate was 2ml/min, and the permeate was collected.

③ Wash the column with Binding buffer of 10 times the column bed volume at a flow rate of 10 mL/min.

④ Wash the impurities with Wash Buffer at a flow rate of 5ml/min, and collect the eluate.

⑤ Elution Buffer, the flow rate is 2ml/min, and the eluate is collected.

⑥Put the purified protein in a dialysis bag, slowly dialyze it in PBS, buffer (pH=7.4), and collect the dialyzed samples for SDS-PAGE analysis.

Note: Binding Buffer (PBS, 0.5% Triton X-100, pH=7.4)

Wash buffer-10 (PBS, 10 mM imidazole, pH=7.4)

Elution Buffer-500 (PBS, 500mM imidazole, pH=7.4)

The results are shown in Figure 5. After purification, relatively pure target proteins p1, p2, p3, p4 and p5 were obtained.

Embodiment 3. Characteristic analysis of recombinant antigen protein

1. Analysis of the antigenicity of recombinant antigenic protein by Western-blot method

The above purified recombinant antigen proteins p1, p2, p3, p4, p5 are subjected to SDS-PAGE electrophoresis according to conventional methods, and the steps are:

1) Transfer: Cut out 6 pieces of Whatman 3M filter paper and 1 piece of nitrocellulose membrane (NC membrane). The size of the filter paper and membrane should be exactly the same as the size of the gel or slightly smaller than the size of the gel. Mark the corner of the filter membrane with a pencil. Ensure the opposite direction of the membrane and the gel after transfer; soak the nitrocellulose membrane in purified water for 5 minutes; add a small amount of transfer buffer to another shallow tray and soak 6 sheets of Whatman 3M filter paper in it. Then install the transfer electrophoresis tank according to the following steps: lay the base of the graphite electrode (anode) flat, and place 3 layers of 3M filter paper, nitrocellulose membrane, polyacrylamide gel and 3 layers of 3M filter paper in sequence. Completely remove the air bubbles between the layers: fasten the top cover of the transfer electrophoresis tank to the graphite electrode-transfer film gel complex; connect the power supply, and connect it according to the parameters of 0.65mA/cm ² -1.0mA/cm ² according to the area of the gel plate. Electric current was applied, and electrophoretic transfer was performed for 0.5h-2h.

2) Ponceau staining: After the transfer, remove the NC membrane, rinse it in deionized water for 2-3 times, transfer it to Ponceau staining solution for 5min-10min, observe the transfer effect, and mark the protein with a pencil Marker position; rinse the nitrocellulose membrane with deionized water at room temperature until the color fades; place the NC membrane in 5% nonfat milk powder and block it for 2 hours at room temperature; wash the membrane: discard the blocking solution, wash the NC membrane 3 times with 1×TBST, 5min each time.

3) Primary antibody incubation: Put the NC membrane into chicken anti-Apg positive serum diluted with 5% skimmed milk powder (volume ratio 1:50), and incubate at 37° C. for 1 h. Wash the membrane: Take out the NC membrane and wash the membrane three times with 1×TBST for 10 min each time.

The positive serum was prepared by immunizing SPF chickens with a trivalent inactivated vaccine prepared by Apg Hp8\BJ\668 strain in our laboratory, and can be provided for verification experiments.

4) Secondary antibody incubation: transfer the membrane into HRP-labeled goat anti-chicken IgG antibody diluted with 5% skimmed milk powder (volume ratio 1:5000), incubate at 37°C for 2h; wash membrane: remove the NC membrane and wash with 1×TBST Membrane 3 times, 10min each time.

5) Color development: Place the NC membrane in the newly prepared DAB color developing solution, and place it in a dark place for color development. After the color depth of the protein band reaches the requirement, rinse with 1×TBST to terminate the reaction.

2. Preparation of Recombinant Protein Antiserum

1) Preparation of vaccine and immunization test

Preparation of recombinant antigen protein vaccine: The recombinant antigen proteins p1, p2, p3, p4 and p5 were mixed and emulsified with equal volume of Freund's complete adjuvant respectively, so that the final concentration of antigen protein in the vaccine was 20 μg/ml. The vaccine for the second immunization adopts incomplete Freund's adjuvant, and the other components and concentrations are the same as the first immunization.

Immunization method: The described 42-day-old experimental SPF chickens were intramuscularly injected with Freund's complete adjuvant emulsified recombinant antigen protein vaccine (the inoculation amount was 0.5ml/bird); 2 weeks later, the thoracic intramuscular injection of Freund's incomplete adjuvant emulsified The recombinant protein vaccine (the inoculation amount is 0.5ml/vaccine); blood is collected from the fin vein after 10 days interval, and the serum is collected.

Detection of serum specific antibodies: using ELISA method, the specific steps are:

Use 1 μg/100 μl of purified recombinant antigen proteins p1, p2, p3, p4, and p5 to coat the enzyme-linked plate overnight at 4°C, block with 1% BSA at 37°C for 1 h, wash the plate once with the washing solution, and then seal the plate at -20°C. save.

Blood was collected from chickens one week after boosting immunization to separate serum, 100 μl was added to enzyme-linked plate after doubling dilution, and adjuvant control and blank control were set at the same time.

37°C reaction for 30min.

After washing the plate 3 times, add rabbit anti-chicken IgY(H+L)-HRP diluted at a volume ratio of 1:5,000, and react at 37°C for 30 min.

After washing the plate for 5 times, add 100 μl of substrate solution, add 2% H ₂ SO ₄ to stop the reaction, and read at 630 nm after color development in the dark for 10 min.

Serum with the ratio of the OD value of the sample to the control group greater than 2 was judged to be positive for serum ELISA antibody.

The Western-blotting detection results of the recombinant antigen proteins p1, p2, p3, p4, and p5 are shown in Figure 6. The chicken serum positive for chicken infectious rhinitis reacted with each recombinant antigen protein, which was in line with the expected results. This shows that the recombinant antigen protein of the present invention has good antibody binding activity.

Embodiment 4. The immune efficacy test of recombinant antigen protein to SPF chicken

1. Recombinant antigen protein vaccine preparation and immunization method

1.1 Preparation of recombinant antigen protein vaccine

Recombinant antigen proteins p1, p2, p3, p4, p5 were mixed with MONTANIDE ^TM ISA 71VG adjuvant (product of SEPPIC) at a weight ratio of 3:7 (30% weight of antigen, 70% weight of adjuvant) to emulsify (operation The method is shown in the introduction of SEPPIC company's product use method), so that the final concentration of antigenic protein in the vaccine is 20μg/ml (the epidemic threat of chicken infectious rhinitis in different regions is different, and the sensitivity of different chicken breeds to immunogens is also different, which can be adjusted according to actual needs. The final concentration of the antigenic protein is 5-100 μg/ml). The prepared vaccines were named as vp1, vp2, vp3, vp4, vp5 in sequence.

Preparation of whole-bacteria inactivated vaccine: After pure culture of A, B, C type A, B, and C Avian Bacillus paragallinarum Hp8 strains, BJ strains and 668 strains, pick 8 to 10 single colonies and spread them on TSA plates (containing 10 % inactivated bovine serum). After culturing at 37℃ for 16h～18h, wash off the bacterial moss on the plate with sterilized 0.01mol/L PBS, dilute to 7.5×10 ⁹ cfu/ml, inactivate (3‰) with formaldehyde for 4h～48h, and wait for Volume mixing. The mixed bacterial solution was mixed with No. 10 industrial white oil (Hangzhou Refinery) adjuvant in a volume of 1:1, emulsified by a colloid mill, and the final concentration of the obtained trivalent inactivated vaccine antigen reached 1.0×10 ⁹ cfu/ml.

1.2 Immunization methods

210 42-day-old SPF test chickens were divided into subunit vaccine group, conventional inactivated vaccine group and PBS control group. The vaccine group was further divided into five groups, vp1, vp2, vp3, vp4, and vp5, with 30 mice in each group; the conventional trivalent inactivated vaccine group and the PBS control group were divided into 30 mice each. The test chickens in each subunit vaccine group were immunized with vp1, vp2, vp3, vp4, vp5 respectively, the test chickens in the conventional trivalent inactivated vaccine group were immunized with conventional inactivated vaccine, and the control group was inoculated with PBS. Intramuscular injection in the chest, 0.5ml per animal; 4 weeks later, the same dose and the same route were used to boost the immunization. Blood was drawn from the fin vein every two weeks for the monitoring of serum-specific antibodies.

