CN107266538A - Infectious coryza of chicken 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. Provide an immunoprotective antigenic protein of Avian bacilli paragallinarum, characterized in that it includes 16 antigenic epitopes or epitope domains with amino acid sequences as shown in Seq ID No.1-16; preferably, its amino acid sequence is selected from Seq One of ID No.17, 18, 19 and 20. Also provided is a chicken infectious rhinitis subunit vaccine, the immune active component of which includes the immunoprotective antigen protein of Aviparagallinarum. The vaccine can significantly enhance the resistance of chickens to A, B, and C serotypes of Aviobacterium paragallinarum, and its protective effect is better than that of the whole-bacteria inactivated vaccine. It can be used to prevent chicken infection caused by different serotypes of Aviobacterium paragallinarum Sexual rhinitis.

Description

Chicken infectious rhinitis subunit vaccine and preparation method thereof

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

technical field

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.

Background technique

Avian infectious coryza is one of the most important respiratory diseases caused by Avibacterium paragallinarum (Apg) infection. Chickens with avian infectious rhinitis mainly present clinical symptoms such as runny nose, swollen eyelids and watery eyes. Chicken infectious rhinitis can lead to the delay of laying eggs and the decline of egg production, thus causing a lot of economic losses.

Apg is a short Gram-negative bacilli belonging to the family Pasteurellaceae. Page et al. divided Apg into three serotypes, A, B, and C. Studies have shown that Apg of the three serotypes A, B, and C have different degrees of pathogenicity, but the inactivated bacteria of the three do 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, etc. At present, most of the inactivated vaccines commonly used in the world contain A and C types of Apg. As the prevalence and occurrence of a large number of B-type Apgs have been discovered at home and abroad, vaccine companies with greater influence in the world have begun to provide vaccines containing A, B, C three serotype trivalent inactivated vaccine. Because Aviobacterium paragallinarum is a fastidious bacterium, the cost of cultivation is high, resulting in high cost of conventional inactivated vaccines; in addition, Aviobacterium paragallinarum contains toxins such as LPS, and the toxic and side effects caused by mass inoculation will also affect the production of chickens. Eggs and growth, focal necrotic plaques also formed in vaccinated chickens. The preventive effect of chicken infectious rhinitis inactivated vaccine on wild chicken group infection 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 types A and C of Avibacterium paragallinarum, and the obtained recombinant proteins can be used as protective antigens against type A and type C chicken infectious rhinitis infections, respectively. However, when designing recombinant antigens for the preparation of vaccines, it is very difficult to screen the antigenic epitopes shared by the three serotypes Apg, A, B, and C, which have neutralizing activity. , C. Reports on the protective antigens of three serotypes of Avibacterium paragallinarum infection.

Contents of the invention

After in-depth research by the inventors, the present invention provides a novel immunoprotective antigenic protein of Aviola paragallinarum, which has strong immunogenicity, and the subunit vaccine prepared with the antigenic protein as an active ingredient can effectively prevent types A, B and A at the same time. Infection with Avibacterium paragallinarum type C.

The technical scheme that the present invention claims protection is as follows:

An immunoprotective antigenic protein of Avian paragallinarum is characterized in that it comprises 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 Avibacterium paragallinarum is selected from one of Seq ID No.17, 18, 19 and 20.

The gene encoding the immunoprotective antigen protein of Aviparagallinarum.

Preferably, the nucleotide sequence of the gene is selected from one of Seq ID No.22, 23, 24 and 25.

A construct characterized by comprising the gene.

A recombinant cell is 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 is characterized in that its immunologically active components include the immunoprotective antigen protein of Avibacterium paragallinarum.

Preferably, the final concentration of the immunoprotective antigen protein of Avibacterium 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 the immunoprotective antigen protein of Aviparagallinarum;

(2) using the expression vector to transform a matching protein expression system to obtain recombinant cells;

(3) cultivating the recombinant cells, inducing expression to obtain the immunoprotective antigen protein of Aviola paragallinarum and purifying the protein;

(4) Mix the purified immunoprotective antigenic protein of Avian bacillus paragallinarum with an auxiliary reagent, adjust the final concentration of the antigenic protein to 5-100 μg/ml, and obtain the chicken infectious rhinitis subunit vaccine.

