Actinobacillus pleuropneumoniae subunit vaccine

Abstract

The present invention relates to live attenuated bacteria of the genus Actinobacillus pleuropneumoniae that have a mutation in an apxIV gene such that no functional ApxIV toxin can be produced. The invention also relates to methods for the production of such bacteria. Also vaccines comprising such bacteria and methods for the production of such vaccines are part of the invention. The invention further relates to subunit vaccines comprising an ApxIV toxin, to methods for the production of such vaccines and to methods for the protection of animals against infection with bacteria of the genus Actinobacillus pleuropneumoniae. In addition, the invention relates to the promotor of the apxIV gene. Finally, the invention relates to diagnostic test for the selective diagnosis of Actinobacillus pleuropneumoniae infections and to diagnostic tests discriminating between Actinobacillus pleuropneumoniae field strains and vaccine strains.

Description

FIELD OF THE INVENTION

The present invention relates to live attenuated bacteria of the genus Actinobacillus pleuropneumoniae, having a mutation in a gene encoding a toxin, methods for the production of such bacteria, to vaccines comprising such bacteria, methods for the production of such vaccines, to vaccines comprising a toxin, methods for the production of such vaccines and methods for the protection of animals against infection with bacteria of the genus, Actinobacillus pleuropneumoniae.

BACKGROUND OF THE INVENTION

Bacteria belonging to the genus Actinobacillus all produce so-called RTX-toxins. (RTX stands for repeat in toxin).

It is the presence of these RTX-toxins that highly contributes to the pathogenic character of these bacteria.

The RTX-toxins have been extensively reviewed by Braun et al. (Critical Rev. in Microbiol. 18(2): 115-158 (1991)). RTX-toxins in Gram-negative strains have also been reviewed in Welch, R. A. (Molecular Microbiology 5/3: 521-528 (1991)) and in Welch et al. (Inf. Agents and Disease 4: 254-272 (1995)).

All known RTX-toxins display some kind of cytotoxic or cytolytic activity. The target-cell-and host-specificity differ however, depending on the toxin and on differences in acylation (McWhinney et al.; J. Bact. 174: 291-297 (1992) and Hackett et al.; J. Biol. Chem. 270: 20250-20253 (1995)). As a result of the difference in target cells, the various toxins of the RTX-toxin family are known e.g. as haemolysin, cytolysin or cytotoxin. The genus Actinobacillus comprises a number of different species, inter alia, Actinobacillus pleuropneumoniae, A. actinomycetemcomitans, A. suis, A. rossii, A. equuli and A. lignieresii.

Actinobacillus pleuropneumoniae produces serotype-dependent RTX-toxins that are cytotoxic/cytolytic to pig, horse, bovine and human erythrocytes, to rabbit and porcine neutrophils and to porcine alveolar macrophages. (Rosendal et al; Am. J. Vet. Res. 49: 1053-1058 (1988), Maudsley J. R. and Kadis S; Can. J. Microbiol. 32: 801-805 (1986), Frey. J and Nicolet. J; Inf. & Imm. 56:2570-2575 (1988), Bendixon et al; Inf. & Imm. 33: 673-676 (1981), Kamp, E. M. and van Leengoed, L. A. M. G.; J. Clin. Microbiol. 27: 1187-1191 (1989)).

Infections with Actinobacillus in pigs are the cause of severe economic losses to pig industry, due to acute mortality in young pigs and reduced weight gain in older animals.

The genetic organisation of the operons involved in the synthesis, activation and transportation of the RTX toxins in Gram-negative bacteria has been reviewed recently by Coote, J. G. (FEMS Microbiology reviews 88: 137-162 (1992)). Frey has specifically reviewed the known three RTX-toxins in Actinobacillus pleuropneumoniae in Bacterial Protein Toxins, Zbl Bakt. Suppl. 24, p. 322-, Freer et al. (Eds.), Gustaf Fischer, Stutttgart, Jena, New York, 1994.

In Actinobacillus pleuropneumoniae, this kind of RTX-operon contains four genes: the actual Toxin-gene (A), an Activator-gene (C), and two genes (B and D) encoding proteins involved in secretion of the toxin into the surrounding medium. The primary translation-product of the Toxin-gene (A) is a non-toxic protein, of which the toxic activity is activated by the Activator-gene (C) product.

Until recently, it was assumed that only three RTX-toxins, all having the above-described genetic organisation or at least having the Toxin-gene (A) and Activator-gene (C), existed in Actinobacillus species.

These three RTX-toxins, ApxI, Apx-II and Apx-III have respectively a pronounced haemolytic activity (ApxI), a mild haemolytic activity (Apx-II) or a macrophage-cytotoxic activity (Apx-III).

The various toxic activities are fairly randomly divided over the serotypes. There are four subgroups:

a subgroup A, represented by serotypes 1, 5, 9 and 11, producing ApxI and Apx-II,

a subgroup B, represented by serotypes 2, 3, 4, 6 and 8, producing Apx-II and Apx-III,

a subgroup C, represented by serotype 10, producing ApxI only,

a subgroup D, represented by serotype 7 and 12, producing Apx-II only,

It is known, that ApxI, -II, and -III all are essential elements in universal vaccines against Actinobacillus pleuropneumoniae infection: a vaccine not comprising at least ApxI, -II, and -III will not provide protection against all Actinobacillus pleuropneumoniae serotypes. Also, a vaccine not comprising at least the Apx-toxins of one specific serotype will not even induce protection against that single serotype.

Subunit vaccines based on in vitro synthesised RTX-toxins from A. pleuropneumoniae that lost their toxicity have been described earlier, e.g. in European Patent EP No. 0,354,628, in which subunit vaccines based upon a haemolysin and a cytotoxin of A. pleuropneumoniae are disclosed, and in European Patent EP No 0,453,024, in which A. pleuropneumoniae subunit vaccines based upon haemolysins, cytotoxins and outer membrane proteins are disclosed.

There are however four important disadvantages to subunit vaccines in general:

high amounts of antigenic material are needed in order to adequately trigger the immune system.

usually, only B-cell immunity is triggered.

several protective antigens are only triggered in vivo, and therefore can not be present in subunit vaccines.

a live pathogenic bacterium has many important immunogenic molecules, such as Outer Membrane Proteins and capsular polysaccharides, all potentially important for protection and thus to be included in an efficient subunit vaccine.

Next to the obvious problems mentioned under points one and two, especially the fourth point makes it difficult to make an efficient subunit vaccine.

This is e.g. illustrated by the A. pleuropneumoniae subunit vaccine disclosed in European Patent EP No 0,453,024 mentioned above, in which four different subunits (three RTX-toxins and an outer membrane protein) are combined in one vaccine.

It is clear, that in order to overcome the disadvantages of subunit vaccines against Pasteurellacea-infection, a live attenuated vaccine would be highly desirable.

A live attenuated vaccine has the following advantages:

it can be administered in low doses (it is self-replicating)

it closely mimics the natural/wild-type infection

it provides all the possible immunologically important antigens at the same time.

