KR20140093078A - Recombinant protein vaccine for preventing Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae infection diseases - Google Patents

Abstract

Provided are: a fusion protein which includes an N terminal domain of an actinobacillus pleuropneumoniae ApxIIIA protein and a C terminal domain of a mycoplasma hyopneumoniae ciliary absorption protein; a polynucleotide which codes the same; a composition which includes the same; and a method for preventing or treating at least one infection between an actinobacillus pleuropneumoniae and a mycoplasma hyopneumoniae by using the composition.

Description

[0001] The present invention relates to recombinant protein vaccines for the prevention of infectious diseases of Mycoplasma pneumoniae and Mycoplasma hyperonymonia, and to a recombinant protein vaccine for preventing Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae infection diseases.

A protein comprising a Mycoplasma highonimonia P97 protein or fragment thereof, a region comprising a Pleuropneumoniae ApxIIIA protein or fragment thereof, and a region containing a Mycoplasma highonimonia P97 protein or a fragment thereof, A composition comprising a fusion protein comprising a fragment, a polynucleotide encoding the fusion protein, a composition comprising the protein or the fusion protein, a kit comprising the protein, and a composition comprising the fusion protein, , ≪ / RTI > and M. hyunneumonia.

Actinobacillus pleuropneumoniae is a causative organism of swine flu pneumonia, which is a contagious and potentially fatal respiratory disease of swine. The toxicity of pleural pneumoniae is associated with several factors including pleural pneumonia toxin (Apx), lipopolysaccharide, outer membrane protein, and adsorption factors. Of these, Apx is the primary determinant of the toxicity of pleuropneumoniae. ApxI, ApxII, ApxIII, and ApxIX are known as Apx proteins. The ApxIII protein may include ApxIIIA, ApxIIIB, and ApxIIID.

Mycoplasma hyopneumoniae is a causative organism of porcine enzootic pneumonia (PEP), a chronic nonfatal disease that affects pigs of all ages. Primary Miko Plasma infected pigs tend to be a potentially fatal secondary infection by PRRS virus, Pasteurella spp., And Pleuropneumoniae.

The P97 adsorbed protein is highly conserved among other strains of M. hyunneumonia. P97 is essential for M. hyunmoniosis to adsorb to ciliated respiratory endothelial cells, and in the absence of functional P97, it is associated with non-toxicity in some strains of this pathogen. The C-terminus of P97 contains two repeat regions, designated R1 and R2, which play an important role in adsorption.

Despite this prior art, there is a need for materials and / or methods that are effective in preventing or treating one or more infections of Pleurotus pneumoniae and M. hyunneumonia.

One aspect is to provide a fusion protein comprising a region comprising a Pleuropneumoniae ApxIIIA protein or fragment thereof and a region comprising M. hyunmonioca ciliary protein P97 or a fragment thereof.

Another aspect is to provide a polynucleotide encoding said fusion protein.

Another aspect is to provide a composition for the prevention or treatment of infection of any one or more of mammalian pleural pneumococci and M. hyunneumoniae comprising the fusion protein as an active ingredient.

Another aspect is to provide a method for preventing or treating infectious diseases of any one or more of mammalian pleural pneumococci and M. hyunneumonia using the composition.

Another aspect is to provide a protein comprising Pleuropneumoniae ApxIIIA protein or fragment thereof.

Another aspect is to provide a protein comprising M. hyunmonioca ciliad protein P97 or a fragment thereof.

Another aspect is a protein comprising the N-terminal region of the Pleuropneumonitus ApxIIIA protein, including a protein comprising the Pleuropneumonitus ApxIIIA protein or fragment thereof and a protein comprising the M. hyunmonioca ciliary adsorptive protein P97 or fragment thereof, The present invention provides a composition for detecting a protein comprising a C-terminal region of plasma high hyunmonia ciliad protein P97, or a fusion protein thereof.

Another aspect is a protein comprising the N-terminal region of the Pleuropneumonitus ApxIIIA protein, including a protein comprising the Pleuropneumonitus ApxIIIA protein or fragment thereof and a protein comprising the M. hyunmonioca ciliary adsorptive protein P97 or fragment thereof, And a kit for detecting a protein comprising the C-terminal region of the plasmid high hyungmonia ciliad protein P97, or a fusion protein thereof.

One aspect provides a fusion protein comprising a region comprising a Pleuropneumoniae ApxIIIA protein or fragment thereof and a region comprising M. hyunmonioca ciliad protein P97 or a fragment thereof.

The fragment of the pleuropneumonitus ApxIIIA protein may be an N-terminal region. The fragment of M. hyunmonioca ciliad protein P97 may be a C-terminal region.

The Pleuropneumonitus ApxIIIA protein may have the amino acid sequence of SEQ ID NO: 1. The N-terminal region may comprise amino acid residues 21 to 131 of the amino acid sequence of SEQ ID NO: 1 (amino acid sequence of SEQ ID NO: 2). The N-terminal region may be the amino acid sequence of SEQ ID NO: 2. The fusion protein may include the amino acid sequence of SEQ ID NO: 1 in the amino acid sequence 21 to 131 as a sequence containing the N-terminal region of the Pleuropneumonitus ApxIIIA protein. The N-terminal region may be, for example, a continuous amino acid sequence selected from the amino acid sequence of SEQ ID NO: 1, including the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: Wherein the N-terminal region comprises a sequence of amino acids 21 to 131 of the amino acid sequence of SEQ ID NO: 1 and a contiguous amino acid sequence selected from the amino acid sequence of SEQ ID NO: 1 and having a length of from 111 to 1049 amino acids, 111 to 800 amino acids, 111 to 600 amino acids, 111 to 400 amino acids, 111 to 300 amino acids, 111 to 200 amino acids, or 111 to 150 amino acids.

The M. hyunmonioca ciliad protein P97 may have the amino acid sequence of SEQ ID NO: 3. The C-terminal region of the M. hyunmonioca ciliad protein P97 comprises an R1 repeat sequence (amino acid residues 814 to 888 of the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4) and an R2 repeat sequence (amino acid sequence of SEQ ID NO: 3 Amino acid residues 981 to 1020 of SEQ ID NO: 5). The C-terminal region may have the amino acid residues 768 to 1085 (amino acid sequence of SEQ ID NO: 6) or amino acid residues 768 to 1108 of the amino acid sequence of SEQ ID NO: 3. The C-terminal region of the M. hyunmonioca ciliary adsorptive protein P97 may comprise a contiguous amino acid sequence selected from the amino acid sequence of SEQ ID NO: 3 and comprising amino acid residues 814 to 1020 of SEQ ID NO: The C-terminal region comprises an amino acid sequence from amino acid 814 to 1020 of the amino acid sequence of SEQ ID NO: 3, a continuous amino acid sequence selected from the amino acid sequence of SEQ ID NO: 3 and having a length of from 207 to 1108 amino acids, for example, from 207 to 1100 amino acids , 207 to 1000 amino acids, 207 to 800 amino acids, 207 to 600 amino acids, 207 to 400 amino acids, 207 to 300 amino acids, or 207 to 250 amino acids.

The fusion protein may comprise M. hyunmonioca ciliad protein P97 or a fragment thereof, for example, the N-terminal region of its C-terminal region is linked to the Pleuropneumonitus ApxIIIA protein or a fragment thereof, , Or a fragment thereof, for example, the C-terminal side of its C-terminal region may be linked to the Pleuropneumonitus ApxIIIA protein or a fragment thereof, e.g., the N-terminal region thereof.

The fusion protein may be one that is linked by the linker between the Pleuropneumonitus ApxIIIA protein or a fragment thereof, e.g., the N-terminal region thereof and the M. hyunmonioca ciliary adsorptive protein P97, or a fragment thereof, e.g., . The linker may be a peptide linker. The term "peptide linker" includes those in which the amino acid is linked to a peptide bond. The peptide linker may be a peptide linker having 50% or more of glycine in the total amino acids. The linker may be one having an amino acid sequence of GS, GSSG (SEQ ID NO: 7), (GGGGS) ₂ (SEQ ID NO: 8), or a combination thereof. The ApxIIIA protein or a fragment thereof, e.g., an N-terminal region thereof and P97 or a fragment thereof, such as a C-terminal region thereof, may optionally be mediated by a linker, such that the N-terminus of another partner is linked to the C- It may be connected in the opposite way. For example, the fusion protein comprises the linker at the C-terminus of the N-terminal region of the ApxIIIA protein comprising the amino acid sequence of SEQ ID NO: 2, wherein the C-terminal region of P97 comprising the amino acid sequence of SEQ ID NO: 6 is linked Lt; / RTI > The fusion protein may have the amino acid sequence of SEQ ID NO: 9. In SEQ ID NO: 9, the 1-21 amino acid residue is included for purification as containing the polyhistidine region as the pET-28a (+) vector derived sequence. The 22-132 amino acid residue is the sequence of the N-terminal region of ApxIIIA, the 133-134 amino acid residue is the linker sequence and the 135-452 amino acid residue is the C-terminal region of P97. The fusion protein may also be a sequence of amino acid residues 22-452 of SEQ ID NO: 9.

Thus, the fusion protein may contain a sequence necessary for protein separation at its N-terminus, C-terminus, or both termini. The fusion protein may comprise a polyhistidine sequence at the N-terminus, at the C-terminus, or at both termini. The polyhistidine sequence may be 6x His. Such a sequence necessary for protein separation includes not only directly involved in the separation such as the polyhistidine sequence but also a sequence indirectly involved in separation such as a sequence which helps the sequence directly involved in separation to be exposed to the outside.

Another aspect provides a polynucleotide encoding the amino acid sequence of the fusion protein. The above fusion proteins are as described above.

The polynucleotide may be one in which one or more of the codon sequences has been modified to a codon suitable for expression of the host cell. The host cell may be E. coli, yeast or a combination thereof. The polynucleotide may encode the amino acid sequence 22-452 of SEQ ID NO: 9 or SEQ ID NO: 9. For example, the polynucleotide may have the nucleotide sequence of SEQ ID NO: 20.

The polynucleotide may be operably linked to a regulatory sequence suitable for expression of the host cell. The regulatory sequence may be a promoter, an operator, a polyadenylate, a ribosome binding sequence, or a combination thereof.

Another aspect provides a vector comprising said polynucleotide. The vector may comprise a replication origin that allows replication in the host cell. The vector may be a plasmid vector, a viral vector, or a combination thereof.