Preferably, the expression vector is selected from pET, pQE and pGEX series carriers; 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 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 analyzed for epitope. According to the prediction results and experimental experience, several peptides with strong immunogenicity were selected, and after multiple sequence optimizations, 16 core antigenic epitopes/epitope domains were determined, and their amino acid sequences are shown in Seq ID No.1-16. Show.

In some embodiments, several recombinant antigenic proteins are obtained from the combination of the above 16 antigenic epitopes/epitope domains.

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

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

Experimental analysis is carried out to the antigenic characteristic of the recombinant antigenic protein of gained, and Western Blotting experiment proves that the recombinant antigenic protein of the present invention gained has stronger immunogenicity; After using this antigenic protein immunization test chicken, can protect test chicken not to be free from secondary chicken poultry bacillus infection.

Immunization experiment data shows that the recombinant antigenic protein obtained in the present invention can produce immune protection against multiple strains of different serotypes of Avibacterium paragallinarum, such as Hp8, 221, 0083, BJ, 222, 668, Modesto and other types A and B and C-type Apg strains, and the protective effect is better than that of whole-bacteria inactivated vaccines. In one embodiment of the present invention, the challenge experiment shows that adopting the obtained recombinant antigenic protein of the present invention (taking p1, p4, p5 as an example) to prepare the chicken flocks of vaccine immunization in A, B or C type Avibacterium paragallinarum After the challenge, all chickens did not have symptoms of rhinitis, and the protection rate was 100%. After the chicken flock immunized with the vaccine prepared by p2 and p3, all chickens did not show symptoms of rhinitis, and the protection rate was 100%. %, after challenged with B-type BJ strain and C-type Modesto strain, only 10% of the chickens had symptoms of rhinitis, and the protection rate was 90%; 2-3 chickens showed clinical symptoms, and the protection rate was 70-80%; while the chickens in the control group without immunization all showed symptoms of rhinitis after challenge, and the protection rate was 0. There was a significant difference in the protection rate between the immune group and the non-immune group.

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

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

The recombinant cells may be cells used for cloning and preserving plasmids, or cells used for protein expression. The expression system can adopt prokaryotic expression system or eukaryotic expression system. The prokaryotic expression system can choose E. coli BL21, DH5ɑ, Top10, JM109 and other strains, preferably Escherichia coli BL21 (DE3); cells or plant cells. Correspondingly, the matching expression vector, transformation condition, expression condition and protein extraction method are selected to prepare the antigenic protein.

The subunit vaccine prepared by using the recombinant antigenic protein of the present invention can significantly enhance the resistance of chickens to three serotypes of A, B, and C serotypes of Avian bacilli paragallinarum, and effectively prevent infection of Avian bacillus paragallinarum. In some embodiments, the subunit vaccine includes 1, 2, 3, 4, 5 or more amino acid sequences of the recombinant antigenic protein of Avibacterium 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 make a combined vaccine against multiple diseases. 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; Bug disease.

In the subunit vaccine, the final concentration of the immunoprotective antigen protein of Aviola paragallinarum is preferably 5-100 μg/ml, more preferably 10-50 μg/ml, 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 vectors : whole gene synthesis of DNA encoding recombinant antigenic proteins, said DNA encoding fusion peptides shown in SEQ ID No.17-21 or fusion peptide mutants in which one or several amino acids are deleted, added or replaced; design The PCR primers amplify the DNA in large quantities. For the convenience of connecting with the expression vector, add suitable enzyme cutting sites at the 5' ends of the upstream and downstream primers. The obtained DNA fragment encoding the recombinant antigenic protein is connected to a suitable expression vector, and the expression vector may be a variety of commercially available expression vectors with trp promoter, T7 promoter, cspA promoter and the like. Such expression vectors include pET series vectors, such as pET-28a; pQE series vectors, such as pQE30; pGEX series vectors, such as pGEX-6-1.

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

Induced protein expression : culture host cells carrying recombinant antigen protein expression vectors, add inducers to induce protein expression, collect induced expression host cells by centrifugation, suspend them in a fixed volume of distilled water or PBS, and break them by ultrasonic cracking , and centrifuged to collect the precipitate and supernatant. SDS-PAGE gel electrophoresis was performed on the recovered fixed amount of supernatant and precipitate, and after staining with Coomassie brilliant blue, the expression of the target protein was confirmed by the position 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.

