MXPA99009688A - New proteins of antinobacillus pleuropneumon - Google Patents

Abstract

The present invention is directed to five new low molecular weight proteins from Actinobacillus pleuropneumoniae (APP) which are capable of inducing, or contributing to the induction of, a protective immune response in pigs against APP, the present invention is also directed to polynucleotides having nucleotide sequences encoding the proteins, as well as vaccines comprising the proteins or polynucleotide molecules, and methods for preparing and using them

Description

NEW PROTEINS OF Actinobacillus Pleuropneumoniae 1. FIELD OF THE INVENTION The present invention is in the field of animal health, and is directed to vaccines that protect pigs against Actinobacillus pleuropneumoniae. More particularly, the present invention is directed to novel antigenic proteins shared by multiple serotypes of A. pleuropneumoniae, to DNA molecules that encode the proteins, to APP vaccines comprising the proteins, and to reagents for diagnosis. 2. BACKGROUND OF THE INVENTION A. pleuropneumoniae (hereinafter referred to as "APP") is a Gram-negative cocbacterium recognized as one of the most important pneumonic pathogens for pigs (Shope, RE 1964, J. Exp. Med. 1 19: 357-368; Sebunya, TNK and Saunders, J: R., 1983, J. Am. Vet. Med. Assocr 182: 1331-1337). Twelve different serotypes have been recognized that vary in their geographical distribution (sebunya, TNK and Saunders, JR, 1983, earlier, Nielsen, R., 1985, Proc. An. Assoc. Swine Pract. 18-22; Nielsen, R. , 1986, Acta Vet., Scand., 27: 453-455). Immune responses to a vaccination against APP have been mainly specific for certain serotypes, suggesting that immunity induced by vaccines is directed to specific capsular antigens for certain serotypes (Maclnnes, Jl and Rosendal, S., 1988, Can. Vet J. 29: 572-574, Fedorka-Cray, PJ et al., 1994, Comp. Cont. Educ. Pract. Vet. 16: 1 17-125; Nielsen R., 1979, Nord. Vet. Med. 31: 407-413; Rosendal, S. et al., 1986, Vet. Microbiol., 12: 229-240). In contrast, the natural immunity for any serotype seems to confer an important protection with respect to a disease caused by other serotypes, suggesting that natural exposure induces a cross-reactive immunity for shared antigens (Sebunya, TNK and Saunders, J: R. 1983, earlier, Maclnnes, Jl and Rosendal, S., 1988, earlier, Fedorka-Cray, PJ et al, 1994 ^ previous, Nielsen R., 1979, earlier, and Rosendal, S. et al., 1986, earlier). Virulence factors that may contribute to cross-protection have been proposed as potential candidates for vaccines, including exotoxins (Apx) (Nakai, T., et al., 1983, Am. J. Vet. Res. 44; 344-347; Frey, J., et al., 1993, J. Gen. Microbiol., 139; 1723-1728, Fedorka-Cray, PJ et al., 1993, Vet. Microbiol. 37: 85-100); capsular antigens (Rosendal, S., et al., 1986, op cit.); outer membrane proteins (OMP) (Denee, H. and Potter, A., 1989, Infect.Immune 57; 798-804; Niven, DF, et al., 1989, Mol.Microbioi., 3; 1083-1089; González, G., et al., 1990, Mol Microbiol 4; 1 173-1 179; Gerlach, GF, et al., 1992, Infect. Immun.60; 3253-3261); and lipopolysaccharides (LPS) (Fenwick, B.W. and Osborn, B., 1986, Infect. Immun 54; 575-582). However, cross-reactivity and cross-protection tables, induced by these components, do not cover all of the twelve APP serotypes. In addition, immunization with individual isolated components or combinations of individual components from APP has not been able to confer protection with respect to stimulation with some heterologous serotypes (unpublished observations). Therefore, it can be postulated that cross-protective responses, induced by a natural infection, are limited to specific groupings of serotypes. Alternatively, it is possible that some of the antigens responsible for the cross-protection observed after a natural infection have not yet been identified. The largest part of the studies concerning APP antigens have focused on the characterization of immunodominant antigens detected in the serum of convalescents using antibodies. This approach does not allow the identification of possible differences in the specificities for antibodies represented during primary responses with respect to those represented during secondary responses, nor does it allow the identification of dominant specificities at the site of infection, which are likely to be responsible for the protection after a secondary encounter with the pathogen.

It is generally accepted that lymphocytes are educated during primary infections, so that when there is secondary exposure to a pathogen the host is better able to avoid disease (MacKay, CR, 1993, Adv. Immunol., 53; 217-240). . The memory cells responsible for this activity (T and B lymphocytes skilled in antigens) persist for long periods of time, and are capable of reactivation after an appropriate subsequent encounter with the antigen. In contrast to the inexperienced cells (candidas) they generally show a faster response time, a localization in specialized tissues, and a more efficient recognition of antigens as well as effector functions (MacKay, CR, 1993, op cit; Linton, P and Klinman, NR, 1992, Sem. Immun., 4; 3-9; Meeusen, ENT, et al., 1991, Eur. J. Immunoi., 21, 2269-2272). During the generation of a secondary response, the frequency of precursor cells capable of responding to the particular antigen is higher than that which is present during the primary response. The traffic boxes of the subsets of ^ cells with memory following secondary responses are also different from those of inexperienced cells. Inexpert cells migrate relatively homogeneously to secondary lymphoid tissues, but they harbor poorly in non-lymphoid tissues. In contrast, memory cells display heterogeneous traffic and, in some cases, migration has been shown to be restricted to certain secondary lymphoid tissues and non-lymphoid sites (MacKay, CR, 1993, op cit., Gray, D ., .993, Ann. Rev. Immunol., 11; 49-77; Picker, LS, et al., 1993, J. Immunol. 150.1 122-1 136). Studies in both rodents and lambs have indicated that lymphocytes from the intestine migrate preferentially back to the intestine, while cells that are drained from the skin or from lymph nodes migrate back to the skin or lymph nodes (Gray , D., 1993, op cit., Picker, LS, et al., 1993, op cit.). Therefore, when a secondary encounter with a pathogen occurs, specific effector cells for cell-mediated immunity and antibody secretion can be more effectively housed at sites of infection and local lymph nodes (Meeusen, ENT, et al., 1991, op. cit.). As a result, infiltrating lymphocytes will proliferate rapidly and their specifications will predominate during early stages of reinfection. Recovery of local B cells from draining tissues and lymph nodes soon after re-infection has allowed some investigators to obtain antibodies with a narrower range of specificities (Meeusen ^ ENT and Brandon, M. 1994, J. Immunol Meth. 172: 71-76). Such antibodies have been used successfully to identify potential protective antigens for various pathogens (Meeusen, ENT and Brandon, M., 1994, op cit; Meeusen, ENT and Brandon, M., 1994, Eur. J. Immunol. 469-474; Bowles, VM, et al., 1995, Immunol., 84: 669-674). The invention described hereinafter is based on a modification of this approach, in which probes of antibody-secreting cells (ASCs) that were associated with local memory responses, produced after stimulation, were recovered. homologous and heterologous of APP. Antibodies obtained from cultures of bronchial lymph nodes (BLN) after heterologous stimulation recognized four previously unrecognized proteins present in the totality of the twelve APP serotypes. Partial sequences of amino acids were obtained for each protein and were used to generate PCR primers (from Polymerase Chain Reaction) that allowed the identification of five new APP proteins and the polynucleotide molecules that encode them. 3. SUMMARY OF THE INVENTION The present invention provides five novel low molecular weight proteins, isolated from the APP, which are designated in the present context, respectively, as "Omp20", "OmpW", "Omp27", "OmpA1" and "OmpA2". These "APP proteins", and the polynucleotide molecules that encode them, are useful either as antithelial components in a vaccine to protect pigs against the APP or as diagnostic reagents intended to identify pigs that are, or have been, infected with APP, or that have been vaccinated with a vaccine of the present invention. . The amino acid sequence of Omp20 is encoded by the ORF encoding Omp20 of plasmid pER416 which is present in host cells of strain Pz416 (ATCC 98926), and its deduced amino acid sequence is presented as SEQ ID NO: 2, comprising a signal sequence from amino acid residues 1 to 19. The amino acid sequence of OmpW is encoded by the ORF encoding OmpW of plasmid pER418 which is present in host cells of strain Pz418 (ATCC 98928), and its deduced amino acid sequence is presented as SEQ ID NO: 4, comprising a signal sequence from amino acid residues 1 to 21. The amino acid sequence of Omp27 is encoded by the ORF encoding Omp27 of plasmid pER417 which is present in host cells of strain Pz417 (ATCC 89827), and its The deduced amino acid sequence is presented as SEQ ID NO: 6, which comprises a signal sequence from amino acid residues 1 to 27. The amino acid sequence of OmpA1 is encoded by the lDRF encoding OmpA1 of plasmid pER419 which is present in host cells of strain Pz419 (ATCC 98929), and its deduced amino acid sequence is presented as SEQ ID NO: 8, which comprises a signal sequence from the residues of amino acids 1 to 19. The amino acid sequence of OmpA2 is encoded by the ORF encoding OmpA2 of plasmid pER420 which is present in host cells of strain Pz420 (ATCC 98930), and its deduced amino acid sequence is presented as SEQ ID NO: 10, which comprises a signal sequence from amino acid residues 1 to 19. Each of these APP proteins, in substantially purified form, is provided by the present invention. The present invention also provides substantially purified polypeptides that are homologous to any of the aforementioned APP proteins of the present invention. The present Invention also provides peptide fragments of any of the APP proteins, or their homologous polypeptides of the present invention. The present invention further provides fusion proteins comprising an APP protein, a homologous polypeptide, or a peptide fragment of the present invention, which has been linked to a carrier or a partner in a fusion. The present invention further provides analogous compounds to, and derivatives of, an APP protein, a homologous polypeptide, a peptide fragment, or a fusion protein of the present invention. The present invention further provides an isolated polynucleotide molecule, comprising a nucleotide sequence encoding the APP protein, Omp20, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding Omp20, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 1 from about nt 329 to about nt-r 790. In a most preferred embodiment, the Isolated polynucleotide molecule encoding Omp20, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 1 from about nt 272 to about nt 790. The present invention further provides an isolated polynucleotide molecule, comprising a sequence of nucleotides encoding the APP protein, OmpW, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding OmpW, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 3 from about nt 439 to about nt x ----- 1, 023. In a more preferred embodiment, the isolated polynucleotide molecule encoding OmpW, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 3 from about nt 376 to about nt 1, 023. The present invention further provides an isolated polynucleotide molecule, comprising a nucleotide sequence encoding the APP protein, Omp27, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding Omp27, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 5 from about nt 238 to about nt 933. In a most preferred embodiment, the isolated molecule of polynucleotide encoding Omp27, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 5 from about nt 157 to about nt 933.

The present invention further provides an isolated polynucleotide molecule, comprising a nucleotide sequence encoding the APP protein, OmpAI, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding OmpA1, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 7 from about nt 671 to about nt 1, 708. In a more preferred embodiment, the isolated polynucleotide molecule encoding OmpAI, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 7 from about nt 614 to about nt 1, 708. The present invention further provides an isolated polynucleotide molecule, comprising a nucleotide sequence encoding the APP protein, OmpA2, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding OmpA2, of the present invention, comprises the nucleotide sequence SEQ ID NO: 9 from about nt 254 to about nt 1, 306. In a more preferred embodiment, the isolated polynucleotide molecule encoding OmpA2, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 9 from about nt 197 to about nt 1, 306. The present invention further provides an isolated polynucleotide molecule, which is homologous to any of the aforementioned polynucleotide molecules of the present invention. The present invention further provides an isolated polynucleotide molecule, comprising a nucleotide sequence encoding a polypeptide that is homologous with any of the APP proteins of the present invention. The present invention further provides an isolated polynucleotide molecule, which consists of a nucleotide sequence that is a substantial portion of any of the aforementioned polynucleotide molecules of the present invention. In a non-limiting embodiment, the substantial portion of a polynucleotide molecule of the present invention encodes a peptide fragment of an APP protein or a homologous polypeptide of the present invention. The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising a APP protinin, a homologous polypeptide or a peptide fragment of the present invention, which has been linked to a carrier or a participant. in a fusion. The present invention further provides polynucleotide molecules that are useful as primers for amplifying any of the polynucleotide molecules of the present invention, or as diagnostic reagents. Specific, but not limiting, embodiments of said oligonucleotide molecules include oligonucleotide molecules having nucleotide sequences selected from the group consisting of any of SEQ ID NOS: 15-47 and 49-93.

The present invention further provides compositions and methods for cloning and expressing any of the polynucleotide molecules of the present invention, including recombinant cloning vectors and recombinant expression vectors comprising a polynucleotide molecule of the present invention, host cells transformed with any of said vectors, and cell lines derived therefrom. The present invention further provides a recombinantly expressed APP protein, a homologous polypeptide, a peptide fragment, or a fusion protein encoded by a polynucleotide molecule of the present invention. The present invention further provides a vaccine for protecting pigs against APP, comprising an immunologically effective amount of one or more antigens of the present invention, selected from the group consisting of an APP protein, a homologous polypeptide, a peptide fragment. , a fusion protein, an analogous compound, a derivative or a polynucleotide molecule of the present invention, capable of inducing, or contributing to the induction of, a protective response against APP in pigs; and a veterinarily acceptable vehicle or diluent. The vaccine of the present invention may further comprise an adjuvant or other immunomodulatory component. In a non-limiting embodiment, the present invention vaccine may be a combination vaccine to protect pigs against APP and, optionally, one or more other diseases or pathological conditions that may afflict pigs, whose combination vaccine has a first component comprising an immunologically effective amount of one or more antigens of the present invention, selected from the group consisting of an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogous compound, a derivative, or a polynucleotide molecule of the present invention, capable of inducing, or contributing to the induction of, a protective response against APP in pigs; a second component comprising an immunologically effective amount of a different antigen, capable of inducing, or contributing to the induction of, a protective response to a disease or pathological condition that may afflict pigs, and a veterinarily acceptable vehicle or diluent. The present invention further provides a method for preparing a vaccine that can protect pigs against APP, which comprises combining an immunologically effective amount of one or more antigens of the present invention, selected from the group consisting of an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogous compound, a derivative, or a polynucleotide molecule of the present invention, capable of inducing, or contributing to the induction of, a protective response against APP in pigs, with a veterinarily acceptable vehicle or diluent, in a form suitable for administration to pigs.

The present invention further provides a method for vaccinating pigs against APP, which comprises administering a vaccine of the present invention to a pig. The present invention further provides a vaccine kit for vaccinating pigs against APP, comprising a container which in turn comprises an immunologically effective amount of one or more antigens of the present invention, selected from the group consisting of an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogous compound, a derivative, or a polynucleotide molecule of the present invention, capable of inducing, or contributing to the induction of, a protective response against APP in pigs. The case may further comprise a second container which in turn comprises a veterinarily acceptable vehicle or diluent. The present invention further provides antibodies that specifically bind to an APP protein of the present invention. The present invention also provides diagnostic kits. In a non-limiting embodiment, the diagnostic kit comprises a first container which in turn comprises an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogous compound, or a derivative of the present invention, which will bind specifically to porcine antibodies directed against an APP protein; and a second container comprising a secondary antibody directed against porcine anti-APP antibodies.

The secondary antibody preferably comprises a detectable label. Said diagnostic kit is useful for detecting pigs that at a given time are, or have previously been, infected with APP, or that have seroconverted as a result of a vaccination with a vaccine of the present invention. In a different non-limiting embodiment, the diagnostic kit comprises a first container which in turn comprises a primary antibody that binds to an APP protein of the present invention; and a second container comprising a secondary antibody that binds to a different epitope of the APP protein, or that binds to the primary antibody. The secondary antibody preferably comprises a detectable label. In a different non-limiting embodiment, the diagnostic kit comprises a container which in turn comprises a polynucleotide molecule or an oligonucleotide molecule of the present invention, which is useful for specifically amplifying a specific polynucleotide molecule for APP of the present invention . These last two diagnostic kits are useful for detecting pigs that are infected with APP at any given time. 4. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Western blot analysis of antibodies present in (a) a serum, and (b) in supernatants from explants of bronchial lymph node (BLN) tissue from pig no. 803 stimulated with serotype 5 of APP and again stimulated heterologously with serotype 7 of APP. All supernatant materials from BLN tissue explants, collected after 24 or 48 h of incubation, contained antibodies that specifically recognized APP proteins. Antibodies from the supernatant materials of BLN tissue explants highlighted several low molecular weight proteins present in serotypes 1, 5 and 7 of APP. Figure 2. Western blot analysis of the cross-reactivity of antibodies present in supernatant materials of BLN tissue explants from swine no. 803 against whole bacterial cell antigens from each of the twelve different APP serotypes, demonstrating that at least three of the low molecular weight proteins, recognized by antibodies induced by heterologous restimulation, were present in all of the twelve serotypes of APP. The antibodies present in this particular BLN supernatant also recognized other protein bands. Figure 3. Western blot analysis demonstrating that the antibody reactivity in supernatant materials of BLN tissue explants from the pig no. 803 against proteins of low molecular weight, is restricted to proteins present in cellular sediments (Cells) instead of supernatants of bacterial cells (Sups).

Figure 4. Western blot analysis of the reactivity of (a) a BLN tissue explant supernatant and (b) a serum from pig no. 803, against purified proteins with respect to serotype 7 of APP by continuous flow electrophoresis. Four protein bands with molecular weights of approximately 19-20, approximately 23, approximately 27 and approximately 29 kDa, respectively, were identified using this method using this method. Figure 5. Alignment of deduced sequences of amino acids of the OmpAI proteins of APP and OmpA2 of APP. The two proteins share an amino acid identity in 73.1%. Figure 6. Alignment of the OmpW protein from Vibrio cholerae and the OmpW protein from APP. The two proteins share an amino acid identity by 44.9%.

. DETAILED DESCRIPTION OF THE INVENTION . 1. New proteins shared by multiple APP serotypes The present invention is based on the discovery of five new low molecular weight proteins, derived from APP (hereinafter referred to as "APP proteins"). These APP proteins are designated in the current text, respectively, as "Omp20", "OmpW", "Omp27", "Omp l" and "OmpA2".

The amino acid sequence of Omp20 is encoded by the ORF encoding Omp20 of plasmid pER416 which is present in host cells of strain Pz416 (ATCC 98926). The deduced amino acid sequence of Omp20 is presented as SEQ ID NO: 2. The first 19 amino acids of the protein shown in SEQ ID NO: 2 represent a signal sequence, and the present invention encompasses both an Omp20 protein having only the residues 20 to 172 of amino acids of SEQ ID NO: 2 (ie lacking the signal sequence) as an Omp20 protein having the sequence of SEQ ID NO: 2 (ie, including the signal sequence). The present invention therefore provides a substantially purified protein comprising the amino acid sequence from about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2. The present invention further provides a substantially purified protein comprising the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of OmpW is encoded by the ORF encoding OmpW of plasmid pER418 which is present in host cells of strain Pz418 (ATCC 98928). The deduced amino acid sequence of OmpW is presented as SEQ ID NO: 4. The first 21 amino acids of the protein shown in SEQ ID NO: 4 represent a signal sequence, and the present invention encompasses both an OmpW protein having only residues 22 to 215 of amino acids of SEQ ID NO: 4 (ie lacking the signal sequence) as an OmpW protein having the sequence of SEQ ID NO: 4 (ie, including the signal sequence). The present invention therefore provides a substantially purified protein comprising the amino acid sequence from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4. The present invention further provides a substantially purified protein comprising the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of Omp27 is encoded by the ORF encoding Omp27 of plasmid pER417 which is present in host cells of strain Pz417 (ATCC_98927). The deduced amino acid sequence of Omp27 is presented as SEQ ID NO: 6. The first 27 amino acids of the protein shown in SEQ ID NO: 6 represent a signal sequence, and the present invention encompasses both an Omp27 protein having only residues 28 to 258 amino acids of SEQ ID NO: 6 (ie lacking the signal sequence) as an Omp27 protein having the sequence of SEQ ID NO: 6 (ie, including the signal sequence). The present invention therefore provides a substantially purified protein comprising the amino acid sequence from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6. The present invention further provides a substantially purified protein comprising the amino acid sequence of SEQ ID NO: 6.

The amino acid sequence of OmpAI is encoded by the ORF encoding OmpAI of plasmid pER419 which is present in host cells of strain Pz419 (ATCC 98929). The deduced amino acid sequence of OmpAI is presented as SEQ ID NO: 8. The first 19 amino acids of the protein shown in SEQ ID NO: 8 represent a signal sequence, and the present invention encompasses both an OmpAI protein having only the 20 to 364 amino acid residues of SEQ ID NO: 8 (ie lacking the signal sequence) as an OmpAI protein having the sequence of SEQ ID NO: 8 (ie, including the signal sequence) ^. The present invention therefore provides a substantially purified protein comprising the amino acid sequence from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8. The present invention further provides a substantially purified protein comprising the amino acid sequence of SEQ ID NO: 8. The amino acid sequence of OmpA2 is encoded by the ORF encoding OmpA2 of plasmid pER420 which is present in host cells of strain Pz420 (ATCC 98930). The deduced amino acid sequence of OmpA2 is presented as SEQ ID NO; 10. The first 19 amino acids of the protein shown in SEQ ID NO: 10 represent a signal sequence, and the present invention encompasses both an OmpA2 protein having only the 20 to 369 amino acid residues of SEQ ID NO: 10 ( that is, lacking the signal sequence) as an OmpA2 protein having the sequence of SEQ ID NO: 10 (ie, including the signal sequence). The present invention therefore provides a substantially purified protein comprising the amino acid sequence from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10. The present invention further provides a substantially purified protein comprising the amino acid sequence of SEQ ID NO: 10. The APP proteins of the present invention, that is, Omp20, OmpW, Omp27, OmpAI and OmpA2, have molecular weights of about 19-20, about 23, about 27, about 29 and about 29 kDa, respectively, based on their electrophoretic mobility, and about 20, about 23, about 27, about 35 and about 35 kDa, respectively, based on their deduced amino acid sequences without the signal sequences. The present invention further provides polypeptides that are homologous with an APP protein of the present invention. As used in the present context to refer to polypeptides, the term "homolog" refers to a polypeptide that otherwise has an amino acid sequence that is selected from the group of amino acid sequences consisting of SEQ ID NOS: 2 , 4, 6, 8, and 10, or the same amino acid sequences but without their native signal sequences, in which one or more amino acid residues have been substituted conservatively by a different amino acid residue, in which the homologous polypeptide has a sequence of amino acids having a sequence identity of about 70%, more preferably about 80% and most preferably about 90%, as determined by any classical amino acid sequence analysis algorithm (such as one of the algorithms) BlastP of GENBANK), with respect to a polypeptide having an amino acid sequence that is selected from the group of secu amino acid gums that consist of the SEQ ID NOS: 2, 4, 6, 8 and 10, and in that the resulting homologous polypeptide is useful for practicing the present invention. Conservative amino acid substitutions are well known in the art. The rules for producing such substitutions include those that have been described by Dayhof, M.D., 1978, Nat. Biomed. Res. Found., Washington, D.C., Volume 5, Supplement 3, among other appointments. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in terms of acidity, polarity or voluminousness of their side chains. The genetically encoded amino acids are generally divided into four groups: (1) the acidic ones = aspartate, glutamate; (2) those of a basic nature = lysine, arginine, histidine; (3) those of non polar character = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) those of polar character without loading = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The amino acids phenylalanine, tryptophan and tyrosine are also classified together as aromatic amino acids. One or more replacements within any particular group, p. eg, that of a leucine for an isoleucine or valine, or that of an aspartate for a glutamate, or that of a threonine for a serine, or that of any other amino acid residue for a structurally related amino acid residue, p. eg, an amino acid residue with similar characteristics of acidity, polarity, bulking of the side chains, or with similarity in some combination of these characteristics, will generally have a negligible effect on the function or immunogenicity of the polypeptide. As used in the present context, a polypeptide is "useful for practicing the present invention" when the polypeptide: (a) is immunogenic, i.e., it can be used in a vaccine composition, either alone to induce a response Protein in pigs against APP, or in combination with other antigens of the present invention to contribute to the induction of a protective response in pigs against APP, or (b) can be used to induce the production of APP-specific antibodies when administered to pigs. a member of a mammalian species, whose antibodies are useful as diagnostic reagents; or (c) can be used as a reagent for diagnosis in order to detect the presence in a sample of blood or serum from a pig of anti-APP antibodies, resulting either from an infection with APP or a vaccination with a vaccine of the present invention.

The present invention also provides peptide fragments of an APP protein or a homologous polypeptide of the present invention. As used in the present context, a "peptide fragment" means a polypeptide that consists of less than the complete amino acid sequence of the corresponding full-length APP protein, either with or without the signal sequence, or a polypeptide homologue thereof, but comprising a sub-sequence of at least about , more preferably at least about 20, and most preferably at least about 30 amino acid residues of the amino acid sequence thereof, and which is useful for practicing the present invention, as the utility is defined above for the polypeptides. A peptide fragment of the present invention may comprise more than one sub-sequence of a full-length APP protein or of a homologous polypeptide of the present invention. For example, two or more different sub-sequences from the full-length APP protein or from a homologous poiypeptide can be pooled and made contiguous with each other in the peptide fragment, when they were not contiguous in the APP protein or in the polypeptide counterpart. In a preferred embodiment, a peptide fragment of the present invention comprises one or more sub-sequences that represent one or more epitopes of the APP protein or homologous polypeptide, or multiple copies of an epitope, against which antibodies can be elicited.

In a non-limiting embodiment, the present invention provides a peptide fragment of an APP protein of the present invention, which peptide fragment comprises the native signal sequence of the APP protein. In a preferred embodiment, the peptide fragment consists of an amino acid sequence that is selected from the group consisting of about amino acid residue 1 to about amino acid residue 27 of SEQ ID NO: 6 (Omp 27), from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 2 (Omp20), from about amino acid residue 1 to about amino acid residue 21 of SEQ ID NO: 4 (OmpW), from about amino acid residue 1 to about the amino acid residue 19 of SEQ ID NO: 8 (OmpAI), and from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 10 (OmpAI). Said signal sequences, and the polynucleotide molecules that encode them, are useful for a variety of purposes, including that of directing the cellular traffic of recombinant proteins expressed in APP or in other bacterial host cells, or with diagnostic probes to detect a polynucleotide molecule specific for APP in a sample of fluid or tissue from an infected animal. The present invention also provides full-length APP proteins or homologous polypeptides, in which sub-sequences thereof are arranged in a different relative order with respect to each other as compared to what is found in the native molecule, such that increases, alters or otherwise improves the antigenicity of the polypeptide. As used in the present context, the terms "antigen", "Antigenic" and the like, refer to a molecule that contains one or more epitopes that stimulate a host's immune system to produce a humoral and / or cellular response specific to certain antigens.