Another aspect provides a composition for the prophylaxis or treatment of infection of any one or more of mammalian pleural pneumococci and M. hyunmoniasia comprising the above-mentioned fusion protein as an active ingredient.

The composition may further comprise a pharmaceutically acceptable carrier or adjuvant. "Active ingredient" means a component required for alleviating or eliminating the symptom to be treated or prevented, and a required amount thereof, and those skilled in the art can appropriately select it.

The carrier is used as a meaning including a diluent, a buffer, a preservative and the like. The carrier may contain one or more components selected from the group consisting of excipients, disintegrants, binders and glidants known in the art. In addition, the adenovirus is a substance used for enhancing the immunogenicity of an antigen, and may include, for example, a complete or incomplete adenovirus.

The composition of the present invention may be in any form known to those skilled in the art, for example, in the form of liquid solutions and injections, but is not limited thereto. For liquids or injections, it may contain 10-40% propylene glycol if necessary, and sodium chloride in an amount sufficient to prevent hemolysis (e.g., about 1%). The liquid or injectable solution may contain any diluents or buffers known in the art.

The mammal may be selected from the group consisting of humans, cows, pigs, goats, and sheep. For example, the mammal is a pig.

Another aspect provides a method of preventing or treating an infectious disease of any one or more of mammalian pleural pneumococci and M. hyunmoniasis, comprising the step of administering the above composition to a mammal other than a human.

In the method of the present invention, the mammal may be selected from bovine, porcine, dog, chlorine and sheep. For example, the mammal is a pig.

In the method of the present invention, the infectious disease may be any symptom caused by infection by one or more of the following: pleural pneumonia and M. hyunmonioma, for example, .

In the methods of the invention, the administration can be by any of the administration means known in the art. For example, the administration can be administered directly into an individual by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The administration may be systemically or locally administered.

In the method of the present invention, the composition of the present invention may be administered in a therapeutically or prophylactically effective amount. The "therapeutically or prophylactically effective amount" can be appropriately selected in consideration of the severity of symptoms, sex, age, weight and the like of a person skilled in the art. The therapeutically or prophylactically effective amount may be, for example, 1 pg to 1 g of the fusion protein per kg of subject to be administered.

Another aspect provides a protein comprising Pleuropneumoniae ApxIIIA protein or fragment thereof, e.g., the N-terminal region thereof. Proteins comprising the Plefiomyces pneumoniae ApxIIIA protein or fragments thereof are as described above.

Another aspect provides a protein comprising mycoplasma high hygromycin ciliary protein P97 or a fragment thereof, e. G., Its C-terminal region. The proteins containing the mycoplasma hyperonymonia ciliary adsorptive protein P97 or fragments thereof are as described above.

Another aspect is a protein comprising the N-terminal region of Pleuropneumonitus ApxIIIA protein, comprising a protein comprising Pleuropneumoniae ApxIIIA protein or fragment thereof and a protein comprising mycoplasma hyperonymonia ciliary adsorptive protein P97 or fragment thereof, There is provided a composition for detecting an antibody that binds to a protein comprising a C-terminal region of plasma high-hyunmonia ciliad protein P97, or a fusion protein of these proteins.

Another aspect is a protein comprising the N-terminal region of Pleuropneumonitus ApxIIIA protein, comprising a protein comprising Pleuropneumoniae ApxIIIA protein or fragment thereof and a protein comprising mycoplasma hyperonymonia ciliary adsorptive protein P97 or fragment thereof, There is provided a kit for detecting an antibody that binds to a protein comprising the C terminal region of plasma high hyunmonia ciliad protein P97, or a fusion protein of these proteins.

For example, a protein comprising the N-terminal region of Pleuropneumonitus ApxIIIA protein or a fragment thereof, a mycoplasma hyperonymonium ciliate adsorption protein P97 or a fragment thereof, for example, a protein comprising the C-terminal region thereof, and Pleuropneumoniae ApxIIIA protein Or a fragment thereof, for example, a fusion protein comprising a N-terminal region thereof and a mycoplasma high-mucilage ciliary protein P97 or a fragment thereof, for example, a C-terminal region thereof, is as described above.

The detection may be detection by ELISA. Such detection can be accomplished, for example, by detecting the ApxIIIA protein or a fragment thereof, e. G., A protein comprising its N-terminal region, and a mycoplasma hyperonymonia ciliate protein P97 or fragment thereof, e. Are coated on a solid substrate, respectively, and the pleuropneumoniae ApxIIIA protein or a fragment thereof, for example a protein comprising its N-terminal region, mycoplasma hyperonymonia ciliary adsorptive protein P97 or a fragment thereof, A sample containing an antibody that binds to a protein of interest, or an antibody that binds to a fusion protein of these proteins, is added to the solid substrate to bind to the protein, respectively, and the bound conjugate is labeled with a labeled antibody specific material (e.g., HRP- 2 antibody), and then confirming the result.

According to one aspect, the fusion protein comprising the N-terminal region of the pleuropneumoniae ApxIIIA protein and the C-terminal region of the M. hyunomonias ciliary adsorptive protein P97 has both immunogenicity of ApxIIIA and P97 protein.

According to another aspect, the polynucleotide encoding the fusion protein can encode the fusion protein.

The composition comprising the fusion protein according to another aspect as an active ingredient can be used for effectively preventing or treating an infection of any one or more of mammalian pleural pneumococci and M. hyunmoniota.

According to another aspect of the method for preventing or treating infection of any one or more of mammalian pleural pneumococci and M. hyunneumonia according to another aspect, infection of any one or more of mammalian pleural pneumococci and M. hyunneumonia Can be efficiently prevented or treated.

According to another aspect, a protein comprising the N-terminal region of the Plefiomyosus ApxIIIA protein, or a protein comprising the C-terminal region of the M. hyunomonia ciliad protein P97, comprises the N-terminal region of the Pleuropneumonitus ApxIIIA protein Protein, a protein comprising the C-terminal region of M. hyunmonioca ciliad protein P97, or an antibody that binds to a fusion protein thereof.

Comprising an N-terminal region of Pleuropneumonitus ApxIIIA protein according to another aspect and a protein comprising a C-terminal region of mycoplasma hyperonymonia ciliary adsorptive protein P97. According to the composition or kit for detecting the protein comprising the C-terminal region of the myoplasma hypercoagulinaemia ciliate protein P97, or the antibody binding to the fusion protein thereof, the N-terminal region of the Pleuropneumonitus ApxIIIA protein , A protein comprising the C-terminal region of mycoplasma hyperonymonia ciliated protein P97, or an antibody that binds to the fusion protein thereof can be efficiently detected.

Figure 1 shows the expression and purification of ApxN, P97C and Ap97 recombinant proteins.
Figure 2 shows the serum titer of total IgG, IgG1, and IgG2a isotype antibodies to ApxN and P97C in mice immunized with ApxN, P97C and Ap97 vaccines.
Figure 3 shows the cytokine profile of spleen cells from immunized mice.
Figure 4 shows the recognition of ApxIII of Pleuropneumoniae by antiserum from mice immunized with Ap97 or ApxN recombinant protein.
Figure 5 shows recognition of M. hyunneumonia P97 by antiserum from mice immunized with Ap97 or P97C recombinant protein.
Figure 6 shows the recognition of M. hyunneumonia P97 and pleuropneumoniae ApxIII by sera isolated from pigs immunized by the Ap97 vaccine.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Materials and methods

Unless otherwise stated, the following examples used the following materials and methods.

(1) Reagent

Except as otherwise described, all of the reagents used in the following examples used high purity products sold by Sigma-Aldrich (St. Louis, Mo., USA).

(2) Culture of the strain

Pleuropneumoniae Strain 1536 (serotype 2) and M. hyunneumonia strain J were purchased from the American Type Culture Collection (ATCC) (Manassas, Va.). Mycoplasma yeast pneumoniae wild type isolate was isolated from porcine papillary tissues suffering from enzootic pneumonia.

The pleural fluid pneumoniae strains contained 10 μg / ml of nicotinamide dinucleotide (NAD), 260 μg / ml of L-cysteine hydrochloride, 0.6 mM of L-glutamine, 1 mg / ml of dextrose, 10 μg / ml of L-cysteine dihydrochloride, (BD Bioscience, Sparks, Md.) Supplemented with 0.1% (v / v) 80% ethanol at 37 ° C. To obtain whole cell lysates, pleural pneumococci were cultured to mid-log level and harvested by centrifugation at 3,000xg for 10 min. The cell pellet was washed with phosphate buffered saline (PBS, pH 7.4), disrupted with disruption buffer (20 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1 M EDTA, 1% SDS) Respectively.

 Proteins secreted by Pleuropneumoniae in the culture medium were precipitated by adding 0.02% (v / v) deoxycholic acid and 15% (v / v) trichloracetic acid to the supernatant. After overnight incubation at 4 ° C, the precipitate was collected by centrifugation, and the resulting pellet was washed with cold ethanol and centrifuged. After removing the supernatant by inhalation, the pellet was dried and dissolved in PBS.

Mycoplasma high-ornithonia strains were cultured in ATCC medium 1699 at 37 ° C with continuous agitation to medium-log step (medium pH range, 6.8-7.2). Cell membranes were separated by osmotic lysis and sonication. Briefly, cell pellets were washed with PBS, resuspended in deionized water, and incubated for 30 min at 37 < 0 > C. The suspension was destroyed by sonication (30-2-s burst at 200-300 W with 2-s cooler between each burst) and then centrifuged at 10,000xg for 20 minutes at 37 < 0 > C. The supernatant was centrifuged at 100,000xg for 1 hour at 4 ° C to recover the cell membrane. The cell membrane pellet was dissolved in resuspension buffer (10 mM potassium phosphate, 50 mM NaCl, 20% glycerol (v / v)) and stored at -70 C until use. The ATCC medium 1699 was prepared as follows. 7.5 g of heart infusion broth (BD238400) and 660 ml of distilled water were mixed, adjusted to pH 7.4, and then sterilized at 121 ° C. The following filter-shaped components were aseptically added: NaHCO 3 or phenol red (Hank's Balanced Salt Solution) (1.4 g of CaCl2, 4.0 g of KCl, 0.6 g of KH2PO4, 1.0 g of MgCl2x6H2O, 1.0 g of MgSO4x7H2O, 80.0 g of NaCl, 0.9 g of Na2PO4x7H2O, 10.0 g of glucose, , 100.0 ml of 5% lactalbumin hydrolyzate, TC (BD259962) and 1 ml of yeast extract solution (GIBCO 18180) were added thereto, ) 20.0 ml.