Purification of protein : centrifuge the recombinant Escherichia coli after induced expression, and collect the bacterial pellet. The collected Escherichia coli is disrupted by chemical substances, surfactants, lysozyme or ultrasonic lysis, so as to release the recombinant protein outside the cells. Use the principle of nickel agarose affinity chromatography to treat the bacterial cell supernatant after ultrasonic lysis. If the target protein exists in the form of inclusion bodies, the solution containing inclusion bodies is recovered in the form of precipitates by centrifugal concentration, and denatured and recombined. Purified by affinity chromatography. When purifying inclusion body proteins, 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 the reducing agent can be phosphate buffer, Tris buffer, glycine buffer, etc. at pH 7-8. The recombinant protein is refolded by dialysis to restore the normal spatial structure. The dialysis solution can use the same kind of buffer, temperature and pH as used to solubilize 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 : the purified antigenic protein is mixed with auxiliary reagents, and the final concentration of the antigenic protein is adjusted to 20 μg/ml. The auxiliary reagents 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 with Freund's complete adjuvant or incomplete adjuvant in equal volumes to prepare the Aviparagallinarum subunit vaccine. The immunization route of the subunit vaccine of the present invention is not particularly limited, and can be administered subcutaneously, intradermally or intramuscularly.

Verification of immune effect: use the subunit vaccine to immunize chickens, collect the serum of the immunized chickens, and measure the antibody titer of Avibacterium paragallinarum in the serum; or attack the immunized chickens with a virulent Apg strain and observe the survival and clinical symptoms of the chickens.

Description of drawings

Figure 1. PET28a plasmid map used in exemplary embodiments of the present invention.

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

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

Fig. 3. SDS-PAGE analysis figure 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 precipitates; 3. pET28a-p3 induces cleavage and precipitates; 4. pET28a-p4 induces cleavage and precipitates; 5. Precipitation after induced lysis of pET28a-p5.

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

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

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

Wherein, M is 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 antigenic protein of the present invention,

Wherein, M is 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 the outer membrane protein of Avibacterium paragallinarum 221 strain.

detailed description

The present invention will be described in further detail below in conjunction with specific examples. It should be declared that the following examples are only for explanation and illustration, 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 field, which can be obtained commercially or prepared by conventional methods in the field, and the specifications can be laboratory pure grade.

Example 1, Prediction of Antigenic Epitopes of the Outer Membrane Protein of Aviola paragallinarum

Outer membrane protein: its amino acid sequence is shown in Seq ID No.27, which is derived from the outer membrane protein coding gene of GenBank No. KJ867498.1 Avibacterium 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 and hydrophobicity of the outer membrane protein. 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 concentrated region for sequence splicing; Based on the prediction results and accumulated experimental experience, the selected epitope sequence was optimized multiple times, and part of the epitope region was intercepted, so that the spliced antigenic protein had strong immunogenicity and was suitable for protein expression and animal immunization. Finally, 16 core antigenic epitopes/epitope domains were determined, the amino acid sequences of which are shown in Seq ID No.1-16.

Several recombinant antigenic proteins are obtained from the combination of the above 16 antigenic epitopes/epitope domains.

Several recombinant antigenic proteins are also obtained through sequence splicing with selected other protein sequences such as connecting peptides or other antigenic epitopes or epitope domains.

Among them, five kinds of recombinant antigenic proteins of Avibacterium paragallinarum are respectively named p1, p2, p3, p4 and p5, and their amino acid sequences are shown in Seq ID No.17, 18, 19, 20 and 21.

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

1. Materials

1.1 Plasmids and strains

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

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

The strains of Aviobacterium paragallinum used in the present invention are Hp8, 221, 0083, BJ, 222, Modesto, and 668 strains, which are purchased from China Veterinary Drug Control Institute in Beijing, China, and belong to commercial strains.

1.2 Antigen protein of Avibacterium paragallinarum

The five recombinant antigen proteins p1, p2, p3, p4 and p5 of Avibacterium paragallinarum obtained by prediction, analysis and splicing by bioinformatics software in Example 1.

1.3 Main reagents and consumables

Sodium chloride, EDTA disodium salt, ethanol, methanol, Ponceau S, and trichloroacetic acid (TCA) are products of Sinopharm.

Tris Base (Tris-HCL), Dithiothreitol (DTT), Glycerin, Sodium Dodecyl Sulfate (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.

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

Proteinase K (20 mg/ml in stock solution, 1 mg/ml in use solution) was purchased from Shanghai Huashun Bioengineering Co., Ltd.