Nevertheless, in spite of the clear advantages, no live vaccines based on Actinobacillus pleuropneumoniae were commercially available prior to the present invention.

The reason for this lies in the following paradox: as mentioned before, ApxI, -II, and -III all are essential elements of universal vaccines against Actinobacillus pleuropneumoniae infection. Live vaccines therefore have to produce these three RTX-toxins. These three RTX-toxins are however strong virulence factors in all Actinobacillus species (see e.g. Coote, J. G.; FEMS Microbiology reviews 88: 137-162 (1992), Tascon et al.; Mol. Microbiol. 14: 207-216 (1994)), Jansen et al.; Inf. & Imm. 63: 27-37 (1995)).

Deletion of the RTX-toxins in order to attenuate the virulence of live App strains is technically feasible, but this does not provide a solution for the dilemma: such RTX-negative strains would be useless as live attenuated vaccine strains since they do no longer induce immunity in the host against the haemolytic/cytotoxic activity of Actinobacillus pleuropneumoniae field strains.

Virulence factors that, although important in the induction of immunity, do play a less important role in building up immunity than ApxI, -II and -III, and thus can in principle be deleted are however currently not known.

It would thus be highly desirable to have a site on the genome of App that attributes to virulence and therefore leads to an attenuated App strain when modified, whereas at the same time it is, although useful in triggering immunity, dispensable from a vaccine point of view. No such sites are however currently known. Moreover, it would be highly desirable if such a site would be universally present in all App strains, instead of being restricted to certain serotypes. Such a site would then allow all different serotypes to be attenuated by deletion of that same site.

It is one of the objectives of the present invention to provide such an attenuation site, universally present in all Actinobacillus pleuropneumoniae strains regardless of their serotype.

Recently, a new gene was found in a serotype 1 strain of Actinobacillus pleuropneumoniae (Thesis T. J. Anderson November 1995).

Although this gene does not resemble the known Actinobacillus ApxI, -II and -III genes, it bears resemblance to RTX-toxin genes known from bacteria belonging to Neisseria meningitidis, for which reason it was named RTX-gene apxlV. The gene however differs in almost all aspects from the three known RTX-toxin genes apxI, -II and -III present in the various species of the Actinobacillus family as described above. First of all, the genomic organisation is completely different. Secondly, there is no activator-mechanism as is found for the known Apx-toxins. In the third place, no specific in vivo haemolytic or cytotoxic activity could at that time be attributed to the gene, or it's possible gene product.

SUMMARY OF THE INVENTION

It was now surprisingly found that this gene, fully in contrast with the three known RTX-genes, is present in all bacteria of the species Actinobacillus pleuropneumoniae, regardless their serotype. This was determined by hybridisation of a probe comprising apxIV coding sequences with restriction fragments of the DNA from Actinobacillus pleuropneumoniae of all serotypes as described in Example 6 and 7.

Unexpectedly it was found now that apxIV deletion mutants are viable, but they behave less virulent compared to their apxIV-possessing parent strains.

Therefore, it was determined that the gene product, the ApxIV toxin is a virulence factor in all Actinobacillus pleuropneumoniae strains. This is an unexpected conclusion, since up until now, no effects at all, let alone effects possibly influencing virulence had been attributed to the gene product in vivo. In fact, up until now there was not even proof that the gene was expressed in Actinobacillus pleuropneumoniae in vivo or in vitro anyway.

It therefore is one of the merits of the invention that it was found that:

the apxIV gene is present in all A. pleuropneumoniae strains regardless the serotype,

the apxIV gene product is a virulence factor in all A. pleuropneumoniae serotypes,

A. pleuropneumoniae strains with a deletion in the apxIV gene are still viable but have a decreased virulence without significantly impairing the immunogenic properties of the strains,

Therefore, the invention provides for the first time live attenuated bacteria of the species Actinobacillus pleuropneumoniae, that do not produce a functional ApxIV toxin.

DETAILED DESCRIPTION OF THE INVENTION

A functional ApxIV toxin is considered to be a protein that has all the characteristics of the ApxIV toxin as expressed in a wild-type bacterium, and is expressed at the wild-type level. Therefore, a non-functional ApxIV toxin is considered to be a toxin that lacks some or all of the characteristics of the ApxIV toxin as expressed in a wild-type bacterium, and/or is expressed at a level, insufficient to obtain wild-type effects of ApxIV toxin.

The inability to produce the ApxIV toxin can e.g. be due to modifications in the coding sequence encoding the ApxIV toxin. It may also be e.g. the result of modifications in regions known to be involved in transcription of the apxIV gene, such as the promotor region, or of modifications in regions involved in translation, such as the ribosome binding site.

The overall structure of the apxIV gene is given in FIG. 1 .

In this figure, the direct repeat regions, characteristic for ApxIV toxin are indicated by dashed boxes, whereas the also ApxIV-specific glycine-rich nonapeptide regions are indicated by black arrows. The repeats are found at the C-terminal part of ApxIV. These characteristic features are present in all Actinobacillus pleuropneumoniae serotypes. The nucleic acid sequence and amino acid sequence of two serotypes are represented in SEQ. ID. No. 1-4. SEQ. ID. NO. 1 shows the nucleic acid sequence of the apxIV gene of App serotype 1, and SEQ. ID. NO. 2 shows the matching amino acid sequence of the serotype 1 ApxIV toxin. SEQ. ID. NO. 3 shows the nucleotide sequence of the apxIV gene of App serotype 3, whereas SEQ. ID. NO. 4 shows the matching amino acid sequence of the serotype 3 ApxIV toxin. FIG. 2 shows the strikingly high level of conservation at amino acid level, especially in the N-terminal 650 amino acids, between the Apx-toxins of the various Actinobacillus pleuropneumoniae serotypes. This is also a remarkable characteristic of the apxIV genes. It is clear from FIG. 1 , that a variation in the number of repeats at the C-terminal part of the toxin may occur, depending on the serotype. This variation accounts for the difference in size of the genes and encoded toxins obtained from the various serotypes.

There may be some variation in nucleic acid sequence even between apxIV genes isolated from different isolates of Actinobacillus pleuropneumoniae, belonging to the same serotype. This is due to natural variation well known in the art to exist in all organisms. It is possible that some amino acids in the ApxIV toxin encoded by the apxIV gene are replaced by others in the ApxIV toxin of another serotype, while the polypeptide is not altered in its function. For instance, a polypeptide containing Asp at a certain site, and its variant containing Asn at the comparable site still have the same properties. This process in which an amino acid is replaced by an functionally analogous amino-acid is called functional displacement. In this case the variant proteins are called functional variants.

Another cause of variation is the phenomenon of degeneracy of the genetic code. Shortly, it means, that e.g. the amino acid glutamic acid is coded for by both GAT and GAA. This phenomenon holds for all amino acids, except Met and Trp. Thus, it is obvious, that e.g. the ApxIV toxin of serotype 1, as given in the present invention can not only be coded for by the nucleotide sequence given in SEQ ID NO: 1 but also by a very large variety of other sequences, still all giving the same or functionally the same polypeptides.