Escherichia coli DH5α and BL21 (DE3) strains (Stratagene, La Jolla, Calif.) Were used for plasmid DNA amplification and recombinant protein production, respectively. Luria-Bertani (LB) Lt; / RTI >

(3) Production of protein expression vector

In order to prepare a pET-ApxN plasmid vector, a fragment of apxIIIA gene was subjected to PCR using genomic DNA of Pleuropneumoniae as a template and ApxN-F of SEQ ID NO: 10 and ApxN-R of SEQ ID NO: 11 as primers to obtain ApxIIIA (GenBank Accession No. L12145) was amplified. Table 1 shows the primers used for amplification. The resulting PCR product (SEQ ID NO: 17) was cloned into pGEM-T Easy vector (Promega, Madison, Wis.). Next, this TA-cloned vector was digested with BamHI and XhoI and the insert was ligated to linearized pET28a (+) (Novagen, Cambridge, UK) with an N-terminal histidine tag to generate pET-ApxN A plasmid vector was prepared. Here, ApxN represents a protein having an amino acid sequence comprising the N-terminal region of ApxIII of SEQ ID NO: 16. In SEQ ID NO: 16, the 1-21 amino acid residue is a polyhistidine containing sequence derived from the pET28a (+) vector, and the 22-139 amino acid residue is the N-terminal sequence of ApxIIIA. The polynucleotide encoding the ApxN protein has the nucleotide sequence of SEQ ID NO:

primer SEQ ID NO: Target sequence
accession number Target gene Amplification product
Length (bp) Remarks ApxN-F 10 L12145 apxIIIA 315 BamHI site included ApxN-R 11 L12145 apxIIIA 315 XhoI site included P97C-F 12 U50901 P97 954 BamHI site included P97C-R 13 U50901 P97 954 XhoI site included Ap97-F 14 L12145 apxIIIA 333 With NdeI site Ap97-R 15 L12145 apxIIIA 333 BamHI site included

The pET-P97C vector was constructed to express the C-terminal region of P97 (GenBank Accession No. U50901). First, a DNA fragment in which the nucleotide sequence of P97 was modified and the 5'-TGA-3 'codon was replaced with 5'-TGG-3' was synthesized so as to produce a codon preferred by E. coli without changing the amino acid sequence. 5'-TGA-3 'is read as tryptophan in mycoplasma, but causes early termination of translation in other eubacteria such as E. coli. This fragment was used as a template and the 954 bp product containing the R1 and R2 regions of P97 was amplified by PCR using P97C-F of SEQ ID NO: 12 and P97C-R of SEQ ID NO: 13 as primers. The resulting PCR product (SEQ ID NO: 19) was cloned into pGEM-T Easy vector (Promega, Madison, Wis.). Next, this TA-cloned vector was cut with BamHI and XhoI and the insert was ligated to linearized pET28a (+) (Novagen, Cambridge, UK) with an N-terminal histidine tag to generate pET-P97C A plasmid vector was prepared. Here, P97C represents a protein having an amino acid sequence comprising the C-terminal region of P97 of SEQ ID NO: 18. In SEQ ID NO: 18, the 1-21 amino acid residue is a polyhistidine containing sequence derived from the pET28a (+) vector, and the 22-339 amino acid residue is the C-terminal sequence of P97. The C-terminal sequence comprises the R1 region and the R2 region. The polynucleotide encoding the P97C protein has the nucleotide sequence of SEQ ID NO: 19. The nucleotide sequence of SEQ ID NO: 19 was used to express P97C in E. coli. In SEQ ID NO: 19, the 192-366 residue codes for the R1 region and the 703-822 residue codes for the R2 region. The guanine nucleotide of the 924 residue is substituted for the adenine nucleotide to prevent premature translation termination.

The pET-Ap97 vector was used to express the chimeric protein Ap97, which contains ApxN and P97C at the N-terminus and C-terminus, respectively. The nucleotide sequence located at the N-terminal region of the ApxIIIA gene (GenBank Accession No. L12145) was amplified by PCR using Ap97-F of SEQ ID NO: 14 and Ap97-R of SEQ ID NO: 15 as primers, Respectively. The resulting PCR products were cloned into pGEM-T Easy vector (Promega, Madison, Wis.). The Gly-Ser peptide linker in the chimeric protein is encoded by 5'-GGATCC-3 'in the Ap97-R primer. Next, the TA-cloned vector was digested with NdeI and BamHI, and the insert was ligated to the linearized pET-P97C to prepare a pET-Ap97 plasmid vector. The 5'-TAG-3 nucleotide sequence for the leucine residue in the Ap97-F and Ap97-R primers was replaced with 5'-CAG-3 '.

The fusion protein Ap97 has the amino acid sequence of SEQ ID NO: 9. In SEQ ID NO: 9, the 1-21 amino acid residue is included for purification as containing the polyhistidine region as the pET-28a (+) vector derived sequence. The 22-132 amino acid residue is the sequence at the N-terminal region of ApxIIIA, the 133-134 amino acid residue is the linker sequence and the 135-454 amino acid residue is the sequence at the C-terminal region of P97, including the R1 and R2 regions. And a nucleotide sequence of polynucleotide SEQ ID NO: 20 which encodes the fusion protein Ap97.

(4) protein expression and purification

Escherichia coli BL21 (DE3) was transformed with each pET-ApxN, pET-P97C, and pET-Ap97 vectors designed to express ApxN, P97C and Ap97 using a heat shock protocol. Selected colonies were cultured in LB medium supplemented with 100 [mu] g / ml kanamycin at 37 [deg.] C with stirring.

The overnight cultures (diluted 1: 100) were incubated with stirring at 37 ° C to the mid-log phase indicated by A600 of 0.5-0.7, followed by incubation with a final 1 mM concentration of isopropyl- beta -thiogalactopyranose After adding isopropyl-β-thiogalactopyranoside (IPTG), the protein expression was induced by incubation at 37 ° C. for 4 hours.

Both the P97C and Ap97 proteins were purified from the E. coli inclusion body. Cells were washed twice with ice-cold PBS (pH 7.4) and then resuspended in buffer A (100 mM sodium phosphate, 10 mM Tris-HCl, 100 g / ml lysozyme and 5 mM phenylmethane sulfonyl fluoride (PMSF) And crushed for 30 minutes. The lysate was centrifuged at 10,000 × g for 20 min and the resulting pellet was resuspended in buffer B (8 M urea, 10 mM Tris-HCl, 100 mM sodium phosphate and 5 mM PMSF, pH 8.0) for 1 hour at room temperature . Next, the solubilized extract was loaded on a Ni-nitrilotriacetic acid (NTA) column (Qiagen, Chatsworth, Calif.). The column was washed with buffer C (8M urea, 10mM Tris-HCl, 100mM sodium phosphate and 5mM PMSF; pH 6.3) and buffer D (8M urea, 10mM Tris-HCl, 100mM sodium phosphate and 5mM PMSF; pH 5.9) and buffer E (8M urea, 10 mM Tris-HCl, 100 mM sodium phosphate and 5 mM PMSF; pH 4.5). Each protein contained polyhistidine at its N-terminus and was purified using this.

To purify ApxN, the cells were washed with ice-cold PBS and incubated for 30 min on ice with buffer F (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, 1 mg / ml lysozyme and 5 mM PMSF; pH 8.0) And sonicated on ice using a 200-300 W to 6 10-s burst with a 10-s cooler between bursts. Cell debris was pelleted by centrifugation at 10,000 xg for 20 minutes and the resulting supernatant was applied to a Ni-NTA column. The column was washed twice with buffer G (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole and 5 mM PMSF, pH 8.0). The protein bound to the Ni-NTA column was eluted with buffer H (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, and 5 mM PMSF, pH 8.0) according to the manufacturer's protocol.

The purified protein was dialyzed at 4 ° C for 36 hours with serially diluted urea or imidazole in phosphate-buffered saline (PBS) containing 150 mM sodium chloride and 5% glycerol. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL).

(5) Mouse immunization experiment

All procedures for handling the mice used in this example were reviewed and approved by the Laboratory Animal Resource Institute of Seoul National University and conducted in an ethical and human manner under veterinary supervision.

Six to eight week old female Balb / C mice (SLC Japan, Shizuoka, Japan) were housed in laboratory animal facilities with veterinary drugs at Seoul National University. Mice were kept in a cage accessible to water and food for 2 weeks until the first immunization. Mice were anesthetized with a mixture of Zoletil (Virbac, Carros, France) -Lomopun (Bayer, Seoul, Korea) and then injected with 25 μg immunogen (Ap97, ApxN, or P97C) on days 0, 14, 28 and 42 Subcutaneously. PBS-immunized mice were included as negative control. Immunizators or PBS were administered in complete Freunds adjuvant on day 0 and in incomplete Freund's adjuvant on days 14, 28, and 42.

Serum samples were obtained by retro-orbital bleeding under anesthesia prior to immunization on days 0, 14, 28, and 42. Mice were sacrificed by cardiac puncture 3 days after the first immunization (Day 63) and splenectomy was performed to allow primary culture of splenocytes.

(6) Splenocyte  And Cytokine  analysis

Splenocytes primed with Ap97, ApxN, and P97C vaccine were incubated at 37 ° C in a humidified atmosphere of 5% CO _{2 at} 37 ° C with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / And then cultured in RPMI 1640 medium supplemented with 50 [mu] M 2-mercaptoethanol. For cytokine production assay, spleen cells (4x10 ⁶ cells / ml) were re-stimulated with 1.25 μg / ml of Ap97, ApxN, and P97C dissolved in PBS for 72 hours. At the end of re-stimulation, cell culture supernatants were harvested and analyzed for IFN-γ and IL-4 using a bead-based multiplex assay kit (Procarta cytokine assay, Panomics, Fremont, CA) according to the manufacturer's protocol.