This term is also used interchangeably with the term "immunogen". As used in the present context, the term "epitope" or "epitope region" refers to a site on an antigen or hapten to which a specific antibody molecule binds. This term is also used interchangeably with that of "antigenic determinant". The present invention further provides fusion proteins, comprising a participating APP protein with or without a native signal sequence, a polypeptide homologous thereto, or a peptide fragment of the present invention, which has been linked to a carrier or a participant. in a fusion, whose fusion proteins are useful for practicing the present invention, as the utility is defined above for the polypeptides. See the} section 5.4.1 given below regarding examples of participants in a merger. Fusion proteins are useful for a variety of reasons, including that of increasing the stability of recombinantly expressed APP proteins, as antigenic components in an APP vaccine, to incite antisera against the particular APP participe protein, in order to study the biochemical properties of the protein of APP participant, to genetically engineer APP proteins with different or enhanced antigenic properties, to serve as diagnostic reagents, or to aid in the intensification or purification of expressed participant APP protein, as described, e.g. ., section 5.4.1 below. The fusion proteins of the present invention can be further engineered, using classical techniques to contain specific protease cleavage sites in such a way that the particular participant APP protein can be released from the carrier or fusion partner by treatment. with a specific protease. For example, a fusion protein of the present invention may comprise a dissociation site by thrombin or factor Xa, among others. The present invention further provides analogous compounds to, and derivatives of, an APP protein, a homologous polypeptide, a peptide fragment or a fusion protein of the present invention, when such analogs and derivatives are useful for practicing the present invention, such as the utility is defined above for the polypeptides. Manipulations that result in the production of analogous compounds can be carried out either at the level of the genes or at the level of the proteins, or at both levels, to improve or otherwise alter the biological or immunological characteristics of the particular polypeptide. from which the analogous compound is prepared. For example, at the level of the genes, a cloned DNA molecule, which is modified by an APP protein of the present invention, a compound analogous to that protein can be modified.

Such modifications include, but are not limited to, digestion with endonucleases, and mutations that create or destroy translation, initiation, or termination sequences, or that create variations in the coding region, or a combination thereof. Such techniques are described among other citations, in those of Maniatis et al., 1989, Molecular Cloninq, A Laboratorv Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloninq: A Laboratorv Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Innis and collaborators (editing coordinators), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Eriich (editing coordinator), PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference. Alternatively or additionally, an analogous compound of the present invention can be prepared by modification of an APP protein or other polypeptide of the present invention at the level of the protein. One or more chemical modifications of the protein can be carried out using known techniques including, but not limited to, one or more of the following; substitution of one or more L-amino acids of the protein by corresponding D-amino acids, amino acid-like compounds or amino acid mimics, so as to produce, eg, carbazones or tertiary centers; or a specific chemical modification, such as a proteolytic cleavage with, e.g., trypsin, chymotrypsin, papain or V8 protease, or a treatment with NaBH or cyanogen bromide, or acetylation, formylation, oxidation or reduction, etc. . An APP protein or other polypeptide of the present invention can be derivatized by conjugation with it or it of one or more chemical groups, including but not limited to, acetyl groups, bridging groups with sulfur, glycosyl groups, lipids, and phostates, and / or a second APP protein or other polypeptide of the present invention, or another protein such as, eg, serum albumin, keyhole limpet hemocyanin, or a commercially activated BSA, or a poly (amino acid) (p. .ej., polylysine), or a polysaccharide (e.g., sepharose, agarose, or modified or unmodified cellulases) among other compounds. Such conjugation is preferably carried out by covalent bonding with amino acid side chains and / or at the N-terminus or C-terminus of the APP protein. The methods for carrying out such conjugation reactions are well known in the field of protein chemistry. Derivatives useful for practicing the claimed invention also include those in which a water-soluble polymer, such as, e.g., polyethylene glycol, is conjugated to an APP protein or other polypeptide of the present invention or a compound analogous thereto. or it, thereby providing additional desirable properties, while retaining, at least in part, or enhancing the immunogenicity of the APP protein. These additional desirable properties include, eg, increased solubility in aqueous solutions, increased storage solubility, increased storage resistance, increased resistance to proteolytic degradation, or increased in vivo half-life. Water-soluble polymers are suitable for conjugation with an APP protein or with another polypeptide of the present invention, include, but are not limited to, polyethylene glycol homopolymers, propylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, in which said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, polyoxyethylated polyols, polo (vinyl alcohol), polysaccharides, poly (vinyl ethyl ethers) , and, β-poly [2-hydroxy-ethyl] -DL-aspartamide. Polyethylene glycol is particularly preferred. Methods for producing polymer conjugates of polypeptides, soluble in water, are known in the art and are described, inter alia, in the United States patent. 3,788,948; the patent of the United States. 3,960,830; the patent of the United States. 4,002,531; the patent of the United States. 4,055,635; the patent of the United States. 4,179,337; U.S. Patent 4,261,973; the patent of the United States. 4,412,989; the patent of the United States. 4,414,147; the patent of the United States. 4,415,665; the patent of the United States. 4,609,546; the patent of the United States. 4,732,863; the patent of the United States. 4,745,180; European Patent Documents (EP) 152,847; and EP 98,180; and Japanese Patent (JP) 5,792,435, the patents of which are incorporated herein by reference. It is intended that all subsequent references to "APP proteins" and the like include APP proteins, homologous polypeptides, peptide fragments, fusion proteins, analogs, and derivatives of the present invention, as previously defined these terms, unless otherwise indicated. . 2 Polynucleotide Molecules Encoding New APP Proteins The present invention further provides isolated polynucleotide molecules comprising a nucleotide sequence encoding an APP protein. As used in the present context, the terms "polynucleotide molecule", "polynucleotide sequence", "coding sequence", "open reading frame (ORF)" and the like are intended to be the same. refer to both DNA and RNA molecules, which may be single-stranded or double-stranded, which may include one or more prokaryotic sequences, cDNA sequences, sequences, genomic DNA sequences (including exons and introns), or chemically synthesized DNA and RNA sequences; and that can include both "the same way" and "anti-sense" strings. As used in the present context, the term "ORF" refers to the minimum nucleotide sequence that is required to encode a particular APP protein of the present invention without involving termination codons of any kind. The boundaries of the polynucleotide coding sequence are generally determined by the presence of an initiation codon at the 5 'end (of amino) and of a translation stop codon at the 3' terminal end (of carboxy). The production and manipulation of the polynucleotide molecules and the oligonucleotide molecules described herein below are within the skill in the art and can be carried out in accordance with the recombinant techniques that are described, inter alia, in those of Maniatis et al., 1989, op cit .; Ausubel et al., 1989, op cit .; Sambrook et al., 1989, op cit .; Innis et al., 1995, op cit., And Eriich, 1992, op cit. . 2.1 Polynucleotide molecules encoding Omp20 It is intended that references hereinafter to sequences of nucleotides from SEQ ID NO: 1, and to selected and substantial portions thereof, also refer to the corresponding nucleotide sequences related to Omp20. of plasmid pER416 which is present in host cells of strain Pz416 (ATCC 98926), unless otherwise indicated. In addition, it is intended that references hereinafter to amino acid sequences shown in SEQ ID NO: 2, and to peptide fragments thereof, also refer to the corresponding amino acid sequences encoded by the nucleotide sequence related to Omp20 of the Plasmid pER416, unless otherwise indicated. The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, Omp20, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding Omp20, the present invention, comprises the nucleotide sequence of SEQ ID NO: 1 from about nt 329 to about nt 790. In a more preferred embodiment, the isolated molecule of The polynucleotide encoding Omp20, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 1 from about nt 272 to about nt 790. In a non-limiting embodiment, the isolated polynucleotide molecule encoding Omp20, of the present invention, comprises the nucleotide sequence of SEQ ID NO: 1. The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule encoding Omp20 of APP of the present invention. The term "homologous", when used to refer to a polynucleotide molecule encoding Omp20, means a polynucleotide molecule having a nucleotide sequence: (a) encoding the same amino acid sequence as the nucleotide sequence of SEQ ID. NO: 1 from nt 329 to nt 790, but including one or more dumb changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) hybridizing with the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 20 to 172 of SEQ ID NO: 2 'under moderately stringent conditions, i.e., hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% sodium dodecylsulfate (SDS), EDTA 1 mM at 65 ° C, and washed at 0.02xSSC / 0.1% SDS at 42 ° C (see Ausubel et al. (Editing coordinators), 1989, Current Protocols in Molecular Bioloqy, Volume I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, on page 2.10.3), and that it is useful to practice the present invention. In a preferred embodiment, the polynucleotide homologous molecule hybridizes with the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 20 to 172 of SEQ ID NO: 2 under highly stringent conditions, i.e., hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C, and washed in 0.1xSSC / 0.1% SDS at 68 ° C (Ausubel et al., 1989, above), and it is useful for practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule is hybridized under highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 1 from nt 329 to nt 790, and is useful to practice the present invention.

As used in the present context, a polynucleotide molecule is "useful for practicing the present invention" when: (a) the polynucleotide molecule encodes a polypeptide that can be used in a vaccine composition or to induce itself , or to contribute in combination with one or more antigens other than the induction of, a protective response in pigs against APP, or (b) the polynucleotide molecule can be used directly in a DNA vaccine composition to induce by itself, or to contribute in combination with one or more different polynucleotide molecules or one or more antigens other than the induction of a protective response in pigs against APP; or (c) the polynucleotide molecule encodes a polypeptide that can be used to induce the production of antibodies specific for APP when administered to a member of a mammalian species, whose antibodies are useful as diagnostic reagents; or (d) the polynucleotide molecule encodes a polypeptide that can be used as a diagnostic reagent to detect the presence of antibodies specific for APP in a blood or serum sample from a pig, or (e) the polynucleotide molecule is it can be used as a diagnostic reagent to detect the presence of a polynucleotide molecule specific for APP in a sample of fluid or tissue from a pig infected by APP. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to the Omp20 protein of the present invention, as defined by "homologous polypeptides" above in section 5.1. The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned Omp20-related polynucleotide molecules of the present invention. As used in the present context, a "substantial portion" of an Omp20-related polynucleotide molecule means a polynucleotide molecule that consists of less than the complete nucleotide sequence of the particular full length Omp20-related polynucleotide molecule, but comprising at least 10%, and more preferably at least about 20%, of the nucleotide sequence of the particular full length Omp20-related polynucleotide molecule, and which is useful for practicing the present invention, as defined the "utility" above for polynucleotide molecules. In a non-limiting embodiment, the substantial portion of the Omp20-related polynucleotide molecule encodes a peptide fragment of any of the Omp20-related proteins or polypeptides., mentioned above of the present invention, as defined above, the term "peptide fragment". The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding the native Omp20 signal sequence from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 2 In a preferred embodiment, even if not limiting, the polynucleotide molecule encoding the Omp20 signal sequence comprises from about nt 272 to about nt 328 of SEQ ID NO: 1. The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising the Omp20 protein, a homologous polypeptide, or a peptide fragment, fused to a carrier or a fusion partner. . 5.2. Polynucleotide Molecules Which Code OmpW It is intended that references hereinafter to nucleotide sequences from SEQ ID NO: 3, and to selected and substantial portions thereof, also refer to the corresponding nucleotide sequences related to OmpW of the Plasmid pER418 which is present in host cells of strain Pz418 (ATCC 98928), unless otherwise indicated. In addition, it is intended that references hereinafter to amino acid sequences shown in SEQ ID NO: 4, and peptide fragments thereof, also refer to the corresponding amino acid sequences encoded by the nucleotide sequence related to OmpW of the Plasmid pER418, unless otherwise indicated.

The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, OmpW, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding OmpW, the present invention, comprises the nucleotide sequence of SEQ ID NO.3 from about nt 439 to about nt 1.023. In a more preferred embodiment, the isolated polynucleotide molecule encoding OmpW, the present invention, comprises the nucleotide sequence of SEQ ID NO: 3 from about nt 376 to about nt 1, 023. In a non-limiting embodiment, the isolated polynucleotide molecule encoding OmpW, the present invention, comprises the nucleotide sequence of SEQ ID NO: 3. The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule that encodes OmpW of APP of the present invention. The term "homologous", when used to refer to a polynucleotide molecule encoding OmpW, means a polynucleotide molecule having a nucleotide sequence: (a) encoding the same amino acid sequence as the nucleotide sequence of SEQ ID. NO: 3 from nt 439 to nt 1, 023, but including one or more dumb changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) which hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 22 to 215 of SEQ ID NO: 4 under moderately stringent conditions, i.e., hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM ETDA at 65 ° C, and washing in 0.02 x SSC / 0.1% SDS at 42 ° C (Ausubel et al., 1989, above), and which is useful for practicing the present invention. In a preferred embodiment, the polynucleotide homologous molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 22 to 215 of SEQ ID NO.

NO: 4 under highly stringent conditions, ie, hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C, and washing in 0.1xSSX / 0.1% SDS at 68% C (Ausubel et al., 1989, supra), and that it is useful to practice the present invention. In a preferred embodiment, the polynucleotide homologous molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 3 from nt 439 to nt 1, 023, and is useful for practicing the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous with the OmpW protein of the present invention, as defined by the "homologous polypeptides" above. The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned OmpW-related polynucleotide molecules of the present invention. As used in the present context, a "substantial portion" of an OmpW-related polynucleotide molecule means a polynucleotide molecule that consists of less than the entire nucleotide sequence to the particular full length OmpW-related polynucleotide molecule, but comprising at least 10%, and more preferably at least about 20%, of the nucleotide sequence of the particular full length OmpW-related polynucleotide molecule, and which is useful for practicing the present invention, as defined the "utility" above for polynucleotide molecules. In a non-limiting embodiment, the substantial portion of the OmpW-related polynucleotide molecule encodes a peptide fragment of any of the aforementioned OmpW-related proteins or polypeptides of the present invention, as defined above. "peptide fragment". The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes the native OmpW signal sequence from about amino acid residue 1 to about amino acid residue 21 of SEQ ID NO: 4. In a preferred embodiment, even if not limiting, the polynucleotide molecule encoding the OmpW signal sequence comprises from about nt 376 to about nt 438 of SEQ ID NO: 3.

The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising the OmpW protein, a homologous polypeptide, or a peptide fragment, fused to a carrier or a fusion partner. . 2.3. Polynucleotide Molecules that Code Omp27 It is intended that references hereinafter to nucleotide sequences from SEQ IDN: 5, and to selected and substantial portions thereof, also refer to the corresponding nucleotide sequences related to Omp27 of the plasmid pER417 which is present in host cells of strain Pz417 (ATCC 98927), unless otherwise indicated. Furthermore, it is intended that references hereinafter to amino acid sequences shown in SEQ ID NO: 6, and to peptide fragments thereof, also refer to the corresponding amino acid sequences encoded by the nucleotide sequence related to Omp27 of the Plasmid pER417, unless otherwise indicated. The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, Omp27, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding Omp27, the present invention, comprises the nucleotide sequence of SEQ ID NO: 5 from about nt 238 to about nt 933. In a more preferred embodiment, the isolated molecule of polynucleotide that encodes Omp27, the present invention, comprises the nucleotide sequence of SEQ ID NO: 5 from about nt 157 to about nt 933. In a non-limiting embodiment, the isolated polynucleotide molecule encoding Omp27, the present invention, comprises the nucleotide sequence of SEQ ID NO: 5. The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule encoding Omp27 of APP of the present invention. The term "homologous", when used to refer to a polynucleotide molecule encoding Omp27, means a polynucleotide molecule having a nucleotide sequence: (a) encoding the same amino acid sequence as the nucleotide sequence of SEQ ID. NO: 5 from nt 238 to nt 933, but including one or more dumb changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) which hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 28 to 258 of SEQ ID NO: 6 under moderately stringent conditions, i.e., hydridation to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM ETDA at 65 ° C, and washing in 0.02 x SSC / 0.1% SDS at 42 ° C (Ausubel et al., 1989, above), and which is useful for practicing the present invention. In a preferred embodiment, the polynucleotide homologous molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding the amio acid residues 28 to 258 of SEQ ID NO: 6 under highly stringent conditions, i.e., hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C, and washed in 0.1xSSC / 0.1% SDS at 68 ° C (Ausubel et al., 1989, above), and it is useful for practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule is hybridized in highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 5 from nt 238 to nt 933, and is useful to practice the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is homologous to the Omp27 protein of the present invention, as the "homologous polypeptides" are defined above. The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the aforementioned Omp27-related polynucleotide molecules of the present invention. As used in the present context, a "substantial portion" of a polynucleotide molecule related to Omp27 means a polynucleotide molecule that consists of less than the complete nucleotide sequence of the particular full length Omp27-related polynucleotide, but comprising at least 10%, and more preferably at least about 20%, of the nucleotide sequence of the particular full length Omp27-related polynucleotide, and which is useful for practicing the present invention, as defined by " utility "above for polynucleotide molecules. In a non-limiting embodiment, the substantial portion of the Omp27-related polynucleotide molecule encodes a peptide fragment of any of the aforementioned Omp27-related proteins or polypeptides of the present invention, as defined above. "peptide fragment". The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding the native Omp27 signal sequence from about amino acid residue 1 to about amino acid residue 27 of SEQ ID NO: 6. In a preferred embodiment, although not limiting, the polynucleotide molecule encoding the Omp27 signal sequence comprises from about nt 157 to about nt 237 of SEQ ID NO: 5. The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising the Omp27 protein, a homophobic polypeptide, or a peptide fragment, fused to a carrier or a fusion partner. . 2.4. Molecule of Polynucleotides that Code OmpAI The references hereinbelow are intended to refer to nucleotide sequences from SEQ ID NO: 7, and to selected and substantial portions thereof, refer to the corresponding nucleotide sequences related to OmpAI of the plasmid pER419 which is present in host cells of strain Pz419 (ATCC 98929), unless otherwise indicated. Furthermore, it is intended that references hereinafter to amino acid sequences shown in SEQ ID NO: 8, and to peptide fragments thereof, also refer to the corresponding amino acid sequences encoded by the nucleotide sequence related to OmpAI of the Plasmid pER419, unless otherwise indicated. The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, OmpAI, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding OmpAI, the present invention, comprises the nucleotide sequence of SEQ ID NO: 7 from about nt 671 to about nt 1, 708. In a more preferred embodiment, the isolated polynucleotide molecule encoding OmpAI, the present invention, comprises the nucleotide sequence of SEQ ID NO: 7 from about nt 614 to about nt 1, 708. In a non-limiting embodiment, the isolated polynucleotide molecule encoding OmpAI, the present invention, comprises the nucleotide sequence of SEQ ID NO: 7. The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule encoding APP OmpAI of the present invention. The term "homologous", when used to refer to a polynucleotide molecule encoding OmpAI means a polynucleotide molecule having a nucleotide sequence: (a) encoding the same amino acid sequence as the nucleotide sequence of SEQ ID NO: 7 from nt 671 to nt 1, 708, but including one or more silent changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 20 to 364 of SEQ ID NO: 8 under moderately stringent conditions, i.e., hybridization to a DNA bound to a filter in NaHP04 0.5M, 7% SDS, 1 mM EDTA at 65 ° C, and washing in 0.02xSSC / 0.1% SDS at 42 ° C (Ausubel et al., 1989, above), and which is useful for practicing the present invention. In a preferred embodiment, the polynucleotide homologous molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 20 to 364 of SEQ ID NO: 8 under highly stringent conditions, i.e., hybridization to a DNA attached to a filter in 0.5 M NaHP04. 7% SDS, 1 mM EDTA at 65 ° C, and washed in 0.1xSSC / 0.1% SDS at 68 ° C (Ausubel et al., 1989, above), and is useful for practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 7 from nt 671 to nt 1,708, and it is useful for practicing the present invention. The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous with the OmpAI protein of the present invention, as defined by "homologous polypeptides" above. The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the OmpAI-related polynucleotide molecules referred to above., of the present invention. As used in the present context, a "substantial portion" of an OmpAI-related polynucleotide molecule means a polynucleotide molecule that consists of less than the entire nucleotide sequence of the particular full length OmpAI-related polynucleotide molecule, but comprising at least 10%, and more preferably at least about 20%, of the nucleotide sequence of the particular full length OmpAI-related polynucleotide molecule, and which is useful for practicing the present invention, as defined by "utility" above for polynucleotide molecules. In a non-limiting embodiment, the substantial portion of the OmpAI-related polynucleotide molecule encodes a peptide fragment of any of the aforementioned OmpAI-related proteins or polypeptides of the present invention, as defined above. "peptide fragment". The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes the native OmpAI signal sequence from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 8. In a preferred embodiment, although not limiting, the polynucleotide molecule encoding the OmpAI signal sequence comprises from about nt 614 to about nt 670 of SEQ ID NO: 7. The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising the OmpAI protein, a homologous polypeptide, or a peptide fragment, fused to a carrier or a fusion partner. . 2.5. Polynucleotide molecules encoding OmpA2 It is intended that references hereinafter to nucleotide sequences from SEQ ID NO: 9, and to selected and substantial portions thereof, also refer to the corresponding nucleotide sequences related to OmpA2 of the pER420 plasmid that is present in host cells of the strain Pz420 (ATCC 98930), unless otherwise indicated. Furthermore, it is intended that references hereinafter to amino acid sequences shown in SEQ ID NO: 10, and to peptide fragments thereof, also refer to the corresponding amino acid sequences encoded by the nucleotide sequence related to OmpA2 of the Plasmid pER420, unless otherwise indicated. The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the APP protein, OmpA2, with or without a signal sequence. In a preferred embodiment, the isolated polynucleotide molecule encoding OmpA2, the present invention, comprises the nucleotide sequence of SEQ ID NO: 9 from about nt 254 to about nt 1, 306. In a more preferred embodiment, the isolated polynucleotide molecule encoding OmpA2, the present invention, comprises the nucleotide sequence of SEQ ID NO: 9 from about nt 197 to about nt 1, 306. In a non-limiting embodiment, the isolated polynucleotide molecule encoding OmpA2, the present invention, comprises the nucleotide sequence of SEQ ID NO: 9. The present invention further provides an isolated polynucleotide molecule that is homologous with a polynucleotide molecule encoding APP OmpA2 of the present invention. The term "homologue", when used to refer to a polynucleotide molecule encoding OmpA2, means a polynucleotide molecule encoding OmpA2, means a polynucleotide molecule having a nucleotide sequence: (a) encoding the same sequence of mino acids that the nucleotide sequence of SEQ ID NO: 9 from nt 254 to nt 1, 306, but including one or more dumb changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) which hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 20 to 369 of SEQ ID NO: under moderately stringent conditions, ie, hybridization to a DNA bound to a filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C, and washing in 0.02xSSC / 0.1% SDS at 42 ° C ( Ausubel et al., 1989, supra), and which is useful for practicing the present invention. In a preferred embodiment, the polynucleotide homologous molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence encoding amino acid residues 20 to 369 of SEQ ID NO: 10 under highly stringent conditions, i.e., hybridization to a DNA bound to a filter in 0.5 M NaHP0, 7% SDS, 1 mM EDTA at 65 ° C, and washed in 0.1xSSC / 0.1% SDS at 68 ° C (Ausubel et al., 1989, above), and it is useful for practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule is hybridized under highly stringent conditions to the complement of a polynucleotide molecule consisting of the nucleotide sequence of SEQ ID NO: 9 from nt 254 to nt 1, 306 and is useful for practicing the present invention.

The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the OmpA2 protein of the present invention, as defined by the "homologous polypeptides" above. The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial portion of any of the OmpA2-related ponucleotide molecules that are mentioned above., of the present invention. As used in the present context, a "substantial portion" of a polynucleotide molecule related to OmpA2 means a polynucleotide molecule that consists of less than the complete nucleotide sequence of the particular full length OmpA2-related polynucleotide molecule, but comprising at least 10%, and more preferably at least about 20%, of the nucleotide sequence of the particular full length OmpA2-related polynucleotide molecule, and which is useful for practicing the present invention, as defined the "utility" above for polynucleotide molecules. In a non-limiting embodiment, the substantial portion of the OmpA2-related polynucleotide molecule encodes a peptide fragment of any of the aforementioned OmpA2-related proteins or polypeptides of the present invention, as defined above. "peptide fragment".

The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding the native OmpA2 signal sequence from about amino acid residue 1 to about amino acid residue 19 of SEQ ID.

NO: 10. In a preferred embodiment, even if not limiting, the polynucleotide molecule encoding the OmpA2 signal sequence comprises from about nt 197 to about nt 253 of SEQ.