Therefore, a variant apxIV sequence encoding a polypeptide that is functionally comparable to the ApxIV toxin falls within the scope of the present invention.

Live attenuated bacteria according to the invention can be obtained in several ways. One possible way of obtaining such bacteria is by means of classical methods such as the treatment of wild-type Actinobacillus pleuropneumoniae bacteria with mutagenic agents such as base analogues, treatment with ultraviolet light or temperature treatment. Strains that do not produce a functional ApxIV toxin do not or to a lesser extend induce anti-ApxIV toxin antibodies, and therefore can easily be selected in animal tests.

The necessary antiserum can be obtained as described below in Example 3.

Another possibility is to deliberately introduce, using recombinant DNA-technology, a well-defined mutation in the gene encoding the ApxIV toxin. Such a mutation may be an insertion, a deletion, a replacement of one nucleotide by another one or a combination thereof, with the only proviso that the mutated gene no longer encodes a functional ApxIV toxin. It can easily be seen, that especially mutations introducing a stop-codon in the open reading frame, or mutations causing a frame-shift in the open reading frame are very suitable to obtain a strain which no longer encodes a functional ApxIV toxin. Such mutations can e.g. be made by means of in vitro site directed mutagenesis using the Transformer® kit sold by Clontech. Many other standard recombinant DNA techniques such as digestion of the gene with a restriction enzyme, followed by endonuclease treatment and religation, are equally applicable.

Therefore, in a preferred form, this embodiment of the invention relates to live attenuated bacteria in which the gene encoding the ApxIV toxin comprises a mutation.

Well-defined mutations involving the deletion of fragments of the apxIV gene or even the whole gene, or the insertion of heterologous DNA-fragments, when compared to classically induced mutations, have the advantage that they will not revert to the wild-type situation.

Thus, in a more preferred form, this embodiment of the invention refers to live attenuated bacteria in which the gene encoding the ApxIV toxin comprises an insertion and/or a deletion.

Given the large amount of vaccines given nowadays to pigs, it is clear that combined administration of several vaccines would be desirable, if only for reasons of decreased vaccination costs. It is therefore very attractive to use live attenuated vaccine strains as a recombinant carrier for heterologous genes, encoding antigens selected from other pathogenic micro-organisms or viruses. Administration of such a recombinant carrier has the advantage that after administration of such a carrier, immunity is induced against two or more diseases at the same time. The live attenuated bacteria according to the present invention provide a very suitable carrier for heterologous genes, since the gene encoding the ApxIV toxin can be used as an insertion site for such heterologous genes. The use of the apxlV gene as an insertion site has the advantage that at the same time the apxIV gene is inactivated, and the newly introduced heterologous gene can be expressed in accordance with the homologous Actinobacillus pleuropneumoniae genes. The construction of such recombinant carriers can be done routinely, using standard molecular biology techniques such as homologous recombination. Therefore, in an even more preferred embodiment, the present invention relates to live attenuated bacteria of the species Actinobacillus pleuropneumoniae that do not produce a functional ApxIV toxin, and in which there is a heterologous gene inserted in the apxIV gene. Such a heterologous gene can, as mentioned above, e.g. be a gene encoding an antigen selected from other pathogenic micro-organisms or viruses. Another possibility is to insert a gene encoding a protein involved in triggering the immune system, such as an interleukine or an interferone.

In a still even more preferred form of the invention, the heterologous gene encodes one or more antigens selected from the group consisting of Porcine Reproductive Respiratory Syndrome (PRRS) virus, Pseudorabies virus, Porcine Influenza virus, Porcine Parvovirus, Transmissible Gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Streptococcus suis.

There is however a serious pitfall in expression of heterologous genes in recombinant carriers: it is known that several proteins are toxic if they are expressed in heterologous bacteria. Therefore, genes encoding such proteins can never be introduced in heterologous carriers, since successful recombinants will eventually die as a result of the expression a certain amount of the heterologous gene. The P2-protein of Haemophilus influenzae, to name just one example, can simply not be expressed in E. coli. (Munson et al., Infect. & Immun. 57: 88-94 (1989)). It is one of the objectives of the present invention to offer a recombinant live carrier that does not have this drawback. It was unexpectedly found, that although the apxIV gene is efficiently expressed in vivo (see Example 5), it is not expressed in vitro (see Example 4). This was concluded from the failure to show the presence of ApxIV toxin in in vitro grown A. pleuropneumoniae cultures. This means that ApxIV expression is switched on or off, depending on the environment in which A. pleuropneumoniae is grown. This feature offers an unexpected advantage over known live recombinant carriers: if the expression of the heterologous gene is brought under the control of the apxIV promoter, the live attenuated P. pleuropneumoniae carrier according to the invention can be grown in vitro to high densities, regardless the inserted heterologous gene, since the foreign gene will not be expressed under these conditions. After administering a number of bacteria to the host, the expression of the heterologous gene will start and at some time during replication or after the death of the bacterium it will become available to the immune system of the host. The heterologous gene to be expressed can be functionally linked to the apxIV promoter by e.g. replacing the coding sequence of the apxIV gene by the coding region of the heterologous gene. It is not necessary to replace the whole apxIV gene: it suffices to replace the ATG-codon of ApxIV by the coding region of the heterologous gene including its stop-codon. It is also possible to express a heterologous gene under the influence of the apxIV promoter by making a fusion construct. This can be made by inserting the heterologous gene in frame with the apxIV reading frame downstream of the apxIV ATG codon.

The wording “functionally linked to the apxIV promoter” means that transcription of the heterologous gene starts at the apxIV promoter.

It goes without saying that each location of the inserted heterologous gene in which it is functionally linked to the apxIV promotor falls within the scope of the invention.

Therefore, the most preferred form of this embodiment relates to live attenuated bacteria according to the present invention, carrying a heterologous gene that is functionally linked to the promotor region of the apxIV gene.

The surprising finding that the native apxIV promotor is a switchable promotor that is switched off in vitro and switched on in vivo makes this promotor a very versatile expression tool both in it's natural host and as a heterologous promotor in other bacteria. When used as a heterologous promotor in other bacteria, the DNA comprising the promotor can be isolated from its host and transferred to a bacterium other than Actinobacillus pleuropneumoniae. Another option that has now become feasible, is the cloning of several copies of the apxIV promotor each controlling the expression of another gene. This can be done in the host bacterium Actinobacillus pleuropneumoniae, but this principle of multiple copies is equally applicable to other bacteria. As mentioned above, the promotor can be used for the selective in vivo expression of one or more heterologous genes encoding antigens selected from other pathogenic micro-organisms or viruses. The promotor can also be used for the expression of a heterologous DNA sequence encoding a cytokine such as an interleukin, Tumor Necrosis Factor or an interferon. Several cytokines, e.g. interferons are known to play an important role as immune modulators. Thus it may be advantageous to express such genetic information under the control of the apxIV promotor.