(7) Enzyme-Linked Immunosorbent Assay ELISA )

Immunoreactivity of mouse serum samples to ApxN and P97C was measured using an indirect ELISA. Briefly, wells of 96-well plates were coated with 0.5 μg ApxN or P97C in 50 μl carbonate buffer (pH 9.6) overnight at 4 ° C. The wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T), blocked with PBS-T containing 5% skim milk for 2 hours at 37 ° C, serially diluted with serum samples Lt; RTI ID = 0.0 > 37 C < / RTI > for 2 hours. The wells were washed and incubated with horseradish peroxidase (HRP) -conjugated anti-mouse IgG, IgG1, or IgG2a (diluted 1: 1,000 in PBS-T) for 1 hour at 37 ° C . After washing, O-phenylenediamine (Amresco, Solon, OH) was added to each well, reacted at 37 ° C for 30 minutes, and the reaction was stopped with 3M HCl. The absorbance at 450 nm was measured for each well using a micro-plate spectrophotometer (Molecular Devices, Sunnyvale, Calif.). The cut-off values for the analysis were calculated as mean specific optical density (OD) plus 3 standard deviations (SD) using serum samples of unimmunized mice (0 day) analyzed at 1: 100 dilution. The titer was established as the reciprocal of the last serum dilution to generate a higher OD than the cut-off.

(8) gel electrophoresis and Western Blotting  analysis

Protein samples were separated by SDS-PAGE electrophoresis (8-10% acrylamide) using a minigel apparatus (Bio-Rad, Hercules, Calif.) And stained with Coomassie blue. For Western blotting analysis, the proteins in the SDS-PAGE gel were transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany) in a blotting apparatus (Bio-Rad). The membranes were blocked by incubation with 5% skim milk-containing TBS-T for 1 hour and then incubated overnight at 4 ° C with antiserum obtained from mouse or pig and diluted 1: 500. After washing, the cells were incubated with HRP-conjugated anti-mouse IgG or anti-pig IgG (Biomedicals, Solon, OH) secondary antibody (diluted 1: 5,000) for 1 hour and chemiluminescence reagent ).

(9) Pig immunization experiment

All procedures for handling the pigs used in this example were reviewed and approved by the Laboratory Animal Resources Institute of Seoul National University and conducted in an ethical and human manner under veterinary supervision.

Twelve 4-6 kg 3 week old male pigs were obtained from sterile farms. The pigs were randomly divided into four groups and fed in a room with a slatted floor and a rubber mat in separate rooms and provided with free access to water and food. After one week of adaptation, each pig group was immunized with muscle (0 day) with 700 μg of Ap97 adsorbed on aluminum hydroxide or 2 ml of PBS containing aluminum hydroxide as negative control. In a similar manner, the booster was inoculated on the 14th day. Blood samples for immuno - serological analysis were collected by a vena cava cranialis puncture 24 days after the first immunization and serum was obtained by centrifugation for 15 minutes at 1,500xg. In the attacking process carried out after blood collection (treatments challenge), pigs 2x10 pleural pneumoniae (serotype 2) ⁸ colony-forming units (colony-forming unis) and M. high ohnyu ammonia of 1.5x10 ⁸ colony-forming units Were each inoculated intranasally or by intratracheal administration.

Clinical symptoms such as rectal temperature, cough, dyspenia, tachypnea, depression, and nasal discharge were monitored and recorded 2 weeks after challenge (38 days). Three weeks after challenge (45 days) pigs were euthanized using current and a complete autopsy was performed. The macroscopic lung lesion score was determined as previously described (Halbur PG et al. (1995) Vet. Pathol. 32: 648-660).

(10) Statistics

Values for antibody titers, cytokine production, and macrophage damage were expressed as means ± SD. A paired or unpaired t-test was performed using SPSS 15.0 (SPSS, Chiago, IL) to compare pairs of selected groups.

Example  One

(1) Production of recombinant protein

SDS-PAGE analysis of purified proteins after expressing the protein by culturing Escherichia coli BL21 (DE3) transformed with pET-ApxN, pET-P97C, and pET-Ap97 vector resulted in about 15 kDa, 45 kDa, A band of 60 kDa was identified (Figure 1A). These values corresponded to the expected molecular weights of ApxN, P97C and Ap97 (chimeric proteins including ApxN and P97C). These findings indicate that the recombinant protein was expressed as expected. Figure 1 shows the expression and purification of ApxN, P97C and Ap97 recombinant proteins. Figure 1A shows a Coomassie blue-stained SDS-PAGE gel showing ApxN, P97C and Ap97 purified from E. coli BL21 (DE3) using His-tag affinity chromatography. 1A, lanes 1, 2, and 3 represent proteins purified from E. coli BL21 (DE3), pET-ApxN, pET-P97C, and pET-Ap97 vectors, respectively, namely ApxN, P97C and Ap97. Figures 1B, 1C and 1D show the Western blot analysis of ApxN, P97C and Ap97 using antiserum from mice immunized with the Ap97 (B), ApxN (C) and P97C (D) vaccines, respectively. Mouse antisera were collected at 3 weeks (63 days) after the last immunization. 1B, 1C, and 1D, lanes 1, 2, and 3 represent ApxN, P97C, and Ap97, respectively.

(2) immunogenicity of recombinant protein in mouse

Antisera generated in mice immunized with Ap97 vaccine were used to test the ability of Ap97 to induce the production of antibodies specific for each of the component subunits ApxN and P97C as well as for the whole Ap97 . Antisera against Ap97 vaccine reacted with ApxN, P97C and Ap97, indicating that immunization of Ap97 induces the production of antibodies that bind to all three recombinant proteins (see FIG. 1B). Thus, Ap97 retained the antigenic properties of the two individual components. The antisera immunized with the ApxN and P97C vaccines were Ap97 and ApxN; And P97C and ApxN, respectively. However, the antiserum obtained from mice immunized with ApxN did not cross-react with P97C and vice versa (see Figures 1C and 1D).

Immunization of Ap97 and ApxN vaccine induced an intense IgG response to ApxN in mice. The IgG logistic regime reached 3.22 and 3.22 at 14 days after the first immunization of Ap97 and ApxN vaccines, respectively, and 5.87 and 5.82 at 3 weeks after the final immunization (Fig. 2A; P < 0.01).

Immunization of the Ap97 and P97C vaccines also increased the level of anti-P97C IgG antibody in mice. The IgG log levels reached 5.55 and 5.23, respectively, at 3 weeks (63 days) after the final immunization of the Ap97 and P97C vaccines. These values were also higher than those for antisera obtained from a given mouse (FIG. 2B; P < 0.01).

Levels of IgG1 and IgG2a isotype for ApxN and P97C were measured in serum collected three weeks (63 days) after the last immunization (see Figures 2C and 2D). Levels of IgG1 and IgG2a isotype for ApxN and P97C were higher than levels in the corresponding PBS-treated control (P < 0.01 or P < 0.05), indicating a mixed Th1-Th2 response. However, the average ratios of IgG2a / IgG1 to ApxN were 7.12 and 9.53, respectively, in the antiserum from mice given Ap97 and ApxN vaccines, indicating a predominance of IgG2a production relative to IgG1, a Th1-biased response to ApxN. In contrast, the average ratio of IgG2a / IgG1 to P97C was 0.78 and 1.68, respectively, in the antiserum obtained from mice given the Ap97 and P97C vaccines. Indicating that Ap97 and P97C elicit relatively balanced IgG2a and IgG1 responses to P97C. This difference in the proportion of IgG isotype generated for the two components of Ap97 is interesting, but the mechanism is unclear.

Figure 2 shows the serum titer of total IgG, IgG1, and IgG2a isotype antibodies to ApxN and P97C in mice immunized with ApxN, P97C and Ap97 vaccines. Blood samples were obtained at the first immunization 0, 14, 28, 42, and 63 days. Figure 2A shows the total IgG titers for ApxN, Figure 2B shows the total IgG titers to P97C, Figure 2C shows the IgG isotype titers to ApxN, and Figure 2D shows the IgG isotype titers to P97C. IgG1 and IgG2a reverse transcripts were measured in serum collected three weeks (63 days) after the last immunization. The numbers on the column represent the average IgG2a / IgG1 ratios (C and D). Data are expressed as mean SD (n = 5). * P < 0.05 and ** P < 0.01, significantly different from the corresponding anti-PBS serum control (language-paired t-test).

(3) a recombinant protein-induced Cytokine  secretion

Re-stimulated spleen mononuclear cells (spleen cells) from mice immunized with Ap97, ApxN, and P97C vaccines were analyzed for IFN-y and IL-4 production (Fig. 3). The concentrations of IFN-y and Th1 cytokines in the culture medium of spleen cells re-stimulated in vitro with Ap97, ApxN and P97C were 123.02 ± 8.23 pg / ml, 42.89 ± 15.81 pg / ml, and 61.51 ± 25.36 pg / ml. These levels were 80.9, 29.4, and 50.8-fold higher, respectively (Fig. 3A; P < 0.01 or P < 0.05), than were re-stimulated with PBS (control).

The concentrations of IL-4 and Th2 cytokines produced by splenocytes re-stimulated with in vitro with Ap97, ApxN and P97C were 18.21 ± 5.08 pg / ml, 5.36 ± 2.83 pg / ml, and 9.77 ± 2.04 pg / ml. These levels were 43.4, 7.66, and 16.3 times higher (Fig. 3B; P < 0.01 or P < 0.05, respectively) than re-stimulated with PBS (control).

Stimulation with concanavalin A, a potent T cell stimulator, also induced spleen cells (including controls) to produce these two cytokines (data not shown). These findings indicate that all three recombinant proteins induced production of IFN-y and IL-4 by splenocytes, suggesting induction of a mixed Th1-Th2 immune response.

Figure 3 shows the cytokine profile of spleen cells from immunized mice. The mice were primed with the Ap97, ApxN, or P97C vaccine. Splenocytes were isolated from each mouse and re-stimulated with Ap97, ApxN, or P97C dissolved in PBS. Data represent the mean ± SD (n = 4). * P < 0.05 and ** P < 0.01 are significantly different from corresponding PBS-treated controls (paired t-test).

(4) ApxIII Wow P97 Of the recombinant protein with mouse antiserum

Western blotting was performed to investigate the reaction of natural ApxIII with the mouse antisera generated against Ap97 and ApxN vaccines (Fig. 4). Protein samples precipitated from the culture medium of Pleural Pneumoniae were used for the analysis (see "Materials and Methods").