ID NO: 9. The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising the OmpA2 protein, a homologous polypeptide, or a peptide fragment, fused to a carrier or a participant in the fusion. . 3. Oligonucleotide Molecules The present invention further provides oligonucleotide molecules that hybridize to any of the aforementioned polynucleotide molecules of the present invention, or hybridize to a polynucleotide molecule having a nucleotide sequence that is the complement of any of the aforementioned polynucleotide molecules of the present invention. Said oligonucleotide molecules preferably have a length of at least about 10 to 15 nucleotides, but may be extended to the length of any sub-sequence of SEQ ID NOS: 1, 3, 5, 7 or 9, or of a homologous polynucleotide molecule. of them, and can hybridize with one or more of the aforementioned polynucleotide molecules under highly stringent conditions. For shorter oligonucleotide molecules, an example of highly stringent conditions includes washing in 6xSSC / 0.5% sodium pyrophosphate at about 37 ° C for -14 bases oligos, at about 48 ° C for -17 bases oligos, and at about 55 ° C for oligos of -20 bases, and approximately 60 ° C for oligos of -23 bases. For longer oligonucleotide molecules (ie, greater than about 100 nts) examples of highly stringent conditions are provided in section 5.2 above. Other suitable hybridization conditions can be determined and adjusted as is known in the art, depending on the particular oligonucleotide and polynucleotide molecules that are used. In a preferred embodiment, an oligonucleotide molecule of the present invention hybridizes under highly stringent conditions to a polynucleotide molecule that consists of a nucleotide sequence selected from SEQ ID NOS: 1, 3, 5, 7 or 9, or with a A polynucleotide molecule consisting of a nucleotide sequence that is the complement of a nucleotide sequence selected from SEQ ID NOS: 1, 3, 5, 7 or 9. In a non-limiting embodiment, an oligonucleotide molecule the present invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 15-47 and 49-93 and the complements of said sequences. In a non-limiting embodiment, the oligonucleotide molecule consists of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 15-47 and 49-93 and the complements of said sequences. The oligonucleotide molecules of the present invention are useful for a variety of purposes, including as primers in the amplification of a polynucleotide molecule that encodes a protein of APP to be used, p. eg, in a differential diagnosis of diseases, or to code or act as anti-sense molecules in gene regulation. The amplification can be used to detect the presence of a polynucleotide molecule that encodes an APP protein in a tissue or fluid sample, e.g. eg, in mucous or bronchial fluid, from an infected animal. The production of a specific amplification product can help confirm a diagnosis of a bacterial infection by APP, while the lack of an amplified product may indicate a lack of such an infection. The oligonucleotide molecules described herein can also be used to isolate homologous genes from other species or strains of Actinobacillus, or from other bacteria. The amplification can be carried out using appropriately designed oligonucleotide molecules in conjunction with classical techniques, such as Polymerase Chain Reaction (PCR), even though other amplification techniques known in the art can be used, eg, the ligase chain reaction. For example, for a PCR, a mixture is prepared comprising appropriately engineered primers, a template comprising the nucleotide sequence to be amplified, and appropriate PCR enzymes and buffers as are known in the art, and made from according to classical protocols for amplifying a specific polynucleotide sequence related to template APP. Methods to perform the PCR are described, among other appointments, in those of Innis and collaborators (editing coordinators), 1995, op cit .; and Eriich (editing coordinator), 1992 op cit .. . 4. Recommandative Expression Systems . 4.1. Cloning and Expression Vectors The present invention further provides compositions for cloning and expressing any of the polynucleotide molecules of the present invention, including recombinant cloning vectors and recombinant expression vectors, comprising a polynucleotide molecule of the present invention, host cells transformed with any of these vectors, and cell lines derived from them. The recombinant vectors of the present invention, particularly the expression vectors, are preferably constructed in such a way that the coding sequence of the polynucleotide molecule (hereinafter referred to as the "APP coding sequence") is in operative association with one or more regulatory elements necessary for transcription and translation of the APP coding sequence in order to produce a polypeptide. As used in the present context, the term "regulatory element" includes, but is not limited to, nucleotide sequences encoding inducible and non-inducible promoters, enhancers, operators and other elements known in the art which serve to direct and / or regulate the expression of polynucleotide coding sequences. Also, as used in the present context, the APP coding sequence is in "operative association" with one or more regulatory elements when the regulatory elements regulate and efficiently allow the transcription of the coding sequence or the translation of the coding sequence. your mRNA, or both. Methods for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements, including in vitro recombinant techniques, synthesis techniques, and in vivo genetic recombination are well known in the art. See, p. eg, the techniques described in the citations of Maniatis et al., 1989, op cit .; Ausubel et al., 1989, op cit; Sambrook et al., 1989, op cit .; Innis et al., 1995, op cit .; and Eriich, 1992, op cit. A variety of expression vectors are known in the art which can be used to express any of the APP coding sequences of the present invention, including baceteriophage DNA expression vectors, plasmid DNA and cosmid DNA, which contain a APP coding sequence. Typical prokaryotic expression vector plasmids, which can be engineered to contain an APP coding sequence of the present invention, include pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond, CA), pPL and Pkk223 (Pharmacia, Piscataway, NJ), pQE50 (Qiagen, Chatsworth, CA), and serial pGEX plasmids (Pharmacia), among many others. Typical eukaryotic expression vectors, which can be engineered to contain an APP coding sequence of the present invention, include an ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, CA), intensifier-based systems. cytomega-lovirus promoters (Promega, Madison, Wl; Stratagenen, La Jolla, Ca; Invitrogen); baculovirus-based expression systems (Promega) and plant-based expression systems, among others. The regulatory elements of these and other vectors may vary in their intensity and in their specificities. Depending on the host system and vector used, any of a number of appropriate transcription and translation elements may be used. For example, when cloning into mammalian cell systems, promoters isolated from the genome of mammalian cells, eg, the mouse metallothionein promoter, or from viruses growing on these cells, can be used, .ej., the 7.5 k promoter of the vaccinia virus or the long terminal repeat of Moloney murine sarcoma virus. Promoters obtained by recombinant DNA or synthesis techniques can also be used to provide transcription of the inserted coding sequence.

In addition, expression from certain promoters may be elevated in the presence of particular inducers, eg, zinc and cadmium atoms for metallothionein promoters. Non-limiting examples of transcriptional regulatory regions or promoters include, for bacteria, the β-gal promoter, the T7 promoter, the TAC promoter, the left and right promoters of β, promoters of trp and lac, promoters of fusion of trp with lap, etc; for yeasts, glycolytic enzyme promoters, such as promoters of ADH-I and II, the GPK promoter, the PGI promoter, the TRP promoter, etc; and for mammalian cells, the early and late promoters of SV40, the late major promoters of adenovirus, among others. Specific initiation signals are also required for a sufficient translation of inserted coding sequence. These signals typically include an ATG start codon and adjacent sequences. In cases where the APP coding sequence of the present invention, including its own initiation codon and adjacent sequences, are inserted into the appropriate expression vector, no additional translation control signals are needed. However, in cases where only a portion of an APP coding sequence is introduced, exogenous translation control signals, including the ATG start codon, may be required. These exogenous signals of translation control and these initiation codons can be obtained from a variety of sources, both natural and synthetic. In addition, the initiation codon must be in phase with the reading frame of the coding regions in order to ensure the translation within the frame of the entire insert. Expression vectors can also be constructed that express a fusion protein comprising any of the APP-related polypeptides of the present invention, fused to a carrier or fusion partner. Such fusion proteins can be used for a variety of purposes, such as to increase the stability of a recombinantly expressed APP protein, to incite antisera against an APP protein, to study the biochemical properties of an APP protein, to treat by genetic engineering an APP protein exhibiting altered immunological properties, or to aid in the identification or purification of a recombinantly expressed APP protein. Possible fusion protein expression vectors include, but are not limited to, vectors that incorporate sequences encoding a protective peptide, such as the one described later in section 8.2, as well as fusions of β-galactosidase and trpE, fusions of maltose-binding protein, glutathione-S-transferase (GST) fusions, and polyhistidine fusions (carrier regions). Methods that can be used to construct expression vectors encoding these and other fusion proteins are well known in the art. The fusion proteins may be useful to aid in the purification of the expressed protein. In non-limiting embodiments, e.g., a protein fusion protein of APP-maltose binding protein can be purified using an amylose resin; a protein fusion protein of APP-GST can be purified using glutathione and agarose globules; and an APP-polyhistidine protein fusion protein can be purified using a divalent nickel resin. Alternatively, antibodies against a protein or a vehicle peptide can be used for purification by affinity chromatography of the fusion protein. For example, a nucleotide sequence encoding the target epitope of a monoclonal antibody can be genetically engineered into the expression vector in operable association with the regulatory elements and can be positioned such that the expressed epitope is fused to a protein of APP of the present invention. In a non-limiting embodiment, a nucleotide sequence encoding the FLAG® epitope tag (International Biotechnologies Inc.), which is a hydrophilic label peptide, can be introduced by classical techniques into the expression vector at a site corresponding to the terminal amino or carboxyl terminus. of the APP protein. The FLAG® epitope fusion product and a polypeptide can then be detected and affinity purified using commercially available anti-FLAG® antibodies.

The expression vector of the present invention can also be engineered to contain polylinker sequences encoding specific sites of protease dissociation, such that the expressed APP protein can be detached from the carrier or participant region. in fusion by treatment with a specific protease. For example, the vector for the fusion protein may include a sequence of nucleotides encoding a dissociation site by thrombin or by factor Xa, among others. A signal sequence located upstream with respect to the APP coding sequence, and in reading frame with it, can be genetically engineered into the expression vector by known methods to direct trafficking and secretion of the APP polypeptide voiced. Non-limiting examples of signal sequences, include those that are not? native to the APP proteins of the present invention, as described herein as well as signal sequences derived from factor-a, immunoglobulins, outer membrane proteins, penicillinase and T cell receptors, among others. To help make the selection of host cells transformed or transfected with a recombinant vector of the present invention, the vector can be engineered which further comprises a coding sequence for a reporter gene product and another selectable marker. Said coding sequence is preferably in operative association with the regulatory elements, as described above. Reporter genes that are useful for practicing the invention are well known in the art and include those encoding chloramphenicol acetyltransferase (CAT), the green fluorescent protein, firefly luciferase and a human growth hormone, among others. Nucleotide sequences encoding selectable markers are well known in the art, and include those that encode gene products that confer resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those which encode thymidine kinase activity, or resistance to methotrexate, ampicillin, kanamycin, chloramphenicol, zeocin, tetracycline and carbenicillin, among many other substances. In specific, though not limiting, embodiments, the present invention provides the following plasmid cloning vectors, constructed as described below in section 11, which are present in host cells that have been deposited in the American Type Culture culture collection. Collection (ATCC): the plasmid pER416 which is present in host cells of strain Pz416 (ATCC 98926), and whose plasmid comprises the ORP of omp20; plasmid pER418 which is present in host cells of strain Pz418 (ATCC 98928), and whose plasmid comprises the ORP of omp \ N; plasmid pER417 which is present in host cells of strain Pz417 (ATCC 98927), and whose plasmid is the ORF of omp27; the plasmid pE419 which is present in host cells of strain Pz419 (ATCC 98929), whose plasmid comprises the ompM ORF; plasmid pER420 which is present in host cells of strain Pz420 (ATCC 98930), whose plasmid comprises Omp A2 ORF.

Transformation of host cells The present invention further provides host cells transformed with a polynucleotide molecule or a recombinant vector of the invention, and the cell lines that are derived therefrom. Host cells useful for practicing the present invention can be either prokaryotic or eukaryotic. Said transformed host cells include, but are not limited to, microorganisms, such as bacterial cells transformed with bacteriophage DNA expression vectors, plasmid DNA or cosmid DNA; or yeast cells transformed with recombinant expression vectors; or animal cells, such as insect cells infected with recombinant virus expression vectors, eg, baculovirus, or mammalian cells infected with recombinant virus expression vectors, eg, adenovirus or vaccinia virus, among others Bacterial cells are generally preferred as host cells. An E. coli strain, such as, eg, strain DH5a, available from Gibco BRL, Life Technologies (Gaithersburg, MD), or E. coli strain LW14, can be typically used, as described below. Cells from eukaryotic hosts, including yeast cells, and mammalian cells, such as those from a mouse, pig, cow, monkey, or human hamster cell line can also be effectively used. Examples of eukaryotic host cells that can be used to express the recombinant protein of the invention include Chinese hamster ovary (CHO) cells (e.g., accession number ATCC CCL61) and mouse embryo cells NIH Swiss NIH / 3T3 (eg with the access number in ATCC CRL 1658). A recombinant vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous cell culture. The vector is generally introduced into host cells according to known techniques such as, eg, by calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, transfection with DEAE-dextran, transduction, conjugation, or bombardment with microprojectiles. The selection of transformants can be performed by classical methods, such as by selection for cells expressing a selectable marker, eg, of antibiotic resistance, associated with the recombinant vector. Once the vector has been introduced into the host cell, the integration and maintenance of the APP coding sequence either in the host cell genome or episomally, can be confirmed by classical techniques, eg, by analysis by Southern hybridization, restriction enzyme analysis, PCR analysis, including reverse transcriptase PCR (rt-PCR, reverse transcriptase-PCR) or by immunological analysis to detect the expected protein product. Host cells that contain and / or express the APP coding sequence can be identified by any of at least four general approaches, which are well known in the art, including: (i) DNA hybridization with DNA, DNA with RNA or RNA with anti-sense RNA; (I) detection of the presence of gene functions "markers"; (ni) determining the level of transcription, as measured by the expression of mRNA transcripts specific for APP in the host cell; and (iv) detection of the presence of a protein product of Mature APP, as measured, eg, by immunoassay. . 4.3 Expression of recombinant polypeptides Once the APP coding sequence has been introduced in a stable manner into the appropriate host cell, the transformed host cell is propagated locally, and the resulting cells are grown under conditions that lead to maximum production of the APP protein. Such conditions typically include growing said cells to a high density. When the expression vector comprises an inducible promoter, suitable induction conditions such as, eg, temperature shift, nutrient depletion, addition of free inductors (e.g. carbohydrate-like compounds, such as ε-propyl-β-D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic byproducts, or the like. When the APP protein, recombinantly expressed, is retained within the host cells, the cells are harvested and used, and the APP protein is purified or substantially isolated from the lysed material under known extraction conditions in the technology to minimize degradation. of proteins, such as, eg at 4 ° C, or in the presence of protease inhibitors, or both at the same time. When the recombinantly expressed APP protein is secreted from the host cells, the spent nutrient medium can simply be picked up and the APP polypeptide can be substantially purified or isolated therefrom. The recombinantly expressed APP protein can be purified or partially or substantially isolated from cell lysates or from a culture medium, as appropriate, using classical methods, including, but not limited to, any combination of the following methods : ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. The increasing purity of the APP polypeptide of the present invention can be determined based, eg, on size, or on reactivity with an antibody specific for the APP polypeptide, or on the presence of a fusion tag. For use in practicing the present invention, e.g., in a vaccine composition, the APP protein can be used in an unpurified state, such as has been secreted into the culture fluid, or as is present in host cells or in a cell lysate material, or in a substantially purified or isolated form. As used in the present context, an APP protein is "substantially purified" when the protein constitutes more than about 20% by weight of the protein in a particular preparation.

Also, as used in the present context, an APP protein is "isolated" when this protein constitutes at least about 80% by weight of the protein in a particular preparation. The present invention therefore provides a method for preparing a APP protein, a homologous polypeptide, a peptide fragment or a fusion protein of the present invention, which comprises culturing a host cell transformed with a recombinant expression vector, said vector comprising said vector. of recombinant expression a polynucleotide molecule which in turn comprises a nucleotide sequence encoding: (a) an APP protein comprising the amino acid sequence of SEQ ID NO: 2,4,6,8 or 10 with or without its native signal sequence; or (b) a polypeptide that is homologous with the APP protein from (a); or (c) a peptide fragment of the APP protein from (a) or a homologous polypeptide from (b); or (d) a fusion protein comprising the APP protein from (a), the homologous polypeptide of (b) or the peptide fragment from (c), fused to a fusion partner; whose polynucleotide molecule is in operative association with one or more regulatory elements that control the expression of the polynucleotide molecule in the host cell, under conditions that lead to the production of the APP protein, the homologous polypeptide, the peptide fragment or the fusion protein, and recover the APP protein, the homologous polypeptide, the peptide fragment or the fusion protein, from the cell culture. Once an APP protein of the present invention has been obtained in sufficient purity, it can be characterized by classical methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, serological reactivity, etc. The amino acid sequence of the APP protein can be determined using classical peptide sequencing techniques. The APP protein can be further characterized using a hydrophilicity analysis (see, eg, Hopp and Woods, 1981, Proc Nati Acad Sci USA 78: 3824); or algorithms of analog software logic systems, to identify hydrophobic regions in hydrophilic. A structural analysis can be carried out to identify regions of the APP protein that adopt specific secondary structures. Biophysical methods, such as X-ray crystallography (Engstrom, 1974), Biochem. Exp. Biol. 1 1: 7-13), computer modeling (Fletterick and Zoller (editing coordinators), 1986, in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), nuclear magnetic resonance (NRM) and mass spectrometry, can also be used to characterize the protein. The information obtained from this studies can be used, eg, to design more effective vaccine compositions, or to select vaccines comprising only specific portions of the APP protein. An APP protein that is useful for practicing the present invention is a polypeptide that: (a) is immunogenic, i.e., capable of inducing on its own, or capable of contributing in combination with other APP proteins or other antigens related to APP to the induction of a protective response against APP when administered to pigs: or (b) is capable of inducing the production of anti-APP antibodies when administered to a member of a mammalian species; or (c) can be used as a diagnostic reagent for the purpose of detecting the presence of anti-APP antibodies in a blood or serum sample from a pig resulting from an infection with APP or a vaccination with a vaccine. the present invention. Said protein, once it has been prepared, it can be identified using routine screening procedures known in the art. For example, the ability to induce, or contribute to the induction of, a protective immune response against APP can be identified by administering the APP protein, alone or in combination with other APP proteins or other antigens related to APP, respectively. to a pig, and testing for the resulting induction of APP neutralizing antibodies, or for the resulting ability of the vaccinated animal to withstand subsequent stimulation with APP compared to an unvaccinated control. The ability to induce the production of APP-specific antibodies can be identified by administering the APP protein to a model animal, such as a mouse, pig, lamb, goat, horse, cow, etc., and testing the serum of the animals in regarding the presence of antibodies specific for APP with the use of classical techniques. The ability to use the APP protein as a diagnostic reagent can be determined by exposing the APP protein to a sample of blood or serum from an animal, which previously or at that time was infected with APP, or had previously been vaccinated with a vaccine of the present invention, and detecting the binding to the protein of APP of specific antibodies to APP from the sample, using classical techniques, such as with an ELISA. . 5. APP vaccines The present invention further provides a vaccine for protecting pigs against APP, comprising an immunologically effective amount of one or more of the following substances: (a) an APP protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2,4,6,8 or 10, with or without its native signal sequence; (b) a polypeptide that is homologous with the APP protein from (a); (c) a peptide fragment consisting of a subsequence of the APP protein from (a) or the homologous polypeptide from (b); (d) a fusion protein comprising the APP protein from (a), a homologous polypeptide from (b), or a peptide fragment from (c), fused to a partner in the fusion; (e) a compound analogous to, or derived from, the APP protein from (a), a homologous polypeptide from (b), a peptide fragment from (c), or a fusion protein from (d); or (f) a polynucleotide molecule comprising a nucleotide sequence encoding the APP protein from (a), a homologous polypeptide from (b), a peptide fragment from (c), a fusion protein from (d), or an analogous compound or derivative from (e); whose substances, APP protein, homologous polypeptide, peptide fragment, fusion protein, analogous compound, derivative or polynucleotide molecule, can induce by themselves, or in combination with one or more other antigens of such type contribute to the induction of , a protective response against APP in pigs; and an acceptable vehicle in veterinary medicine. As used in the present context, the term "immunologically effective amount" refers to the amount of antigen that is capable of inducing, or contributing to the induction of, a protective immune response in pigs against one or more APP serotypes after from a single administration or after multiple administrations. The phrase "capable of inducing a protective immune response" is used in its broad sense in the present context to include the induction of, or the increase in, any response based on immunity in a pig in response to a vaccination, inclusive or either an antibody or an immune response mediated by cells or antibodies, or both, which serves to protect the vaccinated animal against APP. The terms "protective immune response", "protect", and the like, as used in the present context, are not limited to the absolute prevention of a porcine pneumonia associated with APP or to the absolute prevention of an infection of pigs by APP , but are intended to refer also to any reduction in the degree or regime of infection by the pathogen, or any reduction in the severity of the disease or in any symptom or condition resulting from an infection with the pathogen, including, . eg, any detectable decrease in the pathology of the lungs, as compared to that which occurs in an infected control animal, without vaccination. The vaccine compositions of the present invention can be formulated following an accepted convention and using standard buffers, carriers, stabilizers, diluents, preservatives and solubilizers, and can also be formulated to facilitate prolonged release. The diluents may include water, saline, dextrose, ethanol, glycerol and the like. Additives to confer isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose, among others. The stabilizers include albumin, among others. Adjuvants may be optionally employed in the vaccine.

Non-limiting examples of adjuvants include the RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel, oil-in-water type emulsions, water-in-oil type emulsions such as, e.g. eg, complete and incomplete adjuvants of -Freund, a block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), the adjuvant AMPHIGEN®, saponin, Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), or other fractions of saponins, SEAM-1, monophosphoryl lipid A, the lipid-amine adjuvant Avridine, an unstable enterotoxin against heat. from E. coli (recombinant or otherwise), cholera toxin, or a muramildipeptide, among many others. The vaccine may further comprise one or more other immunomodulatory agents such as, e.g. eg, interleukins, interferons or other cytokines. Suitable vehicles, carriers and additives for vaccines acceptable in veterinary medicine are known or will be apparent to those skilled in the art, see, p. eg, Remington's, Pharmaceutical Science. 18th edition 1990, Mack Publishing, which is incorporated into the present by its reference. The vaccine may be stored as a solution, or alternatively in lyophilized form to be reconstituted with a sterile diluent solution before administration. The present invention further provides vaccine formulations for the prolonged release of the antigen. Examples of such formulations for prolonged release include the antigen in combination with biocompatible polymer composite materials, such as, eg, poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and others similar. The structure, selection and use of degradable polymers in vehicles for drug delivery have been compiled in various publications, including that of A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated in the present for your reference. An additional guide for selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (editing coordinators), 1990, "Biodegradable Polymers as Drug Delivery Systems" in: Druqs and the Pharmaceutical Sciences, volume 45, M. Dekker, NY, which is also incorporated herein by reference. Alternatively, or additionally, the antigen can be microencapsulated in order to improve its administration and efficacy. Methods for microencapsulating antigens are well known in the art, and include the techniques described, inter alia, in U.S. Pat. 3,137,631; U.S. patent 3,959,457; U.S. patent 4,205,060; U.S. patent 4,606,940; U.S. patent 4,744,933; U.S. patent 5.132.1 17; and the international patent publication WO 95/28227, all of which are incorporated herein by reference. Liposomes and liposome derivatives (e.g., cloceados and vesicles) can also be used to provide a prolonged release of the antigen. Details about how to prepare and use liposome formulations can be found, among other places, in U.S. Patent Documents. 4,016,100; U.S. patent 4,452,747; U.S. patent 4,921,706; U.S. patent 4,927,637; U.S. patent 4,944,948; U.S. patent . 008,500; and U.S. patent 5,009,956, all of which are incorporated herein by reference. In a non-limiting embodiment, the vaccine of the present invention may be a combination vaccine to protect pigs against APP and, optionally, one or more other diseases or distinct pathological conditions that may afflict pigs, whose combination vaccine comprises a first component which in turn comprises an immunoiogically effective amount of an antigen of the present invention, selected from the group consisting of an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogue compound, a derivative, or a polynucleotide molecule of the present invention, which is capable of inducing, or contributing to the induction of, a protective response against APP in pigs; a second component which in turn comprises an immunologically effective amount of an antigen that is different from the antigen existing in the first component, and which is capable of inducing, or contributing to the induction of, a protective response to a disease or pathological condition that can afflict pigs; and a vehicle or diluent acceptable in veterinary medicine. The second component of the combination vaccine is selected based on its ability to induce, or contribute to the induction of, a protective response against APP or another pathogen, another disease or other pathological condition that may afflict pigs, as known in The technique. Any immunogenic composition now known, or to be determined in the future as being useful in a pig vaccine composition, can be used in the second component of the combination vaccine. Such immunogenic compositions include, but are not limited to, those that provide protection against Actinobacillus suis, Pasteurella multocida, Salmonella chioresuis, Streptococcus suis, Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus hyicus, Haemphilus parasuis, Bordetella bronchiseptica, Mycoplasma hyopneumoniae, Lawsonia intracellularis, Escherichia coli, porcine reproductive and respiratory syndrome virus, swine influenza virus, transmissible gastroenteritis virus, porcine parvovirus, encephalomyocarditis virus, coronavirus, pseudorabies virus and circovirus. In a non-limiting example, the combination vaccine comprises a combination of components that includes one or more APP proteins of the present invention, and one or more bacterial components of different APP, such as Apxl, Apxll or OmIA. The antigen comprising the second component can optionally be covalently linked to the antigen of the first component to produce a chimeric molecule. In a non-limiting embodiment, the antigen of the second component comprises a hapten, whose immunogenicity is detectably increased by conjugation with the antigen of the first component. Chimeric molecules, comprising antigens covalently linked to the first and second components of the combination vaccine, can be synthesized using one or more techniques known in the art. For example, a chimeric molecule can be produced synthetically using a commercially available peptide synthesizer using classical methods of chemical synthesis (see, e.g., Merrifield, 1985, Science, 232: 341-347).

Alternatively, the separated antigens can be synthesized separately and then linked together by the use of chemical linkers, as is known in the art. Alternatively, a chimeric molecule can be produced using recombinant DNA technology according to which, eg, separate polynucleotide molecules, which have sequences encoding the different antigens of the chimeric molecule, are spliced together in frame and expressed in an appropriate transformed host molecule, for subsequent isolation of the chimeric fusion polypeptide. When the vaccine of the invention comprises a polynucleotide molecule instead of a polypeptide, the spliced polynucleotide molecule can be used by itself in the vaccine composition. A broad guide for carrying out such recombinant techniques is provided, inter alia, in those of Maniatis et al., 1989, supra; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Eriich, 1992, earlier. The present invention further provides a method of preparing a vaccine to protect pigs against APP, which comprises combining an immunologically effective amount of one or more antigens of the present invention, selected from the group consisting of an APP protein, a polypeptide homologue, a peptide fragment, a fusion protein, an analogous compound, a derivative or a polynucleotide molecule of the present invention that is capable of inducing, or contributing to the induction of, a protective response against APP in pigs, with a acceptable vehicle or diluent in veterinary medicine, in a form suitable for administration to pigs. The present invention further provides a method for vaccinating pigs against APP, which comprises administering a vaccine of the present invention to a pig. The amount of antigen administered depends on factors such as age, weight, health status and general physical characteristics of the animal being vaccinated, as well as the particular composition of vaccine to be administered. The determination of the optimal dosage for each parameter can be made using routine methods, for example, from empirical studies. The amount of APP protein administered will preferably range between about 0.1 μg and about 10 mg of polypeptide, more preferably between about 10 μg and about 1 mg, and most preferably between about 25 μg and about 0.1 mg. For a DNA vaccine, the amount of a polynucleotide molecule will preferably range between about 0.05 μg and about 500 mg, more preferably between about 0.5 μg and about 50 mg. In addition, the typical dose volume of the vaccine will range between about 0.5 ml and about 5 ml per dose and per animal.