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

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 Baobio (Dalian) Co., Ltd. products.

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

Substrate solution preparation: Substrate solution A: 0.006% H ₂ O ₂ buffer solution; Substrate solution B: Take 14.2g Na ₂ HPO ₄ .12H ₂ O , 10.5g citric acid, dilute to 500mL with double distilled water into 0.1 phosphate citrate buffer (pH5.0), and then add benzidine diamine (TMB). When using, mix equal volumes of liquid A and liquid B, use within 5 minutes after mixing, and use immediately.

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

Paragallinia culture medium TSB, TSA, Freund's complete adjuvant and Freund's incomplete adjuvant were all purchased from Sigma.

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

PBS buffer solution: NaCl 8.0g, KCl 0.2g, KH ₂ PO ₄ 0.24g, Na ₂ HPO ₄ 12H ₂ O 3.628g, dissolved in 800ml distilled water, adjusted to pH 7.4 with hydrochloric acid, distilled water to 1000ml, Autoclave at 121°C for 20 minutes 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, weigh 2.9 g of glycine, 5.8 g of Tris base, 0.37 g of SDS, add 200 mL of methanol, and add ddH ₂ O to a total of 1000 mL.

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

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

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

1.4 Experimental animals: 6-week-old SPF chickens, purchased from Beijing Merial Verton 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, and the gene coding sequences were sent 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 sequence is Seq ID No. 28-37.

3. Construction of recombinant expression plasmids

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

Using the synthetic product obtained in step 2 as a template, the synthetic primers were used to carry out PCR,

PCR reaction system:

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

PCR reaction program: After reacting at 98°C for 1 minute, perform 30 cycles of "denaturation (95°C, 10 seconds), annealing (56°C, 15 seconds) and extension reaction (72°C, 120 seconds)", and then repair the extension Reaction (72°C, 7 minutes).

3.2 Digestion and recovery of PCR products

The above PCR product was digested with NcoI and XhoI,

Enzyme cutting system:

PCR product fragment digestion system (50ul):

The above system was placed in a constant temperature water bath at 37°C for 2 hours.

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

3.3 Digestion and recovery of expression vector

The expression vector PET28a was digested with Nco I and XhoI and the products were recovered. The method and steps were the same as 3.2 PCR product digestion and recovery.

3.4 The target fragment is connected to the carrier

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

The ligation system is: 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.

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

3.5 Transformation and screening of clones

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

3.6 Enzyme digestion and identification of recombinant plasmids

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

Bacteria were collected, and recombinant plasmids were extracted using a common plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the specific operation steps were carried out according to the kit instructions. Use NcoI+XhoI enzyme digestion to identify the recombinant plasmid, and the foreign fragment and vector fragment of the expected size appear after digestion, which is the correct recombinant plasmid.

4. Construction of recombinant expression strains

Take 100 μl of competent cell BL21 (DE ₃ ) and add it to a 1.5ml EP tube, and add 0.5 μl of the correctly identified recombinant plasmids pET28a-p1, pET28a-p2, pET28a-p3, pET28a-p4, pET28a-p5 to their respective EP tubes tube and mix well. After placing on ice for 30 minutes, heat shock at 42°C for 90 seconds, and ice bath for 3-5 minutes. This was spread on LB agar plates containing 25 μg/ml kanamycin. Place at 37°C for 1 hour, then turn the plate over, and incubate it upside down 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 E. coli

5.1 Induced expression of target gene: inoculate positive clones containing recombinant plasmids pET28a-p1, pET28a-p2, pET28a-p3, pET28a-p4, pET28a-p5 in 3 mL LB liquid medium containing 25 μg/ml kanamycin , Shaking culture 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, culture it with shaking at 37°C for about 3 hours, and when the OD ₆₀₀ reaches 0.6-1.0, add IPTG to the final concentration It was 0.8mmol/L, and the bacterial cells were collected after continuing to cultivate for 3h.

5.2 SDS-PAGE electrophoresis analysis of expression products

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

Table 1 SDS-PAGE gel preparation method

Preparation of 12% separating gel: After adding each component, mix quickly, add to the rubber plate, and add purified water on top. Then, prepare a 5% stacking gel: add the relevant components in the table and mix quickly, add to the top of the separation gel of the gel plate (first pour out the purified water on the separation gel), insert the sample comb after filling. After the concentrated gel has solidified, 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 into the sample well: the electrophoresis voltage is 200V, the current is in the range of 20mA-40mA, electrophoresis for 1h, Stop electrophoresis until bromophenol blue swims out of the bottom of the gel.