Therefore, another embodiment of the invention relates to a nucleotide sequence harbouring the promotor controling the expression of the apxIV gene.

The switchable promotor that in the native situation controls the expression of the apxIV gene, was now found to be located in the DNA fragment between position 451 and 1132 of SEQ ID NO: 5. It is clear, that those parts of this DNA fragment that are not essential promotor elements need not necessarily be present in the fragment. Thus, shorter fragments of this DNA fragment in which the promotor activity is retained, are equally suitable for the expression of heterologous genes. Therefore, a more preferred form of this embodiment relates to a nucleotide sequence comprising the DNA fragment from position 451 to 1132 of SEQ ID NO: 5 or a subfragment thereof still having promotor activity.

Bacterial promotors all share two consensus regions, the so-called −10 and the −35 region. Although the flanking sequence of these consensus regions may to a certain extend influence the efficiency of the promotor, it can be advantageous to use only that part of the promotor region that comprises the DNA fragment between −35 and the ATG codon. This DNA fragment is located between position 617 and position 641 of SEQ ID NO: 5.

Therefore, in a more preferred form of this embodiment the invention relates to a nucleotide sequence comprising the DNA fragment from position 617 to 641 of SEQ ID NO: 5.

The present invention also relates to ApxIV toxin as a subunit vaccine component.

Subunit vaccines will most probably comprise the three known Apx-toxins. This was mentioned above. Since it was unexpectedly found, that the ApxIV toxin is however present in all A. pleuropneumoniae serotypes as mentioned above, it is a desirable additional component of subunit vaccines: neutralising antibodies raised against the ApxIV toxin provide protection against the ApxIV toxin produced by each and every Actinobacillus pleuropneumoniae strain, regardless the serotype. Therefore, another embodiment of the invention relates to subunit vaccines for the protection of animals against infection with a bacterium of the species Actinobacillus pleuropneumoniae, that comprise purified ApxIV toxin. The ApxIV toxin can be administered alone, or in combination with any or all of the toxins ApxI, -II and -III mentioned above and/or e.g. in combination with Outer Membrane Proteins (OMPS) of Actinobacillus pleuropneumoniae. Such vaccines can easily be prepared by admixing ApxIV toxin in an amount sufficient to induce an immune response, and a pharmaceutically acceptable carrier. Production of the ApxIV toxin is possible by introducing the apxIV gene in a suitable expression vector, expression of the gene and isolation of the toxin. Many versatile expression systems are known in the art, such as bacterial expression systems, baculovirus expression systems and mammalian cell expression systems. In Example 3 it is described how to obtain the ApxIV toxin by expression of the gene in E. coli.

Still another embodiment of the invention relates to live attenuated vaccines comprising live attenuated bacteria as described above for the protection of animals against infection with a bacterium of the species Actinobacillus pleuropneumoniae. Such vaccines can be obtained by admixing live attenuated bacteria with a pharmaceutically acceptable carrier. These vaccines comprise at least an immunogenically effective amount of the live attenuated producing bacterium according to the invention. Immunogenically effective means that the amount of live attenuated bacterium administered at the moment of vaccination is sufficient to induce in the host an effective immune response to virulent forms of the RTX-toxin producing bacterium. The useful dosage to be administered will vary depending on the age, weight and animal vaccinated, the mode of administration and the type of pathogen against which vaccination is sought. The vaccine may comprise any dose of bacteria, sufficient to evoke an immune response. Doses ranging between 10 3 and 10 10 bacteria are e.g. very suitable doses.

The pharmaceutically acceptable carrier may be as simple as water, but it may e.g. also comprise culture fluid in which the bacteria were cultured. Another suitable carrier is e.g. a solution of physiological salt concentration. Other examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilisers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. phosphate buffer).

Optionally, one or more compounds having adjuvant activity may be added to the vaccine. Adjuvantia are non-specific stimulators of the immune system. They enhance the immune response of the host to the invading pathogen. Examples of adjuvantia known in the art are Freunds Complete and Incomplete adjuvans, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes, cf. for instance European Patent EP 109942), Saponins, mineral oil, vegetable oil, and Carbopol (a homopolymer). Adjuvantia, specially suitable for mucosal application are e.g. the E. coli heat-labile toxin (LT) or Cholera toxin (CT).

Other suitable adjuvants are for example aluminium hydroxide, phosphate or oxide, oil-emulsions (e.g. of Bayol F (R) or Marcol 52 (R) , saponins or vitamin-E solubilisate.

Therefore, in a preferred form, the vaccines according to the present invention comprise an adjuvant.

For administration to animals, the vaccine according to the presentation can be given inter alia intranasally, intradermally, subcutaneously, by aerosol or intramuscularly.

There are several ways to store both subunits and live organisms. Storage in a refrigerator is e.g. a well-known method. Also often used is storage at −70° C. in a buffer containing glycerol. Bacteria can also be kept in liquid nitrogen. Freeze-drying is another way of conservation. Freeze-dried bacteria can be stored and kept viable for many years. Storage temperatures for freeze-dried bacteria may well be above zero degrees, without being detrimental to the viability. Freeze-drying is equally applicable for subunits.

Freeze-drying can be done according to all well-known standard freeze-drying procedures. Optional beneficial additives, such as e.g. skimmed milk, trehalose, gelatin or bovine serum albumin can be added in the freeze-drying process.

Therefore, in a more preferred embodiment, the vaccine according to the present invention is in a freeze-dried form.

In an even more preferred form of this embodiment, the vaccine additionally comprises one or more antigens selected from other pathogenic micro-organisms or viruses. Such a vaccine can be obtained by adding one or more antigens selected from other pathogenic bacteria or viruses to the live attenuated bacterium according to the invention and a pharmaceutically acceptable carrier as described above.

Of course, it is possible to add not only one or more antigens, but also one or more of the whole pathogens as such, in an inactivated or live form.

It can alternatively be obtained by cloning the genetic information encoding one or more antigens selected from other pathogenic micro-organisms or viruses into the live attenuated bacterium, using known recombinant DNA technology as described above.

Such vaccines are of course less stressing for the animal to be vaccinated than separate vaccinations with each of the pathogens, both from a medical and a physical point of view.

In a still even more preferred form, these antigens are selected from the group consisting of Porcine Reproductive Respiratory Syndrome (PRRS) virus, Pseudo-rabies virus, Porcine Influenza virus, Porcine Parvovirus, Transmissible Gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Streptococcus suis.

The invention also relates to methods for the preparation of a live attenuated bacterium of the species Actinobacillus pleuropneumoniae that is not capable of producing a functional ApxIV toxin. These method comprise the introduction of a mutation in the gene encoding the apxIV protein. Both classical mutation techniques, using mutagenic agents, and recombinant DNA techniques well-known in the art for insertion, replacement or deletion of genetic information from the apxIV gene are applicable.

In a preferred form, the above mentioned methods are used for the introduction of a deletion.