The antisera generated in the mice immunized with the Ap97 and ApxN vaccine reacted with the precipitated protein of about 120 kDa in size corresponding to the molecular weight of ApxIII, whereas no protein signal was detected in the control medium. This indicates that the antibody generated by the response to the immunization of Ap97 and ApxN vaccine easily binds to ApxIII secreted by Pleural Efficiency. These data indicate not only ApxN, but also that Ap97 has the antigenic property of ApxIII, which is recognized by the mouse immune system.

The antiserum obtained from the mice immunized with the Ap97 and P97C vaccines was also analyzed for the reaction with P97 found in the membrane fraction of M. hyunmonia (Fig. 5). The antiserum detected a band of about 97 kDa, the predicted size of P97. These data indicate that both Ap97 and P97 retain the antigenic properties of native P97. Moreover, sera from pigs that were artificially infected with M. hyunimumiore reacted with the 97 kDa protein. All three antisera were also reacted with two different proteins (30 and 60 kDa), and the porcine antiserum detected an additional band of 70 kDa. Based on previous studies confirming cleavage products of different sizes of P97, the non-97 kDa protein may show cleavage products of P97. These protein bands, including P97, were not recognized by control sera.

Figure 4 shows the recognition of ApxIII of Pleuropneumoniae by antiserum from mice immunized with Ap97 or ApxN recombinant protein. Western blotting assays were performed with the ApxN-vaccinated (A) and Ap97-vaccinated (B) mouse sera against pleuropneumoniae 1536 culture medium (lane 1) and the protein precipitated from the control medium. The arrows indicate the target band size.

Figure 5 shows recognition of M. hyunneumonia P97 by antiserum from mice immunized with Ap97 or P97C recombinant protein. The cell membrane fraction from M. hyunmoniore was incubated with the Ap97-vaccinated mouse serum (lane 1), the P97C-vaccinated mouse serum (lane 2), the serum of pigs experimentally infected with M. hyunmonioma (Lane 3), and PBS-vaccinated control mouse serum (lane 4). The arrows indicate the target band size.

(5) In the pig Ap97 Immunogenicity of

We also examined the reactivity of Ap97 vaccine-treated porcine sera against native P97 and ApxIII (Figure 6). The antisera for the Ap97 vaccine recognized the expected protein band of molecular weight; 97 kDa in the cell membrane fraction of M. hyunumonia and 120 kDa in the culture medium and cell lysate of pleural pneumoniae. Moreover, sera from pigs treated with PBS did not react with P97 or ApxIII. These data indicate that antibodies derived from an immunized inoculated with Ap97 efficiently react with P97 and ApxIII and that the two domains of the Ap97 protein are immunogenic in pigs.

Figure 6 shows the recognition of M. hyunneumonia P97 and pleuropneumoniae ApxIII by sera isolated from pigs immunized by the Ap97 vaccine. A shows that the TC97-precipitated protein from the cell membrane fraction (lane 1) of M. hyonumonii (lane 1), the culture medium of pleural pneumoniae (lane 2) and the cell lysate of pleuropneumoniae (lane 3) Western blotting analysis was performed with porcine serum. B is the result of Western blotting analysis of the protein samples described in Panel A with PBS-vaccinated pig serum. The arrows indicate the target band size.

(6) In the pig Ap97 Protective efficacy of

Since pigs immunized with Ap97 produced antibodies against P97 and ApxIII (FIG. 6), the pigs were directly challenged with M. hyunneumonia and pleural pneumoniae (Table 2).

Immunogen Attack inoculation Body temperature a Number of pigs with clinical symptoms / Number of pigs inoculated with attack Lung damage score b
(Mean ± SD)
cough Respiratory disturbance
(Empty breath) depressed Biru Ap97 Pleuropneumoniae - 1/3 0/3 0/3 0/3 4.33 ± 2.08 * PBS Pleuropneumoniae ++ 3/3 3/3 3/3 3/3 67.83 + 1.89 Ap97 M. Haiunmonia - 1/3 0/3 0/3 0/3 8.67 ± 6.43 * PBS M. Haiunmonia + 2/3 2/3 2/3 2/3 73.83 + - 9.28

In Table 2, it shows the protection of the pigs against the Ap97 vaccine against Pleuropneumoniae and M. hyunmonioma infections. a represents the maximum measured rectal temperature, - represents 40 deg. C, + represents less than 40 to 42 deg. C, and ++ represents 42 deg. b is the total area of the lung affected by any type of pneumonia infection (0-100%). * P < 0.01, significantly different from the corresponding PBS-treated group (unphased t-test).

As shown in Table 2, all three PBS vaccine-treated control pigs challenged by intranasal administration of pleural pneumoniae showed elevated body temperature (greater than 42 ° C), including cough, dyspnea, depression, and rash , And clinical symptoms. According to post-mortem examination, all three PBS-immunized pigs had typical lung injury of swine fluvularmonia. The lung injury score of these pigs was significantly higher than that of the Ap97-vaccinated pigs. In contrast, Ap97-vaccinated pigs had no clinical symptoms of infection, except for one pig with mild cough.

Two of the three PBS-vaccinated pigs challenged by intratracheal administration of M. hyunneumonia also developed clinical symptoms (cough, dyspnea, depression, and rash), and all three pigs had 40- Showed an increased body temperature of 42 ° C. In addition, a number of important pathological changes associated with M. hyunneumonia have been observed in PBS-vaccinated pigs, but the Ap97-vaccinated pig group (except for having a mild cough in one pig) No symptoms or lung injury was observed.