(3) Polyacrylamide gel staining and decolorization: unload the gel, stain with Coomassie Brilliant Blue R250 staining solution for 30 minutes, and decolorize with decolorization solution for 1 minute, and observe the results.

The results are shown in Figure 2. After the Escherichia coli containing the positive recombinant plasmid was induced to express at 37°C with 1mM IPTG, the protein target band appeared at the expected position by SDS-PAGE detection, and the expected protein size was consistent. Bacteria do not have this protein.

5.3 Preparation and purification of recombinant protein

1) Pick the correct positive clone identified in step 4, inoculate it in 3 mL of LB medium containing 25 μg/ml kanamycin, activate overnight at 37°C, and dilute 1:100 into LB medium containing 25 μg/ml kanamycin In the culture medium, shake at 37°C and 230r/min until the logarithmic growth phase (OD ₆₀₀ 0.6~1), add IPTG with a final concentration of 1mmol/L, and induce culture at 37°C and 230r/min for 6-8h . Take out 1mL at different times before induction and after induction, collect the bacteria by centrifugation at 5,000r/min for 10min, discard the supernatant, add 2×SDS loading buffer, boil for 10min, and use 12% sodium dodecyl sulfate-polymer Acrylamide gel electrophoresis (SDS-PAGE) was used to check, and Quantity One-4.3.1 (BIO-RAD) software was used to analyze the expression yield.

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

Refer to Fig. 3 and Fig. 4 for the results. The two figures are SDS-PAGE after ultrasonic lysis after the induced expression of the recombinant plasmid constructed by the present invention. Fig. 3 is the precipitate after lysis. It can be seen that most of p1, p2, p4 and p5 are in the precipitate; Fig. 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 in the lysed supernatant after induction is higher than that of the precipitate, the lysed supernatant can be used directly for purification, eliminating the step of inclusion body denaturation and renaturation, and using the principle of nickel agarose affinity chromatography to treat the cells after ultrasonic lysis Supernatant, the specific operation is as follows:

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

②The sample (lysate supernatant) is put on the column at a flow rate of 2ml/min, and the breakthrough solution is collected.

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

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

⑤ Elution Buffer elution, flow rate 2ml/min, collect eluate.

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

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

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

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

See Figure 5 for the results. After purification, relatively pure target proteins p1, p2, p3, p4 and p5 were obtained.

Embodiment 3, characteristic analysis of recombinant antigenic protein

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

Carry out SDS-PAGE electrophoresis with the above-mentioned purified recombinant antigenic protein p1, p2, p3, p4, p5 according to conventional methods, 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 relative direction of the membrane and 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 pieces of Whatman 3M filter paper in it. Then install the transfer electrophoresis tank according to the following steps: lay the base (anode) of the graphite electrode flat, and place 3 layers of 3M filter paper, nitrocellulose membrane, polyacrylamide gel and 3 layers of 3M filter paper in sequence. Thoroughly eliminate the air bubbles between the layers: buckle the upper cover of the transfer electrophoresis tank on the graphite electrode-transfer membrane glue complex; connect the power supply, and connect according to the parameters of 0.65mA/cm ² -1.0mA/cm ² Apply current and electrophoresis transfer for 0.5h-2h.

2) Ponceau staining: After the transfer, remove the NC membrane, rinse in deionized water for 2-3 times, transfer to Ponceau staining solution and stain 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% skimmed milk powder, and block it at room temperature for 2 hours; wash the membrane: discard the blocking solution, wash the NC membrane 3 times with 1×TBST, 5 minutes 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 hour. Membrane washing: Take out the NC membrane and wash the membrane 3 times with 1×TBST, 10min each time.

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

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

5) Color development: place the NC membrane in the newly prepared DAB color development 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) Vaccine preparation and immune test

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

Immunization method: give the recombinant antigen protein vaccine (inoculation amount is 0.5ml/ only) of the chest intramuscular injection of Freund's complete adjuvant emulsification to described 42 days old test SPF chicken; After 2 weeks, the chest intramuscular injection of Freund's incomplete adjuvant emulsification The recombinant protein vaccine (vaccination volume is 0.5ml/only); after an interval of 10 days, blood was collected from the wing vein, and the serum was collected.