Serum antibodies were detected by ELISA, and the steps were the same as those in Example 3.

1.3 Challenge test of immunized chickens

All the test chickens after boosting immunization for 4 weeks were subjected to the challenge test. Each experimental group was then challenged with A type (Hp8 strain), B type (BJ strain) and C type (668 strains) Apg, 10 in each group, inoculated in the infraorbital sinus, and the doses were A type (Hp8 strain). ) 1×10 ⁶ CFU, type B (strain BJ) 5×10 ⁵ CFU, type C (strain 668) 1×10 ⁶ CFU. After one week of continuous observation, the clinical morbidity of the experimental chickens, including runny nose, swollen eyelid, and epiphora, were recorded. The immune protection of the recombinant subunit vaccine was evaluated, and the results are shown in Table 2.

Table 2 The protective effect of chicken infectious rhinitis subunit vaccine immunization chicken of the present invention to Apg strain challenge

Note: The challenge dose was 1×10 ⁶ CFU for type A Hp8 strain, 5×10 ⁵ CFU for BJ strain for type B, and 1×10 ⁶ CFU for type C 668 strain.

*Compared with the PBS control group, the number of test chickens obtained immune protection in the subunit vaccine immunization group was significantly different.

Table 2 lists the results of the challenge experiments of the A-type Hp8 strain, the B-type BJ strain and the C-type 668 strain. During the experiment, 5 subunit vaccines prepared by the preferred 5 recombinant proteins of the present invention were used to immunize the test chickens. After 2 immunizations, three serotypes of A, B, and C were used for virulent challenge. All experiments in the non-immunized control group were performed. Chickens get sick one after another. In the subunit vaccine immunization group, only a few chickens showed symptoms of rhinitis after challenge, and the protection rate was 90% to 100%; in the whole bacteria trivalent inactivated vaccine immunization group, 2-3 chickens showed clinical symptoms within 3 days after challenge. , the protection rate was 70-80%; all the experimental chickens in the non-immune control group showed severe symptoms of rhinitis.

The results of the challenge experiment using Apg of A type 221 strain, 0083 strain, B type 222 strain and C type Modesto strain were similar to those of Hp8 strain, BJ strain and 668 strain. Vp1, Vp4 and Vp5 experimental group The protection rate was 100%, and the protection rates of Vp2 and Vp3 experimental groups were 90%-100%. It can be seen that the vaccine prepared by using the recombinant antigen protein provided by the present invention has a remarkable immunization effect, and can effectively prevent chickens from being infected with Apg of three serotypes A, B and C at the same time.

In addition, the protection rate of the protein subunit vaccine was better than that of the whole-bacteria trivalent inactivated vaccine, and there was a very significant difference compared with the non-immunized group. Since the purified protein does not contain components such as lipopolysaccharide carried by the whole cell, the subunit vaccine prepared by the present invention has no side effects, while the whole cell inactivated vaccine will have side effects such as transient appetite loss and lethargy. In terms of preparation cost, subunit vaccines are also lower than conventional inactivated vaccines due to the characteristics of easy culture induction, cheap culture medium, and easy purification.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

SEQUENCE LISTING

<110> Beijing Academy of Agriculture and Forestry

<120> Chicken infectious rhinitis subunit vaccine and preparation method thereof

<130> P170303/NLK

<160> 37

<170> PatentIn version 3.3

<210> 1

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<213> Avibacterium paragallinarum

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Ile Gly Gly Thr Gly Gln Asp Thr Ile

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Lys Lys Gly Asp Thr Thr Asn Gly Ser Thr Thr Thr Phe Ala

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Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala Gln

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Phe Ser Val Lys Asn Gly Ser

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Ala Lys Ile Gly Gly Thr Glu Lys Thr Thr

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Asn Ile Asp Ala Ser Val Glu Asp

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Ser Gly Ser Gly Asn Asp Arg

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Ala Gly Asn Ala Ala Thr Asp Gly Ile

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Val Lys Ala Gly Thr Ala Asp Thr Asp Ala Val Asn Lys

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Ala Val Thr Lys Asp Pro Asn Gln Thr Ser

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Asn Gly Thr Ala Pro Thr Thr Phe Lys Asp

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Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Val Ala Asp Gly Asp

1 5 10 15

Ile Ser Pro Thr Ser Gly Asp Val Val

20 25

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Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr Ala Pro Thr Ala Ser

1 5 10 15

Ser Thr Gln Gly Gly Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala

20 25 30

Pro Ala Gly Asn Val Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Thr

35 40 45

Leu Pro Glu

50

<210> 17

<211> 359

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<213> Artificial Sequence

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<223> Amino acid sequence of the recombinant antigenic protein p1 of F. paragallinarum

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Val Thr Ala Gly Gly Lys Pro Ser Ile Ala Leu Gly Gln Asp Ser Thr

1 5 10 15

Val Ala Asn Ser Ala Ile Ser Arg Thr Ser Ser Ala Thr Ile Asn Phe

20 25 30

Ala Gly Ser Pro Glu Thr Leu Ala Gly Lys Ile Ser Gln Thr Ser Thr

35 40 45

Glu Ala Ile Asn Asn Phe Gly Asn Leu Ala Ile Gly Gly Thr Gly Gln

50 55 60

Asp Thr Ile Glu Val Val Ser Ala Lys Pro Thr Thr Val Ser Ala Glu

65 70 75 80

Ala Asn Lys Gly Ile Lys Val Thr Gly Glu Thr Ser Ser Asn Thr Thr

85 90 95

Gly Asn Ser Asn Leu Gly Glu Asp Gly Lys Asn Ala Asn Ser Pro Ile

100 105 110

Thr Val Glu Ser Ser Thr Asp Asn Asn Lys Lys Asp Lys Ser Gly Gln

115 120 125

Asp Pro Asn Gln Val Thr Gly Arg Lys Lys Gly Asp Thr Thr Asn Gly

130 135 140

Ser Thr Thr Thr Phe Ala Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala

145 150 155 160

Gln Thr Phe Ser Val Lys Asn Gly Ser Asp Glu Ser Lys Leu Ala Asn

165 170 175

Asn Thr Thr Gly Lys Asp Gly Ser Ile Thr Ala Gly Ser Lys Asp Ala

180 185 190

Val Asn Gly Gly Ala Lys Ile Gly Gly Thr Glu Lys Thr Thr Ala Asp

195 200 205

Asn Lys Asn Ser Lys Asn Ile Asp Ala Ser Val Glu Asp Ser Val Thr

210 215 220

Val Gly Gly Glu Thr Ser Gly Ser Gly Asn Asp Arg Ala Gly Asn Ala

225 230 235 240

Ala Thr Asp Gly Ile Val Lys Ala Gly Thr Ala Asp Thr Asp Ala Val

245 250 255

Asn Lys Ala Val Thr Lys Asp Pro Asn Gln Thr Ser Asn Gly Thr Ala

260 265 270

Pro Thr Thr Phe Lys Asp Asn Thr Gly Trp Gly Gly Ile Asp Ala Gly

275 280 285

Asn Lys Lys Ile Ser Asn Val Ala Asp Gly Asp Ile Ser Pro Thr Ser

290 295 300

Gly Asp Val Val Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr Ala

305 310 315 320

Pro Thr Ala Ser Ser Thr Gln Gly Gly Ala Thr Thr Ala Asn Thr Ala

325 330 335

Gly Gly Val Ala Pro Ala Gly Asn Val Ala Thr Gly Asp Ile Ala Pro

340 345 350

Thr Gln Pro Thr Leu Pro Glu

355

<210> 18

<211> 405

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of recombinant antigenic protein p2 of Avian bacillus paragallinarum