<110> SNU R & DB FOUNDATION <120> Recombinant protein vaccine for preventing Actinobacillus          pleuropneumoniae and Mycoplasma hyopneumoniae infection diseases <130> PN099639 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 1049 <212> PRT <213> Actinobacillus pleuropneumoniae serovar 2 <400> 1 Met Ser Thr Trp Ser Ser Met Leu Ala Asp Leu Lys Lys Arg Ala Glu   1 5 10 15 Glu Ala Lys Arg Gln Val Lys Lys Gly Tyr Asp Val Thr Lys Asn Gly              20 25 30 Leu Gln Tyr Gly Val Ser Gln Ala Lys Leu Gln Ala Leu Ala Ala Gly          35 40 45 Lys Ala Val Gln Lys Tyr Gly Asn Lys Leu Val Leu Val Ile Pro Lys      50 55 60 Glu Tyr Asp Gly Ser Val Gly Asn Gly Phe Phe Asp Leu Val Lys Ala  65 70 75 80 Ala Glu Glu Leu Gly Ile Gln Val Lys Tyr Val Asn Arg Asn Glu Leu                  85 90 95 Glu Val Ala His Lys Ser Leu Gly Thr Ala Asp Gln Phe Leu Gly Leu             100 105 110 Thr Glu Arg Gly Leu Thr Leu Phe Ala Pro Gln Leu Asp Gln Phe Leu         115 120 125 Gln Lys His Ser Lys Ile Ser Asn Val Val Gly Ser Ser Thr Gly Asp     130 135 140 Ala Val Ser Lys Leu Ala Lys Ser Gln Thr Ile Ile Ser Gly Ile Gln 145 150 155 160 Ser Val Leu Gly Thr Val Leu Ala Gly Ile Asn Leu Asn Glu Ala Ile                 165 170 175 Ile Ser Gly Gly Ser Glu Leu Glu Leu Ala Glu Ala Gly Val Ser Leu             180 185 190 Ala Ser Glu Leu Val Ser Asn Ile Ala Lys Gly Thr Thr Thr Ile Asp         195 200 205 Ala Phe Thr Thr Gln Ile Gln Asn Phe Gly Lys Leu Ala Glu Asn Ala     210 215 220 Lys Gly Leu Gly Gly Val Gly Arg Gln Leu Gln Asn Ile Ser Gly Ser 225 230 235 240 Ala Leu Ser Lys Thr Gly Leu Gly Leu Asp Ile Ser Ser Leu Leu                 245 250 255 Ser Gly Val Thr Arg Ser Phe Ala Leu Arg Asn Lys Asn Ala Ser Thr             260 265 270 Ser Thr Lys Val Ala Gly Phe Glu Leu Ser Asn Gln Val Ile Gly         275 280 285 Gly Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Leu Arg     290 295 300 Ala Gly Leu Ser Thr Thr Gly Pro Ala Ala Ala Leu Ile Ala Ser Ser 305 310 315 320 Ile Ser Leu Ala Ile Ser Pro Leu Ala Phe Leu Arg Val Ala Asp Asn                 325 330 335 Phe Asn Arg Ser Lys Glu Ile Gly Glu Phe Ala Glu Arg Phe Lys Lys             340 345 350 Leu Gly Tyr Asp Gly Asp Lys Leu Leu Ser Glu Phe Tyr His Glu Ala         355 360 365 Gly Thr Ile Asp Ala Ser Ile Thr Thr Ile Ser Thr Ala Leu Ser Ala     370 375 380 Ile Ala Gla Aly Ala Gla Ala Gla Ala Gla Ala 385 390 395 400 Pro Ile Thr Leu Leu Val Thr Gly Ile Thr Gly Leu Ile Ser Gly Ile                 405 410 415 Leu Glu Phe Ser Lys Gln Pro Met Leu Asp His Val Ala Ser Lys Ile             420 425 430 Gly Asn Lys Ile Asp Glu Trp Glu Lys Lys Tyr Gly Lys Asn Tyr Phe         435 440 445 Glu Asn Gly Tyr Asp Ala Arg His Lys Ala Phe Leu Glu Asp Ser Phe     450 455 460 Ser Leu Leu Ser Ser Phe Asn Lys Gln Tyr Glu Thr Glu Arg Ala Val 465 470 475 480 Leu Ile Thr Gln Gln Arg Trp Asp Glu Tyr Ile Gly Glu Leu Ala Gly                 485 490 495 Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp             500 505 510 Tyr Phe Gln Glu Gly Lys Leu Leu Glu Lys Lys Pro Asp Asp Phe Ser         515 520 525 Lys Val Val Phe Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser     530 535 540 Gln Thr Ser Thr Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly 545 550 555 560 Thr Glu Ser Arg Glu Arg Thr Gln Thr Gly Lys Tyr Glu Tyr Ile Thr                 565 570 575 Lys Leu Val Val Lys Gly Lys Asp Lys Trp Val Val Asn Gly Val Lys             580 585 590 Asp Lys Gly Ala Val Tyr Asp Tyr Thr Asn Leu Ile Gln His Ala His         595 600 605 Ile Ser Ser Ser Ala Arg Gly Glu Glu Tyr Arg Glu Val Arg Leu     610 615 620 Val Ser His Leu Gly Asn Gly Asn Asp Lys Val Phe Leu Ala Ala Gly 625 630 635 640 Ser Ala Glu Ile His Ala Gly Glu Gly His Asp Val Val Tyr Tyr Asp                 645 650 655 Lys Thr Asp Thr Gly Leu Leu Val Ile Asp Gly Thr Lys Ala Thr Glu             660 665 670 Gln Gly Arg Tyr Ser Val Thr Arg Glu Leu Ser Gly Ala Thr Lys Ile         675 680 685 Leu Arg Glu Val Ile Lys Asn Gln Lys Tyr Ala Val Gly Lys Arg Glu     690 695 700 Glu Thr Leu Glu Tyr Arg Asp Tyr Glu Leu Thr Gln Ser Gly Asn Ser 705 710 715 720 Asn Leu Lys Ala His Asp Glu Leu His Ser Val Glu Glu Ile Gly Ser                 725 730 735 Asn Gln Arg Asp Glu Phe Lys Gly Ser Lys Phe Arg Asp Ile Phe His             740 745 750 Gly Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp Ile         755 760 765 Leu Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn     770 775 780 Asp Gln Leu Tyr Gly Gly Glu Gly Asp Asp Lys Leu Leu Gly Gly Asn 785 790 795 800 Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gln Val                 805 810 815 Leu Gly Asn Gly Phe Asn Val Leu Arg Gly Gly Lys Gly Asp Asp Lys             820 825 830 Leu Tyr Gly Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly Asn         835 840 845 Asp Tyr Leu Glu Gly Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg Ser     850 855 860 Thr Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ala Ser Asp Ser 865 870 875 880 Asp Lys Leu Tyr Leu Ser Asp Leu Ser Phe Asp Asn Ile Leu Val Lys                 885 890 895 Arg Val Asn Asp Asn Leu Glu Phe Arg Ser Asn Asn Asn Ser Asn Ser             900 905 910 Gly Val Leu Thr Ile Lys Asp Trp Phe Lys Gly Gly Asn Ser Tyr Asn         915 920 925 His Lys Ile Glu Gln Ile Val Asp Lys Asn Gly Arg Lys Leu Thr Ala     930 935 940 Gly Asn Leu Gly Asn Asn Phe His Asp Thr Gln Gln Ala Ser Ser Leu 945 950 955 960 Leu Lys Asn Val Thr Gln Glu Gln Asn Glu Ser Asn Leu Ser Ser Leu                 965 970 975 Lys Thr Glu Leu Gly Lys Ile Ile Thr Asn Ala Gly Asn Phe Gly Val             980 985 990 Ala Lys Gln Gly Asn Thr Gly Ile Asn Thr Ala Ala Leu Asn Asn Glu         995 1000 1005 Val Asn Lys Ile Ile Ser Ser Ala Asn Thr Phe Ala Thr Ser Gln Leu    1010 1015 1020 Gly Gly Ser Gly Met Gly Thr Leu Pro Ser Thr Asn Val Asn Ser Met 1025 1030 1035 1040 Met Leu Gly Asn Leu Ala Arg Ala Ala                1045 <210> 2 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> N-terminal region of ApxIIIA <400> 2 Gln Val Lys Lys Gly Tyr Asp Val Thr Lys Asn Gly Leu Gln Tyr Gly   1 5 10 15 Val Ser Gln Ala Lys Leu Gln Ala Leu Ala Ala Gly Lys Ala Val Gln              20 25 30 Lys Tyr Gly Asn Lys Leu Val Leu Val Ile Pro Lys Glu Tyr Asp Gly          35 40 45 Ser Val Gly Asn Gly Phe Phe Asp Leu Val Lys Ala Ala Glu Glu Leu      50 55 60 Gly Ile Gln Val Lys Tyr Val Asn Arg Asn Glu Leu Glu Val Ala His  65 70 75 80 Lys Ser Leu Gly Thr Ala Asp Gln Phe Leu Gly Leu Thr Glu Arg Gly                  85 90 95 Leu Thr Leu Phe Ala Pro Gln Leu Asp Gln Phe Leu Gln Lys His             100 105 110 <210> 3 <211> 1108 <212> PRT <213> Mycoplasma hyopneumoniae <400> 3 Met Ser Lys Lys Ser Lys Thr Phe Lys Ile Gly Leu Thr Ala Gly Ile   1 5 10 15 Val Gly Leu Gly Val Phe Gly Leu Thr Val Gly Leu Ser Ser Leu Ala              20 25 30 Lys Tyr Arg Ser Glu Ser Pro Arg Lys Ile Ala Asn Asp Phe Ala Ala          35 40 45 Lys Val Ser Thr Leu Ala Phe Ser Pro Tyr Ala Phe Glu Thr Asp Ser      50 55 60 Asp Tyr Lys Ile Val Lys Arg Trp Leu Val Asp Ser Asn Asn Asn Ile  65 70 75 80 Arg Asn Lys Glu Lys Val Ile Asp Ser Phe Ser Phe Phe Thr Lys Asn                  85 90 95 Gly Asp Gln Leu Glu Lys Ile Asn Phe Gln Asp Pro Glu Tyr Thr Lys             100 105 110 Ala Lys Ile Thr Phe Glu Ile Leu Glu Ile Ile Pro Asp Asp Val Asn         115 120 125 Gln Asn Phe Lys Val Lys Phe Gln Ala Leu Gln Lys Leu His Asn Gly     130 135 140 Asp Ile Ala Lys Ser Asp Ile Tyr Glu Gln Thr Val Ala Phe Ala Lys 145 150 155 160 Gln Ser Asn Leu Leu Val Ala Glu Phe Asn Phe Ser Leu Lys Lys Ile                 165 170 175 Thr Glu Lys Leu Asn Gln Gln Ile Glu Asn Leu Ser Thr Lys Ile Thr             180 185 190 Asn Phe Ala Asp Glu Lys Thr Ser Ser Gln Lys Asp Pro Ser Thr Leu         195 200 205 Arg Ala Ile Asp Phe Gln Tyr Asp Leu Asn Thr Ala Arg Asn Pro Glu     210 215 220 Asp Leu Asp Ile Lys Leu Ala Asn Tyr Phe Pro Val Leu Lys Asn Leu 225 230 235 240 Ile Asn Arg Leu Asn Asn Ala Pro Glu Asn Lys Leu Pro Asn Asn Leu                 245 250 255 Gly Asn Ile Phe Glu Phe Ser Phe Ala Lys Asp Ser Ser Thr Asn Gln             260 265 270 Tyr Val Ser Ile Gln Asn Gln Ile Pro Ser Leu Phe Leu Lys Ala Asp         275 280 285 Leu Ser Gln Ser Ala Arg Glu Ile Leu Ala Ser Pro Asp Glu Val Gln     290 295 300 Pro Val Ile Asn Ile Leu Arg Leu Met Lys Lys Asp Asn Ser Ser Tyr 305 310 315 320 Phe Leu Asn Phe Glu Asp Phe Val Asn Asn Leu Thr Leu Lys Asn Met                 325 330 335 Gln Lys Glu Asp Leu Asn Ala Lys Gly Gln Asn Leu Ser Ala Tyr Glu             340 345 350 Phe Leu Ala Asp Ile Lys Ser Gly Phe Phe Pro Gly Asp Lys Arg Ser         355 360 365 Ser His Thr Lys Ala Glu Ile Ser Asn Leu Leu Asn Lys Lys Glu Asn     370 375 380 Ile Tyr Asp Phe Gly Lys Tyr Asn Gly Lys Phe Asn Asp Arg Leu Asn 385 390 395 400 Ser Pro Asn Leu Glu Tyr Ser Leu Asp Ala Ala Ser Ala Ser Leu Asp                 405 410 415 Lys Lys Asp Lys Ser Ile Val Leu Ile Pro Tyr Arg Leu Glu Ile Lys             420 425 430 Asp Lys Phe Phe Ala Asp Asp Leu Tyr Pro Asp Thr Lys Asp Asn Ile         435 440 445 Leu Val Lys Glu Gly Ile Leu Lys Leu Thr Gly Phe Lys Lys Gly Ser     450 455 460 Lys Ile Asp Leu Pro Asn Ile Asn Gln Gln Ile Phe Lys Thr Glu Tyr 465 470 475 480 Leu Pro Phe Phe Glu Lys Gly Lys Glu Glu Gln Ala Lys Leu Asp Tyr                 485 490 495 Gly Asn Ile Leu Asn Pro Tyr Asn Thr Gln Leu Ala Lys Val Glu Val             500 505 510 Glu Ala Leu Phe Lys Gly Asn Lys Asn Gln Glu Ile Tyr Gln Ala Leu         515 520 525 Asp Gly Asn Tyr Ala Tyr Glu Phe Gly Ala Phe Lys Ser Val Leu Asn     530 535 540 Ser Trp Thr Gly Lys Ile Gln His Pro Glu Lys Ala Asp Ile Gln Arg 545 550 555 560 Phe Thr Arg His Leu Glu Gln Val Lys Ile Gly Ser Asn Ser Val Leu                 565 570 575 Asn Gln Pro Gln Thr Thr Lys Glu Gln Val Ser Ser Ser Leu Lys Ser             580 585 590 Asn Asn Phe Phe Lys Asn Gly His Gln Val Ala Ser Tyr Phe Gln Asp         595 600 605 Leu Leu Thr Lys Asp Lys Leu Thr Ile Leu Glu Thr Leu Tyr Asp Leu     610 615 620 Ala Lys Lys Trp Gly Leu Glu Thr Asn Arg Ala Gln Phe Pro Lys Gly 625 630 635 640 Val Phe Gln Tyr Thr Lys Asp Ile Phe Ala Glu Ala Asp Lys Leu Lys                 645 650 655 Phe Leu Glu Leu Lys Lys Lys Asp Pro Tyr Asn Gln Ile Lys Glu Ile             660 665 670 His Gln Leu Ser Phe Asn Ile Leu Ala Arg Asn Asp Val Ile Lys Ser         675 680 685 Asp Gly Phe Tyr Gly Val Leu Leu Leu Pro Gln Ser Val Lys Thr Glu     690 695 700 Leu Glu Gly Lys Asn Glu Ala Gln Ile Phe Glu Ala Leu Lys Lys Tyr 705 710 715 720 Ser Leu Ile Glu Asn Ser Ala Phe Lys Thr Thr Ile Leu Asp Lys Asn                 725 730 735 Leu Leu Glu Gly Thr Asp Phe Lys Thr Phe Gly Asp Phe Leu Lys Ala             740 745 750 Phe Phe Leu Lys Ala Ala Gln Phe Asn Asn Phe Ala Pro Trp Ala Lys         755 760 765 Leu Asp Asp Asn Leu Gln Tyr Ser Phe Glu Ala Ile Lys Lys Gly Glu     770 775 780 Thr Thr Lys Glu Gly Lys Arg Glu Glu Val Asp Lys Lys Val Lys Glu 785 790 795 800 Leu Asp Asn Lys Ile Lys Gly Ile Leu Pro Gln Pro Pro Ala Ala Lys                 805 810 815 Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Thr Thr Lys Pro             820 825 830 Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Glu Ala Ala Lys Pro Val         835 840 845 Ala Ala Lys Pro Ala Lys Pro Ala Ala Lys Pro Glu Ala     850 855 860 Ala Lys Pro Ala Lys Pro Ala Lys Pro 865 870 875 880 Lys Pro Glu Ala Ala Lys Pro Val Ala Thr Asn Thr Gly Phe Ser Leu                 885 890 895 Thr Asn Lys Pro Lys Glu Asp Tyr Phe Pro Met Ala Phe Ser Tyr Lys             900 