Detection of serum-specific antibodies: ELISA method is used, and 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 hour, wash the plate once with washing solution, encapsulate it, and store it at -20°C save.

One week after the booster immunization, the chickens were blood-separated to separate the serum, and 100 μl was added to the enzyme-linked plate after doubling dilution, and an adjuvant control and a blank control were set at the same time.

React at 37°C for 30 minutes.

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 minutes.

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

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

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

Embodiment 4, recombinant antigenic protein is to SPF chicken immune efficacy test

1. Preparation of recombinant antigenic protein vaccine and immunization method

1.1 Preparation of recombinant antigenic protein vaccine

Recombinant antigenic protein p1, p2, p3, p4, p5 were mixed with MONTANIDE ^TM ISA 71VG adjuvant (SEPPIC company product) according to 3:7 weight ratio (30% by weight of antigen, 70% by weight of adjuvant) emulsification (operation For the method, please refer to the introduction of the product use method of SEPPIC Company), so that the final concentration of antigenic protein in the vaccine is 20 μg/ml (the threat of chicken infectious rhinitis epidemic 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 are named vp1, vp2, vp3, vp4, vp5 in sequence.

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

1.2 Immunization method

210 42-day-old SPF chickens were divided into subunit vaccine group, conventional inactivated vaccine group and PBS control group. The vaccine group was further divided into five groups of vp1, vp2, vp3, vp4 and vp5, with 30 rats in each group; the conventional trivalent inactivated vaccine group and the PBS control group had 30 rats each. The test chickens in each subunit vaccine group were immunized with vp1, vp2, vp3, vp4, and vp5 respectively, the test chickens in the conventional trivalent inactivated vaccine group were immunized with conventional inactivated vaccines, and the test chickens in the control group were vaccinated with PBS. Chest intramuscular injection, 0.5ml/monkey; 4 weeks later, the same dose, the same way to boost immunization. During this period, blood was collected from the wing vein every two weeks for the monitoring of serum-specific antibodies.

The detection of serum antibodies adopts the ELISA method, and the steps are the same as in Example 3.

1.3 Immune Chicken Challenge Test

The challenge test was carried out on all the test chickens 4 weeks after the booster immunization. Each test group was challenged with A-type (Hp8 strain), B-type (BJ strain) and C-type (668 strains) Apg respectively, 10 in each group, inoculated in the infraorbital sinus, and the dosage was followed by A-type (Hp8 strain). ) 1×10 ⁶ CFU, type B (strain BJ) 5×10 ⁵ CFU, type C (strain 668) 1×10 ⁶ CFU. Observe continuously for a week, and record the clinical morbidity of the test chickens, including runny nose, swollen eyelids, and epiphora. 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 bacterial strain challenge virus

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

*The number of experimental chickens that obtained immune protection in the subunit vaccine immunization group was significantly different from that in the PBS control group.

Table 2 lists the challenge experiment results of A-type Hp8 strain, B-type BJ strain and C-type 668 strain. During the experiment, respectively use 5 kinds of subunit vaccine immunization test chickens prepared by the preferred 5 recombinant proteins of the present invention, and use A, B, and C three serotypes of Apg virulent attack after 2 immunizations, all tests of the non-immune control group Chickens are getting sick. In the subunit vaccine immunization group, only a few test 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 experimental chickens in the non-immune control group showed severe symptoms of rhinitis.

The results of the challenge experiments using the Apg of A-type 221 strains, 0083 strains, B-type 222 strains and C-type Modesto strains were similar to those of Hp8 strains, BJ strains and 668 strains. The protection rate was 100%, and the protection rate of Vp2 and Vp3 experimental groups was 90%-100%. It can be seen that the vaccine prepared by adopting the recombinant antigen protein provided by the invention has remarkable immune 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 significant difference compared with the non-immune group. Since the purified protein does not contain lipopolysaccharide and other components carried by the whole bacteria, the subunit vaccine prepared by the present invention has no side effects, while the whole bacteria inactivated vaccine has side effects such as transient loss of appetite and listlessness. In terms of production cost, subunit vaccines are also lower than conventional inactivated vaccines due to the characteristics of easy cultivation and induction, cheap medium, and easy purification.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered Be the protection scope of the present invention.