<400> 18

Ser Thr Val Ala Asn Ile Ser Arg Thr Ser Ser Ala Phe Ala Gly Ser

1 5 10 15

Pro Glu Thr Thr Ala Gly Lys Glu Gly Asn Ile Ser Gln Thr Ser Thr

20 25 30

Glu Ala Ile Asn Lys Asn Asn Phe Gly Gly Asn Ala Asn Leu Ala Thr

35 40 45

Asp Ile Gly Gly Thr Gly Gln Asp Thr Ile His Glu Val Val Ser Lys

50 55 60

Ser Ala Lys Pro Thr Thr Val Ser Ala Glu Ala Asn Lys Gly Ile Lys

65 70 75 80

Val Thr Gly Glu Thr Ser Ser Asn Thr Thr Gly Asn Ser Ser Asn Leu

85 90 95

Gly Glu Asp Gly Lys Asn Ala Lys Ala Asn Ser Pro Ile Thr Val Glu

100 105 110

Ser Ser Thr Asp Asn Asn Lys Lys Lys Asp Lys Ser Gly Gln Asp Pro

115 120 125

Asn Gln Val Thr Gly Arg Met Lys Lys Gly Asp Thr Thr Asn Gly Ser

130 135 140

Thr Thr Thr Phe Ala Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala Gln

145 150 155 160

Phe Ser Val Lys Asn Gly Ser Asp Glu Ser Lys Leu Ala Ser Ile Gly

165 170 175

Ala Glu Asn Ala Glu His Val Glu Val Asn Asn Thr Thr Gly Lys Ser

180 185 190

Ser Ile Thr Ala Thr Ala Gly Asp Gly Ser Ile Thr Ala Gly Ser Lys

195 200 205

Asp Ala Val Asn Gly Ala Lys Ile Gly Gly Thr Glu Lys Thr Thr Ile

210 215 220

Ser Glu Lys Ala Asp Asn Lys Asn Ser Lys Thr Val Lys Asn Phe Thr

225 230 235 240

Thr Thr Asn Ile Asp Ala Ser Val Glu Asp Ser Leu Asn Val Thr Val

245 250 255

Gly Gly Glu Thr Glu Ser Gly Ser Gly Asn Asp Arg Thr Leu Gly Ala

260 265 270

Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Val Lys Ala Gly Thr Ala

275 280 285

Asp Thr Asp Ala Val Asn Lys Gly Leu Gly Leu Ala Val Thr Lys Asp

290 295 300

Pro Asn Gln Thr Ser Ile Pro Ile Asn Gly Thr Ala Pro Thr Thr Phe

305 310 315 320

Lys Asp Asn Thr Gly Trp Gly Asp Gly Ile Asp Ala Gly Asn Lys Lys

325 330 335

Ile Ser Asn Val Ala Asp Gly Asp Ile Ser Pro Thr Ser Gly Asp Val

340 345 350

Val Gly Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr Ala Pro Thr

355 360 365

Ala Ser Ser Thr Gln Gly Gly Ala Thr Thr Ala Asn Thr Ala Gly Gly

370 375 380

Val Ala Pro Ala Gly Asn Val Ala Thr Gly Asp Ile Ala Pro Thr Gln

385 390 395 400

Pro Thr Leu Pro Glu

405

<210> 19

<211> 430

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of recombinant antigenic protein p3 of Avian bacillus paragallinarum

<400> 19

Gln Asp Ser Thr Val Ala Asn Ser Ala Ile Ser Arg Thr Ser Ser Ala

1 5 10 15

Thr Phe Ala Gly Ser Pro Glu Thr Thr Ala Gly Lys Glu Arg Lys Gly

20 25 30

Asn Ile Ser Gln Thr Ser Thr Glu Ala Ile Asn Lys Asn Asn Phe Gly

35 40 45

Gly Asn Ala Asn Leu Ala Thr Asp Gly Thr Ile Ile Gly Gly Thr Gly

50 55 60

Gln Asp Thr Ile His Glu Val Val Ser Lys Ser Ala Lys Pro Thr Thr

65 70 75 80

Val Ser Ala Glu Ala Asn Lys Gly Ile Lys Val Thr Gly Glu Thr Ser

85 90 95

Ser Asn Thr Thr Gly Asn Ser Ser Asn Leu Gly Glu Asp Gly Lys Asn

100 105 110

Ala Lys Ala Asn Ser Pro Ile Thr Val Glu Ser Ser Thr Asp Asn Asn

115 120 125

Lys Lys Lys Thr Phe Asp Lys Ser Gly Gln Asp Pro Asn Gln Val Thr

130 135 140

Gly Arg Met Phe Lys Lys Gly Asp Thr Thr Asn Gly Ser Thr Thr Thr

145 150 155 160

Phe Ala Glu Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala Gln Phe Ser

165 170 175

Val Lys Asn Gly Ser Asp Glu Ser Lys Leu Ala Pro Ser Ile Gly Ala

180 185 190

Glu Asn Ala Glu His Val Glu Val Asn Asn Thr Thr Gly Lys Ser Ser

195 200 205

Ile Thr Leu Ala Thr Ala Gly Val Ala Asp Gly Ser Ile Thr Ala Gly

210 215 220

Ser Lys Asp Ala Val Asn Gly Gly Ala Lys Ile Gly Gly Thr Glu Lys

225 230 235 240

Thr Thr Ile Ser Glu Tyr Lys Ala Asp Asn Lys Asn Ser Lys Thr Val

245 250 255

Lys Asn Phe Thr Ser Thr Thr Asn Ile Asp Ala Ser Val Glu Asp Ser

260 265 270

Thr Val Thr Val Gly Gly Glu Thr Glu Ser Gly Ser Gly Asn Asp Arg

275 280 285

Thr Leu Ser Gly Ala Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Val

290 295 300

Lys Ala Gly Thr Ala Asp Thr Asp Ala Val Asn Lys Gly Leu Ala Val

305 310 315 320

Thr Lys Asp Pro Asn Gln Thr Ser Ile Phe Asn Pro Ile Asn Gly Thr

325 330 335

Ala Pro Thr Thr Phe Lys Asp Thr Ala Val Asn Thr Gly Trp Gly Ser

340 345 350

Asp Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Val Ala Asp Gly

355 360 365

Asp Ile Ser Pro Thr Ser Gly Asp Val Val Gly Asp Glu Val Ser Pro

370 375 380

Thr Lys Thr Gln Thr Thr Ala Pro Thr Ala Ser Ser Thr Gln Gly Gly

385 390 395 400

Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala Pro Ala Gly Asn Val

405 410 415

Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Thr Leu Pro Glu

420 425 430

<210> 20

<211> 487

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of recombinant antigenic protein p4 of Avian bacillus paragallinarum