905 910 Leu Glu Tyr Thr Asp Glu Asn Lys Leu Ser Leu Lys Thr Pro Glu Ile         915 920 925 Asn Val Phe Leu Glu Leu Val His Gln Ser Glu Tyr Glu Glu Gln Glu     930 935 940 Ile Ile Lys Glu Leu Asp Lys Thr Val Leu Asn Leu Gln Tyr Gln Phe 945 950 955 960 Gln Glu Val Lys Val Thr Ser Asp Gln Tyr Gln Lys Leu Ser His Pro                 965 970 975 Met Met Thr Glu Gly Ser Ser Asn Gln Gly Lys Lys Ser Glu Gly Thr             980 985 990 Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala Pro Asn Gln Gly Lys Lys         995 1000 1005 Ala Glu Gly Thr Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala Pro Ser    1010 1015 1020 Gln Gln Ser Pro Thr Thr Glu Leu Thr Asn Tyr Leu Pro Asp Leu Gly 1025 1030 1035 1040 Lys Lys Ile Asp Glu Ile Ile Lys Lys Gln Gly Lys Asn Trp Lys Thr                1045 1050 1055 Glu Val Glu Leu Ile Glu Asp Asn Ile Ala Gly Asp Ala Lys Leu Leu            1060 1065 1070 Tyr Phe Ile Leu Arg Asp Asp Ser Lys Ser Gly Asp Pro Lys Lys Ser        1075 1080 1085 Ser Leu Lys Val Lys Ile Thr Val Lys Gln Ser Asn Asn Asn Gln Glu    1090 1095 1100 Pro Glu Ser Lys 1105 <210> 4 <211> 75 <212> PRT <213> Artificial Sequence <220> <223> R1 region of P97 <400> 4 Ala Ala Lys Pro Ala Lys Pro Ala Lys Pro Glu Thr   1 5 10 15 Thr Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Glu Ala Ala              20 25 30 Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys          35 40 45 Pro Glu Ala Ala Ala Ala Lys Pro      50 55 60 Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val  65 70 75 <210> 5 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> R2 region of P97 <400> 5 Gly Ser Ser Asn Gln Gly Lys Lys Ser Glu Gly Thr Pro Asn Gln Gly   1 5 10 15 Lys Lys Ala Glu Gly Ala Pro Asn Gln Gly Lys Lys Ala Glu Gly Thr              20 25 30 Pro Asn Gln Gly Lys Lys Ala Glu          35 40 <210> 6 <211> 318 <212> PRT <213> Artificial Sequence <220> <223> C-terminal region of P97 <400> 6 Lys Leu Asp Asp Asn Leu Gln Tyr Ser Phe Glu Ala Ile Lys Lys Gly   1 5 10 15 Glu Thr Thr Lys Glu Gly Lys Arg Glu Glu Val Asp Lys Lys Val Lys              20 25 30 Glu Leu Asp Asn Lys Ile Lys Gly Ile Leu Pro Gln Pro Pro Ala Ala          35 40 45 Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Thr Thr Lys      50 55 60 Pro Val Ala Ala Lys Pro Glu Ala Ala Ala Lys Pro  65 70 75 80 Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu                  85 90 95 Ala Ala Lys Pro Ala Lys Pro Ala Lys Pro Val Ala             100 105 110 Ala Lys Pro Glu Ala Ala Lys Pro Ala Thr Asn Thr Gly Phe Ser         115 120 125 Leu Thr Asn Lys Pro Lys Glu Asp Tyr Phe Pro Met Ala Phe Ser Tyr     130 135 140 Lys Leu Glu Tyr Thr Asp Glu Asn Lys Leu Ser Leu Lys Thr Pro Glu 145 150 155 160 Ile Asn Val Phe Leu Glu Leu Val His Gln Ser Glu Tyr Glu Glu Gln                 165 170 175 Glu Ile Ile Lys Glu Leu Asp Lys Thr Val Leu Asn Leu Gln Tyr Gln             180 185 190 Phe Gln Glu Val Lys Val Thr Ser Asp Gln Tyr Gln Lys Leu Ser His         195 200 205 Pro Met Met Thr Glu Gly Ser Ser Asn Gln Gly Lys Lys Ser Glu Gly     210 215 220 Thr Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala Pro Asn Gln Gly Lys 225 230 235 240 Lys Ala Glu Gly Thr Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala Pro                 245 250 255 Ser Gln Gln Ser Pro Thr Thr Glu Leu Thr Asn Tyr Leu Pro Asp Leu             260 265 270 Gly Lys Lys Ile Asp Glu Ile Ile Lys Lys Gln Gly Lys Asn Trp Lys         275 280 285 Thr Glu Val Glu Leu Ile Glu Asp Asn Ile Ala Gly Asp Ala Lys Leu     290 295 300 Leu Tyr Phe Ile Leu Arg Asp Asp Ser Lys Ser Gly Asp Pro 305 310 315 <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide <400> 7 Gly Ser Ser Gly   One <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 <210> 9 <211> 452 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > A fusion protein of N-terminal region of ApxIIIA and C-terminal          of P97 <400> 9 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Gln Val Lys Lys Gly Tyr Asp Val Thr Lys Asn              20 25 30 Gly Leu Gln Tyr Gly Val Ser Gln Ala Lys Leu Gln Ala Leu Ala Ala          35 40 45 Gly Lys Ala Val Gln Lys Tyr Gly Asn Lys Leu Val Leu Val Ile Pro      50 55 60 Lys Glu Tyr Asp Gly Ser Val Gly Asn Gly Phe Phe Asp Leu Val Lys  65 70 75 80 Ala Ala Glu Glu Leu Gly Ile Gln Val Lys Tyr Val Asn Arg Asn Glu                  85 90 95 Leu Glu Val Ala His Lys Ser Leu Gly Thr Ala Asp Gln Phe Leu Gly             100 105 110 Leu Thr Glu Arg Gly Leu Thr Leu Phe Ala Pro Gln Leu Asp Gln Phe         115 120 125 Leu Gln Lys His Gly Ser Lys Leu Asp Asp Asn Leu Gln Tyr Ser Phe     130 135 140 Glu Ala Ile Lys Lys Gly Glu Thr Thr Lys Glu Gly Lys Arg Glu Glu 145 150 155 160 Val Asp Lys Lys Val Lys Glu Leu Asp Asn Lys Ile Lys Gly Ile Leu                 165 170 175 Pro Gln Pro Pro Ala Ala Lys Pro Glu Ala Ala Lys Pro Ala Ala             180 185 190 Lys Pro Glu Thr Lys Pro Lys Pro Ala Lys Pro Glu Ala Ala Lys         195 200 205 Pro Glu Ala Ala Ala Ala Lys Pro     210 215 220 Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu 225 230 235 240 Ala Ala Lys Pro Ala Lys Pro Ala Lys Pro Val Ala                 245 250 255 Thr Asn Thr Gly Phe Ser Leu Thr Asn Lys Pro Lys Glu Asp Tyr Phe             260 265 270 Pro Met Ala Phe Ser Tyr Lys Leu Glu Tyr Thr Asp Glu Asn Lys Leu         275 280 285 Ser Leu Lys Thr Pro Glu Ile Asn Val Phe Leu Glu Leu Val His Gln     290 295 300 Ser Glu Tyr Glu Glu Glu Glu Ile Ile Lys Glu Leu Asp Lys Thr Val 305 310 315 320 Leu Asn Leu Gln Tyr Gln Phe Gln Glu Val Lys Val Thr Ser Asp Gln                 325 330 335 Tyr Gln Lys Leu Ser His Met Met Met Thr Glu Gly Ser Ser Asn Gln             340 345 350 Gly Lys Lys Ser Glu Gly Thr Pro Asn Gln Gly Lys Lys Ala Glu Gly         355 360 365 Ala Pro Asn Gln Gly Lys Lys Ala Glu Gly Thr Pro Asn Gln Gly Lys     370 375 380 Lys Ala Glu Gly Ala Pro Ser Gln Gln Ser Pro Thr Thr Glu Leu Thr 385 390 395 400 Asn Tyr Leu Pro Asp Leu Gly Lys Lys Ile Asp Glu Ile Ile Lys Lys                 405 410 415 Gln Gly Lys Asn Trp Lys Thr Glu Val Glu Leu Ile Glu Asp Asn Ile             420 425 430 Ala Gly Asp Ala Lys Leu Leu Tyr Phe Ile Leu Arg Asp Asp Ser Lys         435 440 445 Ser Gly Asp Pro     450 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ApxN-F primer <400> 10 ggggatccgg ctacgatgta actaaaaatg gt 32 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> ApxN-R primer <400> 11 ggctcgagtt attgtaagaa ctgatccagt tgcgg 35 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> P97C-F primer <400> 12 cgggatccaa actggatgac aacctccaa 29 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> P97C-R primer <400> 13 ggctcgagtt aaggatctcc ggatttgctg tcgtc 35 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Ap97-F primer <400> 14 ggccatatgc aagttaaaaa aggctacgat gtaac 35 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Ap97-R primer <400> 15 ggcggatcca tgtttttgtt agaactgatc cagttg 36 <210> 16 <211> 139 <212> PRT <213> Artificial Sequence <220> ApxN protein <400> 16 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Gly Ser Gly Tyr Asp Val Thr Lys Asn Gly Leu Gln Tyr Gly Val Ser          35 40 45 Gln Ala Lys Leu Gln Ala Leu Ala Ala Gly Lys Ala Val Gln Lys Tyr      50 55 60 Gly Asn Lys Leu Val Leu Val Ile Pro Lys Glu Tyr Asp Gly Ser Val  65 70 75 80 Gly Asn Gly Phe Phe Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile                  85 90 95 Gln Val Lys Tyr Val Asn Arg Asn Glu Leu Glu Val Ala His Lys Ser             100 105 110 Leu Gly Thr Ala Asp Gln Phe Leu Gly Leu Thr Glu Arg Gly Leu Thr         115 120 125 Leu Phe Ala Pro Gln Leu Asp Gln Phe Leu Gln     130 135 <210> 17 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding ApxN protein <400> 17 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccggctacga tgtaactaaa 120 aatggtttgc aatatggggt gagtcaagca aaattacaag cattagcagc tggtaaagcc 180 gttcaaaagt acggtaataa attagtttta gttattccaa aagagtatga cggaagtgtt 240 ggtaacggtt tctttgattt agtaaaagca gctgaggaat taggcattca agttaaatat 300 gttaaccgta atgaattgga agttgcccat aaaagtttag gtaccgcaga ccaattcttg 360 ggtttaacag aacgtggact tactttattt gcaccgcaac tggatcagtt cttacaataa 420                                                                          420 <210> 18 <211> 339 <212> PRT <213> Artificial Sequence <220> <223> P97C protein <400> 18 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Lys Leu Asp Asp Asn Leu Gln Tyr Ser Phe Glu              20 25 30 Ala Ile Lys Lys Gly Glu Thr Thr Lys Glu Gly Lys Arg Glu Glu Val          35 40 45 Asp Lys Lys Val Lys Glu Leu Asp Asn Lys Ile Lys Gly Ile Leu Pro      50 55 60 Gln Pro Pro Ala Ala Ala Ala Ala Lys Pro Ala Ala Lys  65 70 75 80 Pro Glu Thr Thr Lys Pro Ala Lys Pro Glu Ala Ala Lys Pro                  85 90 95 Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val             100 105 110 Ala Ala Lys Pro Ala Lys Pro Ala Ala Lys Pro Glu Ala         115 120 125 Ala Lys Pro Ala Lys Pro Ala Lys Pro     130 135 140 Asn Thr Gly Phe Ser Leu Thr Asn Lys Pro Lys Glu Asp Tyr Phe Pro 145 150 155 160 Met Ala Phe Ser Tyr Lys Leu Glu Tyr Thr Asp Glu Asn Lys Leu Ser                 165 170 175 Leu Lys Thr Pro Glu Ile Asn Val Phe Leu Glu Leu Val His Gln Ser             180 185 190 Glu Tyr Glu Glu Glu Glu Ile Ile Lys Glu Leu Asp Lys Thr Val Leu         195 200 205 Asn Leu Gln Tyr Gln Phe Gln Glu Val Lys Val Thr Ser Asp Gln Tyr     210 215 220 Gln Lys Leu Ser His Pro Met Met Thr Glu Gly Ser Ser Asn Gln Gly 225 230 235 240 Lys Lys Ser Glu Gly Thr Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala                 245 250 255 Pro Asn Gln Gly Lys Lys Ala Glu Gly Thr Pro Asn Gln Gly Lys Lys             260 265 270 Ala Glu Gly Ala Pro Ser Gln Gln Ser Pro Thr Thr Glu Leu Thr Asn         275 280 285 Tyr Leu Pro Asp Leu Gly Lys Lys Ile Asp Glu Ile Ile Lys Lys Gln     290 295 300 Gly Lys Asn Trp Lys Thr Glu Val Glu Leu Ile Glu Asp Asn Ile Ala 305 310 315 320 Gly Asp Ala Lys Leu Leu Tyr Phe Ile Leu Arg Asp Ser Ser Ser Ser Ser                 325 330 335 Gly Asp Pro             <210> 19 <211> 1017 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding P97C protein <400> 19 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaaactgg atgacaacct ccaatactcc ttcgaagcga tcaaaaaagg cgaaaccact 120 aaagagggca aacgcgagga ggtggacaag aaggtgaaag agctcgacaa caaaatcaaa 180 ggtatccttc cgcaaccgcc ggcggcgaaa cctgaggcgg caaagccggt cgccgccaaa 240 ccagaaacta ccaaaccagt cgcagccaag ccggaagcag cgaaaccaga ggccgcgaaa 300 cccgtcgccg caaagccgga agcagccaaa ccggtggcag ccaaacccga agcggcaaaa 360 cctgtagcag caaaaccaga agcggcaaaa cctgtggccg ccaagccaga agcagccaaa 420 cccgtagcga ccaatacagg cttctcattg accaacaaac caaaagaaga ctactttccg 480 atggctttta gctataaatt agaatacaca gacgagaata aactgtccct gaaaacccct 540 gaaattaatg tctttctcga actggtacat caaagtgaat atgaagaaca agaaatcatt 600 aaagaattag acaaaaccgt attaaacttg cagtatcagt ttcaagaagt aaaagtgacg 660 agcgatcaat accagaaact gtcccaccct atgatgacag aaggcagctc aaaccaaggc 720 aaaaaaagcg aaggcacacc gaaccaaggc aaaaaagctg agggagcccc caaccaaggt 780 aagaaggccg aaggcacccc gaatcaaggt aaaaaagcag aaggtgcacc cagccagcag 840 agcccaacaa ccgagctgac aaactattta ccggacctgg gtaaaaaaat tgacgaaatt 900 atcaaaaaac agggcaaaaa ctggaaaacc gaagttgaac tcatcgaaga caacattgcg 960 ggtgatgcta aattattgta tttcattctg cgcgacgaca gcaaatccgg agatcct 1017 <210> 20 <211> 3 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding a fusion protein of the N-terminal region of          ApxIIIA and C-terminal region of P97 <400> 20 atg 3