Animals can be vaccinated at any appropriate time, including within one week after birth, or at the weaning age, just before or at the time of breeding or breeding, or at the time of infection by APP begins to appear for the first time in one or more members of a population of animals. Supplementary administrations, or revaccinations, may be required to achieve full protection. Methods for determining whether adequate immune protection has been achieved in an animal are well known in the art, and include, e.g., determining seroconversion. The vaccine can be administered by any appropriate route, such as, for example, by administration by the oral, intranasal, intramuscular, intra-lymph node, intradermal, intraperitoneal, subcutaneous, rectal or vaginal routes, or by a combination of these routes. . The skilled artisan will readily be able to formulate the vaccine composition according to the chosen route. The present invention further provides a vaccine kit for vaccinating pigs against an infection or disease caused by APP, comprising a first container which in turn comprises an immunologically effective amount of one or more antigens of the present invention, selected from the group which consists of an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogous compound, a derivative or a polynucleotide molecule of the present invention, which is capable of inducing, or contributing to the induction of, a protective response against APP in pigs. The kit may optionally further comprise a second container comprising in turn a vehicle or diluent acceptable in veterinary. The vaccine composition can be stored in the first container either in solution or in lyophilized form, to be reconstituted using the vehicle or diluent of the second container. . 6. Anti-APP Antibodies The present invention further provides isolated antibodies that bind to an APP protein of the present invention. Such antibodies are useful for a variety of purposes, including, eg, as affinity reagents to purify the APP protein, or to detect the presence of the APP protein in a cell or in a tissue or fluid sample that has been collected from an animal infected by APP, eg, by use of an ELISA or Western blot analysis, or as a therapeutic agent to prevent, suppress or treat an APP infection. The antibodies against an APP protein of the present invention can be incited according to known methods by administration of an appropriate antigen of the present invention to a host animal selected from pigs, cows, horses, rabbits, goats, lambs and mice, among others. Various adjuvants, such as those described above, can be used to enhance the production of antibodies. The antibodies of the present invention can be either polyclonal or monoclonal. Polyclonal antibodies can be separated and isolated from the serum of immunized animals and assayed for APP anti-protein specificity using classical techniques. Alternatively, monoclonal antibodies against an APP protein can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, originally described by Kohier and Milstein (Nature, 1975, 356: 495-497); the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc.Nat.Acid Sci. USA 80: 2026-2030); and the EBV hybridoma technique (Colé et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). Alternatively, the techniques described for the production of single-chain antibodies (see, e.g., U.S. Pat. 4,946,778) can be adapted to produce single-chain antibodies specific for APP proteins. Antibody fragments that contain specific binding sites for an APP protein of the present invention are also encompassed within the scope of the present invention. Such fragments include, but are not limited to F (ab ') 2 fragments that can be generated by digestion with pepsin from an intact antibody molecule and Fab fragments, which can be generated by reducing the disulfide bridges of the F fragments ( ab ') 2. Alternatively, libraries of Fab and / or scFv expression can be constructed (see, eg, Huse et al., 1989, Science, 246: 1275-1281), to allow rapid identification of fragments having the desired specificity. for an APP protein of the present invention. Techniques for the production and isolation of monoclonal antibodies and fragments of such antibodies are well known in the art and are further described, inter alia, in Harlow's and Lane, 1988, Antibodies: A Laboratorv Manual, Cold Spring Harbor Laboratory, and J.W. Goding, 1986, Monoclonal Antibodies: Principies and Practice, Academic Press, London. All the aforementioned publications are incorporated herein by reference. . 7. Diagnostic kits The present invention additionally provides diagnostic kits. In a non-limiting embodiment, the diagnostic kit of the present invention comprises a first container which in turn comprises an APP protein, a homologous polypeptide, a peptide fragment, a fusion protein, an analogous compound, or a derivative of the present invention , which can be specifically linked to antibodies directed against the APP protein; and a second container comprising a secondary antibody directed against porcine antibodies. The secondary antibody preferably comprises a detectable label. Said diagnostic kit is useful for detecting pigs that at a given time are, or have been, previously infected with APP, or that have been seroconverted as a result of a vaccination with a vaccine of the present invention. In an alternative embodiment, the present invention provides a diagnostic kit comprising a first container comprising a primary antibody that binds to an APP protein and a second container that comprises a secondary antibody that binds to a different epitope on the protein of APP, or that is directed against the primary antibody. The secondary antibody preferably comprises a detectable label. In an alternative embodiment, the diagnostic kit comprises a container which in turn comprises a polynucleotide molecule or an oligonucleotide molecule of the present invention that can specifically hybridize, or can amplify, to a polynucleotide molecule specific for APP. These last two diagnostic kits are useful for detecting pigs that at a given time are infected with APP. . 8. Anti-sense Oligonucleotides and Ribozymes The present invention further provides oligonucleotide molecules that include anti-sense oligonucleotides, phosphorothioates and ribozymes that act to bind, degrade and / or inhibit the translation of an mRNA that encodes an APP protein.

Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules act to directly block the translation of an mRNA by binding to a targeted mRNA and thereby preventing translation of the protein. For example, anti-sense oligonucleotides of at least about 15 bases and complementary to singular regions of the transit sequence of MRNA, which encode an APP protein, can be synthesized, e.g. eg, by conventional techniques of phosphors and esters. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific dissociation of an RNA. The mechanism of action of a ribozyme involves a specific hybridization for a sequence of the ribozyme molecule with the complementary target RNA, followed by an endonucleolytic dissociation. The molecules of ribozymes with the motif or model of "hammerhead", engineered, which specifically and efficiently catalyze the endonucieolitic dissociation of mRNA sequences of the APP protein, are also within the scope of the invention. Dissociation sites by specific ribozymes within any potential RNA target are initially identified by screening the target molecule for ribozyme cleavage sites including the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences that have between about 15 and 20 ribonucleotides that correspond to the region of the target gene containing the cleavage site can be evaluated for predicted structural features such as secondary structure, which can make the oligonucleotide sequences unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides using, e.g. eg, analysis of ribonuclease protection. Both the anti-sense oligonucleotides and the ribozymes of the invention can be prepared by known methods. These include techniques for chemical synthesis such as p. eg, chemical synthesis with phosphoramidite in solid phase. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Said DNA sequences can be incorporated in a wide variety of vectors incorporating appropriate promoters of RNA polymerases such as the promoters of the T7 or SP6 polymerases. Various modifications in the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking ribonucleotide or deoxyribonucleotide sequences to the 5 'and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl instead of the phosphod iesterase bonds within the framework of the oligonucleotide. The following examples are illustrative only and are not intended to limit the scope of the present invention. 6. EXAMPLE Identification of new APP proteins The results of the following experiment demonstrate the specificities of local antibody responses, induced when pigs that had previously been stimulated with APP serotype 5 were heterologously reactivated with APP serotype 7. The specificities for antibodies were used in order to identify APP proteins that had not previously been recognized, in three of which (Omp20, OmpW, Omp27) was demonstrated by Western blot analysis that were present in the twelve APP serotypes. The two additional new proteins (OmpAI, OmpA2) were identified following the isolation and concentration of protein fractions (see section 6.1.6, below). 6. 1. Materials and Methods 6. 1.1. Stimulation with bacteria The culture of APP serotype 5 (strain K-17), used to prepare porcine stimulation material, was obtained from Dr. R.A. Schuitz, Avoca, IA, USA The culture of APP serotype 7 (strain WF-83), used to prepare porcine stimulation material, was obtained from Dr. E. Jones, Swedeland, PA, USA Crossbred pigs 7-8 weeks old, clinically healthy, were obtained from a herd in Nebraska with no previous history of APP infection and housed in isolation facilities at Pfizer Animal Health, Lincoln, NE, in accordance with guidelines of the IACUC. The animals were examined by a veterinarian to determine their health status before the initiation of the study. Following a 2-week acclimation period, the pigs were anesthetized using a combination of 100 mg / ml telazol, 50 mg / ml xylazine and 50 mg / ml ketamine, which were administered at a rate of approximately 1 ml /22.5 kg (50 pounds) of body weight, and inoculated intranasally with 2.6 x 106 cfu (colony forming units) of serotype 5 of APP. At 75 days after primary stimulation, six of the surviving pigs, who had demonstrated APP disease on a clinical scale but had recovered, were anesthetized again as described above. The first and second of these 6 pigs were re-stimulated intranasally with 1 x 107 cfu of APP serotype 5 (homologous restimulation), the third and fourth of these pigs were stimulated intranasally with 1 x 108 cfu of APP serotype 7 (heterologous restimulation); and the fifth and sixth pigs were inoculated intranasally with growth medium of bacteria only (controls). All stimulation nodules were administered in 1 ml volumes (0.5 ml / nostril = nostril). All pigs were sacrificed at 48 hours after restimulation, their sera and organs were collected and pieces of tissue from the organs were cultured in vitro for 24 or 48 h, as described in sections 6.1.2 and 6.1 .3 next. Supernatant materials containing antibodies, from cultures of tissue explants, were used to compare an antibody profile with memory produced by heterologous restimulation with that produced by homologous restimulation or single stimulation (control). The specificity of antibodies produced by tissue explants in vitro was determined by Western blot analysis against a set of whole bacterial cell preparations representing all 12 APP serotypes. This analysis demonstrated the presence of an antibody profile following a heterologous restimulation, which was different from the antibody profile following a homologous restimulation or a single stimulation (control). Reference cultures of serotypes of APP serotype 1 (strain 4074), serotype 2 (strain 4226), serotype 3 (strain 1421), serotype 4 (strain M62), serotype 5 (strain K-17), serotype 6 (strain Femo Fe-171 D)), serotype 7 (strain WF-83), serotype 8 (strain 405), serotype 9 (strain CVJ-13261), serotype 10 (strain 13039), serotype 11 (strain 56153) and serotype 12 ( strain 8329), used to prepare material for Western blots, were obtained from Dr. B. Fenwick, Kansas State University, Manhatta, KS, USA. 6. 1.2. Tissue collection Samples of sera from pigs were obtained before restimulation and again before necropsy (at 48 h after restimulation). The pigs were euthanized at 48 h after restimulation with an overdose of intravenous pentobarbital. The lungs were removed and examined for characteristic lesions as a whole, attributable to an infection by APP and a complete joint examination was carried out on all major organs. Tissue samples were collected from lungs, lymph nodes (mesenteric, popliteal and bronchial), Peyer's patches and tonsils, washed with 70% ethanol and rinsed 3 times in RPMI transport media (RPMI medium (Gibco / BRL, Grad Island, NY) supplemented with 10 mM HEPES, 5% FBS, 50 U / ml penicillin and 50 μg / ml streptomycin). After washing, the tissues were placed in 50 ml centrifuge tubes containing 10 ml of transport media and placed on ice until treated in the laboratory (within 3 hours of collection). Additional tissue samples were frozen in liquid nitrogen for future mRNA isolation and for immunohistochemistry, or fixed in formalin for histopathology. A sample of lung tissue was also given to the diagnostic laboratory at Lincoln University of Nebraska for identification of bacteria. 6. 1.3. Cultures of tissue explants Tissues were placed in individual Petri dishes containing approximately 5 ml of transport media. Small pieces of tissue about 2 x 2 mm in size were cut from the original sample with a scalpel blade and / or scissors, and placed in individual wells of 12 or 24 well plates (Costar, Cambridge, MA) containing 2 ml of wash medium (Hank's balanced salt solution free of Ca2 + and Mg2 + (HBSS) supplemented with HEPES mM and 50 μg / ml gentamicin). The tissue pieces were rinsed in the washing media and transferred to another well containing washing means. The washing and rinsing operation was repeated four times, and the tissue pieces were then transferred to wells containing RPMI media supplemented with 10% FBS, 10 mM HEPES, 2 mM glutamine and 50 μg / ml gentamicin, 60 μg / ml. ml of amphotericin B, 40 μg / ml of sodium deoxycholate, 50 U / ml of penicillin and 50 μg / ml of streptomycin. The plates were incubated at 38.5 ° C for 24 or 48 h in a humidified chamber containing 5% C02. After incubation, the supernatant fluids were removed and frozen at -70 ° C. 6. 1.4. Western blot analysis The specificity of the antibodies recovered in supernatant materials from tissue explants was examined by Western blot analysis in the following manner. Representative isolates of each of the 12 APP serotypes were each separately grown to generate the whole bacterial cell antigen in order to test the supernatants. Each layer was cultivated (planted at 1%) in the minimum-3 medium (MM) (1.8% Bacterin HP), 1.7% lactic acid, 0.3% glycerol, 0.05 M HEPES, L-glutamic acid (monosodium sai) 0.01 1 M, nicotinamide 5 x 10"5 M, and 0.2% casamino acids), supplemented with 10 μg / ml of β-nicotinamido-adenine-dinucleotide (β-NAD), for 5-6 h at 37 ° C and 180 rpm until reaching at an optical density DO560 of -0.5-0.6. The cells were pelleted by centrifugation at 12,000 x g for 10 min, the medium was reserved for analysis, and the pellet was resuspended in 5 ml of Dulbecco's phosphate buffered saline (DPBS). Prior to protein analysis, the resuspended pellet was frozen at -20 ° C and then thawed in order to lyse any intact bacterial cells. The protein concentration of each preparation was determined using a kit of reagents for analysis of BCA proteins (Pierce, Rockford, IL). Preparations of APP antigens (5 μg / lane) were loaded onto a 4-20% Tris-glycine gel (Novex, San Diego, CA), and the proteins were separated by electrophoresis at room temperature with an electric current intensity constant 20 mA. The separated proteins were transferred to membranes ProBlot® (Applied Biosystems, Foster City, CA) using an electrotransfer apparatus with semi-dry graphite (Milliblot, Millipore, Seattle, WA). The transfer was carried out at room temperature for 30 min at a constant electric current intensity of 200 mA. After the transfer was complete, the membranes were blocked by incubating overnight at room temperature with Buffer A (50 mM Tris HCl, 150 mM NaCl, pH 7.4 and dry milk without 5% fat (w / v). The blocking buffer was then decanted and replaced either with serum (dilution a 1: 100) or culture supernatants from tissue explants (dilution 1: 3) in Buffer A, and the membranes were incubated for 1 h at room temperature, followed by a wash for 10 min in Buffer B (Buffer A containing 0.2% (v / v) of Triton X-100) and two washes for 10 min in Buffer A. After the wash was complete, the membranes were incubated for 1 h at room temperature with goat antibodies. anti-pig, conjugated with phosphatase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1: 1000 in buffer A. The membranes were then washed in buffer A for 10 min and incubated for 15 min in a substrate system of 5-bromo-4-chloro-3-indolyl- Tetrazolium phosphate and nitro-blue (BCIP / NBT) (Kirkegaard &Perry Laboratories). 6. 1.5. Preparation of membranes with serotype 7 of APP An aliquot of serotype 7 of APP was seeded (1%) in MM supplemented with 10 μg / ml of β-NAD and cultured overnight 37 ° C (180 rpm). A portion of the culture overnight was inoculated into a fresh medium (the bacterial inoculum was 3% of the total volume) and incubated for 5-6 h or up to a culture density of 274 Klett units. The cells were pelleted by centrifugation at 4,500 rpm for 40 min at 10 ° C, the supernatant was removed, and the pellet was resuspended in 5 ml of 50 mM Tris-HCl, pH 8.0, with sufficient amount of PMSF (phenyl-methylsulfonyl fluoride) to result in a final concentration of 1 mM PMSF. Bacterial cells were used using a French Press press under 1, 120,000 g / cm2 (16,000 lb / in2) in a 40K PSI pressure cell (Sin Aminco., Rochester, NY). The broken cells were centrifuged at 1,000 x g for 15 min to eliminate large bacterial debris. The crude total membranes were collected by centrifugation at 45,000 rpm for 60 min at 18 ° C. The supernatant was discarded, the pellet resuspended in 50 mM Tris-HCl, pH 8.0, and the protein determined using standard Bradford protein analysis. To 15 mg of a crude membrane in a volume of 3 ml were added 30 μl of 100 mM PMSF and 750 μl of 2.5% sarcosyl, and the total volume was mixed thoroughly. After an incubation for 30 min on ice, the membranes were pelleted by centrifugation at 200,000 x g for 15 min at 10 ° C. The supernatant was removed from the pelleted membrane fraction and the pellet resuspended in 3 ml of 50 mM Tris-HCl / 100 mM NaCl, pH 8.0. This membrane preparation, which represented the membrane antigen of APP serotype 7, was then stored at -20 ° C. 6. 1.6 Fractionation and purification of membrane protein The purification of APP proteins for their sequence at the N-terminus was achieved by continuous elution on SDS-PAGE using a BioRad Model 491 Prep Cell (BioRad, Richmond, CA). A volume of 10 ml (4.5 mg of total protein) of the membrane protein fraction of APP serotype 7 was mixed with an equal volume of buffer for non-reducing sample (125 mM Tris-HCl, pH 6.8, 4 SDS %, glycerol at % and bromophene blue 0.1%). The protein and buffer mixture was boiled for 5 min and applied to an SDS-polyacrylamide gel with 3% stacking and 15% separation. The samples were subjected to electrophoresis with a constant electric current intensity of 20 mA (initial voltage 175-250 V, final voltage 200-300 V) for 72 h. Approximately 800 fractions of 5 ml each were collected at a flow rate of 1 ml / min throughout the batch, and analyzed in terms of their protein content by spectrophotometry at A2so- One out of every 10 fractions was analyzed by SDS- PAGE and silver staining (Bio-Rad, Richmond, CA). Fractions that putatively contained the same protein, as determined by molecular weight, were pooled and stored at 4 ° C. The pooled samples were desalinated and concentrated in preparation for the N-terminal sequencing. Desalination was performed by applying aliquots of pooled sample to a Presto® desalination column (Pierce, Rockford, IL) with a bed volume of 10 ml. . Aliquots of 3 ml of each protein grouping were applied to separate columns and eluted in ddH20 (deionized and destifed) in 10 fractions of 2 ml each. This was repeated 10 times for each protein cluster until 30 ml had been desalted. As determined by Western blot analysis, the majority of the de -iinized protein was found in fraction No. 2. Therefore, the second fractions from each of the 10 elutions were pooled for each individual protein.

The resulting 20 ml samples were lyophilized and resuspended in 0. 5 ml of ddH20 for sequencing at the N-terminus. The N-terminal sequences were obtained at the facilities of Pfizer Central Research Molecular Sciences Sequence Facility. 6. 2. Results 6. 2.1.-Clinical signs and pathological findings after renewed stimulation The pigs show no signs of clinical disease either after homologous restimulation (with serotype 5) or heterologous restimulation (with serotype 7). The pathological examination confirmed that the animals had not developed lung lesions that were compatible with an acute APP infection following a restimulation. However, the bronchial lymph nodes of the animals reactivated with serotype 7 were hemorrhagic and enlarged in comparison with those from animals restimulated homologously with serotype 5 or the present control animals. 6. 2.2. Specificity of antibodies produced by restimulation The specificity of antibodies present in serum and supernatants of tissue explants was checked by Western blot analysis as described above. All supernatants derived from tissues, collected after incubation for 24 or 48 h, contained antibodies that specifically recognized APP proteins. In general, the reactivity against serotype 5 antigens was greater than the reactivity against serotype 7 or serotype 1 antigens. However, the reactivity of most tissue-derived supernatants was less intense and more narrow in that the spectrum than serum reactivity (FIGURE 1a). In general, the reactivity of supernatant materials from tissue explants did not have any particular model. However, the Western blot reactivity model of supernatant materials from tissue explants from a specific animal (No. 803, restimulated heterologously with serotype 7) was strong, and highlighted several low molecular weight proteins present in serotypes 1, 5 and 7 of APP (FIGURE 1b). This supernatant was used as a source of antibodies to further characterize the degree of cross-reactivity of the secondary antibody response (with IS 97 memory) produced by heterologous restimulation with APP serotype 7 in pigs that had been initially stimulated with the serotype 5 of APP. The degree of cross-reactivity of the antibodies in the supernatant materials of BLN tissue explants from the Pig N ° 803 was determined by Western blot analysis using whole bacterial cell antigens, prepared from each of the twelve different APP serotypes. This analysis showed that three of the low molecular weight proteins, recognized by the antibodies, were present in the twelve serotypes (FIGURE 2). The antibodies present in this BLN tissue explant supernatant also recognized other protein bands. A band of high molecular weight, present in serotypes 1, 2,3,4,5,6,7, 8, and 9, which may correspond to the exotoxin, Apx II (see Nakai, 1983, earlier), and other Protein band present in serotypes 2,5,8 and 10, represent the most reactive models in a cross-way in second place. Western blots of sediments and supernatants of APP cells revealed that the reactivity of the antibodies in BLN tissue explant supernatants for low molecular weight proteins was restricted to proteins present in cell pellets (FIGURE 3), indicating that the proteins are associated with the bacterial cell and are not secreted. 6. 2.3. Proteins recognized by cross-reactive antibodies Low molecular weight proteins. were purified as described above, providing particularly purified preparations containing the protein of interest as identified by Western blot analysis using: (a) BLN tissue explant supernatant fluids from Pig No. 803; or (b) whey from the pig No. 803 (FIGURE 4). After fractionation of the membrane proteins, four protein bands with molecular weights of approximately 19-20, approximately 23, approximately 27 and approximately 29 kDa, respectively, were identified using this method. Sequence analyzes of the N-terminus of the proteins in the four bands yielded a primary sequence and tentative residues (in parentheses) as shown in TABLE 1, below, and designated therein as "Pep-1" (SEQ. ID NO: 1 1), "Pep-2" (SEQ ID NO: 12), "Pep-3" (SEQ ID NO: 13 and "Pep-4" (SEQ ID NO: 14) Occasional secondary signals were observed and they were probably due to the presence of minor contaminants (data of which are not shown) The partial sequences of the N-terminus shown in TABLE 1 were used to design probes and primers in order to obtain the primary DNA sequences encoding Cross-reactive APP proteins: Comparisons of sequence homologies emerged that the four proteins recognized with the dominant local antibody response produced after heterologous restimulation had not been previously described for APP.

TABLE 1 Amino acid sequences at the N-terminal end of APP proteins of low molecular weight Xaa indicates that the amino acid residue in the particular position could not be determined 7. EXAMPLE Molecular cloning of the DNA encoding APP proteins 7. 1. Isolation of chromosomal DNA and construction of qenomic libraries The genomic DNA from each of the twelve APP serotypes was isolated separately or by the hexadecyltrimethylammonium bromide (C ) -proteinase K method (Ausubel and collaborators 1988, Curr. Protocols Mol. Biol. Wiley Interscience, NY) or by the isolating genomic DNA isolation reagent (Genosys Biotechnologies, Inc., the Woolands, Texas). The APP DNA was dissolved in a TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at < 1 μg / ml and quantified by UV spectrophotometry. To facilitate the cloning of APP gene sequences encoding Omp20, OmpW, Omp27 and OmpA, several genomic libraries were constructed. These libraries were specifically modified by ligation of a known sequence (Vectorette II®, Genosys Biotechnologies Inc., The Woodlands, TX) with the 5 'and 3' experts of restricted DNA fragments, essentially as recommended by the supplier. Therefore, Vectorette libraries were constructed by separately directing two μg of chromosomal DNA from APP 7-1 (serotype 7, passage 1) with one of the restriction endonucleases Bam \, BglU, HindlW, EcoRI, Dral or Hpa \ at 37 ° C overnight. The reaction mixture was then labeled and sampled with fresh additional restriction enzyme and adjusted to a final concentration of 2 mM ATP and 2 mM DDT. The addition of Vectorette tails was carried out by the addition of T4 DNA (400 U) plus 3 pMol of the appropriate compatible Vectorette eniazador (Vectorette for BamHl: BamHl, Bglll; Vectorette for Hindlll: Hindlll; Vectorette for EcoRI: EcoRI; Vectorette for blunt end: Dral, Hpal). The mixture was incubated for three cycles at 20 ° C, for 60 min; at 37 ° C, for 30 min to complete the queue addition reaction, and then adjusted to 200 μl with dH20 and stored at -20 ° C. 7. 2. Molecular cloning of omp20 Scrutiny of the Vectorette libraries was carried out to obtain DNA fragments encoding Omp20 and flanking regions. The degenerate oligonucleotide ER49 (SEQ ID NO: 39) was designed based on the amino acid sequence (aa) of the N-terminus of this protein (TABLE 1, Pep-Í (SEQ ID NO: 11) aa 1-9) . For PCR amplification of a fragment of the omp2Q gene, oligonucleotide ER49 (SEQ ID NO: 39) was used in combination with a primer specific for Vectorette, ER70 (SEQ ID NO: 48) in 50 μl reaction runs containing 1x PCR buffer II (Perkin Elmer), 1.5 mM MgCl 2, 200 μM of each of the deoxy-NTP, 100 pMol of each of the primers and 2.5 U of the thermostable AmpliTaq Gold polymerase (Perkin Elmer). Multiple individual reactions were performed with 5 μl of the Vectorette libraries as DNA template. The amplification was carried out in the following manner: denaturation (at 95 ° C, for 9 min), 35 cycles of denaturation (95 ° C, 30 s), annealing (55 ° C, 1 min) and polymerization (72 ° C) C, for 3 min) followed by a final prolongation (at 72 ° C, for 7 min).