<400> 20

Lys Pro Ser Ile Ala Leu Gly Gln Asp Ser Thr Val Ala Asn Ser Ala

1 5 10 15

Ile Ser Arg Thr Ser Ser Ala Thr Phe Ala Gly Ser Pro Glu Thr Thr

20 25 30

Ala Gly Lys Glu Arg Gly Asn Ile Ser Gln Thr Ser Thr Glu Ala Ile

35 40 45

Asn Lys Asn Asn Phe Gly Gly Asn Ala Asn Leu Ala Thr Asp Thr Ile

50 55 60

Ile Gly Gly Thr Gly Gln Asp Thr Ile His Glu Val Val Ser Lys Ser

65 70 75 80

Ala Lys Pro Thr Thr Val Ser Ala Glu Ala Asn Lys Gly Ile Lys Val

85 90 95

Thr Gly Glu Thr Ser Ser Asn Thr Thr Gly Asn Ser Ser Asn Leu Gly

100 105 110

Glu Asp Gly Lys Asn Ala Lys Ala Asn Ser Pro Ile Thr Val Glu Ser

115 120 125

Ser Thr Asp Asn Asn Lys Lys Lys Thr Phe Asp Lys Ser Gly Gln Asp

130 135 140

Pro Asn Gln Val Thr Gly Arg Met Phe Lys Lys Gly Asp Thr Thr Asn

145 150 155 160

Gly Ser Thr Thr Thr Phe Ala Glu Asp Gly Leu Thr Ile Asp Ser Thr

165 170 175

Thr Asn Ser Ala Gln Phe Ser Val Lys Asn Gly Ser Asp Glu Ser Lys

180 185 190

Leu Ala Pro Thr Lys Leu Ser Ile Gly Ala Glu Asn Ala Glu His Val

195 200 205

Glu Val Asn Asn Thr Thr Gly Lys Ser Ser Ile Thr Leu Ala Thr Ala

210 215 220

Gly Val Ala Asp Gly Ser Ile Thr Ala Gly Ser Lys Asp Ala Val Asn

225 230 235 240

Gly Gly Gln Gly Ala Lys Ile Gly Gly Thr Glu Lys Thr Thr Ile Ser

245 250 255

Glu Ala Ile Ser Asp Val Lys Gln Ala Tyr Lys Ala Asp Asn Lys Asn

260 265 270

Ser Lys Thr Val Lys Asn Phe Thr Ser Thr Thr Asn Ile Asp Ala Ser

275 280 285

Val Glu Asp Ser Leu Asn Ala Asn Ile Ile Ala Gly Thr Val Thr Val

290 295 300

Gly Gly Glu Thr Glu Gly Ile Val Leu Thr Lys Ser Gly Ser Gly Asn

305 310 315 320

Asp Arg Thr Leu Ser Leu Ser Gly Ala Gly Asn Ala Ala Thr Asp Gly

325 330 335

Ile Lys Val Ser Gly Val Lys Ala Gly Thr Ala Asp Thr Asp Ala Val

340 345 350

Asn Lys Gly Gln Asp Ala Leu Gly Thr Thr Asp Leu Ala Val Thr Lys

355 360 365

Asp Pro Asn Gln Thr Ser Ile Phe Asn Pro Ile Asn Gly Thr Ala Pro

370 375 380

Thr Thr Phe Lys Asp Ala Val Asp Lys Leu Thr Thr Ala Val Asn Thr

385 390 395 400

Gly Trp Gly Ser Ile Asp Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser

405 410 415

Asn Val Ala Asp Gly Asp Ile Ser Pro Thr Ser Gly Asp Val Val Thr

420 425 430

Arg Val Tyr Gly Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr Ala

435 440 445

Pro Thr Ala Ser Ser Thr Gln Gly Gly Ala Thr Thr Ala Asn Thr Ala

450 455 460

Gly Gly Val Ala Pro Ala Gly Asn Val Ala Thr Gly Asp Ile Ala Pro

465 470 475 480

Thr Gln Pro Thr Leu Pro Glu

485

<210> 21

<211> 575

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of recombinant antigenic protein p5 of Avian bacillus paragallinarum

<400> 21

Leu Ala Thr Asp Gly Thr Ile Thr Phe Thr Asn Ile Gly Gly Thr Gly

1 5 10 15

Gln Asp Thr Ile His Asp Ala Ile Asn Asn Val Leu Thr Lys Leu Ile

20 25 30

Ser Leu Ser Ala Thr Glu Glu Glu Glu Val Val Ser Gly Glu Ala Val

35 40 45

Tyr Asp Ala Leu Lys Gly Ala Lys Pro Thr Val Ser Ala Glu Ala Asn

50 55 60

Lys Gly Ile Thr Gly Leu Val Asp Val Val Lys Lys Ala Asn Ser Pro

65 70 75 80

Ile Thr Val Glu Pro Ser Thr Asp Asn Asn Lys Lys Lys Thr Phe Thr

85 90 95

Val Gly Leu Met Lys Asp Ile Glu Gly Val Asn Ser Ile Thr Phe Asp

100 105 110

Lys Ser Gly Gln Asp Leu Asn Gln Val Thr Gly Arg Met Ser Ser Ala

115 120 125

Gly Leu Thr Phe Lys Lys Gly Asp Thr Thr Asn Gly Ser Thr Thr Thr

130 135 140

Phe Ala Glu Asp Gly Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala Gln

145 150 155 160

Thr Asn Leu Val Lys Val Ser Arg Asp Gly Phe Ser Val Lys Asn Gly

165 170 175

Ser Asp Glu Ser Lys Leu Ala Ser Thr Lys Leu Ser Ile Gly Ala Glu

180 185 190

Asn Ala Glu His Val Glu Val Thr Lys Ser Gly Ile Ala Leu Lys Ala

195 200 205

Asp Asn Thr Ser Asp Lys Ser Ser Ile Thr Leu Ala Gln Asp Ala Ile

210 215 220

Thr Leu Ala Gly Asn Ala Thr Gly Thr Ala Ile Lys Leu Thr Gly Val

225 230 235 240

Ala Asp Gly Asn Ile Thr Val Asn Ser Lys Asp Ala Val Asn Gly Gly

245 250 255

Gln Leu Arg Thr Leu Leu Gly Val Asp Ser Gly Ala Lys Ile Gly Gly

260 265 270

Thr Glu Lys Thr Thr Ile Ser Glu Ala Ile Ser Asp Val Lys Gln Ala

275 280 285

Leu Thr Asp Ala Thr Leu Ala Tyr Lys Ala Asp Asn Lys Asn Gly Lys

290 295 300

Thr Val Lys Leu Thr Asp Gly Leu Asn Phe Thr Ser Thr Thr Asn Ile

305 310 315 320

Asp Ala Ser Val Glu Asp Asn Gly Val Val Lys Phe Thr Leu Lys Asp

325 330 335

Lys Leu Thr Gly Leu Lys Thr Ile Ala Thr Glu Ser Leu Asn Ala Ser

340 345 350

Gln Asn Ile Ile Ala Gly Gly Thr Val Thr Val Gly Gly Glu Thr Glu

355 360 365

Gly Ile Val Leu Thr Lys Ser Gly Ser Gly Asn Asp Arg Thr Leu Ser

370 375 380

Leu Ser Gly Ala Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Ser Gly

385 390 395 400

Val Lys Ala Gly Thr Ala Asp Thr Asp Ala Val Asn Lys Gly Gln Leu

405 410 415

Asp Lys Leu Phe Lys Ala Ile Asn Asp Ala Leu Gly Thr Thr Asp Leu

420 425 430

Ala Val Thr Lys Asp Pro Asn Gln Thr Ser Ile Phe Asn Pro Ile Asn

435 440 445

Gly Thr Ala Pro Thr Thr Phe Lys Asp Ala Val Asp Lys Leu Thr Thr

450 455 460

Ala Val Asn Thr Gly Trp Gly Ser Lys Val Gly Ile Leu Ala Thr Gly

465 470 475 480

Ile Asp Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Val Ala Asp

485 490 495

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

500 505 510

Tyr Ala Leu Met Gln Lys Gly Ile Arg Val Tyr Gly Asp Glu Val Ser

515 520 525

Pro Thr Lys Thr Gln Thr Thr Ala Pro Thr Ala Ser Ser Thr Gln Gly

530 535 540

Gly Ala Thr Thr Ala Asn Thr Ala Gly Gly Val Ala Pro Ala Gly Asn

545 550 555 560

Val Ala Thr Gly Asp Ile Ala Pro Thr Gln Pro Thr Leu Pro Glu

565 570 575

<210> 22

<211> 1077

<212> DNA

<213> Artificial Sequence

<220>

<223> The gene coding sequence of the recombinant antigenic protein p1 of Avian bacillus paragallinarum