Methods for the preparation of a live attenuated vaccine according to the invention, that comprise admixing bacteria according to the invention with a pharmaceutically acceptable carrier are also part of the invention.

Also falling within the scope of the invention are methods for the preparation of a subunit vaccine. Such methods comprise the mixing of purified ApxIV toxin with a pharmaceutically acceptable carrier.

Another generally acknowledged problem in the field of vaccination with live vaccines is the following: the presence of antibodies against a certain pathogen in the serum of a host animal indicates that the host has been infected with the pathogen, either in a virulent or attenuated form. It is however impossible to discriminate between field-infected animals and animals vaccinated with a live vaccine strain. The live attenuated Actinobacillus pleuropneumoniae according to the present invention offers a solution to this problem as follows:

As described in Example 3, the apxIV gene of Actinobacillus pleuropneumoniae serotype 1 has been isolated and expressed in a heterologous host cell. This expression product was subjected to PAGE and then used for Western-blotting. The blots were incubated with convalescent serum obtained from a deliberately Actinobacillus pleuropneumoniae -infected pigs, and sera from field-strains. It was found that the apxIV gene is expressed in vivo in all Actinobacillus pleuropneumoniae field strains tested. This implicates, that pigs infected with Actinobacillus pleuropneumoniae will always have antibodies against the strain with which they were infected, regardless the serotype of the infectious strain.

The live attenuated bacteria according to the present invention can, due to the deletion of the apxIV gene, no longer make ApxIV toxin. Therefore animals vaccinated with a live attenuated Actinobacillus pleuropneumoniae strain according to the invention will not have antibodies against ApxIV toxin in their serum.

In a comparative test, e.g. an ELISA test, such sera will therefore react with all immunogenic Actinobacillus pleuropneumoniae -proteins such as e.g. ApxI, II and/or III, but not with ApxIV. Sera from pigs infected with an Actinobacillus pleuropneumoniae field strain however will react with all immunogenic Actinobacillus pleuropneumoniae -proteins, including ApxIV. Therefore, the live attenuated Actinobacillus pleuropneumoniae according to the present invention turns out to be a very suitable marker vaccine, i.e. a vaccine strain that can be discriminated from a field strain.

A diagnostic test for the discrimination between vaccine strains and field strains can be a simple ELISA-test in which purified ApxIV toxin is coated to the wall of the wells of an ELISA-plate. Incubation with serum from pigs to be tested, followed by e.g. incubation with a labelled anti-pig antibody can then reveal the presence or absence of antibodies against ApxIV toxin.

Another example of a diagnostic test system is e.g. the incubation of a Western blot comprising purified ApxIV toxin with serum of pigs to be tested, followed by detection of specific anti-ApxIV antibodies.

Therefore, diagnostic test for the discrimination between sera from pigs infected with Actinobacillus pleuropneumoniae field strains and from pigs vaccinated with a vaccine comprising live attenuated vaccine Actinobacillus pleuropneumoniae strains according to the invention, that comprise purified ApxIV toxin. also fall within the scope of the invention.

Still another problem seen in pig health care is the following: It is difficult to determine in a both quick and unambiguous manner if a pig is infected with Actinobacillus pleuropneumoniae or A. suis, or possibly a combination of both. Diagnostic tests for the specific detection of A. suis are currently not available. This is mainly due to the fact that A. pleuropneumoniae and A. suis share so many antigens. As an example, two highly antigenic Apx-toxins, ApxI and ApxIII have highly conserved homologues in e.g. A. suis (Van Ostaayen et al., submitted for publication).

The known RTX-genes, encoding the ApxI, -II and -III toxins or homologues are found in practically all members of the genus Actinobacillus, such as A. pleuropneumoniae, A. suis, A. rossii and A. equuli. Thus, it was initially assumed by the inventors, that the new RTX-toxin ApxIV would also be common to all members of the genus Actinobacillus.

It was however found after testing a the swine-pathogenic Actinobacillus, again surprisingly in contrast with the known three RTX-genes, that this novel RTX-gene apxIV is only present in the swine-pathogen Actinobacillus pleuropneumoniae. It is absent in all other common swine pathogenic Actinobacillus species, and therefore it is also absent in Actinobacillus suis. l See Example 6 and 7.

Therefore, it was surprisingly noticed that the presence of antibodies against ApxIV in the serum of a pig is a quick and unambiguous proof that the pig has been infected with A. pleuropneumoniae , and not with A. suis or any other swine-pathogen Actinobacillus species.

Thus the present invention also provides a diagnostic test based on the presence or absence of antibodies against ApxIV, and therefore a discriminating test for specifically distinguishing an infection with A. pleuropneumoniae from an infection with A. suis

Such a test can e.g. be an ELISA test that comprises in separate wells the ApxI and -II toxins, present in both A. pleuropneumoniae and A. suis and the purified ApxIV toxin. Serum from A. suis -infected animals will react only with the wells comprising the ApxI and -II whereas A. pleuropneumoniae -infected animals will also react with the well comprising the purified ApxIV toxin.

EXAMPLE 1

Cloning and analysis of the apxIV gene of A. pleuropneumoniae serotype 1

Standard molecular biological procedures (plasmid DNA isolation, restriction digestion, agarose gel electrophoresis, Southern blotting, ligation, transformation, electroporation) were, unless stated otherwise, essentially performed as described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) or Ausubel et al., (Current Protocols in Molecular Biology. John Wiley & Sons, N.Y., 1987). PCR was performed essentially as described in Innis et al., (PCR protocols, A guide to Methods and Applications, Academic Press Inc., San Diego, 1990). Chromosomal DNA isolation was performed according to Pitcher et al., (Lett. Appl. Microbiol., 8;151-156, 1989). The origin of all A. pleuropneumoniae reference strains (serotype 1: strain 4074; serotype 2: strain S1536: serotype 3: S1421; serotype 4 M62; serotype 5a: K17; serotype 5b: L20; serotype 6: femø; serotype 7: WF83; serotype 8: 405; serotype 9: CVI13261; serotype 10: 13039; serotype 11: 56153 and serotype 12: 8329) is described by Frey and Nicolet, (J. Clin. Microbiol., 28;232-236, 1990). A. pleuropneumoniae serotype 3 strain HV114 is a field isolate (i.e. one of the serotype 3 strains tested in Beck et al., J. Clin. Microbiol., 32;2749-2754, 1994). Other Actinobacillus strains used; A. rossii: ATCC 27072; A. equuli: ATCC 19392; A. suis: ATCC 15558. Pasteurella haemolytica type 1 strain ATCC 14003 was used.

E. coli host strains used: XL1-blue (Stratagene, La YolIa, Calif.; genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacl q ZDM15 Tn10 (Tet r )]) and HMS174(DE3) (AMS Biotechnology Ltd, Switzerland; genotype: F − recA (r K12− m K12+ ) rif r IDE3).