The amplified products were visualized by separation on a 1.2% agarose gel (Sigma). A product of 433 bp (base pairs) resulted from the amplification of the Vectorette library for EcoRl. The fragment was cloned into the PCR cloning vector pGEM®-T Easy (Promega, Madison, Wl) and sequenced. Sequence analysis confirmed that the identity of the fragment was that it partially encoded Omp20 based on the amino acid sequence of the N-terminal (Pep-1). Based on the newly identified sequence of this partial gene, specific primers ER67 (SEQ ID NO: 46) and ER68 (SEQ ID NO: 47) were designed to obtain additional flanking sequences at 5 'and 3' for a second round of screening. Vectorette libraries by PCR amplification, as described above. Screening of the Vectorette libraries for EcoRI, Hindlll, Dral and Hapl by PCR, using ER68 (SEQ ID NO: 47) plus ER70 (SEQ ID NO: 48), resulted in a satisfactory amplification of a fragment of approximately 600 bp from from the Vectorette library for Dral. This fragment was sequenced to determine the 3 'end of the omp20 gene. Since no single products were observed during the screening of these libraries using ER67 (SEQ ID NO: 46) plus ER70 (SEQ ID NO: 48), the additional specific primers ER71 (SEQ ID NO: 49), ER72 (SEQ) were designed ID NO: 50), ER76 (SEQ ID NO: 52) and ER77 (SEQ ID NO: 53), to obtain DNA fragments located 5 'from the ER49 binding site. Such "walk through the genome" by amplification from numerous DNA libraries Vectorette was reiterated until the outer boundaries of the ORF, that is to say the codons of initiation and stoppage of the translation as well as the flanking sequences of nucleotides, were characterized. Generally, the PCR products were sequenced directly or cloned into the cloning vector for PCR pGEM®-T Easy before the analysis of the sequences. 7. 3. Molecular cloning of omp27 The screening of the Vectorette libraries for omp27 was carried out essentially as described above for omp20, except that the degenerate oligonucleotide ER50 (SEQ ID NO: 40), which was designed based on the amino acid sequence of the N-terminal end of the purified Omp27 protein (TABLE 1, Pep-3) (SEQ ID NO: 13), aa 13-24). Following PCR amplification of Vectorette libraries, as described above, it was confirmed that a 152 bp fragment encoded a portion of Omp27 based on the amino acid sequence of the γ terminal of Pep-3 (SEQ ID NO: 13). A second round of genome walking, using the Vectorette libraries for BamHl, Bgl l, Hindlll, EcoRI, / - / pal and Dral and the specific primers ER88 (SEQ ID NO: 64) and ER89 (SEQ ID NO: 65) , resulted in a PCR amplification of an approximately 2 kb fragment from the Dral library, and a fragment of approximately 1.5 kb from the Hindlll library. These DNA fragments were cloned into the PCR cloning vector pGEM®-T Easy, which allowed to obtain the 5 'and 3' nucleotide sequences of the omp27 gene, respectively. 7. 4. Molecular cloning of ompAl and ompA2 The amino acid sequence at the N-terminus, obtained from the purified 29 kDa protein (TABLE 1, Pep-4- (SEQ ID NO: 14)), was used to design the degenerate end-terminal primers of N RA22 (SEQ ID NO: 73) and RA34 (SEQ ID NO: 77) for use in the direct PCR amplification of the gene encoding the 29 kDa protein from APP chromosomal DNA. The Pep-4 sequence was analyzed using a comparison by a BlastP computer program (National Center for Biotechnology Information) versus the GenBak protein database (Altschul et al., 1990, J. Mol. Biol. 215: 403 -10), and was found to exhibit homology with OmpA proteins from several different eubacteria, such as Pasteurella multocida, P. haemolytica, Haemophilus ducreyi, H.sommus, H. influenzae, Actinobacillus actinomycetemcomitans, E. coli, Shigella sp, and Salmoella.sp of the N-terminus of the OmpA-related proteins from Pasteurellacea were aligned to produce the consensus sequence Ala-Pro-Gln-Ala / Glu-Asn-Thr-Phe-Tyr-Ala / Val-Gly-Ala- Lys-Ala (SEQ ID NO: 94). These alignments were analyzed and used to design several additional degenerate primers at the terminal ends of N. The oligonucleotide primers RA53 (SEQ ID NO: 81), RA54 (SEQ ID NO: 82), RA55 (SEQ ID NO: 83), RA56 (SEQ ID NO: 84), and RA57 (SEQ ID NO: 85) overlap each other, and were designed to join the region encoding the aa 4-1 1, aa 5-12, aa 3-10, aa 1 -8 and aa 1-7 , respectively, of this consensus peptide. Other degenerate oligonucleotide primers were designed after alignment of the C-terminal regions of the OmpA-related proteins. This alignment indicated a highly conserved region within the terminal end of C, which included the amino acid sequence Cys-Leu-Ala-Pro-Asp-Arg-Arg-Val-Glu-lle (SEQ ID NO: 95). Both reverse primers RA49 (SEQ ID NO: 78) and RA50 (SEQ ID NO: 79) were designed to bind to the negative strand of DNA in this region of the OmpA. These reverse primers were applied in a two dimensional matrix, in which each RA49 (SEQ ID NO: 78) and RA50 (SEQ ID NO: 79) were combined pairwise with RA22 (SEQ ID NO: 73), RA34 (SEQ ID NO: 77), RA53 (SEQ ID JMO: 81), RA54 (SEQ ID NO: 82), RA55 (SEQ ID NO: 83), RA56 (SEQ ID NO: 84), RA57 (SEQ ID NO: 85), in HotStart 50® tubes (Molecular Bioproducts Inc., San Diego, CA) with the combined Klen Tag / and Pfu polylmerases. The following references describe the methods of "hot initiation": D'Aquila et al., 1991, Nucí. Acids Res. 19: 3749; and Horton et al., 1994, Biotechniques 16: 42-43. The cycle program for PCR was a variant of the "contact PCR" protocols and was carried out as follows: 3C 106 denaturation (94 ° C, 5 min) 30 cycles of denaturation (94 ° C) C, 30 s), annealing (59 ° C, initial cycle 30 s, then at -0.1 ° C for each additional cycle), and polymerization at (72 ° C, 1 min), followed by a final extension (at 72 ° C). C, 15 min.) The following references describe the "contact PCR" protocols: Roux, 1994, BioTechniques, 16: 812-814; and Hecker and Roux, 1996, BioTechniques 20: 478-485. Among the PCR products generated, a band of approximately 950 bp was produced from reactions or with RA49 (SEQ ID NO: 78) or with RA50 (SEQ ID NO: 79), in which each was combined in pairs with forward primers RA34 (SEQ ID NO: 77), RA53 (SEQ ID NO: 81), RA56 (SEQ ID NO: 84) or RA57 (SEQ ID NO: 85). These DNA fragments were cloned into the PCR cloning vector pGEM®T Easy and sequenced. The analysis of these fragments sequenced indicated the existence of two different sequences of variants. The products derived from RA49 (SEQ ID NO: 78) / RA57 (SEQ ID NO: 85) and from RA50 (SEQ ID NO: 79), RA56 (SEQ ID NO: 84), represented the A1 variant, and the protein partially coded was designated as "OmpAI". The products derived from RA50 (SEQ ID NO: 79) RA34 (SEQ ID NO: 77), and from RA50 (SEQ ID NO: 79) / RA53 (SEQ ID NO: 81), represented the A2 variant, and the protein partially encoded was designated as "OmpA2". The two partial DNA sequences of ompA, similar but distinct, were expanded to include the ORF integers and flanking sequences 5 'and 3' by walk genome by application of Vectorette libraries as described previously herein .

The alignment of the DNA partial sequences of o / t? PA1 and ompA2 allowed the design of specific oligonucleotide primers capable of differentiating between these closely related gene sequences.

, ER58 (SEQ ID NO: 42), and for ompA2, i.e. ER59: differentiators primers, which were drawn out, specific for the 5 'and 3' ompAI, ie ER55 (41 SEQ ID NO) were used (SEQ ID NO: 43), ER62 (SEQ ID NO: 44), respectively, for probing Vectorette libraries, as described above. Singular fragments of approximately 1, 100, 400, 450 and 280 pb respectively were obtained from a Vectorette library for EcoRI when probed as ER70 (SEQ ID NO: 48), plus one of ER55 (SEQ ID NO: 41) , ER58 (SEQ ID NO: 42), ER59 (SEQ ID NO: 43) or ER62 (SEQ ID NO: 44), respectively. The sequence analysis of the resulting fragments allowed the determination of the regions of endpoints and flanking of both ORFs ompA? and omp A2. 7. 5 Molecular Cloning of omp \ N The amino acid sequence of the N-terminus, obtained from the purified 23 kDa protein (TABLE 1, Pep-2 (SEQ ID NO: 12) was used to design the RA20 (SEQ ID NO: 72), a degenerate oligonucleotide primer corresponding to amino acids 1-8 of Pep-2. Purified APP DNA was used as a template in a variant of the "PCR with gene walk" methods. PCR primer RA 20 (SEQ ID NO: 72) was used in "warm start" reactions with KlenTaq // polymerases (Ab Peptides, Inc. St. Louis, MO) and Pfu (Strategene, Inc., La Jolla, CA) The cycle program for PCR was a variant of the protocols "contact PCR (touchdown) and was carried out as follows: denaturation (94 ° C, 5 min), 40 cycles of denaturation (94 ° C) C, 1 min), annealed at (63 ° C, initial cycle 2 min, then -0.2 ° C and -2 for each additional cycle) and polymerization (72 ° C, 1.5 min), sec was removed by a final extension (72 ° C, 10 min). Among the numerous PCR products generated, a product of approximately 220 bp was obtained and cloned into the PCR cloning vector pGEM®T Easy. Analysis of the sequence of this plasmid insert confirmed that the cloned PCR product encoded amino acids corresponding to a portion of the 23 kDa protein, based on the amino acid sequence of the N-terminus of Pep-2 (SEQ ID NO: 12). Based on this newly identified sequence, a specific primer RA23 (SEQ ID NO: 74) was generated for the amplification of the sequences located downstream. The genomic DNA mini-libraries of APP serotype 7 were constructed by limited digestion with Ta? Fal or HinP I, both of which create 5'-CG overhangs. This DNA was ligated into a pUC21 or pUCI28 vector cut with BspDI and transformed into E. coli DH5a to provide the genomic libraries. Minipreparations of these genomic libraries carried in plasmids were used, as molds in a "gene walk PCR" using "hot initiation" methods. The specific primer RA23 (SEQ ID NO: 74), together with the sequencing primers in Advance M13 and Reverse M13, specific for vectors, was used with polymerases KlenTag and Pfu combined. The cycle program for PCR was carried out in the following manner: denaturation (94 ° C, 5 min); 32 cycles of denaturation (94 ° C, 30 s), annealing (63 ° C, initial cycle of 30 s, then -0.2 ° C per cycle) and polymerization (72 ° C, 30 s), followed by a final extension ( 72 ° C, 7 min). Among the numerous PCR products generated, a 0.8 kb product and a 1.4 kb product were cloned into the pGEM®T Easy PCR cloning vector and sequenced. The analyzes and the alignments of the resulting sequences, together with those previously obtained, yielded the sequence of the mature protein. In order to obtain the sequence of the 5 'flanking region of the ompW gene, the specific primers RA24 (SEQ ID NO: 75), and RA26 (SEQ ID NO: 76) were used to probe numerous Vectorette libraries, as before. describes The amplification was carried out in the following manner: denaturation (95 ° C, 9 min); 40 cycles of denaturation (95 ° C, 30s), annealing (60 ° C, 1 min) and polymerization (72 ° C, 3 min); followed by a final prolongation (72 ° C, 7 min). Specific products of 600 and 700 bp resulted from probing the Vectorette libraries for Hpa and Dral, respectively. The 700 bp product was sequenced directly to obtain the nucleotide sequence spanning the flanking region in 'and encoded the N-terminus of the 23 kDa protein. Due to the partial similarity between the predicted 23 kDa protein of APP and the predicted Vibrio cholerae OmpW protein, the APP gene fragment was designated as "ompW". 7. 6 Molecular Analysis of Genes Coding APP Proteins 7. 6 .1 DNA Amplification by Specific PCR The results of the cloning and preliminary sequencing of the new APP proteins, as described above, were used to design oligonucleotide primers for the specific amplification of the intact genes omp20, omp27, ompAI , ompA2, ompW, directly from chromosomal DNA of serotype 7 of APP. This approach was preferred based on the desire to eliminate the introduction of sequencing aberrations due to possible mutations that arose during the cloning of the gene fragments in E. coli. Correspondingly, oligonucleotides flanking the above intact APP genes were used to specifically amplify those regions from chromosomal DNA. The 1 1.1 pairs of 5 'and 3' primers used for each gene amplification were the following: for omp20 ,, the primers were ER80 (SEQ ID NO: 56) and ER81 (SEQ ID NO: 57); for omp27, the primers were ER95 (SEQ ID NO: 69) and ER 96 (SEQ ID NO: 70); for ompA1, the primers were ER84 (SEQ ID NO: 60) and ER86 (SEQ ID NO: 62); for ompA2, the primers were ER87 (SEQ ID NO: 63) and ER66 (SEQ ID NO: 45); and for omp \ N, the primers were ER82 (SEQ ID NO: 58) and ER83 (SEQ ID NO: 59). The reactions of PCR were carried out in triplicate, and contained 260 ng of purified chromosomal DNA, 1 x of PC2 buffer (Ab Peptides, Inc.); 200 μM of each of the dNTPs, 100 pMol of each of the primers, thermostable polymerases 7.5 U of KlenTaq / and 0.15 U of Pfu cloned in a final sample volume of 100 μl. The conditions for the amplification consisted of a denaturation (94 ° C, 5 min) followed by 30 cycles of denaturation (95 ° C, 30 s), annealing (65 ° C, 30 s) and polymerization (72 ° C, 2 min. ). A final extension (72 ° C, 7 min) completed the amplification of the intact target gene region. After amplification, each of the triplicate samples was pooled and the specific product was purified by agarose gel electrophoresis and centrifugation chromatography extraction (QIAquick®1 QIAGEN Inc., Santa Clarita, CA) before direct analysis of sequences using the termination reactions of DyeDeoxy in an ABL automated DNA sequencer (Applied Biosystems, Foster City, CA). The synthetic oligonucleotide primers were used to sequence both DNA strands of the amplified products from APP serotype 7. The primers used to sequence the genes of the APP proteins are presented below in TABLE 2. The nucleotide sequence of the omp20 ORF is presented in SEQ ID NO: 1 from pt 272 to 790. The nucleotide sequence of the ORF of ompW is presented in SEQ ID NO: 3 from nt 376 to 1, 023. The nucleotide sequence of the omp27 ORF is presented in SEQ ID NO: 5 from nt 157 to 933. The nucleotide sequence of the ompAI ORF is presented in SEQ ID NO: 7 from nt 614 to 1, 708. The nucleotide sequence of the ompA2 ORF is presented in SEQ ID NO: 9 from nt 197 to 1,306.

TABLE 2 7. 6 .2. Similarity of the OmpAI and OmpA2 Proteins of APP Serotype 7 The amino acid sequences of the OmpAI protein (SEQ ID NO: 8) and the OmpA2 protein (SEQ ID NO: 10) were deduced from SEQ ID NOS: 7 and 9, respectively, and were aligned to compare their similarity. The deduced OmpAI protein has a length of 364 amino acids, which is shorter by 5 amino acids than the deduced OmpA2 protein. The alignment of the APP proteins shown in FIGURE 5 indicates that the two proteins share an amino acid identity of a 73. 1% (270 among 369). 7. 6 .3. Comparison of the OmpW Protein of APP Serotype 7 with the OmpW of Vibrio cholerae The amino acid sequence (SEQ ID NO: 4) deduced from the nucleotide sequence (SEQ ID NO: 3) of the ORF coding for the OmpW protein of APP of 23 kDa, was extremely similar to the OmpW protein described in Vibrio cholerae (Jalajakumari, M.B. et al., 1990, Nucleic Acids Res. 18 (8): 2180). The amino acid sequences of these two proteins were aligned using the Clustal W multiple sequence alignment algorithm (see 1 .4) (Thompson, J.D., et al., 1994, Nucleic Acids Res., 22: 4673-4680). This comparison indicated that the OmpW proteins of APP and OmpW of V. cholerae, which have lengths of 215 and 216 residues, respectively, share an amino acid identity of 44.9% (out of 97 out of 216). The aligned proteins are shown in FIGURE 6. 7. 7. Southern Blot Hybridizations The conservation of DNA sequences encoding the Omp20, OmpW, Omp27, OmpAI and OmpA2 proteins between different APP serotypes was determined by performing Southern blot hybridization using each of the 5 different coding sequences as probes against APP DNAs from the different serotypes. The probes were generated with a probe synthesis kit for PCR DIG® (Boehringer Mannheim, Inc., Indianapolis, Indiana) according to the manufacturer's instructions. For example, the ompW probe was constructed in the following manner. A PCR product encompassing the ompW coding sequence was generated using specific primers for ompW and DNA of APP serotype 7. The genomic DNA of APP serotype 7 (0.2 μg), the MW3 primer 1 μM (SEQ ID NO: 71), the RA52 primer 1 μM (SEQ ID NO: 80), 7.5 μu of KlenTag / polymerase (Ab Peptides , Inc.), 0.075 U of Pfu polymerase (Stratagene), 1 x Klen Tag / buffer, and 0.2 mM of the dNTPS were combined in a volume of 50 μl. The PCR was carried out in the following manner: denaturation (95 ° C, 5 rhin), 35 cycles of denaturation (94 ° C, 30 s), annealing (58 ° C, 30 s), and polymerization (72 ° C) , 1 min); and final prolongation (72 ° C, 7 min). The ompW PCR product with -650 bp was then purified from the agarose gel electrophoresis using a JETsorb® kit (GENOMED, Inc., Research Triangle Park, NC). The purified DNA was quantified using a Low Mass DNA Ladder® mass pattern (GIBCO / BRL, Gaithersburg, MD).

A digoxigenin-labeled probe was generated by PCR amplification of 24 g of the ompW PCR product, produced as described above, using a DIG® PCR probe synthesis kit according to the manufacturer's instructions, and the probe generated by PCR it was stored without purification at -20 ° C. The genomic DNA of APP digested with EcoRI (1.5 μg) obtained from each of the serotypes 1, 2, 5, 7, 8 and 9 of APP was separated by agarose gel electrophoresis. The DNA profiles were transferred to a 0.45 μm Hybond-N nylon membrane (Amersham, Inc., Cleveland, OH) using alkaline transfer with a Turboblotter® kit (Schleicher &Schuell, Inc., Keene, New Hampshire), according to the manufacturer's instructions. DNA was covalently bound to the membrane by UV irradiation using a UV crosslinker Stratalinker® UV Cross-Linker (Stratagene) in the self-crosslinking setting (120 jt? J / cm2). Transfer blots were allowed to dry and stored at room temperature. DNA spots on nylon were incubated in the presence of a probe to allow hybridization in order to detect probe sequences in multiple APP serotypes. The spots were previously hybridized for 2.5 h at 68 ° C using an excess (0.2 ml / cm2) of 1x Prehybridization Solution (GIBCO / BRL). The probe hybridization solution was prepared by adding 5.4 μl of the unpurified digoxigenin-labeled probe to 500 μl of 1x Hybridization Solution (GIBCO / BRL) and boiling at 100 ° C for 10 min. The probe was cooled to 0 ° C for 1 min, and then added to a sufficient amount of 1 x Hybridization Solution to give a total of 0. 025 ml / cm2 of stain. The spots were hybridized in this mixture of hybridization solution with probe at 68 ° C for 16 hours. Thorough washes were washed on the spots in the following manner: (i) 2 washes with an excess of (0.2 ml / cm2) of 2x SSC / 0.1% SDS at 25 ° C for 5 min; (ii) 2 washes with an excess of (0.2 ml / cm2) of 0.1 x SSC / 0.1% 0.1% SDS at 68 ° C for 15 min. The spots were then revealed using a chemiluminescence method with a DIG® DNA Labeling and Detection Starter Kit II Hign Prime DNA Labeling and Detection Starter Kit II and a DIG® Wash and Block Buffer Set Wash and Block Buffer Set ( Boehringer Mannheim, Inc., Indianapoiis, Indiana), according to the manufacturer's instructions. The revealed spots were exposed to an X-ray film for various periods of time in order to detect the bands that hybridized. The probes generated as above against sequences of omp20, ompW, omp27, ompA1 and ompA2 were hybridized with DNA in all APP serotypes tested (serotypes 1, 2, 5, 7, 8 and 9). The sizes of the bands with EcoRI detected were identical in all the serotypes for ompA1 and ompA2, but they were not conserved for omp20, ompWy omp27. The size of the EcoRI fragments hybridized by the omp20 probe in each of the serotypes was as follows: serotypes 1, 2, 7 and 9 gave a fragment of 5.8 kb; serotype 5 gave a 6.1 kb fragment; and serotype 8 gave a 5.0 kb fragment. The size of the EcoRI fragments hybridized by the ompW probe in each of the serotypes was as follows: serotype 1 gave a fragment of 1.15 kb; serotype 2 gave a fragment of 1.1 kb; serotype 5 gave a 1.0 kb fragment; serotype 7 gave a 0.9 kb fragment; serotype 8 gave a fragment of 1.05 kb; and serotype 8 gave a fragment of 1.2 kb. The size of the EcoRI fragments hybridized by the omp27 probe in each of the serotypes was as follows: serotypes 1, 2 and 9 gave a fragment of approximately 9.5 kb; and serotypes 5, 7 and 8 gave a fragment of approximately 10.5 kb. The size of the EcoRI fragments hybridized by the ompA1 probe was 2.3 kb in all serotypes. Fragments of 0.55 kb and 0.85 kb that hybridized more weakly were also detected. The size of the EcoRI fragments hybridized by the ompA2 probe was 0.85 kb in all serotypes. Fragments of 0.55 kb and 2.3 kb that hybridized more weakly were also detected. 8. EXAMPLE Expression of recombinant APP proteins 8. 1 Host strain The E. coli hosts used for the expression of recombinant proteins was E. coli. The genotype of this strain is "IN (rrnD-rrnE) IgalE:: Tn10? C / 857? H1 bio." This strain was provided by SmithKine Beecham Pharmaceuticals, King of Prussia, PA, USA, and contains the repressor ? temperature sensitive,? c / 857, which inhibits expression from the promoters? at 30 ° C. At 42 ° C, the repressor is deactivated and expression is enabled from? promoters, providing a transcription of high level and protein synthesis E.coli LW14 was propagated at 30 ° C. 8. 2 Expression Vectors in Plasmids The expression vector used for the synthesis of recombinant proteins was pEA181, alternatively designated pEA181 KanRBS3. This vector has a size of 6.766 kb, encoded resistance to kanamycin (kan), and contains the promoter? strong PL. The vector contains a site for Ndel just underneath an optimized ribosome binding site; the presence of this site for Ndel allows the exact placement of the Met initiation codon of a protein to obtain optimal expression. The vector also encodes a leader NS1 fusion protein to enable enhanced expression of poorly expressed proteins. This vector was provided by SmithKine Beecham Pharmaceuticals (see also U.S. Patent 4,925,799 and Rosenberg et al., 1983, Meth. Enzymol.101: 123-138). The coding sequences of each of the five APP proteins were amplified by PCR using 5 'specific primers designed to provide a site for Ndel (CATATG) that overlaps the Met initiation codon (ATG), and specific primers for 3 'designed to be cloned on the site for Xbal in 3' within pEA181. The respective PCR products were initially cloned into the PCR cloning vector pGEM®-T Easy (Promega Corp) and transformed into the commercially available competent E. coli DH5a (MAX Efficiency DH5a Competent Cells, GIBCO / BRL). The PCR products were excised from these plasmid codons as fragments of Ndel-Xbal or Ndel-Spel and cloned into pEA181 cut with Ndel / Xbal. Due to the presence of the promoter? strong P > the derivatives of pEA181 could be transformed only in the sine of E. coli LW14 that repressed the expression from the vector by the activity of the repressor? sensitive to temperature? c / 857. Transformation and propagation of carrier transformants of pEA181 and its derivatives were carried out at 30 ° C. E. coli LW14 was made competent by the Hanahan method for the preparation of frozen competent cells (Hanahan, 1985, in: DNA Cloninq: A Practical Approach (Glover, D., editing coordinator) volume 1, pages 109-135 , IRL Press, Oxford, England).

Due to difficulties with the expression of mature OmpW in E.co/i LW14 a peptide leader was used that allows an intensified synthesis of protein. This leader peptide, called a "protective peptide" or "pp", protects the recombinant proteins with respect to the proteolytic degradation, based on information from Sung et al. 1986, Proc. Nati Acad. Sci. USA 83: 561-565; Sung et al., 1987, Meth. Enzymol. 153: 385-389); and U.S. Pat. 5,460,954, whose citations are incorporated herein by reference. The protective peptide, consisting of the amino acid sequence Met-Asn-Thr-Thr-Thr-Thr-Thr-Thr-Ser-Arg (SEQ ID NO: 96), was fused to the N-terminus of each of APP proteins designing PCR primers so as to contain the coding sequence of the protective peptide located upstream from the APP coding sequence. The amplification of the separate APP coding sequences, with said primers generated sequences encoding the protective peptide located at the N-terminus., fused with the first amino acid in the mature APP protein (ie lacking the native signal sequence). A site for Ndel was placed next to the Met codon in the protective peptide, so that it could be ligated into the Ndel site of pEA181. The pairs of primers for the amplification of the protective peptide-protein coding sequences of APP were as follows: for omp \ N, MW3 (SEQ ID NO: 71) and RA52 (SEQ ID NO: 80); for ompA1, RA78 (SEQ ID NO: 88) and RA71 (SEQ ID NO: 87); for ompA2, RA78 (SEQ ID NO: 88) and RA69 (SEQ ID NO: 86); for omp20, ER78 (SEQ ID NO: 54) and ER73 (SEQ ID NO: 51); and for omp27, ER92 (SEQ ID NO: 67) and ER94 (SEQ ID NO: 68). 8. 3 Expression of Recombinant Proteins Transformers of E. coli LW14 carrying pEA181 derivatives that encoded protective peptide fusions with the respective mature APP proteins OmpAI, OmpA2, Omp20, OmpW and Omp27, were propagated overnight at 30 ° C in LB Km50 (Luria broth with 50 μg / ml kanamycin sulfate). The cultures were diluted 1: 100 in 2X medium YT Km50 (1.6% tryptone, 1% yeast extract, 0.5% NaCl, 1.25 mM NaOH, containing 50 μg / ml kanamycin sulfate) and grown at 30 ° C until the Aßoo was 0.8 to 1.0. Then the cultures were displaced at 42 ° C in a water bath incubator and incubated for 3 to 4 h. Wet cells of E. coli transformants LW14 from a 5-liter fermentation batch that had grown in 2X medium YT Km50, and which expressed either pp-OmpA1, pp-OmpA2 or pp-OmpW, were harvested by centrifugation. Cells were suspended in 0.1 M Tris-HCl, pH 8.0 and lysed in a high pressure homogenizer. Inclusion bodies (IB, inclusion bodies) were collected by centrifugation (12,000 RCF, 30 min), and were washed once or twice with 2x RIPA / TET which was in a ratio of 5: 4. The 2x RIPA consists of 20mM Tris (pH 7.4), 0.3M NaCl, 2% sodium deoxycholate, and Igepal CA-630® (Sigma) at 2% (v / v). The TET consists of 0.1 M Tris (pH 8.0), 50 mM EDTA and 2% Triton X-100 (v / v). The inclusion bodies were dissociated in 5 M guanidine hydrochloride, adjusted to > 1.4 mg / ml protein in 2.5 M guanidine hydrochloride, and filter sterilized (0.2 μm). This preparation was used for the vaccination tests described below. Wet cells of transformants of E. coli LW14 from a fermentation batch of 5 liters that had grown in 2X medium YT Km50, and which expressed pp-Omp20, were harvested by centrifugation. The cells were suspended in 25% sucrose - 50 mM Tris-HCl, pH 8.0 with lysozyme (cells dispersed in 0.5 ml of sucrose buffer per 50 ml of culture, for each ml of sucrose buffer, 0.125 ml was added. of a solution of lysozyme at 10 mg / ml) and sonicated (treated by ultrasound). The inclusion bodies were collected by centrifugation (12,000 RCF, 30 min), washed with 2x RIPA / TET as above, collected again by centrifugation, and washed with 0.1 M glycine and Zwittergent 3-14 (Calbiochem) at pH 1 1. The pH was adjusted to 7.0 and the inclusion bodies were collected by centrifugation (12,000 RCF, 30 min), dissolved in 3.5 M guanidine hydrochloride (final protein concentration, 6.36 mg / ml), and sterilized in filter (0.2 μm). This preparation was used for the vaccination tests described below. Wet cells of E. coli transformants LW14 from a 600 ml flask culture, which had grown in 2X medium YT Km50, as described above, and which expressed pp-Omp27, were harvested by centrifugation. The cells were suspended in sucrose at % - 50 mM Tris-HCl, pH 8.0, with lysozyme, as above, then sonicated, washed with 2x RIPA TET as above, and collected by centrifugation. The inclusion bodies were collected by centrifugation (12,000 RCF, 30 min), were dissolved in 5 M guanidine hydrochloride (final concentration of protein 2.46 mg / ml), and sterilized in filter (0.2 μm). This preparation was used for the vaccination tests described below. 9. EXAMPLE Immunological characterization of recombinant APP proteins 9. 1 Materials and Methods 9. 1.1. Preparation and Quantification of Whole Cell Antigens of APP for Western Blotting Antigens of whole bacterial cells were prepared as described above in Section 6.1.4, except that the HP growth medium replaced the MM medium and the cells were suspended in 10 ml. of DPBS instead of 5 ml of DPBS. Protein centrifugation of each preparation was determined using a BCA Protein Analysis kit (from Pierce). Said briefly, each sample was diluted to 1/10, 1/20, 1/40 and 1/80 in deionized and distilled water (ddH2O) sterile. BSA (bovine serum albumin, standard protein) was diluted to concentrations ranging from 200 to 800 μg / ml. A volume of 20 μl of sample or standard was added to wells in triplicate in a 96-well microtiter plate, and 200 μl of Reagent B diluted 1/50 in Reagent A was added to each well.