SEQUENCE LISTING

<110> Beijing Academy of Agriculture and Forestry Sciences

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

<130> P170303/NLK

<160> 37

<170> PatentIn version 3.3

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<213> Avibacterium paragallinarum

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Ile Gly Gly Thr Gly Gln Asp Thr Ile

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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

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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 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

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<223> Amino Acid Sequence of Recombinant Antigen Protein p1 of Avibacterium 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 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

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<223> Amino Acid Sequence of Recombinant Antigen Protein p2 of Avibacterium 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

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<213> Artificial Sequence

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<223> Amino Acid Sequence of Recombinant Antigen Protein p3 of Avibacterium paragallinarum

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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 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 Avibacterium 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 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 Antigen Protein p5 of Avibacterium 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 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 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> Gene Coding Sequence of Recombinant Antigen Protein p1 of Avibacterium 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> Gene Coding Sequence of Recombinant Antigen Protein p2 of Avibacterium 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 ctaacctggtgaagacggt 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 gacctcttc 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 Antigen Protein p3 of Avibacterium 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 caacaaaaaaaaaaccttcg 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

tctacccacca 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 Antigen Protein p4 of Avibacterium 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

accccagccga ccctgccgga a 1461

<210> 26

<211> 1725

<212>DNA

<213> Artificial Sequence

<220>

<223> Gene Coding Sequence of Avibacterium paragallinarum Recombinant Antigen Protein p5

<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 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 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 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 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 used to amplify 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 used to amplify 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 used to amplify 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 used to amplify 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 used to amplify 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 used to amplify 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 used to amplify the gene encoding the antigenic protein p4

<400> 34

gcccatggaa accgtctatc gctctgggt 29

<210> 35

<211> 29

<212>DNA

<213> Artificial Sequence

<220>

<223> Downstream primers used to amplify 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 used to amplify the gene encoding the antigenic protein p5

<400> 36

gcccatggtt agctactgat ggtaccat 29

<210> 37

<211> 29

<212>DNA

<213> Artificial Sequence

<220>

<223> Downstream primers used to amplify the gene encoding the antigenic protein p5

<400> 37

cgctcgagtt ccggcagggt cggctgggt 29

Claims (10)

1. a kind of secondary poultry bacillus immune protective antigen albumen, it is characterised in that including amino acid sequence such as Seq ID No.1 16 epitopes or epitope domain shown in~16.

2. secondary poultry bacillus immune protective antigen albumen according to claim 1, it is characterised in that its amino acid sequence Selected from Seq ID No.17, one of 18,19 and 20.

3. encode the gene of the secondary poultry bacillus immune protective antigen albumen described in claim 1.

4. gene according to claim 3, it is characterised in that its nucleotide sequence is selected from Seq ID No.22,23,24 Hes One of 25.

5. a kind of construct, it is characterised in that include the gene described in claim 3 or 4.

6. a kind of recombinant cell, it is characterised in that obtained as the construct transformed acceptor cell described in claim 5；Optionally Ground, the recipient cell is selected from bacterium, yeast, zooblast and plant cell.

7. a kind of infectious coryza of chicken subunit vaccine, it is characterised in that its immunoactive component includes the institute of claim 1 or 2 The secondary poultry bacillus immune protective antigen albumen stated.

8. infectious coryza of chicken subunit vaccine according to claim 7, it is characterised in that the secondary poultry bacillus is immunized The final concentration of 5-100 μ g/ml of protective antigens albumen.

9. a kind of preparation method of infectious coryza of chicken subunit vaccine, it is characterised in that comprise the following steps：

(1) expression vector of the secondary poultry bacillus immune protective antigen albumen described in claim 1 or 2 is built；

(2) matching protein expression system is converted with the expression vector, recombinant cell is obtained；

(3) recombinant cell is cultivated, induced expression obtains secondary poultry bacillus immune protective antigen albumen and purifying protein；

(4) secondary poultry bacillus immune protective antigen albumen after purification is mixed with auxiliary reagent, adjusts the antigen protein Final concentration of 5-100 μ g/ml, obtain infectious coryza of chicken subunit vaccine.

10. method according to claim 9, it is characterised in that the expression vector is selected from pET, pQE and pGEX series Carrier；The protein expression system is selected from e. coli bl21, DH5 ɑ, Top10 and JM109 bacterial strains；The auxiliary reagent includes Immunopotentiator, stabilizer, preservative, salting liquid and/or distilled water.