<400> 22

gttaccgctg gtggtaaacc gtctatcgct ctgggtcagg actctaccgt tgctaactct 60

gctatctctc gtacctcttc tgctaccatc aacttcgctg gttctccgga aaccctggct 120

ggtaaaatct ctcagacctc taccgaagct atcaacaact tcggtaacct ggctatcggt 180

ggtaccggtc aggacaccat cgaagttgtt tctgctaaac cgaccaccgt ttctgctgaa 240

gctaacaaag gtatcaaagt taccggtgaa acctcttcta acaccaccgg taactctaac 300

ctgggtgaag acggtaaaaa cgctaactct ccgatcaccg ttgaatcttc taccgacaac 360

aacaaaaaag acaaatctgg tcaggacccg aaccaggtta ccggtcgtaa aaaaggtgac 420

accaccaacg gttctaccac caccttcgct ctgaccatcg actctaccac caactctgct 480

cagaccttct ctgttaaaaa cggttctgac gaatctaaac tggctaacaa caccaccggt 540

aaagacggtt ctatcaccgc tggttctaaa gacgctgtta acggtggtgc taaaatcggt 600

ggtaccgaaa aaaccaccgc tgacaacaaa aactctaaaa acatcgacgc ttctgttgaa 660

gactctgtta ccgttggtgg tgaaacctct ggttctggta acgaccgtgc tggtaacgct 720

gctaccgacg gtatcgttaa agctggtacc gctgacaccg acgctgttaa caaagctgtt 780

accaaagacc cgaaccagac ctctaacggt accgctccga ccaccttcaa agacaacacc 840

ggttggggtg gtatcgacgc tggtaacaaa aaaatctcta acgttgctga cggtgacatc 900

tctccgacct ctggtgacgt tgttgacgaa gtttctccga ccaaaaccca gaccaccgct 960

ccgaccgctt cttctaccca gggtggtgct accaccgcta acaccgctgg tggtgttgct 1020

ccggctggta acgttgctac cggtgacatc gctccgaccc agccgaccct gccggaa 1077

<210> 23

<211> 1215

<212> DNA

<213> Artificial Sequence

<220>

<223> The gene coding sequence of the recombinant antigenic protein p2 of Avianbacterium paragallinarum

<400> 23

tctaccgttg ctaacatctc tcgtacctct tctgctttcg ctggttctcc ggaaaccacc 60

gctggtaaag aaggtaacat ctctcagacc tctaccgaag ctatcaacaa aaacaacttc 120

ggtggtaacg ctaacctggc taccgacatc ggtggtaccg gtcaggacac catccacgaa 180

gttgtttcta aatctgctaa accgaccacc gtttctgctg aagctaacaa aggtatcaaa 240

gttaccggtg aaacctcttc taacaccacc ggtaactctt ctaacctggg tgaagacggt 300

aaaaacgcta aagctaactc tccgatcacc gttgaatctt ctaccgacaa caacaaaaaa 360

aaagacaaat ctggtcagga cccgaaccag gttaccggtc gtatgaaaaa aggtgacacc 420

accaacggtt ctaccaccac cttcgctctg accatcgact ctaccaccaa ctctgctcag 480

ttctctgtta aaaacggttc tgacgaatct aaactggctt ctatcggtgc tgaaaacgct 540

gaacacgttg aagttaacaa caccaccggt aaatcttcta tcaccgctac cgctggtgac 600

ggttctatca ccgctggttc taaagacgct gttaacggtg ctaaaatcgg tggtaccgaa 660

aaaaccacca tctctgaaaa agctgacaac aaaaactcta aaaccgttaa aaacttcacc 720

accaccaaca tcgacgcttc tgttgaagac tctctgaacg ttaccgttgg tggtgaaacc 780

gaatctggtt ctggtaacga ccgtaccctg ggtgctggta acgctgctac cgacggtatc 840

aaagttgtta aagctggtac cgctgacacc gacgctgtta acaaaggtct gggtctggct 900

gttaccaaag acccgaacca gacctctatc ccgatcaacg gtaccgctcc gaccaccttc 960

aaagacaaca ccggttgggg tgacggtatc gacgctggta acaaaaaaat ctctaacgtt 1020

gctgacggtg acatctctcc gacctctggt gacgttgttg gtgacgaagt ttctccgacc 1080

aaaacccaga ccaccgctcc gaccgcttct tctacccagg gtggtgctac caccgctaac 1140

accgctggtg gtgttgctcc ggctggtaac gttgctaccg gtgacatcgc tccgacccag 1200

ccgaccctgc cggaa 1215

<210> 24

<211> 1290

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene coding sequence of recombinant antigenic protein p3 of Avian bacillus paragallinarum

<400> 24

caggactcta ccgttgctaa ctctgctatc tctcgtacct cttctgctac cttcgctggt 60

tctccggaaa ccaccgctgg taaagaacgt aaaggtaaca tctctcagac ctctaccgaa 120

gctatcaaca aaaacaactt cggtggtaac gctaacctgg ctaccgacgg taccatcatc 180

ggtggtaccg gtcaggacac catccacgaa gttgtttcta aatctgctaa accgaccacc 240

gtttctgctg aagctaacaa aggtatcaaa gttaccggtg aaacctcttc taacaccacc 300

ggtaactctt ctaacctggg tgaagacggt aaaaacgcta aagctaactc tccgatcacc 360

gttgaatctt ctaccgacaa caacaaaaaa aaaaccttcg acaaatctgg tcaggacccg 420

aaccaggtta ccggtcgtat gttcaaaaaa ggtgacacca ccaacggttc taccaccacc 480

ttcgctgaac tgaccatcga ctctaccacc aactctgctc agttctctgt taaaaacggt 540

tctgacgaat ctaaactggc tccgtctatc ggtgctgaaa acgctgaaca cgttgaagtt 600

aacaacacca ccggtaaatc ttctatcacc ctggctaccg ctggtgttgc tgacggttct 660

atcaccgctg gttctaaaga cgctgttaac ggtggtgcta aaatcggtgg taccgaaaaa 720

accaccatct ctgaatacaa agctgacaac aaaaactcta aaaccgttaa aaacttcacc 780

tctaccacca acatcgacgc ttctgttgaa gactctaccg ttaccgttgg tggtgaaacc 840

gaatctggtt ctggtaacga ccgtaccctg tctggtgctg gtaacgctgc taccgacggt 900

atcaaagttg ttaaagctgg taccgctgac accgacgctg ttaacaaagg tctggctgtt 960

accaaagacc cgaaccagac ctctatcttc aacccgatca acggtaccgc tccgaccacc 1020

ttcaaagaca ccgctgttaa caccggttgg ggttctgacg gtatcgacgc tggtaacaaa 1080

aaaatctcta acgttgctga cggtgacatc tctccgacct ctggtgacgt tgttggtgac 1140

gaagtttctc cgaccaaaac ccagaccacc gctccgaccg cttcttctac ccagggtggt 1200

gctaccaccg ctaacaccgc tggtggtgtt gctccggctg gtaacgttgc taccggtgac 1260

atcgctccga cccagccgac cctgccggaa 1290

<210> 25

<211> 1461

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene coding sequence of recombinant antigenic protein p4 of Avian bacillus paragallinarum

<400> 25

aaaccgtcta tcgctctggg tcaggactct accgttgcta actctgctat ctctcgtacc 60

tcttctgcta ccttcgctgg ttctccggaa accaccgctg gtaaagaacg tggtaacatc 120

tctcagacct ctaccgaagc tatcaacaaa aacaacttcg gtggtaacgc taacctggct 180

accgacacca tcatcggtgg taccggtcag gacaccatcc acgaagttgt ttctaaatct 240

gctaaaccga ccaccgtttc tgctgaagct aacaaaggta tcaaagttac cggtgaaacc 300

tcttctaaca ccaccggtaa ctcttctaac ctgggtgaag acggtaaaaa cgctaaagct 360

aactctccga tcaccgttga atcttctacc gacaacaaca aaaaaaaaac cttcgacaaa 420

tctggtcagg acccgaacca ggttaccggt cgtatgttca aaaaaggtga caccaccaac 480

ggttctacca ccaccttcgc tgaagacggt ctgaccatcg actctaccac caactctgct 540

cagttctctg ttaaaaacgg ttctgacgaa tctaaactgg ctccgaccaa actgtctatc 600

ggtgctgaaa acgctgaaca cgttgaagtt aacaacacca ccggtaaatc ttctatcacc 660

ctggctaccg ctggtgttgc tgacggttct atcaccgctg gttctaaaga cgctgttaac 720

ggtggtcagg gtgctaaaat cggtggtacc gaaaaaacca ccatctctga agctatctct 780

gacgttaaac aggcttacaa agctgacaac aaaaactcta aaaccgttaa aaacttcacc 840

tctaccacca acatcgacgc ttctgttgaa gactctctga acgctaacat catcgctggt 900

accgttaccg ttggtggtga aaccgaaggt atcgttctga ccaaatctgg ttctggtaac 960

gaccgtaccc tgtctctgtc tggtgctggt aacgctgcta ccgacggtat caaagtttct 1020

ggtgttaaag ctggtaccgc tgacaccgac gctgttaaca aaggtcagga cgctctgggt 1080

accaccgacc tggctgttac caaagacccg aaccagacct ctatcttcaa cccgatcaac 1140

ggtaccgctc cgaccacctt caaagacgct gttgacaaac tgaccaccgc tgttaacacc 1200

ggttggggtt ctatcgacgg tatcgacgct ggtaacaaaa aaatctctaa cgttgctgac 1260

ggtgacatct ctccgacctc tggtgacgtt gttacccgtg tttacggtga cgaagtttct 1320

ccgaccaaaa cccagaccac cgctccgacc gcttcttcta cccagggtgg tgctaccacc 1380

gctaacaccg ctggtggtgt tgctccggct ggtaacgttg ctaccggtga catcgctccg 1440

acccagccga ccctgccgga a 1461

<210> 26

<211> 1725

<212> DNA

<213> Artificial Sequence

<220>

<223> The gene coding sequence of the recombinant antigenic protein p5 of Avian bacillus paragallinarum