The plate was incubated at 37 ° C for 30 min. The absorbance of the samples was determined at 560 nm. The protein concentration for each of the samples was calculated by extrapolation using the BSA standard curve. 9. 1.2. Antibodies Secondary antibodies used for Western blots flowed goat anti-porcine IgG conjugated with alkaline phosphatase (H + L) and goat anti-mouse IgG (H + L) (Kirkegaard and Perry Laboratories). These antibodies were used to visualize an antibody specific for an APP protein in serum samples or supernatant material by Western blot analysis. Both antibodies were diluted 1/1, 000 in a buffer for dilution (PBS, 0.05% Tween 20, 5% skimmed milk powder) before use. 9. 1.3. Vaccination Protocol Preparations of recombinantly expressed proteins, produced as described above in Section 8.3, were diluted to 80 μg / ml in DPBS, and then combined to 1: 1 with 2x concentrated SEAM-1 adjuvant (Quil A 80 μg / ml, 16 μg / ml cholesterol, 5% squalene, 1% Span 85, 0.1% vitamin A acetate, 0.1% ethanol and 0.01% thimerosol).

Male CF1 mice were injected by s.c. with 0.25 ml of a protein and adjuvant preparation equivalent to 10 μg of recombinant protein, 10 μg of Quil A and 2 μg of cholesterol. Negative control groups (with adjuvant) received 0.25 ml of DPBS mixed 1: 1 with adjuvant. The mice were vaccinated a second time with the same protein preparation at 20-22 days after the primary vaccination.

Two weeks after the second vaccination, the animals were anesthetized with CO2 and bled either by the brachial artery or by cardiac puncture. The serum was separated from each of the blood samples, and serum pools from mice within the same group were stored at 20 ° C. 9. 1 .4. Western Blot Analysis A volume of lysed material from whole bacterial APP cells (derived from APP serotypes 1, 2, 5, 7 or 9, prepared as above is described in Section 9.1.1.) Corresponding to 10 μg of protein, mixed with water to a final volume of 10 μi, and 2 μl of 5x reducing buffer for sample (Pierce) was added. In a similar manner, an aliquot of recombinant protein was also prepared (see Section 8.3 above) (loading with protein was variable). The samples were heated for 5 min at 100 ° C and the whole volume was loaded in separate wells of a 14% Tris-glycine gel, 1.5 mm thick and 15 wells (Novex).

Wide-range, pre-stained molecular weight markers (5 μl / well) (BioRad) were also included in each of the gels. Proteins separated on selected gels were stained with Coomassie Blue. Proteins separated by SDS-PAGE were transferred to PVDF membranes (bioRad) with a constant electric current intensity of 200 mA for 1.5 h. The spots were either: (i) directly incubated in blocking buffer (skim milk powder % and 0.05% Tween 20 in PBS); or (ii) dried at room temperature, stored in the frozen state at -20 ° C until needed, then rehydrated in methanol, rinsed in water and subsequently incubated in blocking buffer. The membranes were incubated in blocking buffer (also using as a dilution buffer) overnight with gentle shaking. The blocking buffer was removed, and the diluted sample of serum or supernatant was added to the membrane, followed by an incubation for 1 h at room temperature. After having withdrawn the test sample, the membranes were washed 3 times for 5-10 min each time with PBST (PBS with 0.05% Tween 20). Anti-IgG anti-IgG or porcine anti-IgG antibodies, conjugated with alkaline phosphatase (H + L) were diluted, added to the washed membrane, and incubated for 1 h at room temperature. The membranes were washed with PBST, and the BCIP / NBT substrate (Kirkegaard and Perry Laboratories) was added to the membranes and incubated with gentle agitation until an appropriate co-calorimetric reaction developed. The membranes were then rinsed with water to stop the reaction and dried at room temperature. 9. 2. Results The antigenic characteristics of the new APP proteins were determined using the following three methods. The first method used pig antibody probes, that is, sera from convalescent pigs or ASC supernatants, obtained from experimentally infected animals either with serotype 1 of APP or with serotype 5, or with serotype 5 followed by restimulation with serotype 7, as immunological probes in Wstern transfer blots containing the recombinantly expressed APP proteins (TABLE 3). The second method used sera from mice immunized with APP proteins recombinantly expressed to probe Western blots containing APP antigens (whole bacterial cell pellets) (TABLE 4). The third method used sera from mice immunized with APP proteins recombinantly expressed to probe Western blots containing the recombinantly expressed APP proteins (TABLE 5). The results of each of the methods are described below. 9. 2.1. Recognition of Recombinant APP Proteins by Probes Pig Antibodies Generated Against APP Serotypes Antibody probes (sera or ASC supernatants) were obtained from pigs as a continuation of an experimental stimulation with serotype 1 of APP, or with serotype 5, or with the serotype 5 followed by restimulation with serotype 7. The sera were used to originally identify the new proteins of APP (FIGURES 1 -4). TABLE 3 summarizes the reactivity of the antibody probes with APP proteins recombinantly expressed by Western blotting. The ASC probes generated as a continuation of a stimulation with serotype 5 and a restimulation with serotype 7 recognized the recombinant proteins OmpW, OmpAI, OmpA2 and Omp20. The ASC probes do not react immunologically with the recombinant Omp27. In contrast, sera derived from animals that were stimulated with serotype 1, or with serotype 5, or serotype 5 followed by restimulation with serotype 7, only recognized the recombinant proteins OmpAI and OmpA2. An ASC probe obtained from an unstimulated control pig (No. 780) does not react with any of the recombinantly expressed proteins. However, a control pig did not stimulate additional (No. 779) reacted with all recombinantly expressed proteins. In addition, serum from an unstimulated control pig (No. 1421) reacted as OmpAI and OmpA2. These last two animals were suspected to have been infected with APP subclinically 9. 2.2. Recognition of APP Proteins by Mouse Antisera Generated Against APP Proteins Recombinants Antisera from mice immunized with the recombinantly expressed APP proteins were used to probe Western blots containing APP antigens from bacterial cell pellets. The results are compiled in the TABLE. 4. Mice immunized with the pp-OmpW, or pp-OmpA1, or pp-OmpA2, recombinants, produced serum antibodies that recognized specific bands compatible with the predicted molecular weights of the particular APP protein. However, sera from immunized mice do not react specifically with the particular native protein in any of the serotypes tested. 9. 2.3. Recognition of Recombinant APP Proteins by Mouse Antisera Generated Against Recombinant APP Proteins Antisera from mice immunized with the newly expressed recombinant proteins were used to probe Western blots containing the recombinant APP proteins. The results are compiled in TABLE 5. Antisera from mice immunized with recombinant OmpW, as a fusion protein either with GST or with pp, they recognized recombinant OmpW, OmpAI and OmpA2 proteins. Sera from mice immunized either with recombinant OmpAI or with recombinant OmpA2 reacted strongly with both OmpAI and OmpA2 immunogens. Sera from mice immunized with recombinant Omp20 reacted strongly with recombinant Omp20, and to a greater degree with recombinant OmpAI and OmpA2. In contrast, sera from mice vaccinated with recombinant Omp27 do not recognize a recombinant Omp27, but react with recombinant OmpAI and OmpA2. Sera from control mice vaccinated with PBS reacted c very weakly with recombinant OmpAI and OmpA2, and do not recognize recombinant OmpW, Omp20 or Omp27. In summary, pigs that had been experimentally infected with APP produced local antibodies (ASC probes) that recognized recombinant proteins OmpW, OmpAI, OmpA2 and Omp20, whereas serum antibodies only reacted with recombinant OmpAI and OmpA2, which demonstrated by Western blots (TABLE 3). Therefore, it is stated that the serum is much more restricted in terms of immunological reactivity than the ASC probes. Neither the serum nor the ASC probes recognized a recombinant expressed Omp27. It is possible that Omp27 is not recognized in a Western blot analysis due to the denaturing conditions of the analysis The immunological characterization of recombinant OmpW, OmpAI and OmpA2 indicates that these proteins can induce serum antibodies that recognize native proteins (based on predicted molecular weight) found in serotypes 1, 2, 5, 7, 8 and 9 (TABLE 4), as well as recognized in the recombinantly obtained forms of these proteins (TABLE 5), and in that a recombinant Omp20 was also recognized. Sera from mice immunized with recombinant Omp20 or Omp27 were used to probe Western blots containing antigens from whole bacterial cells, derived from serotypes 1, 2, 5, 7, 8 and 9 of APP that had grown in vitro (TABLE 4). These sera do not recognize bands compatible with their native form in any of the APP serotypes that were examined. It is possible that the Omp20 and Omp27 represent antigens of APP that are only expressed in vivo, and therefore are present in sediments of bacterial cells prepared in the laboratory. Alternatively, these two proteins may have been denatured by the Western blot procedure and made unrecognizable to specific antibodies.

TABLE 3 Reactivity of sera and ASC supernatants from convalescent pigs against recombinant APP proteins 1 Each of the recombinant proteins contained protective erpeptide (pp), lacked the native signal sequence, and was derived from inclusion bodies (IB). 2 Serum, diluted 1/125, or ASC probes, diluted 1/4, were assayed for specific antibodies for each of the recombinant proteins. 3 Animals were stimulated with the indicated APP serotype. Animals No. 803 and 808 were stimulated with serotype 5, allowed to recover from infection and then re-stimulated with serotype 7. 4 (+) indicates the presence of a band corresponding to the indicated recombinant protein; (-) indicates that serum from the ASC probe does not react with the specified recombinant protein.

ND = not determined.

TABLE 4 Reactivity of a serum from mice immunized with recombinant APP proteins against whole cell preparations of APP 1 Proteins in whole bacterial cell preparations (10μg per lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with mouse serum (1/50). 2 Mice are immunized twice subcutaneously (s.c.) with a recombinant APP protein preparation or with PBS (control). All of the recombinant APP proteins used for immunizations of mice were solubilized inclusion body preparations. All APP proteins contained the protective peptide except the OmpW that was used either as an OmpW-GST fusion protein or as a pp-OmpW fusion protein. 4 (+) indicates the presence of a band corresponding to the recombinant protein used to immunize the animal; (-) indicates the absence of a specific band.

TABLE 5 Reactivity of a serum from mice immunized with recombinant APP proteins against recombinant APP proteins 1 Recombinant proteins separated by SDS-PAGE were transferred to PVDF membranes and probed with mouse serum (1/50). 2 Mice were immunized twice s.c. with a recombinant protein preparation or with PBS (control). 3 Each of the recombinant APP proteins present in the gels of SDS-PAGE contained the protective peptide (pp) and lacked the native signal sequence. 4 All the recombinant APP proteins used for immunizations of mice came from solubilized inclusion body preparations. All proteins contained the protective peptide except the O p that was used either as an OmpW-GST fusion protein or as a pp-OmpW fusion protein, and all proteins lacked the native signal sequence. 5 (+) indicates that the test serum reacted with the specified recombinant protein; (-) indicates the absence of a specific band; for the control with PBS, (+/-) indicates that these bands were visible but very diffuse compared to a serum from an animal? nmunizadcT (++) indicates a very strong immunoreactivity. 6 ND = not determined.

. EXAMPLE Study of animals to test the efficacy of various combinations of antigens . 1 Materials and Methods 50 apparently healthy cross-bred pigs (with an approximate age of 6.5 weeks) were obtained from a herd without a history of disease by APP or vaccination against APP. The animals were assigned randomly by the litter and by the title of cytolytic neutralization antibodies Apxll to five groups of 10 pigs each (in 98% the animals had neutralization titers of the study). The pigs were acclimated for one week before the initiation of the study. The animals were vaccinated with 2 ml of the appropriate vaccine (APP proteins with pp and without signal sequence) or with a placebo by the intramuscular route (IM, in the muscle of the left neck) on day 0, when the pigs they were approximately 7.5 weeks old. After 3 weeks, the animals were revaccinated with a second dose of 2 ml (LM, in the muscle of the right neck). TABLE 6 identifies the vaccines used for the first and second vaccinations of the 5 groups of pigs.

TABLE 6 All the pigs were observed, for approximately 1 h after the vaccinations, in terms of vomiting, depression, diarrhea, ataxia and lack of coordination, increased respiration and tremors. In addition, observations were made for 3 days following the first and second vaccines. Rectal temperatures were recorded one day before vaccination, immediately before vaccination, at 6 h after vaccination and 1 day after vaccination. Two weeks after the second vaccination, the pigs were stimulated intranasally with a live virulent culture of APP serotype 1 (ATCC strain 27088) which causes a mortality of approximately 50% in non-immune pigs. A dose of 1.0 ml was used (0.5 ml per nostril) of a culture containing 1.5 x 107 cfu / ml. All animals were anesthetized before stimulation with an i.m. which consisted of a combination of 50 mg of telazol, 50 mg of xylazine and 50 mg of ketamine per ml in the regime of 1.0 mi / 22.5 kg (50 pounds) of body weight. . 2. Results No significant increases in temperature were observed after the first or second vaccinations with the recombinant proteins of the present invention. Significant local reactions were observed subsequent to vaccination in animals that received the commercial vaccine compared to all other groups (TABLE). 7. None of the animals that received the new APP proteins alone (Group C), and only one animal that received a second vaccination of the new APP proteins and a combination of Apxl / Apxil / OmIA (5) (Group A), exhibited local reactions subsequent to vaccination.

TABLE 7 * The numbers represent a group average Group A, which had been vaccinated with all the new APP proteins and with Apxl / Apxil / OmIA (5), had lower mortality than any other group, including the commercial vaccine (30% versus 60%) (see CHART) 8). The amount of damage to the lungs (% of injuries) was also less in Group A compared to Groups B, C and Control, but similar to damage to the lungs that had been observed in animals that received the commercial vaccine (Group D).

SQUARE 8 These results indicate that a vaccine, which is composed of the new APP proteins of the present invention in combination with toxin antigens, provides protection against a stimulation by heterologous APP, whose protection is equivalent to or superior to that of a commercial vaccine. 11. EXAMPLE Preparation of plasmids and materials in deposit Artificial structures of separate plasmids were prepared, which encoded each of the APP proteins for deposit in the American Type Culture Collection (ATCC). Each of the artificial structures contains the total ORF encoding the particular APP protein with native signal sequence. The ORF's were inserted into the cloning site with TA of pCR2. 1 Mole in the opposite orientation with relation to the lactose promoter. The ORF's were obtained by PCR from Genomic DNA of APP serotype 7 using the primers listed below in TABLE 9. The host cells were E. coli Top10. Both the host cells and the vector are available from Invitrogen (Carlsbad, CA). All 5 'primers start at the ATG start codon of the respective ORF. The strains prepared as above, and listed in TABLE 9 below, were deposited with the ATCC domiciled in 10801 University Blvd., Manassas, VA, 20110, USA, on October 15, 1998 and were assigned the access numbers that are listed.

TABLE 9 All patents, patent applications and publications that have been previously cited are incorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the specific embodiments described, of which it is intended that they constitute simple illustrations of individual aspects of the invention. Functionally equivalent compositions and functionally equivalent methods are within the scope of the invention.