On the basis of the preliminary sequence data obtained from the thesis of T. J. Anderson (University of Guelph, 1995), two primers, designated APXIVA-1L (5′-TGGCACTGACGGTGATGA-3′ (SEQ ID NO: 8)) and APXIVA-1R (5′-GGCCATCGACTCAACCAT-3′ (SEQ ID NO: 9)) were synthesised. These primers were used in a PCR amplification, with chromosomal DNA from A. pleuropnumoniae serotype 3 strain HV114 and serotype 1 reference strain 4074 as a template. With both strains a fragment of 442 bp was amplified. The fragment derived from the serotype 3 chromosomal DNA was labelled with Digoxigenin-11-dUTP (Boehringer Mannheim) according to the protocol of the manufacturer (this fragment was designated probe APXIVA, see FIG. 4 ). The labelled probe was subsequently used to hybridize a Southern blot containing ClaI digested chromosomal DNA from strain 4074. The probe hybridised with a fragment of approximately 8.0 kb. The apxIV gene from serotype 1 strain 4074 was isolated by ligating ClaI digested chromosomal DNA into ClaI digested pBluescript II SK − (Stratagene USA). E. coli strain XL1-blue was transformed with the ligated DNA and transformants were selected on an LB plate with 100 mg/ml of ampicillin. Clones harbouring the apxIV were selected by colony hybridisation of a nitrocellulose replica of the plate with the APIXVA probe. Thus, a plasmid designated pROK7 was isolated and shown to harbour a ClaI insert of approximately 8.0 kb. The first 6736 bp of the ClaI insert were sequenced (SEQ ID NO: 1) and an open reading frame of 4971 nucleotides was identified encoding a protein of 1657 amino acid residues (SEQ ID NO: 2) with a predicted size of approximately 186 kD. The gene was designated apxIV_varl (see FIG. 3 ).

EXAMPLE 2

Cloning and analysis of the apxIV gene of A. pleuropneumoniae serotype 3

The labelled probe APXIVA (mentioned in example 1) was used to hybridize a Southern blot containing ClaI digested chromosomal DNA from strain HV114. The probe hybridised with a fragment of approximately 7.0 kb. The isolated chromosomal DNA from HV114 was digested with ClaI, and ligated with ClaI digested pBluescript II SK − (Stratagene USA). E. coli strain XL1-blue was transformed with the ligated DNA and transformants were selected on an LB plate with 100 mg/ml of ampicillin. Clones harbouring the apxIV were selected by colony hybridisation of a nitrocellulose replica of the plate with the APXIVA probe. Thus, a plasmid designated pROK5 was isolated and shown to harbour a ClaI insert of approximately 7 kb. The insert was analysed by sequence analysis (SEQID 3). An open reading frame of 4146 bp was identified encoding a protein of 1382 amino acid residues (SEQID 4), with a predicted size of approximately 154 kD. The gene was designated apxIV_var3 (see FIG. 3 ).

EXAMPLE 3

EXPRESSION OF ApxIV var3-polyhistidine fusion proteins in E. coli

From plasmid pROK5, a deletion clone was made which contains the 3′ end of the apxIV gene, starting at the BamHI site (nucleotide No. 2747 in SEQ ID NO: 3) up to the ClaI site at the end of the insert downstream of the apxIV gene. This plasmid was designated pROK1. Using oligonucleotides APXIVAHIA1-L (5′AGCCATATGGGCGATTTAAATTTCAG-3′ (SEQ ID NO: 10)) and APXIVHIS1-R (5′-TATGGATCCTCGTGCTTCTGAGC-3′(SEQ ID NO: 11)) and DNA from plasmid pROK1 as a template, a DNA fragment of 2.1 kb was amplified (see FIG. 4 ) containing the region from bp 3520 to 5643 in apxIV_var3 (SEQ ID NO: 3) flanked with NdeI and BamHI restriction sites at the 5′ and 3′ end respectively. After cloning of the Ndel/BamHI digested PCR fragment in expression vector pETHIS-1, digested with the same enzymes, a plasmid designated pJFFapxIV6/10his-1 was obtained. Plasmid pETHIS-1 is a derivative of pET14b (Novagen Inc., Madison, Wis.) where the multiple cloning site has been extended and a region encoding a histidine decamer has been inserted. Consequently, the pJFFapxIV6/10his-1 plasmid contains a translational fusion encoding a histidine hexamer, followed by amino acid residues 653 up to 1360 from SEQ ID NO: 4, followed by a histidine decamer, under the control of a T7 promoter. The plasmid was transferred to E. coli strain HMS174(DE3) with pLysS which contains an IPTG inducible T7 RNA polymerase gene as well as the T & lysozyme gene for increased stability. The strain was grown in LB medium containing 25 mg/ml of chloramphenicol and 100 mg/ml of ampicillin, up to an OD 650 of 0.5, and induced with isopropyl-b-D-thiogalactopyranoside at a concentration of 10 mM. After the addition of IPTG, the cells were incubated at 37° C. for 2.5 hours, the cells were harvested by centrifugation, and fusion protein with the expected size of 80 kD was isolated in the form of inclusion bodies. The inclusion bodies were solubilized in a solution of 6M guanidine hydrochloride, 300 mM NaCl and 50 mM NaH 2 PO 4 at pH 8.0 and the 80 kD fusion protein was further purified by immobilized Metal Affinity Chromatography (IMAC) (Schmitt et al., Molecular Biology Reports 18;223-230, 1993) using Ni 2+ chelated columns (Qiagen AG, Basel). Pure protein was eluted from the column at pH 5.0. Pooled fractions were dialysed against a solution of 300 mM NaCL and 50 mM NaH 2 PO 4 at pH 8.0. A rabbit was immunised with 500 mg of the polyhistidine fusion product, mixed with 1 volume of Complete Freunds Adjuvant (Difco Labs, Detroit, Mich.). A booster dose of the same amount, mixed with incomplete Freund Adjuvant was given 3 weeks later. Four weeks after the booster, the rabbit was bled and a hyperimmune serum comprising anti-ApxIV toxin antibodies, designated serum 522-409, was obtained.

EXAMPLE 4

Expression of apxIV genes in in vitro grown A. pleuropneumoniae

The A. pleuropneumoniae reference strain from serotype 1 was grown in Columbia broth supplemented with 10 mg/ml of b-NAD and harvested as described (Beck et al., J. Clin. Microbiol., 32;2749-2754, 1994). Adjacent to lanes comprising ApxIA, ApxIIA and ApxIVA-polyhistidine fusion proteins the concentrated culture supernatant was separated by polyacrylamide gel electrophoresis (Laemmli, Nature 227:680-685, 1970) and subjected to a Western blotting procedure (Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979). The Western blot was reacted with anti-ApxIA- and anti-ApxIlA monoclonal antbodies as described by Beck et al., (J. Clin. Microbiol., 32;2749-2754, 1994), and with anti-ApxIV serum 522-409 (see example 3). The isolated RTX toxin fraction of serotype 1 clearly contains ApxIA and ApxIlA. The presence of ApxIVA could not be demonstrated (see FIG. 5 ).