<400> 26

ttagctactg atggtaccat tacatttacg aatatccaag gcactggata ggacaccata 60

catgatgcca ttaacaatgt tttgacaaaa cttatctctc tctccgcaac ggaagaagag 120

gaagtcgtat caggggaggc ggtgtatgac gctctaaagg gtgccaaacc taccgtttcg 180

gcagaagcaa acaagggcat aacgggactg gtcgatgtag tgaaaactgc gaatagtccc 240

attaccgttg agccaagcac agacaacaat aaggttaaaa cgttcactgt cgggttaatg 300

aaggacatcg aaggtgtaaa ctctataacc tttgataaat ccggccaaga cttgaatcag 360

acgacgggac gtatgtcatc ggctgggctt actttcaaga agggtgatac cacaaacggc 420

agtacgacta ccttcgccga ggacggactc acaattgata gcacgactaa ttctgcacaa 480

accaacctag tgaaagtttc ccgcgacggg ttttcagtca agaatggttc ggatgaaagt 540

aaactggcct ctacaaagtt atccatcggc gctgagaacg ccgaacacgt agaggtgacg 600

aaatcaggga tagcattgaa ggcggacaat actagtgata aaagctctat tacccttgct 660

taagacgcca tcacactcgc aggaaacgcg acagggacgg ctattaagct aactggtgtt 720

gccgatggca atatcacagt caactccaaa gacgcggtaa atggagggta gctgcgaacc 780

ttattgggtg tggattcagg cgctaagata ggagggacag aaaaaacgac tatttcggag 840

gccatcagtg acgttaagca agcacttacc gatgcgacac tcgcttacaa agccgacaac 900

aagaatggta aaacggtcaa gctaactgat ggcctgaact tcaccagcac aacgaatata 960

gacgcatctg tagaagataa cggagtggtt aaatttactt taaaggacaa attgaccggg 1020

cttaagacaa ttgcgacgga gtccctcaat gcttcacaga acatcatagc cggtggcact 1080

gtcaccgtag gaggggaaac agagggtatt gtgctaacga aatcgggcag tggaaatgat 1140

cggactctga gcttatctgg ggcaggtaac gcggctaccg acggcatcaa ggtttccgga 1200

gtcaaagccg ggacagcaga tacggacgcg gtaaataagg gttaattgga taaacttttc 1260

aaggctataa acgacgccct cggcactacc gatctagcag tgacaaaaaa tccgaactag 1320

acgtcaattt ttaatcctat caacggaact gcgcccacca cattcaagga cgctgttgat 1380

aaactgacga ctgccgtcaa taccgggtgg ggttcgaagg taggcatatt agcaacagga 1440

attgacggga tcgatgcggg taacaaaaag ataagtaatg tggctgacgg cgatattagc 1500

ccaacgtctg gagacgttgt cactgggaga caattgtatg cccttatgca gaaaggtatc 1560

agggtatacg gcgatgaagt gtccccgacc aagacataaa cgactgcacc taccgcgtca 1620

tcgacatagg gaggggctac caccgctaac accgctggtg gtgttgctcc ggctggtaac 1680

gttgctaccg gtgacatcgc tccgacccag ccgaccctgc cggaa 1725

<210> 27

<211> 2083

<212> PRT

<213> Avibacterium paragallinarum

<400> 27

Met Asn Lys Val Phe Lys Ile Lys Tyr Ser Val Val Lys Gln Glu Met

1 5 10 15

Ile Val Val Ser Glu Leu Ala Asn Asn Lys Asp Lys Thr Ala Ser Gln

20 25 30

Lys Asn Thr His Asn Thr Ala Phe Phe Gln Pro Leu Phe Thr Lys Cys

35 40 45

Thr Tyr Leu Ala Leu Leu Ile Asn Ile Ala Leu Gly Thr Ser Leu Phe

50 55 60

Pro Gln Leu Ala Asn Ala Lys Phe Leu Glu Val Tyr Asn Ser Ser Val

65 70 75 80

Lys Leu Gln His Val Asn Ser Gly Val Pro Ser Asp Ser Val Asn Leu

85 90 95

Asn Pro Ser Gly Ser Glu Asn Val Gly Met Asn Ser Asn Gln Gly Val

100 105 110

Ala Ile Gly Arg Gly Ala Val Asn Ser Tyr Ser Ala Thr Gly Ser Ile

115 120 125

Ala Ile Gly Gln Gly Ala Lys Asn Asp Asn Trp Ala Thr Arg Ser Ile

130 135 140

Ala Ile Gly Gln Gly Ala Lys Asn Glu Ser Ile Ala Ser Asp Ser Val

145 150 155 160

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

165 170 175

Leu Asn Ala Tyr Thr Gln Leu Asp Pro Arg Arg Thr Pro Glu Ser Arg

180 185 190

Gln Gly Ser Val Val Ile Gly Glu Asn Ala Lys Ser Ala Gly Asn Gln

195 200 205

Ser Val Ser Leu Gly Gln Asn Ala Trp Ser Lys Thr Asn Ser Ile Ser

210 215 220

Ile Gly Ala Gly Thr Phe Ala Glu Gly Asn Ser Thr Ile Ala Ile Gly

225 230 235 240

Thr Asp Lys Ile Leu Gly Thr Asn Tyr Asn Asp Lys Leu Pro Ala Pro

245 250 255

Ser Trp Asp Gly Arg Thr Gly Lys Ala Pro Ala Asn Ser Ile Trp Asp

260 265 270

Ile Phe Ser Glu Leu Tyr Met Gly Lys Lys Thr Asn Gly Thr Asp Tyr

275 280 285

Asp Ala Lys Lys Asn Asp Arg Asp Pro Asn Lys Pro Glu Ala Phe Tyr

290 295 300

Thr Tyr Ser Asp Phe Lys Ser Arg Tyr Val Asn Asn Pro Ser Thr Ser

305 310 315 320

Pro Thr Tyr Ala Ala Lys Leu Gly Ala Ile Ala Leu Gly Ser Arg Thr

325 330 335

Ile Ala Ala Gly Glu Met Ser Thr Ala Val Gly Ser Leu Ala Phe Ala

340 345 350

Leu Ala Asp Lys Ser Thr Ala Met Gly Leu Arg Ser Phe Val Ala Lys

355 360 365

Asp Ala Val Gly Gly Thr Ala Ile Gly Glu Glu Ser Arg Thr Phe Ala

370 375 380

Lys Asp Ser Val Ala Ile Gly Asn Lys Thr Glu Ala Ser Asn Ala Gly

385 390 395 400

Ser Met Ala Tyr Gly Tyr Lys Ala Lys Ala Val Gly Ala Gly Ala Ile

405 410 415

Ala Ile Gly Ala Glu Val Thr Ala Gly Ala Glu Phe Asp Ser Ser Gln

420 425 430

Val Gly Asn Leu Leu Leu Asp Arg Gly Ala Tyr Ala Thr Leu Lys Ser

435 440 445

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

450 455 460

Thr Gln Phe Phe Asp Asn Met Leu Thr Gln Gly Ser His Leu Thr Tyr

465 470 475 480

Glu Asn Thr Thr Tyr Leu Thr Thr Ser Ala Gly Asp Ile Lys Lys Thr

485 490 495

Leu Ala Ala Val Gly Asp Gly Gly Lys Asn Ala Ile Ala Ile Gly Asn

500 505 510

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

515 520 525

Leu Ala Ser Ala Gln Asn Ala Phe Ala Leu Gly Ser Tyr Ser Leu Val

530 535 540

Ser Pro Leu Ala Ala Asn Thr Ile Val Ile Gly Val Gly Gly Tyr Ala

545 550 555 560

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

565 570 575

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

580 585 590

His Asp Ser Leu Ala Met Gly Val Asn Ala Phe Ile Gly Asn Gly Ser

595 600 605

Asn Ser Ser Leu Ala Leu Gly Thr Gly Ser Thr Ile Ala Lys Asn Thr

610 615 620

Lys Ser Pro Asp Ser Leu Ala Ile Gly Lys Asp Ser Arg Ile Asp Ala

625 630 635 640

Lys Asp Thr Asp Asn Gly Val Leu Tyr Thr Pro Gln Val Tyr Asp Glu

645 650 655

Thr Thr Arg Ala Phe Arg Thr Phe Asp Glu Asn Lys Asp Tyr Met Arg

660 665 670

Gln Ala Met Ala Leu Gly Phe Asn Ala Lys Val Ser Arg Gly Lys Gly

675 680 685

Lys Met Glu Thr Gly Ile Asn Ser Met Ala Ile Gly Ala Arg Ser Gln