Claims (27)

A fusion protein comprising a region comprising a Pleuropneumoniae ApxIIIA protein or fragment thereof and a region comprising a mycoplasma hyperonymonia ciliate protein P97 or fragment thereof. The fusion protein of claim 1, wherein the fragment of Pleuropneumonitus ApxIIIA protein is an N-terminal region and the fragment of mycoplasma hyperonymonia ciliated protein P97 is a C-terminal region. 3. The fusion protein of claim 2, wherein the N-terminal region of the Pleuropneumonitus ApxIIIA protein comprises the amino acid sequence of SEQ ID NO: 2. The fusion protein according to claim 2, wherein the C-terminal region of the mycoplasma hyunmonia ciliad protein P97 comprises the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 5. 5. The fusion protein of claim 4, wherein the C-terminal region of the mycoplasma hyperonymonia ciliad protein P97 has the amino acid sequence of SEQ ID NO: 6. The fusion protein of claim 2, wherein the fusion protein is linked by a linker between the N-terminal region of Pleuropneumoniae ApxIIIA protein and the C-terminal region of mycoplasma hyperonymonia ciliary adsorptive protein P97. 6. The fusion protein of claim 5, wherein the linker is GS, GSSG, (GGGGS) ₂ , or a combination thereof. The fusion protein of claim 1, further comprising a polyhistidine sequence at the N-terminus, the C-terminus, or at both termini. 9. The fusion protein of claim 8, wherein the polyhistidine sequence is 6x His. The fusion protein of claim 1, having the amino acid sequence of SEQ ID NO: 9. The fusion protein of claim 1, further comprising a sequence necessary for protein separation at the N-terminus. A polynucleotide encoding a fusion protein according to any one of claims 1 to 11. 13. The polynucleotide of claim 12 wherein at least one of the polynucleotides is replaced with a higher codon used in E. coli. The polynucleotide of claim 12, which has the nucleotide sequence of SEQ ID NO: 20. A composition for the prophylaxis or treatment of infection of any one or more of mammalian pleural pneumococci and mycoplasma hyperonymonia comprising the fusion protein of any one of claims 1 to 11 as an active ingredient. 16. The composition of claim 15, wherein the mammal is a pig. A method for preventing or treating an infectious disease of any one of mammalian pleural pneumococci and mycoplasma hyperonymonia, comprising administering the composition according to claim 15 to a mammal other than a human. 18. The method of claim 17, wherein the mammal is a pig and the infectious disease is a respiratory disease including pneumonia. Protein comprising Pleuropneumoniae ApxIIIA protein or fragment thereof. The protein according to claim 19, wherein the fragment of the Pleuropneumonitus ApxIIIA protein is an N-terminal region. 21. The protein of claim 20, wherein the N-terminal region of the Pleuropneumonitus ApxIIIA protein comprises the amino acid sequence of SEQ ID NO: 2. A protein comprising mycoplasma hyperonymonia ciliad protein P97 or a fragment thereof. 23. The protein of claim 22, wherein the fragment of the mycoplasma hyperonymonia ciliad protein P97 is the C-terminal region. 24. The protein of claim 23, wherein the C-terminal region of the mycoplasma hyperonymonia ciliated protein P97 comprises the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 5. The protein according to claim 23, wherein the C-terminal region of the mycoplasma hyperonymono-ciliary adsorption protein P97 has the amino acid sequence of SEQ ID NO: 6. A protein comprising the N-terminal region of the pleuropneumonitus ApxIIIA protein comprising any one of the proteins of claims 19 to 21 and the protein of any one of claims 22 to 25, a C-terminal region of the mycoplasma hyperonymonia ciliated protein P97 , Or a fusion protein thereof. A protein comprising the N-terminal region of the pleuropneumonitus ApxIIIA protein comprising any one of the proteins of claims 19 to 21 and the protein of any one of claims 22 to 25, a C-terminal region of the mycoplasma hyperonymonia ciliated protein P97 , Or a fusion protein thereof.

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