LIST OF SEQUENCES < 110 > Ffizsr Proauc 3 Inc. < 120 > NEW PROTEINS FROM ACTINOBAClLUS PIEUROPNEUMINIAE < 130 > PC9854A < 140 > < 141 > < 160 > 96 < 170 > Pateptln Ver. 2.0 < 210 > 1 < 211 > 1018 < 212 > DNA < 213 > Actinobacillus pleuropneumoniae < 220 > < 221 > CDS < 222 > (272) .. (787) < 400 > 1 ggttggaaaa caccttatsa agtttacttc aaaaaatcgt tgcacttggt ttgacaattc 60 aagactaaaa atgaccggtc gtsagctaaa accgcatgac cgtactgtgg atgtgacgat 120 tcgrcgtatt cgtaaacact ttgaagatca ccctaataca ccggaaatca ttgtaaccat 180 tcatggtgaa ggttaccgtt tttgcggcga gttagagtag taattaaacg cctataagcg 240 tttagcatct tctttctaaa aaggacattt t atg aaa aat tta here gtt tta 292 Met Lys Asn Leu Thr Val Leu May 1 ca tta gca ggt tta ttc tet gcg tcg gca ttt gcc gca ccg gtc gga Wing Leu Wing Gly Leu Phe Being Being Wing Phe Wing Wing Pro Val Gly 340 10 15 20 A_n Th_ The? £ g and fa £ J \ f £ ¡£ J "gat ctc acc ac gta aaa 388 25 Y Í Val Giy Val AsP Leu Thr Thr Val Lys tat aaa gtg gac ggt gtg aaa ggt aaa caa ta acc ggt cct gcg tta Tyr Lys Val Asp Gly Val Lys Gly Lys Gln Ser Thr Gly Pro Ala Leu 436 40 45 50 55 gtc gta gat tac ggt atg gat tac ggt gac aat ttt gtc ggt gtt gta Val Val Asp Tyr Gly Met Asp Tyr Gly Asp Asn Phe Val Gly Val Val 484 60 65 70 ca ggt aaa gta aaa gta ggc agt here aaa gta ttt age gat gta aaa Gln Gly Lys Val Lys Val Gly Ser Thr Lys Val Phe Ser Asp Val Lys 532 75 * 80 85 caa aaa act aaa tat act gtc gct tat ca g ca g tat tat gta gct Gln Lys Thr Lys Tyr Thr Val Wing Tyr Gln Gln Gly Tvr Arg Val Wing 580 90 95 100 tet gat tta ctt ccg tat gtc aaa gtc gat gtg gcg caa agt aaa gtc 628 Being Asp Leu Leu Pro Tyr Val Lys Val Asp Val Wing Gln Ser Lys Val 105 110 115 ggc gat acc aat ttc cgt ggt tac ggt tac ggt ggt ggt gct aaa tat 676 Gly Asp Thr Asn Phe Arg Gly Tyr Gly Tyr Gly Wing Gly Wing Lys Tyr 120 125 130 135 gcc gta gta tea agt aat gta gaa gtg ggt gcg gaa tat acg cgc age aat 724 Wing Val Ser Ser Asn Val Glu Val Gly Wing Glu Tyr Thr Arg Ser Asn 140 145 150 tta aga aaa age ggt gct aaa tta aaa ggt aat gaa ttt act gcg aac 772 Leu Arg Lys Ser Gly Ala Lys Leu Lys Gly Asn G lu Ala Phe Thr Asn 155 160 165 ggt cta tac cgt ttc taattatttt tecettatga caagcggtcg Gly Tyr Leu 827 tttcttgcaa Arg Phe 170 aaaatttgcg aaaaacgacc gcttattttt ttattaatac tttatttact gagccatttt 887 ttcagctacg gttagaaaac cgtctgcagt cgcatagatt tcttcaaagc cttgcgcttg 947 tagaatacgg tcggacactt gccctctcca cacgaaatgc ctaatatcca cctgcctttt atccgctttt 1007 g, 1018 < 210 > 2 < 211 > 172 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 400 > 2 Met Lys Asn Leu Thr Val Leu Ala Leu Ala Gly Leu Phe Ser Ala Be 1 5 10 15 Ala Phe Ala Ala Pro Val Gly Asn Thr Phe Thr Gly Val Gly Val Gly 20 25 30 Val Asp Leu Thr Thr Val Lys Tyr Val Lys Asp Gly Val Lys Gly Lys 35 40 45 Gln Ser Thr Gly Pro Wing Leu Val Val Asp Tyr Gly Met Asp Tyr Gly 50 55 60 Asp Asn Phe Val Gly Val Val Gln Gly Lys Val Lys Val Gly Ser Thr 65 70 75 80 Lys Val Phe Ser Asp Val Lys Gln Lys Thr Lys Tyr Thr Val Wing Tyr 85 90 95 Gln Gln Gly Tyr Arg Val Ala Ser Asp Leu Leu Pro Tyr Val Lvs Val 100 105 110 Asp Val Wing Gln Ser Lys Val Gly ASD Thr Asn Phe Arg Gly Tvr Gly 115 120 '125 Tyr Gly Wing Gly Wing Lys Tyr Wing Val Ser Ser Asn Val Glu Val Gly 130 135 140 Wing Glu Tyr Thr Arg Ser Asn Leu Arg Lvs Ser Gly Wing Lys Leu Lys 145 150' 155 160 Gly Asn Glu Phe Thr Wing Asn Leu Gly Tyr Arg Phe 165 170 < 210 > 3 < 211 > 1188 < 212 > DNA < 213 > Actinobacillus pleuropneumoniae < 220 > < 221 > CD? < 222 > (376) .. (1020) < 400 > 3 ctaacgcata aagtaaatgt gccggttcaa tgtagttatt atcttttccg atagctaacg 60 attgggcttc tgcaagggct tcttgcaatt tggtagtaaa tttttcgaaa ttcatatttt 120 tactcctaaa tttcattaat ctgtatcgag cagaatttat accgcttcaa cgttttaata 180 gtcgaactta aatggagcta tttcaagtga aaatgtgaaa aagatcgcaa aaaataaatt 240 agtacctcgt tgtaggtact aaaatggcgt atatttgatt cttgtcaata aaagttagcc 300 gaattgttct tagaatgtta ttaacgtaac gaattggtta cttttttatt tttaagaaaa 360 tattaagagg tcaaa atg aaa aaa gca gta tta gcg gta gca tta ggc ggt 411 Met Lys Lys Ala Val Leu Ala Ala Val Leu Gly Gly 1 May 10 gcg tta tta gcg ggt tcg gca atg gca cat caa gcg ggc gat gtg att 459 Ala Leu Leu Ala Gly Ser Ala Met Ala His Gln Ala Gly Asp Val lie 15 20 25 ttc cgt gcg gcg atc ggt gtg att gca aat tea agt tcg gat tat 507 Phe Arg Wing Gly Wing He Gly Val lie Wing Asn Being Ser Asp Tyr 30 35 40 cag acc ggg gcg gac gta aac tta gat gta aat aat aat att cag ctt 555 Gln Thr Gly Ala Asp Val Asn Leu Asp Val Asn Asn Asn He Gln Leu 45 50 55 60 ggt tta acc ggt acc tat atg tta agt gat aat tta ggt ctt gaa tta 603 Gly Leu Thr Gly Thr Tyr Met Leu Ser ASD Asn Leu Gly Leu Glu Leu 65 70 75 tta gcg gca here ccg ttt tet cac aaa atc acc ggt aag tta ggt gca 651 Leu Ala Wing Thr Pro Phe Ser His Lys He Thr Gly Lys Leu Gly Wing 80 85 90 here gatta ggc gaa gtg gca aaa gta aaa cat ctt ccg ccg age ctt 699 Thr Asp Leu Gly Glu Val Wing Lys Val Lys His Leu Pro Pro Ser Leu 95 100 105 tac tta caa tat tat ttc ttt gat tet aat gcg here gtt cgt cea tac 747 Tyr Leu Gln Tyr Tyr Phe Phe Asp Ser Asn Ala Thr Val Arg Pro Tyr 110 115 120 gtt ggt gcc ggt tta aac tat act cgc ttt ttc agt ct gaa agt tta 795 Val Gly Ala Gly Leu Asn Tyr Thr Arg Phe Phe Ser Ala Glu Ser Leu 125 130 135 140 ccg aaa caa gta tta caa aac tta cgt gtt aaa aaa cat aka gtc gca 843 Lys Pro Gln Leu Val Gln Asn Leu Arg Val Lys Lys His Ser Val Wing 145 150 155 ccg att gcg aat tta ggt gtt gat gtg aaa tta acg gat aat cta tea 891 Pro He Wing Asn Leu Gly Val Asp Val Lys Leu Thr Asp Asn Leu Ser 160 16 5170 ttc aat gcg gca gct tgg tac here cgt att aaa act act gcc gat tat 939 Phe Asn Ala Ala Ala Trp Tyr Thr Arg He Lys Thr Thr Ala ASD Tyr 175 180 185 gat gtt ccg gga tta gg cat gta agt here ccg att act tta gat cct 987 - | Q Asp Val Pro Gly Leu Gly His Val Ser Thr Pro He Thr Leu Asp Pro 190 195 200 gtt gta tta ttc tea ggt att age tac aaa ttc taagtatttt gaaactgtta 1040 Val Val Leu Phe Ser Gly He Ser Tyr Phe Lys 205 210 215 tgagaaaggg agcgttaatc gctccctttt tgttgtaaaa aatccttgaa aaacgaccgc 1100 caaaaatgta ttgttaagca ggatcatttt agtgagcaat tcacgagtcg gctcaataaa 1160 1188 ttttgtttct aaaaattcat ccggctgg < 210 > 4 < 211 > 215 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 400 > 4 Met Lys Lys Wing Val Leu Wing Wing Val Leu Gly Gly Wing Leu Leu Wing 1 5 10 15 Gly Being Wing Met Wing His Gln Wing Gly Asp Val He Phe Arg Wing Gly 20 25 30 Wing He Gly Val He Wing Asn Being Being Asp Tyr Gln Thr Gly Wing 20 35 40 45 Asp Val Asn Leu Asp Val Asn Asn Asn He Gln Leu Gly Leu Thr Gly 50 55 60 Thr Tyr Met Leu Ser Asp Asn Leu Gly Leu Glu Leu Leu Wing Ala Thr 65 70 75 80 Pro Phe Ser His Lvs He Thr Glv Lys Leu Gly Wing Thr Asp Leu Gly 85 '90 95 Glu Val Wing Lys Val Lys His Leu Pro Pro Ser Leu Tyr Leu Gln Tyr 100 105 HO Tyr Phe Phe Asp Ser Asn Wing Thr Val Arg Pro Tyr Val Gly Wing Gly 115 120 125 Leu Asn Tyr Thr Arg Phe Phe Ser Wing Glu Ser Leu Lys Pro Gln Leu 130 135 140 Val Gln Asn Leu Arg Val Lys Lys His Ser Val Wing Pro He Wing Asn 145 150 155 160 Leu Gly Val Asp Val Lys Leu Thr Asp Asn Leu Ser Phe Asn Wing Wing 165 170 175 Wing Trp Tyr Thr Arg He Lys Thr Thr Wing ASD Tyr Asp Val Pro Gly 180 185 190 Leu Gly His Val Ser Thr Pro He Thr Leu Asp Pro Val Val Leu Phe 195 200 205 Ser Gly He Ser Tyr Lys Phe 210 215 < 210 > 5 < 211 > 1171 < 212 > DNA J < 213 > Actinobacillus pleuropneumoniae. < 220 > < 221 > CDS < 222 > (157) .. (930) < 400 > 5 tatttgagct taggctttaa taaagctcga atcctaagcc aggaaatata gaaagtacat 60 taaatataat ttagtattgt attaatagag gataaagcca caaactggca agcaagaatt 120 ggttttactt tttaacctca ctaaaaggag acaact atg aaa cat age aaa ttc 174 Met Lys Hi = Ser Lys Phe 1 5 aaa tta ttt aaa tat tat tta att ttt cct ttt att act ttt gca 222 Lys Leu Phe Lys Tyr Tyr Leu He Ser Phe Pro Phe He Thr Phe Wing 10 15 20 agt aat gtt aat gga gcc gaa att gga ttg gga gga gcc cgt gag agt 270 Ser Asn Val Asn Gly Wing Glu He Gly Leu Gly Gly Wing Arg Glu Ser 25 30 35 agt att tac tat tet aaa cat aaa gta gca here aat ccc ttt tta gca 318 Ser He Tyr Tyr Ser Lys Hi = Lys Val Wing Thr Asn Pro Phe Leu Wing 40 45 50 ctt gat ctt tet tta ggt aat ttt tat atg aga ggg act gca gga att 366 Leu Asp Leu Ser Leu Gly Asn Phe Tyr Met Arg Gly Thr Ala Gly He 55 60 65 70 age gaa ata gga tat gaa caat tet tcc act gac aat ttc age gta tea 414 Ser Glu He Gly Tyr Glu Gln Ser Phe Thr Asp Asn Phe Ser Val 75 75 85 ctg ttt gtt aac cea ttt gat ggt ttt tea att aaa gga aaa gac ttg 462 Leu Phe Val Asn Pro Phe Asp Gly Phe Ser lie Lys Gly Lys Asp Leu 90 95 100 tta cct gga tat ca ata agt att ca act cgc aaa act ca ttt gcc ttt 510 Leu Pro Gly Tyr Gln Ser lie Gln Thr Arg Lys Thr Gln Phe Ala Phe 105 110 115 ggt tgg gga tta aat tat aat ttg gga ggt tta ttc ggc tta aat gat 558 Gl Trp Gly Leu Asn Tyr Asn Leu Gly Gly Leu Phe Gly Leu Asn A = D 120 125 130 act ttt ata t'cc ttg gaa gga aaa age gga aaa cgt ggt gcg agt agt 606 Thr Phe He Ser Leu Glu Gly Lys Ser Gly Lys Arg Gly Ala Ser Ser 135 140 145 150 aat gtc age tta ctt aaa tcg ttt aat atg acg aaa aat tgg aaa gtt 654 Asn Val Ser Leu Leu Lys Ser Phe Asn Met Thr Lys Asn Trp Lys Val 155 160 165 tea cea tat att ggc tea ag tat tea tet aaa t at here gat 702 Ser Pro Tyr He Gly Ser Ser Tyr Tyr Ser Ser Lys Tyr Thr Asp Tyr 170 175 180 tac ttt ggt att aaa caa tcc gaa tta ggt aat aaa att here tcc gta 750 Tyr Phe Gly He Lys Gln Ser Glu Leu Gly Asn Lys He Thr Ser Val 185 190 195 tat aaa cct aaa gca gct tat gca here cac ata ggt att aat act gat 798 Tyr Lys Pro Lys Ala Ala Tyr Ala Thr His lie Gly He Asn Thr Asp 200 205 210 tat gct ttc acg aac aat ctt ggc atg ggt tta tet gtc ggt tgg aat 846 Tyr Ala Phe Thr Asn Asn Leu Gly Met Gly Leu Ser Val Gly Trp Asn 215 220 225 230 aaa tat tet aaa gaa att aag caat tet cct atc ata aaa cga gac tet 894 Lys Tyr Ser Lys Glu He Lys Gln Ser Pro He He Lys Arg ASD Ser 235 240 245 caá ttt act tea tet ctt aaggee cctttt ttaatt ttaatt aaaagg ttttcc taaaatagaa 940 GGllnn PPhhee TThhrr SSeerr SSeerr LLeeau SSe.rT- LT-e.u. TTyyr "T" y-r L 'ys' P "I 250255 tattctaggg agaataetca ttctttatct ttataaagtt aattgtttct ccctgtttct 1000 atattattta gttacttgtt caaaagctae attggttatt ttgtcatttt ataaaagata 1060 ataaggtggt tattttgaaa attaagaaat atattaaata tacectattt actttccttt 1120 taggcatatc atatttatat tttgggggcg aaaacgaaaa ttatcaagag to 1171 < 210 > 6 < 211 > 258 < 212 > 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 400 > 6 Met Lys His Ser Lys Phe Lys Leu Phe Lvs Tyr Tyr Leu He Ser Phe 1 5 Í0 15 Pro Phe He Thr Phe Wing Being Asn Val Asn Giv Wing Giu He Gly Leu 20 25"30 Gly Gly Wing Arg Glu Being Ser He Tyr Tyr Being Lys His Lys Val Wing 35 40 45 Thr Asn Pro Phe Leu Wing Leu Asp Leu Being Leu Gly Asn Phe Tyr Met fifty . 55 60 Arg Gly Thr Wing Gly He Ser Glu He Gly Tyr Glu Gln Ser Phe Thr 65 70 75 80 Asp Asn Phe Ser Val Ser Leu Phe Val Asn Pro Phe Asp Gly Phe Ser 85 90 95 He Lys Gly Lys Asp Leu Leu Pro Gly Tyr Gln Ser He Gln Thr Arg 100 105 110 Lys Thr Gln Phe Wing Phe Gly Trp Gly Leu Asn Tyr Asn Leu Gly Gly 115 120 125 Leu Phe Gly Leu Asn ASD Thr Phe lie Ser Leu Glu Gly Lys Ser Gly 130"135 140 Lys Arg Gly Ala Ser Ser Asn Val Ser Leu Leu Lys Ser Phe Asn Met 145 150 155 160 160 Thr Lys Asn Trp Lys Val Ser Pro Tyr He Gly Ser Ser Tyr Tyr Ser 165 170 175 Ser Lys Tyr Thr Asp Tyr Tyr Phe Gly He Lys Gln Ser Glu Leu Gly 18 ° 185 190 Asn Lys He Thr Ser Val Tyr Lys Pro Lys Wing "Ala Tvr Ala Thr His 195 200 205 He Gly He -Asn Thr ASD Tyr Ala Phe Thr Asn Asn Leu Gly Met Gly 210" 215, 220 Leu Ser Val Gly Trp Asn Lys Tyr Ser Lys Glu He Lys Gln Ser Pro 225 230 235 240 He He Lys Arg Asp Ser Gln Pne Thr Ser Ser Leu Ser Leu Tvr Tyr 245 250 255 Lys Phe < 210 > 7 < 211 > 1922 < 212 > DNA < 213 > Actinobacillus pleuropneumoniae < 220 > < 221 > CDS < 222 > (614) .. (1705) < 400 > 7 acggtaacta cttattcttc tcatgttcca acsccaattc ttgcagagaa attaatcccg 60 atgttacaaa aaggcgactt aggggagccg acacctgctg ctgaaatcga caacgtttac 120 ttacgtgata-- tcaacgatgc aatccgtaac catccggttg aattaatcgg tcaagagtta 180 cgtggttata tgacggatat gaaacgtatt tcatcgcaag gttaattaaa aattaatcaa 240 aagcctactt cgcaagaagt gggctttttg ttattcaagc cgcttaccgc tatcaatggt 300 aagtgatatg cataataget tataaattat aagttgtttt aageaaatat atctctatcg 360 gtaastaaaa aattaattgt aaaacggata agatcattaa aaaaaatcta ttttttgagt 420 taaaaaatga gaattaegea tetaaattat aggtttagtt gtatttttca atttttattt 480 ggtagaatac aactgtaata aaagcttaat tattttgaga tacacataaa ataatttacg 540 gctttattca ttatcccttt taggttaggg atttgtcttt aatagatgac gataaattta 600 aaa gaggatcatc atg aaa aaa tea tta gtt gct tta here gta tta tcg 649 Met Lys Lys Ser Leu Val Ala Leu Thr Val Leu Ser 1 5 10 gct gca gcg gta gct ca gca gcg cea caa ca aat act ttc tac gca 697 Ala Ala Ala Val Ala Gln Ala Ala Pro Gln Gln Asn Thr Phe Tyr Ala 15 20 25 ggt gcg aaa gca ggt tgg gc g tea ttc cat gat ggt atc gaa cata tta 745 Gly Wing Lys Wing Gly Trp Wing Being Phe His Asp Gly He Glu Gln Leu 30 35 40 gat tea gct aaa aac here gat cgc ggt here aaa tac ggt atc aac cgt 793 Asp Ser Ala Lys Asn Thr Asp Arg Gly Thr Lys Tyr Gly He Asn Arg 45 50 55 60 aat tea gta act tac ggc gta ttc ggc ggt tac cata attta aac caa 841 A = n Ser Val Thr Tyr Gly Val Pne ¿íy Gly Tyr Gln He Leu Asn Gln 65 70 75 gac aaa tta ggt tta gcc gct gaa tta ggt tat gac tat ttc ggt cgt 889 Asp Lys Leu Gly Leu Ala Wing Glu Leu Gly Tyr Asp Tyr Phe Gly Arg 80 85 90 gtg cgc ggt tet gaa aaa cea aac ggt aaa gcg gac aag aaa act ttc 937 Val Arg Gly Ser Glu Lys Pro Asn Gly Lys Wing Asp Ly = Lys Thr Phe 95 100 105 cgt cac gct gca cac ggt gcg here atc gca tta aaa cct age tac gaa 985 Arg His Ala Ala His Gly Ala Thr He Ala Leu Lys Pro Ser Tyr Glu 110 115 120 gta tta cct gac tta gac gtt tac ggt aaa gta ggt atc gca tta gta 1033 Val Leu Pro Asp Leu Asp Val Tyr Gly Lys Val Gly He Ala Leu Val 125 130 135 140 c ^ aac aat here tat aaa here ttc aat gca gca ca gag aaa gtg aaa act 1081 Asn Asn Thr Tyr Lys Thr Phe Asn Wing Wing Gln Glu Lys Val Lys Thr 145 150 155 cgt cgt ttc caá agt tet tta att tta ggt gcg ggt gtt gag tac gca 1129 Arg Arg Phe Gln Ser Ser Leu He Leu Gly Ala Gly Val Glu Tyr Wing 160 165 170 att ctt cct gaa tta gcg gca cgt gtt gaa tac cata tgg tta aac aac 1177 He Leu Pro Glu Leu Ala Wing Arg Val Glu Tyr'Gln Trp Leu Asn Asn 175 18O 185 0 gca ggt aaa gca age tac tet act tta aat cgt atg ggt gca act gac 1225 Ala.Gly Lys Wing Ser Tyr Ser Thr Leu Asn Arg Met Gly Wing Thr Asp 190 195 200 tac cgt tcg gat atc agt tcc gta tet gca ggt tta age tac cgt ttc 1273 Tyr Arg Ser Asp He Ser Ser Val Ser Wing Gly Leu Ser Tyr Arg Phe 205 210 215 220 ggt ca ggt gcg gta ccg gtt gc gct ccg gca gtt gaa act aaa aac 1321 Gly Gln Gly Wing Val Pro Val Wing Ala Pro Wing Val Glu Thr Lys Asn 225 230 235 ttc gca ttc age tet gac gta tta ttc gca ttc ggt aaa tea aac tta 1369 5 Phe Wing Phe Ser Ser Asp Val Leu Phe Wing Phe Gly Lys Ser Asn Leu 240 245 250 aaa ccg gct gcg gca here gca tta gat gca atg caw acc gaa atat aat 1417 Lys Pro Ala Ala Ala Thr Ala Leu Asp Ala Met Gln Thr Glu He Asn 255 260 265 aac gca ggt tta tea aat gct gcg atc ca gta aac ggt tac acg gac 1465 Asn Ala Gly Leu Ser Asn Ala Ala He Gln Val Asn Gly Tyr Thr Asp 270 275 280 cgt atc ggt aaa gaa get tea aac tta aaa ctt tea caa cgt cst gcg 1513 Arg He Gly Lys Glu Wing Being Asn Leu Lvs Leu_Ser Gin Ara Arg Wing 285 290"295 '300 gaa here gta gct aac tac atc gtt tet aaa ggt gct ccg gca gct aac 1561 Glu Thr Val Wing Asn Tyr He Val Ser Lvs Gly Wing Pro Ala Ala Asn 305 3Í0 315 gta act gca gta ggt tac ggt gaa gca aac cct__gta acc ggc gca aca 1609 Val Thr Ala Val Gly Tyr GÍy Glu Ala Asn Pro Val Thr Gly Ala Thr 320 325 330 tgt gac aaa gtt aaa ggt cgt aaa gca tta atc gct tgc tta gca ccg 1657 Cys Asp Lys Val Lys Gly Arg Lvs Ala Leu He Ala Cys Leu Ala Pro 335 340 345 gat cgt cgt gtt gaa gtt ca gtt ca ggt act aaa gaa gta act atg 1705 Asp Ara Arg Val Glu Val Gln Val Gln Gly Glu Thr Lys Val Thr Met 350 355 360 taatttagtt aattttetaa agttaaatta gtaaccctct tgcttattta agcaagaggg 1765 ttattttttt gttccatttt aattagtgct actcttcctg tgtttatatt tgtstttatg 1825 ataaactctt cataactttt atteaettat agatgaaaat gaaatacagc ttaacccctt 1885 tccatacctt tcatttagcg gcaaatgcaa caaaatc 1922 < 210 > 8 } < 211 > 364 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 400 > 8 Met Lys Lys Ser Leu Val Wing Leu Thr Val Leu Wing Wing Wing Val 1 5 10 15 Wing Gln Wing Wing Pro Gln Gln Asn Thr Phe Tyr Wing Gly Wing Wing Lys Wing 20 25 30 Gly Trp Wing Being Phe H? Asp Gly He Glu Gln Leu Asp Be Ala Lys 35 40 45 Asn Thr Asp Arg Gly Thr Lvs Tyr Gly He Asn Arg Asn Ser Val Thr 50 55 60 Tyr Gly Val Phe Gly Gly Tvr Gln He Leu Asn Gln Asp Lys Leu Gly 65 70"75 80 Leu Ala Ala Glu Leu Gly Tvr Asp Tyr Phe Gly Arg Val Arg Gly Ser 85"90 95 Glu Lys Pro Asn Gly Lys Wing Asp Lys Lys Thr Phe Arg His Wing Ala 100 105 110 Hrs Gly Wing Thr He Ala Wing Leu Lvs Pro Being Tyr Glu Val Leu Pro Asp 115 120 125 Leu ASD Val Tyr Giy Lys Val Gly He Ala Leu Val Asn Asn Thr Tyr 130 135 140 Lys Thr Phe Asn Wing Wing Gln Glu Lys Val Lys Thr Arg Arg Phe Gln 145 150 155 160 Ser Ser Leu He Leu Gly Ala Gly Val Glu Tyr Ala. He Leu Pro Glu 165 170 175 Leu Ala Ala Arg Val Glu Tyr Gln TrD Leu Asn Asn Wing Gly Lvs Wing 180 185 190 Ser Tyr Ser Thr Leu Asn Arg Met Gly Ala Thr Asp Tyr Arg Be Asp 195 200 205 Be Ser Be Val Be Wing Glv Leu Be Tyr Arg Phe Gly Gln Gly Wing 210 215 220 Val Pro Val Wing Wing Pro Wing Val Glu Thr Lvs Asn Phe Wing Phe Ser 225 230 235 240 Ser Asp Val Leu Phe Wing Phe Gly Lys Ser Asn Leu Lys Pro Wing Wing 245 250 255 Wing Thr Wing Leu Asp Wing Met Gln Thr Glu He Asn Asn Wing Gly Leu 260 265 270 Being Asn Wing Wing Gln Val Asn Gly Tyr Thr Asp Ars He Gly Lys 2 75 280 285 Glu Ala Ser Asn Leu Lys Leu Ser Gln Arg Arg Ala Glu Thr Val Ala 290 295 300 Asn Tyr He Val Ser Lys Gly Ala Pro Ala Ala Asn Val Thr Ala Val 305 310 315 320 Gly Tyr Gly Glu Ala Asn Pro Val Thr Gly Wing Thr Cys Asp Lys Val 325 330 335 Lys Gly Arg Lys Wing Leu He Wing Cys Leu Wing Pro Asp Arg Arg Val 340 345 350 Glu Val Gln Val Gln Glv Thr Lvs Glu Val Thr Met 355 360 < 210 > 9 < 211 > 1319 < 212 > DNA < 213 > Actinobacillus pleuropneumoniae < 220 > < 221 > CDS < 222 > (197) .. (1303) < 400 > 9 atgtaatatt aggactggaa agttcgaaat tacaaattga tattacaaat tgattgtagt 60 tttgcttttt atccttgata attaactctc ttttttctct agtgagacga gagcattaaa 120 tcaaaacttt ggtgccataa gcggtgctga agtgattttg ttttattaat cgatgacaat 180 atcaaa ttagaggatc aaa atg aaa tea tta gtt gct tta gta gca tta tea 232 Met Lvs Lys Ser Leu Val Ala Leu Ala Val Leu Ser i 5 ~~ 10 gct gca gca gta gct caca gca cct cea caca caat aat act ttc tac gca 280 Ala Ala Ala Ala Ala Gln Ala Ala Pro Gln Gln Asn Thr Phe Tyr Ala 15 20 25 ggt gcg aaa stt ggt caa tea tea ttt cac cac ggt gtt aac cata tta 328 Gly Ala Lys Val Gly Gln Ser Ser Phe H_s His Glv Val Asn Gln Leu 30 35 40 aaa tet ggt eac gat gat cgt tat aat gat aaa here cgt aag tat ggt 376 Lys Ser Gly Hi = Asp Asp Arg Tvr Asn Ásp Lvs Thr Arg Lys Tyr GÍy 45 50 55 60 atc aac cgt aac tet gta act tac ggt gta tcc ggt tac cac atc 424 He Asn Arg Asn Ser Val Thr Tyr Gly Val Phe Gly Gly Tyr Gln He 65 70 75 tta aac caá aac aat ttc ggt tta gcg act gaa tta ggt tat gat tac 472 Leu Asn Gl n Asn Asn Phe Gly Leu Wing Thr Glu Leu Gly Tyr Asp Tyr 80 85 90 tac ggt cgt gta cgt ggt aac gat gst gaa ttc cgt sca atg aaa cac 520 Tyr Gly Arg Val Arg Gly Asn Asp GYy Glu Phe Aro the Met Lvs His 95 100"105 tet gct tc gct tta aac ttt gcg tta aaa cea age tac gaa gta tta 566 Ser Ala His Gly Leu Asn Phe the Leu Lys Pro Ser Tyr Glu Val Leu 110 115 120 cct gac tta gac gtt tac ggt aaa gta ggt gtt gcg gtt gtt cgt aac 616 Pro Ásp Leu Asp Val Tyr Gly Lys Val Gly Val Ala Val Val Arg Asn 125 130 135 140 gac tat aaa tcc tat ggt gca gaa aac act aac gaa cea here gaa aaa 664 Asp Tyr Lys Ser Tyr Gly Wing Glu Asn Thr Asn Glu Pro Thr Glu Lys 145 150 155 ttc cat aaa tta aaa gca tea act att tta ggt gca ggt gtt gag tac 712 Phe His Lys Leu Lys Ala Ser Thr He Leu Gly Ala GÍy Val Glu Tyr 160 165 170 gca att ctt cct gaa tta gcg gca cgt gtt gaa tac cac tac tta aac 760 Wing He Leu Pro Glu Leu Wing Wing Arg Val Glu Tyr Gln Tyr Leu Asn 175 180 185 aaa gcg ggt aac tta aat aaa gca tta gtt cgt tea ggc here gat gat 808 Lys Ala Gly Asn Leu Asn Lys Ala Leu Val Arg Ser Gly Thr Gln Asp 190 195 200 gtg gac ttc ca g tat gct cct gat atc cac tet gta here gca ggt tta 856 Val Asp Phe Gln Tyr Ala Pro Asp He His Ser Val Thr Wing Gly Leu 205 210 215 220 tea tac cgt tc ggt ca ggc gct gtac gca cea gtt gtt gag cea gaa 904 Ser Tyr Arg Phe Gly Gln Gly the Val Wing Pro Val Val Glu Pro Glu 225 230 235 gtt gta gta act aaa aac tte gca ttc age tea gac gtt tta ttt gat ttc 952 Val Val Thr Lys Asn Pne Wing Pne Ser Ser Ásp Val Leu Phe Asp Phe 240 245 250 ggt aaa tea age tta aaa cea gca gca gca gct gta tta gac gca gct gct 1000 Gly Lys Ser Ser Leu Lys Pro Ala Ala Ala Thr la Leu Ásp Ala Ala 255 260 265 aac act gaa atc gct aac tta ggt tta gca act cea gct atc ca gtt 1048 Asn Thr Glu He Wing Asn Leu Gly Leu Wing Thr Pro Wing He Gln Val 270 275 280 aac ggt tat ac gac cgt atc ggt aaa gaa gct tea aac tta aaa ctt 1096 Asn Gly Tyr Thr Asp Arg He Gly Lys Glu Wing Being Asn Leu Lys Leu 285 290 295 300 tea caca cgc gg ga gaa act gta gct aac tac tta gtt tet aaa ggt 1144 Ser Gln Arg Arg Ala Glu Thr Val Ala Asn Tyr Leu Va l Ser Lys Gly 305 310 315 caa aac cct gc aac gta act gca gta gta gt gaa gca aac cea 1192 Gln Asn Pro Wing Asn Val Thr Wing Val Gly Tyr Gly Glu Wing Asn Pro 320 325 330 gta acc ggc gca here tgt gac aaa gtt aaa ggt cgt aaa gca tta atc 1240 Val Thr Gly Ala Thr Cvs ASD Lvs Val Lys Gly Arg Lys Ala Leu He 335 340 345 gct tgc tta gca ccg gat cgt cgt gtt gaa gtt ca gta gta gta gct gct 1288 Ala Cys Leu Ala Pro Asp Arg Arg Val Glu Val Gln Val Gln Gly Ala 350 355 360 aaa aac gta gct atg taatatagtg ggtttt 1319 Lys Asn Val Ala Met 365 < 210 > 10 < 211 > 369 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 400 > 10 Met Lys Lys Ser Leu Val Wing Leu Wing Val Leu Ser Wing Wing Wing Val 1 5 10 15 Wing Gln Wing Wing Pro Gln Gln Asn Thr Phe Tyr Wing Gly Wing Lys Val 20 25 30 Gly Gln Ser Being Phe His His Gly Val Asn Gln Leu Lys Ser Gly His 35 40 45 Asp Asp Arg Tyr Asn Asp Lys Thr Arg Lys Tyr Gly He Asn Arg Asn 50 55 60 Ser Val Thr Tyr Gly Val Phe Gly Gly Tyr Gln He Leu Asn Gln Asn 65 70 75 80 Asn Phe Gly Leu Wing Thr Glu Leu Gly Tyr Asp Tyr Tyr Gly Arg Val 85 90 95 - > Arg Gly Asn Asp Gly Glu Phe Arg Wing Met Lys His Ser Wing His Gly 100 105 110 Leu Asn Phe Wing Leu Lys Pro Ser Tyr Glu Val Leu Pro Asp Leu Asp 115 120 125 Val Tyr Gly Lys Val Gly Val Val Wing Val Arg Asn Asp Tyr Lys Ser 130 135 140 Tyr Gly Wing Glu Asn Thr Asn Glu Pro Thr Glu Lys Phe His Lys Leu 145 150 155 160 Lys Wing Being Thr He Leu Gly Wing Gly Val Glu Tyr Wing He Leu Pro 165 170 175 Glu Leu Wing Wing Arg Val Glu Tyr Gln Tyr Leu Asn Lys Wing Gly Asn 180 185 190 Leu Asn Lys Wing Leu Val Arg Ser Gly Thr Gln Asp Val Asp Phe Gln 195 200 205 Tyr Ala Pro Asp He His Ser Val Thr Ala Gly Leu Ser Tyr Arg Phe 210 215 220 Gly Gln Gly Wing Val Wing Pro Val Val Glu Pro Glu Val Val Thr Lys 225 230 235 240 Asn Phe Wing Phe Ser Being Asp Val Leu Phe Asp Phe Gly Lys Ser Ser 245 250 255 Leu Lys Pro Ala Ala Ala Thr > Wing Leu Asp Wing Wing Asn Thr Glu He 260 265 270 Wing Asn Leu Gly Leu Wing Thr Pro Wing He Gln Val Asn Gly Tyr Thr 275"280 285 Asp Arg He Gly Lys Glu Wing Being Asn Leu Lys Leu Ser Gln Arg Arg 290 295 300 Wing Glu Thr Val Wing Asn Tyr Leu Val Ser Lys Gly Gln Asn Pro Wing 305 310 315 320 Asn Val Thr Ala Val Gly Tyr Gly Glu Wing Asn Pro Val Thr Gly Ala 325 330 335 Thr Cys Asp Lys Val Lys Gly Arg Lys Ala Leu He Ala Cys Leu Ala 340 345 350 Pro Asp Arg Arg Val Glu Val Gln Val Gln Gly Ala Lys Asn Val Ala 355 360 365 Met < 210 > 11 < 211 > 20 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 220 > < 221 > SITE < 222 > (18) < 223 > Xaa = unknown amino acid < 400 > 11 Pro Wing Val Gly Asn Thr Phe Thr Gly Val Lys Val Tyr Val Asp Leu 1 5 10 15 Thr Xaa Val Wing 20 < 210 > 12 < 211 > 35 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 220 > < 221 > SITE < 222 > (30) < 223 > Xaa = Asn or Val < 400 > 12 His Gln Wing Gly ASD Val He Phe Arg Wing Gly Wing He Gly Val He 1 5 10 15 Wing Asn Being Being Asp Tyr Gln Thr Gln Wing Asp Val Xaa Leu Asp 20 25 30 Val Asn Asn 35 < 210 > 13 < 211 > 30 < 212 > PRT < 213 > Actinobacillus pleuropneumoniae < 400 > 13 Wing Glu He Gly Leu Gly Gly Wing Arg Glu Being Ser He Tyr Tyr Being 1 5 10 15 Lys His Lys Val Wing Thr Asn Pro Phe Leu Wing Leu Asp Leu 20 25 30 < 210 > 14 < 211 > 19 < 212 > PRT < 213 > Actinobacillus pieuropneumoniae < 220 > < 221 > SITE < 222 > (2) < 223 > Xaa = Asp or Glu < 220 > < 221 > smo < 222 > (14) < 223 > Xaa = unacknowledged amino acid < 220 > < 221 > SITE < 222 > (15) < 223 > Xaa = unknown amino acid < 220 > < 221 > SITE < 222 > (17) < 223 > Xaa = aminoaaao aesconociao < 400 > 14 Ala Xaa Pro Glu Asn Thr Phe Tyr Pro Gly Ala Lys Val Xaa Xaa Ser 10 15 Xaa Phe His < 210 > 15 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 15 aaaaatttgc gaaaaacgac 20 < 210 > 16 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 16 acttctacat tacttgatac 20 < 210 > 17 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence Cebaaor < 400 > 17 tccgtatgtc aaagtcgatg 20 < 210 > 18 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descnation of Artificial Sequence Primer < 400 > 18 taaacaatca accggtcctg 20 < 210 > 19 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence '< 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 19 ttaccttgta caacaccgac 20 < 210 > 20 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 20 aaaagcagta ttagcggcag 20 < 210 > 21 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 21 ttgatgtgcc attgccgaac 20 < 210 > 22 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description aa Cebaaor Artificial Sequence < 400 > 22 160 gttttaaact ttcagcactg 20 < 210 > 23 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 23 agttcgtcca tacgttggtg 20 < 210 > 24 < 211 > 20 < 2Í2 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence Cebaaor < 400 > 24 aatatatctc ratcggtaag 20 < 210 > 25 < 211 > 20 < 212 > DNA f < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 25 ctaaacctat aatttagatc 20 -r < 210 > 26 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 26 tgtttccgca cgacgttgtg 20 < 210 > 27 < 211 > 20 0 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 27 aastaaacss ttacacssac 20 < 210 > 28 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descnocion. Primer < 400 > 28 atcaaccgta attcagtaac 20 < 210 > 29 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Detection. Primer < 400 > 29 atastcataa cctaattcas 20 < 210 > 30 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 30 gtatgggtge aactgactac 20 < 210 > 31 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 31 aacacgtgcc gctaattcag 20 < 210 > 32 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descncction of Artificial Sequence. Primer < 400 > 32 tgcttgagct accgctgcag 20 < 210 > 33 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descpoption of Artificial Sequence • Cebaaor < 400 > 33 gcgaaagrtg gtcaatcatc 20 < 210 > 34 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 34 ataacgatca tcgtgaccag 20 < 210 > 35 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 35 tgcgtctaaa gctgttgctg 20 < 210 > 36 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: CeDaaor < 400 > 36 atcaagctta aaaccagcag 20 < 210 > 37 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence Primer < 400 > 37 ctataaatcc tatggtgcag 20 < 210 > 38 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence '< 220 > < 223 > Description of Cebaaor Artificial Sequence < 400 > 38 tgcacctaaa atasttgatg 20 < 210 > 39 c < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence Cebaaor < 400 > 39 ggggatcc c dccdgtaggn aatacnttt-- cngg 34 < 210 > 40 < 211 > 43 < 212 > DNA 0 <213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence Primer < 400 > 40 ggggatceat ytattatwsw aaacataaag tdgcdacnaa tcc 43 < 210 > 41 < 211 > 30 < 212 > DNA < 212 > Artificial Sequence 5 '< 220 > - < 223 > Artificial Sequence Describing Cebaaor < 400 > 41 ggggatccgt accgcgatct gtstttttag 30 < 210 > 42 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > 0 < 223 > Descption of Artificial Sequence Primer < 400 > 42 ggggatccsg tgctceggca gctaacg 27 < 210 > 43 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Ceoaaor Artificial Sequence < 400 > 43 ggggatccat acttacgtgt tttatcatta taacg 35 < 210 > 44 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence Primer < 400 > 44 ggggatccgg tcaaaaccct gcaaacg 27 < 210 > 45 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence Primer < 400 > Four. Five . tttgcccggg ctcttttatt gatttaagtt act 33 < 210 > 46 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 46 gactaacgca ggaccggttg attg 24 < 210 > 47 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence. Primer < 400 > 47 ggggatccgt gggtgcggaa tatacgcgca g 31 < 210 > 48 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 2 3 > Ceoaaor Artificial Sequence Descpo with < 400 > 48 caacgtggat ccgaattcaa gcttc ~~ 25 < 210 > 49 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 49 gtcaccgtaa tccataccgt aatg 24 < 210 > 50 < 211 > 26 < 212 > DNA < 213 > "Artificial <220> <223> Artificial Sequence Descppciop. Cebaaor <400> 50 gattgtttac ctttcacacc gtccac 26 < 210 > 51 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 51 tcacccggga aaaatatcta gaaacgg _ 27 < 210 > 52 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 52 ccgtggtgag atcaacgcct acgcctac 28 < 210 > 53 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 53 cctttcacac cgtccacttt atattttacc gtggtgas 38 < 210 > 54 < 211 > 68 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence Cebaaor < 400 > 54 gegeatatga acaccaccac caccaccacc tctcgtgcac cggtcggaaa tacctttacc 60 ggcgtagg 68 < 210 > 55 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Arboric Artificial Sequence < 400 > 55 caagactaaa aatgaccgst cgtg 24 < 210 > 56 < 211 > 25 < 212 > DNA < 213 > Arthrial Sequence < 220 > < 223 > Description of Artificial Sequence Primer < 400 > 56 tgaatttacg accacgtaaa tgttt 25 < 210 > 57 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence Primer < 400 > 57 ctatgtgaaa gcaaaagcgg attgg 25 < 210 > 58 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > DescpDCion of Artificial Sequence Primer < 400 > 58 ccgtccggtt gtttgactaa cgc 23 < 210 > 59 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Cebaaor < 400 > 59 tcgaacaagc acaccagccg gatg 24 < 210 > 60 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 60 ggcggaatac ggtaactact tattc 25 < 210 > 61 < 211 > '27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 61 cgcataaaaa at-gatctaaa ttatagg 27 < 210 > 62 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 62 ggggaattca acgattttgc ttgc 24 < 210 > 63 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence. Primer < 400 > 63 gaattcttgc tcgtttgaat tagaag 26 < 210 > 64 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence. < 220 > < 223 > Description of Artificial Sequence Primer < 400 > 64 ggtaattttt atatgagagg sactg 25 < 210 > 65 < 211 > 25 < ? 12 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence Cebaaor < 400 > 65 caaacagtga tacgctgaaa ttgtc 25 < 210 > 66 < 211 > 25 < 212 > DNA 1 Q213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence Primer < 400 > 66 gcacacaca taggtattaa tactg 25 < 210 > 67 < 211 > 66 < 212 > DNA < 213 > Artificial Sequence 1 §220 > < 223 > Artificial Sequence Descction Primer < 400 > 67 gegeatatga acaccaccae caccaccacc tctc tgccs aaattggatt gggaggagcc 60 cgtgag 66 < 210 > 68 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence 20 < 220 > < 223 > Description of S Seecquence Artificial Primer < 400 > 68 gtattetetc tagaatat tattttag 28 < 210 > 69 < 211 > 27 < 212 > DHA __ < 213 > Artificial Sequence. < 220 > < 223 > Description of Artificial Sequence. Ceoaacr < 400 > 69 sccacatsaa aaattattat ttsagct 27 < 210 > 70 < 211 > 25 < 212 > DNA < 213 Artificial Sequence < 220 > < 223 > Designation of Cebaaor Artificial Sequence < 400 > 70 acgtgaaaaa taatctcttg ataat 25 < 210 > 71 < 211 > 60 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 71 cgccatatga acaccaccac caccaccacc tctcgtcatc aggcgggaga tgtgatttrc 60 < 210 > 72 < 211 > 23 < 212 > DNA < 212 > Artificial Sequence 5 < 220 > < 223 > DescpDciop ae Sec Ceoaaor < 400 > 72 caycargcdg ghsat-grdat ytt 23 < 210 > 73 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > 0 < 223 > Descption of Cebaaor Artificial Sequence < 400 > 73 ccagaaccc: araavacdt vta 23 < 210 > 74 < 211 > 22 < 212 DNA < 213 > Artificial Sequence1 < 220 > < 223 > DescriDaop ae - Artificial sequence. Ceoaac- < 400 > 74 accggtacct atatgttaag te 22 < 210 > 75 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Cebaaor < 400 > 75 ataggtaccg gttaaaccaa ge 22 < 210 > 76 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence! < 220 > < 223 > Designation of Arenca-Cebaaor Sequence < 400 > 76 gttgccgcta ataattcaag acc 23 < 210 > 77 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Arthral Sequence Detection Primer < 400 > 77 aaacnttyt ayccdggngc naa 23 < 210 > 78 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Describing Artificial Sequence. Primer < 400 > 78 nacdckdckr tcnggngcna rrca 24 < 210 > 79 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Cebaaor Artificial Sequence < 400 > 79 datytcnacd ckdckrtcng? twenty-one < 210 > 80 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 80 cgctctagag attttttaca acaaaaaggg 30 < 210 > 81 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Cebaaor < 400 > 81 gmnaayacnt tytaygyngg n c 23 < 210 > 82 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 82 aayacnttyt aygynggngc naar 24 < 210 > 83 < 211 > 23 < 212 > DNA < 13 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 83 carg naaya cnttytaygy ngg 23 < 210 > 84 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Artificial Sequence. Primer < 400 > 84 gcnccncar mnaayacntt ta 23 < 210 > 85 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > < 400 > 85 cgcgcnccnc arsmnaayac ntt 23 210 > 86 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 86 cggtcgactg atttaagtta ctaaaaccc 29 < 210 > 87 Q < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 87 cggtcgacgg gttactaat taactttag 29 < 210 > 88 < 211 > € 7 < 212 > DNA 5 < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 88 gcgtcgacca tatgaacacc accaccacca ccacctctcg tgcgccacaa caaaatactt 60 tytacgc 67 < 210 > 89 < 211 > 32 0 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence DescriDtion. Cebaaor < 400 > 89 atgaaaaatt taacagtt t agcattagca gg 32 < 210 > 90 173 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 400 > 90 atgaaacara gcaaattcaa attatttaaa tattattt 38 < 210 > 91 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence. Primer < 400 > 91 atgaaaaaag cagtattagc ggcagtatta gg 32 < 210 > 92 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Descption: Primer < 400 > 92 atgaaaaaat cattagttgc tttaacagta ttatcg 36 < 210 > 93 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Descption of Sec Cebaaor < 400 > 93 atgaaaaaat cattagttgc tttagcagta ttatca 36 < 210 > 94 < 211 > 13 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > N-terminal consensus sequence of reiational proteins with OmpA proceaemes from Pasteureliaceae < 220 > < 221 > smo < 222 > (4) < .223 > Xaa = Ala Glu < 2 0 > < 221 > smo < 222 > (9) 174 < 223 > Xaa = Ala or Val < 400 > 94 Ala Pro Gln Xaa Asn Thr Phe Tyr Xaa Gly Ala Lys Ala 1 5 10 < 210 > 95 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > C-terminal consensus sequence of OmpA-related proteins from Pasteurellaceae < 400 > 95 Cys Leu Ala Pro Asp Arg Arg Val Glu He 1 5 10 < 210 > 96 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence protective peptide < 400 > 96 Met Asn Thr Thr Thr Thr Thr Ser Arg 1 5 10