EXAMPLE 5

Expression of apxIV genes in A. pleuropneumoniae in vivo during infection

A polyacrylamide gel containing the 80 kD polyhistidine-ApxIV_var3 fusion protein (see example 3) was transferred to a nitrocellulose membrane. The membrane was divided into strips which were reacted with (100-fold dilutions of) convalescent field sera against serotype 1 or sera from a pig, experimentally infected with the serotype 1 reference strain (Frey and Nicolet, Vet. Microbiol., 28;61-73, 1991). The reaction was visualised using alkaline phosphatase-labelled conjugate against rabbit IgG (Kirkegaard Perry Inc., Gaithersburg, Md.) and NBT (4-Nitrobluetetrazolium chloride) and BCIP (5-Bromo-4-chloro-3-indolyl-phosphate) colour development (see FIG. 6 ). The serotype 1 field sera, as well as serum from the experimentally infected pig react with the 80 kD polyhistidine-ApxIV_var3 protein. This indicates that the ApxIV protein actually is expressed, is antigenic and induces anti-ApxIV toxin antibodies during A. pleuropneumoniae infection in pigs.

EXAMPLE 6

Presence of apxIV genes in all A. pleuropneumoniae serotypes and the absence thereof in non-pleuropneumoniae Actinobacillus-strains using Southern blotting

To investigate the presence of the apxIV gene in the various A. pleuropneumoniae serotypes and related bacteria, three probes were made (see FIG. 4 ). Probe APXIVA is described in example 1. Probe APXIVA2 contains the 2015 bp DNA fragment between the BamHI and NruI sites. The 758 bp probe APPIVA1 was made by PCR amplification with oligos APPIV1-L (5′-GGGACAGTGGCTCAATTAAG-3′(SEQ ID NO: 12)) and APPIV1-R (5′-AGCTGTAAACTCCACCAACG-3′(SEQ ID NO: 13)). All probes were labelled with Digoxigenin-11-dUTP (Boehringer Mannheim) according to the protocol of the manufacturer and hybridised with Southern blots containing ClaI digested chromosomal DNA of all A. pleuropneumoniae reference strains and the HV114 field strain, Actinobacillus suis (ATCC 15558), Actinobacillus rossii (ATCC 27072) and Actinobacillus equuli (ATCC 19392). All three probes react similarly (see FIG. 7 for the results with the APXIVA2 probe). All A. pleuropneumoniae strains react, whereas no hybridisation is observed with the A. suis, A. equuli and A. rossii strains.

EXAMPLE 7

Presence of apxIV genes in A. pleuropneumoniae and related strains using PCR amplification

With 50 ng of chromosomal DNA from the various A. pleuropneumoniae serotypes, other Actinobacillus species and P. haemolytica as templates and primers APXIVA-1L(5′-TGGCACTGACGGTGATGA-3′ (SEQ ID NO: 8)) and APXIVA-1R (5′-GGCCATCGACTCAACCAT-3′ (SEQ ID NO: 9)) PCR amplification was performed. After analysis of the products on an agarose gel, products with the expected size of 442 bp were observed in all A. pleuropneumoniae samples, but in none of the other Actinobacillus species (FIG. 8 ). This indicates that in addition to the results in example 6, also PCR could be used to discriminate A. pleuropneumoniae from other Actinobacillus species.

EXAMPLE 8

Overexpression of ApxIV-var1 polyhistidine fusion protein

Starting with plasmid pROK-7 (see example 1) as a template and oligonucleotides APX4/II5-L (5′-CGCCATATGACAAAATTAACTATGCAAC (SEQ ID NO: 14)) and APX4II6-R (5′-CGCGAATTCAGCGACACAAGAGATAT(SEQ ID NO: 15) as PCR-primers, a PCT fragment was amplified. A sufficient amount of this fragment was then digested with restriction enzymes NdeI and EcoRI and cloned in expression vector pETHIS-1, digested with the same enzymes as described in Example 3. From the resulting plasmid, designated pJFFApxIVA1His1, a 206 kD polyhistidine fusion protein (MW determined in PAGE) of 1841 amino acid residues was overexpressed in E. coli as described in Example 3. The protein is encoded in the coding region spanning nucleic acid no. 1132 to 6546 as depicted in SEQ ID NO: 5. The amino acid sequence of the protein is given in SEQ ID NO: 6. In Western blot this product was shown to react with specific anti-ApxIV serum 522-409 (antiserum described in example 3).

EXAMPLE 9

Protection of mice by vaccination with ApxIV against APP challenge

The 206 kD polyhistidine-ApxIV fusion protein as described in Example 8 and a comparable 108 kD polyhistidine-ApxIA-fusion protein, both derived from serotype 1 reference strain 4074 genomic material, were overexpressed as described in example 3. The cell pellet of induced E. coli cells was resuspended into PBS buffer (1 g. cell pellet in 6 ml buffer) and sonicated on ice for two times 45 seconds on ice for lysis of the cells. After centrifugation for 20 minutes at 22.000×g at 4° C., the supernatant was discarded and the resulting pellet was washed with a solution of 3M urea (pH 6.3). The urea was removed after centrifugation for 20 minutes at 22.000×g, and the resulting pellet was solubilized in 6M GuanidiniumHCl (pH 8.0). The protein samples were standardised by specific protein content after densitometry of PAGE gels. The samples were diluted with PBS and formulated with an oil adjuvant.

Three groups of 15 mice each, were intraperitoneally immunised with the ApxIV antigen (36.3 microgram), ApxIA antigen (36.3 microgram), or the adjuvant alone. The vaccines were administered in a volume of 0.4 ml. Twenty-four days after the first vaccination, each group of mice was split in two groups of 7 and 8 mice which were boosted with half the amount of antigen. The groups of 7 mice were boosted intrapertoneally in a volume of 0.2 ml and the groups of 8 mice were vaccinated intramuscularly with 0.1 ml in each hind leg. Thirteen days after the booster, the mice were challenged intraperitoneally with 1.5 10 8 cfu of a virulent serotype 1 strain .

EXAMPLE 10

Pore forming capacity of ApxIV

Freshly induced E. coli cells expressing ApxIV were used as the source of protein for testing pore information in artificial lipid bilayers as described by Maier et al., Infect. Immun., 64; 4415-4423(1996). The methods used for black lipid bilayer experiments have been described previously (Benz et al.; Biochim. Biophys. Acta 511: 305-319 (1978)). Membranes were formed from a 1% solution of asolectin (soybean lecitin type IV-S from Sigma, St. Louis Mo.) in n-decane. Zero current membrane potentials were measured with a Keithley 610 C electrometer 5-10 min. after a 10-fold salt gradient was established across the membranes (Benz et al.; Biochim. Biophys. Acta 551: 238-247 (1979)). The presence of ApxIV resulted in a high frequency of pore formation in the presence of 0.5% cholesterol, with an average single channel conductance (G) of 4 nS.

These results indicate that the ApxIV induces pores into artificial bilayers and is therefore toxic to eukaryotic cells and thus is a virulence factor for A. pleuropneumoniae.