690 695 700

Ala Thr Leu Gln Asn Ser Thr Ala Leu Gly Val Asn Ala Lys Thr Asp

705 710 715 720

Tyr Thr Trp Glu Gln Leu Glu Ala Asp Pro Trp Val Ser Lys Gly Ala

725 730 735

Ile Ser Ile Pro Thr Ser Gly Lys Ile Gly Val Ile Ser Val Gly Ser

740 745 750

Lys Gly Ser Glu Arg Arg Ile Val Asn Val Ala Ser Gly Ser Leu Asp

755 760 765

Thr Asp Ala Val Asn Val Ala Gln Leu Lys Thr Ile Glu Glu Arg Phe

770 775 780

Gln Ser Glu Ile Asp Leu Leu Gln Asn Gly Gly Gly Gly Val Gln Tyr Leu

785 790 795 800

Ser Val Glu Lys Thr Asn Ile Asn Gly Glu Ala Gly Arg Val Ala Ser

805 810 815

Gln Ile Arg Lys Gly Glu Ser Tyr Lys Arg Tyr Val Lys Leu Lys Thr

820 825 830

Gln Leu Leu Tyr Leu Asp Ala Arg Lys Lys Leu Asn Gly Glu Lys Phe

835 840 845

Asp Gln Thr Ser Leu Asp Lys Ile Ser Lys Ala Val Gln Glu Leu Glu

850 855 860

Ala Glu Tyr Ser Gly Glu Leu Lys Thr Thr Ala Ser Glu Leu Asn Arg

865 870 875 880

Val Ala Met Gln Leu Asn Ala Glu Thr Thr Val Asn Asn Phe Gly Lys

885 890 895

Phe Asn Gln Tyr Lys Thr Gln Ile Glu Asn Ala Thr Asn Ala Asp Ser

900 905 910

Glu Lys Asn Val Gly Gly Leu Ser Pro Gln Val Ile Ala Gln Leu Lys

915 920 925

Ala Asn Asn Asn Tyr Leu Asn Asp Gly Ala Lys Gly Gln Asp Ser Ile

930 935 940

Ala Phe Gly Trp Gln Ala Lys Thr Ser Glu Ala Asn Asn Gly Leu Ala

945 950 955 960

Gly Lys Gln Ala Ile Ala Ile Gly Phe Gln Ala Asn Ser Ser Ala Glu

965 970 975

Asn Ala Ile Ser Ile Gly Thr Asn Ser Asp Thr Ser Met Thr Gly Ala

980 985 990

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

995 1000 1005

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

1010 1015 1020

Arg Thr Ser Ser Ala Thr Ile Asn Gly Leu Thr Phe Asn Asn Phe

1025 1030 1035

Ala Gly Ser Pro Glu Thr Leu Gly Val Leu Ser Ile Gly Thr Ala

1040 1045 1050

Gly Lys Glu Arg Lys Ile Val Asn Val Ala Ala Gly Asn Ile Ser

1055 1060 1065

Gln Thr Ser Thr Glu Ala Ile Asn Gly Ser Gln Leu Tyr Ala Thr

1070 1075 1080

Asn Phe Met Leu Asn Lys Leu Ala Gln Ser Val Lys Asn Asn Phe

1085 1090 1095

Gly Gly Asn Ala Asn Leu Ala Thr Asp Gly Thr Ile Thr Phe Thr

1100 1105 1110

Asn Ile Gly Gly Thr Gly Gln Asp Thr Ile His Asp Ala Ile Asn

1115 1120 1125

Asn Val Leu Thr Lys Leu Ile Ser Leu Ser Ala Thr Glu Glu Val

1130 1135 1140

Val Ser Gly Glu Ala Val Tyr Glu Ala Leu Lys Ser Ala Lys Pro

1145 1150 1155

Thr Thr Val Ser Ala Glu Ala Asn Lys Gly Ile Lys Val Thr Gly

1160 1165 1170

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

1175 1180 1185

Asp Asp Ala Thr Leu Asn Lys Ile Asn Asn Ala Val Asn Gln Asp

1190 1195 1200

Leu Ser Asn Leu Gly Glu Asp Gly Lys Asn Ala Ile Thr Gly Leu

1205 1210 1215

Val Asp Val Val Lys Lys Ala Asn Ser Pro Ile Thr Val Glu Ser

1220 1225 1230

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

1235 1240 1245

Lys Asn Ile Thr Glu Val Asn Ser Ile Thr Phe Asp Lys Ser Gly

1250 1255 1260

Gln Asp Pro Asn Gln Val Thr Gly Arg Met Ser Ser Ala Gly Leu

1265 1270 1275

Thr Phe Lys Lys Gly Asp Thr Thr Asn Gly Ser Thr Thr Thr Phe

1280 1285 1290

Ala Glu Asp Gly Leu Thr Ile Asp Ser Thr Thr Asn Ser Ala Gln

1295 1300 1305

Thr Asn Leu Val Lys Val Ser Arg Asp Gly Phe Ser Val Lys Asn

1310 1315 1320

Gly Ser Asp Glu Ser Lys Leu Ala Pro Thr Lys Leu Ser Ile Gly

1325 1330 1335

Ala Glu Asn Ala Glu His Val Glu Val Thr Lys Ser Gly Ile Ala

1340 1345 1350

Leu Lys Ala Asn Asn Thr Thr Gly Lys Ser Ser Ile Thr Leu Ser

1355 1360 1365

Asp Ser Ala Ile Thr Leu Ala Ala Ala Thr Ala Gly Asn Ala Ile

1370 1375 1380

Lys Leu Thr Gly Val Ala Asp Gly Ser Ile Thr Ala Gly Ser Lys

1385 1390 1395

Asp Ala Val Asn Gly Gly Gln Leu Arg Thr Leu Leu Gly Val Asp

1400 1405 1410

Ser Gly Ala Lys Ile Gly Gly Thr Glu Lys Thr Thr Ile Ser Glu

1415 1420 1425

Ala Ile Ser Asp Val Lys Gln Ala Leu Thr Asp Ala Lys Leu Ala

1430 1435 1440

Tyr Lys Ala Asp Asn Lys Asn Ser Lys Thr Val Lys Leu Thr Asp

1445 1450 1455

Gly Leu Asn Phe Thr Ser Thr Thr Asn Ile Asp Ala Ser Val Glu

1460 1465 1470

Asp Ser Gly Val Val Lys Phe Thr Leu Lys Asp Lys Leu Ile Gly

1475 1480 1485

Leu Lys Thr Ile Ala Thr Glu Ser Leu Asn Ala Ser Arg Asn Ile

1490 1495 1500

Ile Ala Gly Gly Thr Val Thr Val Gly Gly Glu Thr Glu Gly Ile

1505 1510 1515

Val Leu Thr Lys Ser Gly Ser Gly Asn Asp Arg Thr Leu Ser Leu

1520 1525 1530

Ser Gly Ala Gly Asn Ala Ala Thr Asp Gly Ile Lys Val Ser Gly

1535 1540 1545

Val Lys Ala Gly Thr Ala Asp Thr Asp Ala Val Asn Lys Gly Gln

1550 1555 1560

Leu Asp Lys Leu Phe Lys Ala Ile Asn Asp Ala Leu Gly Thr Thr

1565 1570 1575

Asp Leu Ala Val Thr Lys Asp Pro Asn Gln Thr Ser Ile Phe Asn

1580 1585 1590

Pro Ile Asn Gly Thr Ala Pro Thr Thr Phe Lys Asp Ala Val Asp

1595 1600 1605

Lys Leu Thr Thr Ala Val Asn Thr Gly Trp Gly Ser Lys Val Gly

1610 1615 1620

Ile Leu Ala Thr Gly Ile Asp Gly Ile Asp Ala Gly Asn Lys Lys

1625 1630 1635

Ile Ser Asn Val Ala Asp Gly Asp Ile Ser Pro Thr Ser Gly Asp

1640 1645 1650

Val Val Thr Gly Arg Gln Leu Tyr Ala Leu Met Gln Lys Gly Ile

1655 1660 1665

Arg Val Tyr Gly Asp Glu Val Ser Pro Thr Lys Thr Gln Thr Thr

1670 1675 1680

Ala Pro Thr Ala Ser Ser Thr Gln Gly Gly Ala Thr Thr Ala Asn

1685 1690 1695

Thr Ala Gly Gly Val Ala Pro Ala Gly Asn Val Ala Thr Gly Asp

1700 1705 1710

Ile Ala Pro Thr Gln Pro Thr Leu Pro Glu Met Asn Thr Ala Leu

1715 1720 1725

Val Asn Asp His Leu Ala Val Pro Leu Gly Gly Ser Leu Lys Ile

1730 1735 1740

His Gly Asp His Asn Val Lys Thr Thr Ile Ser Ala Asp Asn Gln

1745 1750 1755

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

1760 1765 1770

Val Ile Gly Ser Asn Lys Pro Glu Lys Ala Lys Leu Ala Ala Gln

1775 1780 1785