Claims (2)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A substantially purified protein comprising an amino acid sequence selected from the group consisting of about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO : 4, from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8, and from about the residue from amino acid 20 to approximately amino acid residue 369 of SEQ ID NO: 10.
2. 2. The protein of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 3. A substantially purified polypeptide that is homologous with a protein comprising an amino acid sequence selected from the group consisting of about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to approximately amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about the residue of amino acid 364 of SEQ ID NO: 8, and from about residue 20 to about amino acid residue 369 of SEQ ID NO: 10. 4. The polypeptide of claim 3, which is homologous with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. 5. A substantially purified polypeptide that is a peptide fragment of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to approximately amino acid residue 364 of SEQ ID NO: 8, and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; or (b) a polypeptide that is homologous with any of the polypeptides from (a). 6. The polypeptide of claim 5, which is a peptide fragment of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. 7. The polypeptide of claim 6, comprising an amino acid sequence selected from the group consisting of about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 2, from about amino acid residue 1 to about amino acid residue 21 of SEQ ID NO: 4, from about amino acid residue 1 to about amino acid residue 27 of SEQ ID NO: 6, from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 8 and from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 10. 8. A fusion protein comprising: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about the amino acid residue 22 to approximately amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8 and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; (b) a polypeptide that is homologous with a polypeptide from (a); or (c) a petidic fragment of a polypeptide from (a) or (b), linked to a carrier or participant in the fusion. 9. The fusion protein of claim 8, wherein the fusion partner is selected from the group consisting of a protective peptide, β-galactosidase, trpE, maltose-binding protein, glutathione-S-transferase and poly- histidine. 10. The fusion protein of claim 9, wherein the fusion partner is a protective peptide comprising the amino acid sequence Met-Asn-Thr-Thr-Thr-Thr-Thr-Thr-Ser-Arg (SEQ. ID NO: 96). 11. An analogous compound or derivative of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to approximately amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about the residue of amino acid 364 of SEQ ID NO: 8, and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; (b) a polypeptide that is homologous with a polypeptide from (a); (c) a peptide fragment of a polypeptide from (a) or (b); or (d) a fusion protein comprising a polypeptide derived from (a) or (b), or a peptide fragment from (c) linked to a carrier or a fusion partner. 12. An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of about amino acid residue 20 to about amino acid residue 20 to about the amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 28 of SEQ ID NO: 6, from approximately amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8, and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10. 13. The isolated polynucleotide molecule of claim 12, which encodes the amino acid sequence from about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, which comprises the nucleotide sequence of SEQ ID NO. : 1 from about nt 329 to about nt 790. 14. - The isolated polynucleotide molecule of the claim 12, which encodes the amino acid sequence is SEQ ID NO: 2. 15. The polynucleotide molecule of claim 14, comprising the nucleotide sequence of SEQ ID NO: 1 from about nt 272 to about nt 790. 16. The isolated polynucleotide molecule of claim 15, comprising the nucleotide sequence of SEQ ID NO: 1. 17.- The isolated polynucleotide molecule of the claim 12, which encodes the amino acid sequence from about amino acid residue 22 to amino acid residue 215 of SEQ ID NO: 4, comprising the nucleotide sequence of SEQ ID NO: 3 from about nt 439 to about nt 1, 023. 18. The isolated polynucleotide molecule of claim 12, which encodes the amino acid sequence of SEQ ID NO: 4. 19. The isolated polynucleotide molecule of claim 18, comprising the nucleotide sequence of SEQ ID NO: 3 from about nt 376 to about nt 1, 023. 20.- The isolated polynucleotide molecule of the claim 19, which comprises the nucleotide sequence of SEQ ID NO: 3. 21.- The isolated polynucleotide molecule of the claim 12, which encodes the amino acid sequence from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, which comprises the nucleotide sequence of SEQ ID NO: 6. NO: 5 from about nt 238 to about nt 933. 22. The isolated polynucleotide molecule of claim 12, which encodes the amino acid sequence is SEQ ID NO: 6. 23. The isolated polynucleotide molecule of claim 22, comprising the nucleotide sequence of SEQ ID NO: 5 from about nt 157 to about nt 933. 24.- The isolated polynucleotide molecule of claim 23, which comprises the nucleotide sequence of SEQ ID NO: 5. 25. The isolated polynucleotide molecule of the claim 12, which encodes the amino acid sequence from about amino acid residue 20 to about residue 364 of SEQ ID NO: 8, which comprises the nucleotide sequence of SEQ ID NO: 7 from about nt 671 to about nt 1, 708 26.- The isolated polynucleotide molecule of the claim 12, which encodes the amino acid sequence of SEQ ID NO: 8. 27.- The isolated polynucleotide molecule of the claim 26, which comprises the nucleotide sequence of SEQ ID NO: 7 from about nt 614 to about nt 1, 708. 28. The isolated polynucleotide molecule of the claim 27, which comprises the nucleotide sequence of SEQ ID NO: 7. 29. The isolated polynucleotide molecule of claim 12, which encodes the amino acid sequence from about the amino acid residue to about 20 amino acid residues. 369 of SEQ ID NO: 10, comprising the nucleotide sequence of SEQ ID NO: 9 from approximately nt 254 to approximately nt 1, 396. 30. The isolated polynucleotide molecule of the claim 12, which encodes the amino acid sequence of SEQ ID NO: 10. 31 .- The isolated polynucleotide molecule of the claim 30, which comprises the nucleotide sequence of SEQ ID NO: 9 from about nt 197 to about nt 1, 306. 32. The isolated polynucleotide molecule of the claim 31, which comprises the nucleotide sequence of SEQ ID NO: 9. 33.- An isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising a nucleotide sequence that encodes Omp20, OmpW, Omp27, OmpA I or OmpA2 of APP. 34. An isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to a protein having an amino acid sequence selected from the group from about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 258 of SEQ ID NO: 6, from about 20 amino acid residue to approximately amino acid residue 364 of SEQ ID NO: 8, and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10. 35.-. An isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of about 20 amino acid residue up to approximately amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 258 of SEQ ID NO: 4 NO: 6, from about amino acid residue 364 of SEQ ID NO: 8, and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; (b) a nucleotide sequence from (a); or (c) a nucleotide sequence encoding a polypeptide that homologous to a polypeptide from (a). 36.- An isolated polynucleotide molecule comprising a nucleotide sequence that encodes a sequence of amino acids selected from the group consisting of about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 2, from about amino acid residue 1 to approximately amino acid residue 21 of SEQ ID NO: 4, from about amino acid residue 1 to about amino acid residue 27 of SEQ ID NO: 6, from about amino acid residue 1 to about the residue of amino acid 19 of SEQ ID NO: 8 and from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 10. 37.- The isolated polynucleotide molecule of the claim 36, in which the nucleotide sequence is selected from the group consisting of from about nt 272 to about nt 328 of SEQ ID NO: 1, from about nt 376 to about nt 438 of SEQ ID NO: 3, from about nt 157 to about nt 237 of SEQ ID NO: 5, from about nt 614 to about nt 670 of SEQ ID NO: 7, and from about nt 197 to about nt 253 of SEQ ID NO: 1 38. An isolated polynucleotide molecule comprising a nucleotide sequence encoding a fusion protein comprising: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of about amino acid residue 1 to about the amino acid residue 19 of SEQ ID NO: 2, from about amino acid residue 1 to about amino acid residue 21 of SEQ ID NO: 4, from about the residue from amino acid 1 to about amino acid residue 27 of SEQ ID NO: 6, from about amino acid residue 1 to about amino acid residue 19 of SEQ ID NO: 8 and from about amino acid residue 1 to about the residue of amino acid 19 of SEQ ID NO: 10; (b) a polypeptide that is homologous with a polypeptide from (a); or (c) a peptide fragment of a polypeptide of (a) or (b); united to a bearer or a participant in the merger. 39.- The isolated polynucleotide molecule of the claim 38, wherein the fusion partner is selected from the group consisting of a protective peptide, β-gaiactosidase, trpE, maltose-binding protein, glutathione-S-transferase and polyhistidine. 40.- The isolated polynucleotide molecule of the claim 39, wherein the fusion partner is a protective peptide comprising the amino acid sequence Met-Asn-Thr-Thr-Thr-Thr-Thr-Thr-Ser-Arg (SEQ ID NO: 96). 41.- An oligonucleotide molecule that can hybridize under highly stringent conditions with a polynucleotide molecule consisting of a nucleotide sequence selected from SEQ ID NOS: 1, 3, 5, 7 or 9, or a polynucleotide molecule that consists of a nucleotide sequence that is the complement of a nucleotide sequence selected from SEQ ID NOS: 1, 3, 5, 7 or 9. 42. - The oligonucleotide molecule of claim 41, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 15-47 and 49-93. 43. A recombinant vector comprising the polynucleotide molecule of claim 12, or a polynucleotide molecule homologous thereto. 44. The recombinant vector of claim 43, comprising a polynucleotide molecule which in turn comprises a nucleotide sequence of SEQ ID NO: 1 from about nt 329 to about nt 790 or a homologous polynucleotide molecule with this . Four. Five-. The recombinant vector of claim 44, which is a plasmid that is the same as plasmid pER416 present in host cells of strain Pz416 (ATCC 98926). 46. The recombinant vector of claim 43, comprising a polynucleotide molecule which in turn comprises a nucleotide sequence of SEQ ID NO: 3 from about nt 439 to about nt 1 .023 or a homologous polynucleotide molecule with this. 47. The recombinant vector of claim 46, which is a plasmid that is the same as the plasmid pER418 present in host cells of strain Pz418 (ATCC 98928). 48. - The recombinant vector of claim 43, comprising a polynucleotide molecule which in turn comprises a nucleotide sequence of SEQ ID NO: 5 from about nt 238 to about nt 933 or a polynucleotide molecule homologous thereto. 49-. The recombinant vector of claim 48, which is a plasmid that is the same as the plasmid pER417 present in host cells of strain Pz417 (ATCC 98927). 50.- The recombinant vector of claim 43, which comprises a polynucleotide molecule which in turn comprises a sequence of nucleotides of SEQ ID NO: 7 from about ei nt 671 to about nt 1, 708 or a polynucleotide molecule homologous thereto. 51-. The recombinant vector of claim 50, which is a plasmid which is the same as plasmid pER419 present in host cells of strain Pz419 (ATCC 98929). 52. The recombinant vector of claim 43, comprising a polynucleotide molecule which in turn comprises a nucleotide sequence of SEQ ID NO: 9 from about nt 254 to about nt 1, 306 or a homologous polynucleotide molecule with this. 53-. The recombinant vector of claim 52, which is a plasmid that is the same as plasmid pER420 present in host cells of strain Pz420 (ATCC 98930). 54.- A recombinant vector comprising the polynucleotide molecule of claim 34, 35, 36 or 38. 55.- A transformed host cell, comprising the recombinant vector of claim 43 or 54. 56.- A vaccine against APP , comprising an immunologically effective amount of an antigen of the present invention, selected from the group consisting of (a) a polypeptide comprising an amino acid sequence selected from the group consisting of from about amino acid residue 20 to about the residue of amino acid 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 258 of SEQ ID NO: 6 , from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8 and from about the residue of am. inoperacid 20 to approximately the amino acid residue 369 of SEQ ID NO: 10; (b) a polypeptide that is homologous with the polypeptide from (a); (c) a peptide fragment of the polypeptide from (a) or (b); (d) a fusion protein comprising the polypeptide from (a) or (b) or the peptide fragment from (c) linked to a carrier or a merged partner; (e) an analogous compound or derivative of the polypeptide from (a) or (b), the peptide fragment from (c) or the fusion protein from (d); or (f) a polynucleotide molecule encoding the polypeptide from (a) or (b), the peptide fragment from (c), the fusion protein from (d) or the analogue compound or derivative from (e) ); whose antigen is able to induce, or contribute to the induction of, a protective response against APP in pigs; and a veterinarily acceptable vehicle or diluent. 57. The vaccine of claim 56, comprising an immunomodulatory component. 58.- The vaccine of claim 57, wherein the immunomodulatory component is an adjuvant. 59. The vaccine of claim 56, further comprising an immunologically effective amount of a different antigen capable of inducing, or contributing to the induction of, a protective response against a disease or pathological condition that may afflict pigs. 60.- A method of preparing a vaccine that can protect pigs against APP, which comprises combining an immunologically effective amount of an antigen of the present invention, selected from the group consisting of: (a) a polypeptide comprising a sequence of amino acids selected from the group consisting of from about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about the amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8 and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; (b) a polypeptide that is homologous with a polypeptide from (a); (c) a peptide fragment of the polypeptide of (a) or (b); (d) a fusion protein comprising the polypeptide from (a) or (b) or the peptide fragment from (c) linked to a carrier or fusion partner; (e) an analogous compound or derivative of the polypeptide from (a) or (b); or the peptide fragment from (c), or the fusion protein from (d); or (f) a polynucleotide molecule encoding the polypeptide from (a) or (b), the peptide fragment from (c), the fusion protein from (d) or the analogue compound or derivative from (e) ); whose antigen is able to induce, or contribute to the induction of, a protective response against APP in pigs; with a vehicle or diluent acceptable in veterinary medicine in a form suitable for administration to pigs. 61.- The use of an immunologically effective amount of an antigen of the present invention, selected from the group consisting of (a) a polypeptide comprising an amino acid sequence selected from the group consisting of about 20 amino acid residue up to approximately amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to amino acid residue 258 of SEQ ID NO: 6, from about the residue from amino acid 20 to about amino acid residue 364 of SEQ ID NO: 8 and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; (b) a polypeptide that is homologous with the polypeptide from (a); (c) a peptide fragment of the polypeptide from (a) or (b); (d) a fusion protein comprising the polypeptide from (a) or (b) or the peptide fragment from (c) linked to a carrier or a merged partner; (e) an analogous compound or derivative of the polypeptide from (a) or (b), the peptide fragment from (c) or the fusion protein from (d); or (f) a polynucleotide molecule encoding the polypeptide from (a) or (b), the peptide fragment from (c), the fusion protein from (d) or the analogue compound or derivative from (e) ); in combination with a vehicle or diluent acceptable in veterinary for the manufacture of a vaccine against APP in pigs. 62.- A vaccine kit for vaccinating pigs against APP, comprising a container that in turn comprises an immunologically aficacious amount of one or more of the antigens of claim 56. 63. - The kit of claim 62, further comprising a second container comprising a vehicle or diluent acceptable in veterinary. 64.- An isolated antibody that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of about amino acid residue 20 to about amino acid residue 172 of SEQ ID NO: 2, from about the residue from amino acid 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about the residue of amino acid approximately 364 of SEQ ID NO: 8, and from about amino acid residue 20 to approximately amino acid residue 369 of SEQ ID NO: 10. 65.- A diagnostic kit comprising a first container comprising: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of approximately the amino residue o acid 20 to about amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to about the amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8 and from about amino acid residue 20 to about amino acid residue 369 of SEQ ID NO: 10; (b) a polypeptide that is homologous with a polypeptide from (a); (c) a peptide fragment of a polypeptide of (a) or (b); (d) a fusion protein comprising the polypeptide from (a) or (b) or the peptide fragment from (c) linked to a carrier or fusion partner; or (e) an analogous compound or derivative of the polypeptide from (a) or (b); or the peptide fragment from (c), or the fusion protein from (d); which will bind specifically to porcine antibodies directed against an APP antigen; and a second container comprising a secondary antibody directed against porcine antibodies. 66.- A diagnostic kit comprising a first container comprising a primary antibody that binds to an APP protein, said APP protein comprising an amino acid sequence selected from the group consisting of approximately the amino acid residue 20 to approximately the amino acid residue 172 of SEQ ID NO: 2, from about amino acid residue 22 to about amino acid residue 215 of SEQ ID NO: 4, from about amino acid residue 28 to about amino acid residue 258 of SEQ ID NO: 6, from about amino acid residue 20 to about amino acid residue 364 of SEQ ID NO: 8 and from about 20 amino acid residue to about the amino acid residue 369 of SEQ ID NO: 10; and a second container comprising a secondary antibody which binds to a different epitope located on the APP protein, or which is directed against the primary antibody. 67.- A diagnostic kit comprising a container comprising a polynucleotide molecule or oligonucleotide molecule of the present invention that can hybridize specifically with a specific polynucleotide molecule for APP, or can amplify it.