Glu Gly Asn Ala Leu Val Ile Thr Asn Lys Asp Asp Gly Asn Ala

1790 1795 1800

Ala Met Val Phe Asn Asn Glu Lys Asn Met Leu Val Leu Ser Asp

1805 1810 1815

Lys Lys Ala Lys Pro Arg Val Leu Leu Asp Gly Gln Asn Gly Ala

1820 1825 1830

Leu Thr Leu Val Gly Asn Asp Asp Ser Gln Val Thr Leu Ser Ser

1835 1840 1845

Lys Lys Gly Lys Asp Ile Asp Gly Asn Asp Leu Ser Arg Leu Ser

1850 1855 1860

Val Thr Thr Glu Arg Thr Asn Ala Asp Gly Gln Leu Glu Lys Val

1865 1870 1875

Glu Thr Ser Phe Ala Thr Met Asp Asp Gly Leu Lys Phe Lys Ala

1880 1885 1890

Asp Gly Asp Lys Val Ile Asn Lys Lys Leu Asn Glu Thr Val Glu

1895 1900 1905

Ile Val Gly Asp Glu Asn Val Thr Thr Ser Ile Thr Asp Asp Asn

1910 1915 1920

Lys Val Lys Val Ser Leu Asn Lys Lys Ile Ala Ile Asp Glu Val

1925 1930 1935

Lys Ile Pro Asn Thr Asp Pro Asp Ala Gln Lys Gly Asp Ser Ile

1940 1945 1950

Val Ile Asn Asn Gly Gly Ile His Ala Gly Asn Lys Val Ile Thr

1955 1960 1965

Gly Val Lys Ala Ser Asp Asp Pro Thr Ser Ala Val Asn Arg Gly

1970 1975 1980

Gln Leu Asn Thr Val Ile Asp Asn Val Gln Asn Asn Phe Asn Gln

1985 1990 1995

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

2000 2005 2010

Ile Ala Gly Ala Met Ala Thr Ala Ser Leu Gln Asn Val Ala Leu

2015 2020 2025

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

2030 2035 2040

Glu Asn Ala Val Ala Ile Gly Met Ser Arg Leu Ser Asp Asn Gly

2045 2050 2055

Lys Val Gly Ile Arg Leu Ser Gly Met Ser Thr Ser Asn Gly Asp

2060 2065 2070

Lys Gly Ala Ala Met Ser Val Gly Phe Thr

2075 2080

<210> 28

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Upstream primers for amplifying the gene encoding antigenic protein p1

<400> 28

gcccatgggt taccgctggt ggtaaaccg 29

<210> 29

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Downstream primers for amplifying the gene encoding antigenic protein p1

<400> 29

cgctcgagtt ccggcagggt cggctgggt 29

<210> 30

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Upstream primers for amplifying the gene encoding antigenic protein p2

<400> 30

gcccatggtc taccgttgct aacatctct 29

<210> 31

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Downstream primers for amplifying the gene encoding antigenic protein p2

<400> 31

cgctcgagtt ccggcagggt cggctgggt 29

<210> 32

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Upstream primers for amplifying the gene encoding antigenic protein p3

<400> 32

gcccatggca ggactctacc gttgctaac 29

<210> 33

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Downstream primers for amplifying the gene encoding antigenic protein p3

<400> 33

cgctcgagtt ccggcagggt cggctgggt 29

<210> 34

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Upstream primers for amplifying the gene encoding antigenic protein p4

<400> 34

gcccatggaa accgtctatc gctctgggt 29

<210> 35

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Downstream primers for amplifying the gene encoding antigenic protein p4

<400> 35

cgctcgagtt ccggcagggt cggctgggt 29

<210> 36

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Upstream primers for amplifying the gene encoding antigenic protein p5

<400> 36

gcccatggtt agctactgat ggtaccatt 29

<210> 37

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Downstream primers for amplifying the gene encoding antigenic protein p5

<400> 37

cgctcgagtt ccggcagggt cggctgggt 29

Claims (9)

1. An avibacterium paragallinarum immunoprotective antigen protein characterized in that its amino acid sequence is selected from one of Seq ID No.17, 18, 19 and 20.

2. A gene encoding the avibacterium paragallinarum immunoprotective antigen protein of claim 1.

3. The gene according to claim 2, characterized in that its nucleotide sequence is selected from one of Seq ID No.22, 23, 24 and 25.

4. A construct comprising the gene of claim 2 or 3.

5. A recombinant bacterium obtained by transforming a host bacterium with the construct according to claim 4.

6. An avian infectious rhinitis subunit vaccine, characterized in that the immunologically active component thereof comprises the avibacterium paragallinarum immunoprotective antigen protein of claim 1.

7. The chicken infectious rhinitis subunit vaccine of claim 6, wherein the avibacterium paragallinarum immunoprotective antigen protein has a final concentration of 5-100 μ g/ml.

8. A preparation method of a chicken infectious rhinitis subunit vaccine is characterized by comprising the following steps:

(1) constructing an expression vector of the avibacterium paragallinarum immunoprotective antigen protein of claim 1;

(2) transforming a protein expression system matched with the expression vector by using the expression vector to obtain a recombinant bacterium;

(3) culturing the recombinant strain, inducing expression to obtain an avibacterium paragallinarum immunoprotection antigen protein, and purifying the protein;

(4) mixing the purified avibacterium paragallinarum immunoprotection antigen protein with an auxiliary reagent, and adjusting the final concentration of the antigen protein to 5-100 mu g/ml to obtain the chicken infectious rhinitis subunit vaccine.

9. The method of claim 8, wherein the expression vector is selected from the group consisting of pET, pQE and pGEX series vectors; the protein expression system is selected from escherichia coli BL21, DH5 alpha, Top10 and JM109 strains; the auxiliary agent comprises an immunopotentiator, a stabilizer, a preservative, a salt solution and/or distilled water.

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