LEGEND TO THE FIGURES

FIG. 1 : Comparison of ApxIVAvar1 and ApxIVvar3 (var stands for serotype). Graphic representation of the different features found in the C-terminal end. Dashed boxes represent the direct repeat regions in ApxIVA. Bold vertical bars indicate the position of glycine rich nonapeptides, and DNA polymerase family B signatures are indicated by black triangles. The amino acid sequence YSDSANSKK (SEQ ID NO: 16) represents the spacer sequence in the ApxIV Avar3 gene. The sequence segment of ApxIV Avar1 which is deleted in ApxIVAvar3 is also indicated in the figure.

FIG 2 : Alignment of the amino acid sequences of direct repeat 1 (A), direct repeat 2 (B) and direct repeat 3 (C) of ApxIVAvar1 and ApxIVAvar3. The copies of each of the direct repeats 1, 2 and 3 are labeled by letters to distinguish them for the sequence comparison. Variant residues are shown in bold letters.

FIG. 3 : Partial restriction maps of pBLac7 (Thesis of T. J. Anderson University of Guelph, 1995), pROK7 and pROK5. The different open reading frames (ORF's) are indicated by arrows. The interrupted arrow of lacZ in pROK7 indicates partial sequencing of the gene. Potential rho-independent transcription terminators are indicated (W). Potential transcription start sites are indicated by a triangle. Restriction sites: A=Asp700; B=BamHI; C=ClaI; E=EcoRV; Ec=EcoRI; H=HindIII; N=NruI; Nd=Ndel; Nh=Nhel; P=PstI; S=SpeI; Sm=SmaI.

FIG. 4 : Location of the various oligonucleotides and probes on the map of the apxIVAvar3 gene.

FIG. 5 : Expression of ApxIV in in vitro cultivated serotype 1 reference strain 4074. Panel A was reacted with the anti-ApxIA monoclonal antibody, panel B with anti-ApxIlA monoclonal antibody and panel C was reacted with anti-ApxIVA serum 522-409. Lane 1 contains ApxIA-polyhistidine fusion protein, lane 2 contains ApxIlA-polyhistidine fusion protein, lane 3 contains strain 4074 concentrated culture supernatant, lane 4 contains ApxIVA polyhistidine fusion protein.

FIG. 6 : Immunoblot showing the reactivities of sera from pigs which were experimentally infected with the reference strain of serotype 1 (lane 1) or pig sera from serotype 1 field infections (lane 2-4) with the 80 kD polyhistidine-ApxIV fusion protein. As a positive control (+), serum 522-409 was used, as a negative control (−), polyclonal rabbit serum against ApxI and ApxIl (Frey et al., Infect. Immun., 57;2050-2056, 1989) was used as a 1000-fold dilution

FIG. 7 : Southern blot of ClaI digested genomic DNA hybridised with probe APXIVA2. Lanes 1-13: A. pleuropneumoniae reference strains; 1: serotype 1; 2: serotype 2; 3: serotype 3; 4: serotype 4; 5: serotype 5a; 6: serotype 5b; 7: serotype 6; 8: serotype 7; 9: serotype 8; 10: serotype 9; 11: serotype 10; 12: serotype 11; 13: serotype 12; 14: HV114 field strain; 15 A. suis (ATCC 15558); 16: A. rossii (ATCC 27072); 17: A. equuli (ATCC 19392). Molecular size markers are indicated (in kilobasepairs) on the left.

FIG. 8 : PCR amplification of apxIV using primers APXIVA-1L and APXIVA-1R. Lane assignments: lanes 1 to 13 contain the A. pleuropneumoniae reference strains from serotypes 1, 2, 3, 4, 5a, 5b, 6, 7, 8, 9, 10, 11 and 12 respectively; lane 14: strain HV114; lane 15: A. suis ATCC 15558; lane 16: A. rossii ATCC 27072; lane 17: A. equuli ATCC 19392; lane 18: A. lignieresii ATCC 49236; lane 19: P. haemolytica type 1 ATCC 14003. Molecular size markers (in kilobasepairs) are indicated on the left.

16 1 6736 DNA Actinobacillus pleuropneumoniae CDS 1576..6549 1atcgatatgc cgccgggtac gggcgatatc caacttactc tttcgcaaca aattccggtt 60accggtgcgg tggtggtaac cactccgcaa gatattgcgt tattagatgc ggtgaaaggt 120atttcaatgt tccaaaaagt gtcggtaccg gtcttaggta tcattgaaaa tatgagcgta 180catatctgcc aaaattgcgg tcaccacgaa gatattttcg gcaccggcgg tgcggagaaa 240gtggcgaaga aatacggtac taaagtatta ggacaaatgc cgttgcatat tcgcttacgt 300caagatttgg atgccggcac accgaccgtc gttgcggcac cggaacacga aaccagccga 360gcctatattg aattagcggc aaaagtcgct tcggaattat actggcaagg ttcggttatc 420ccgtctgaaa ttatgattcg tgaagtaaaa taagttttaa taaccacgaa aacacaaaga 480acacaagcgg tagaatttgc agaaaaattt gcaaatccta ccgctttttt attagtacga 540ttcgctgttg gactgctatt tgatttggtt tgtcaggata ttatgttatt gtaatgaaat 600gttagtgaat tatttttatt aatttgaaag gaaacaaaat gaaaataaaa aaacgttaca 660ttgcgctgtt ggtcttaggt gtcgttatca gctatgcctg gtatcaaaat tatcaatggg 720aacagctgat gttaagcggt tattgtgaaa aggacggaag ttattttgat gataggcata 780cgaagcaaga actgattgat agggcaatta actatatgct ggagcatcaa tctaaaaaaa 840catacgatgc ttatactgat gaacctttag aaataaaacc atatttaaca atagaggaat 900ttaaaaaact caatccaaat tgttgtgaaa ttacctcatg gccagcagat gcagttccac 960aagattggga tgttcgtgtg gaaggtaagg catataggta tgtaatcgta aaatatttaa 1020gaaccttagc aaatagagaa cctgaacgat gggaaactag tattgttttt gataattgcg 1080gcaatcctaa aagagcaagc tacttatatt atttaaagag agaaatttat tatgacaaaa 1140ttaactatgc aagatgtgac caatttatat ttatataaaa cgaaaactct acctaaagat 1200agattggatg attcacttat ttctgaaata ggaaaaggag atgatgatat tgatagaaaa 1260gaatttatgg tggggccggg acgttttgtg accgctgata actttagcgt tgtaagagat 1320ttttttaatg ctgggaaatc acgcattatt gcgccgcaag tcccgcctat tcgttcacag 1380caggaaaaaa tcttggtcgg tttaaaaccg ggcaaatatt ccaaagcgca gatattggaa 1440atgctgggtt atacgaaagg cggagaagtg gtaaatggca tgtttgccgg tgaagtccag 1500acattaggct tttatgacga tggcaaaggg gatttactcg aacgcgccta tatctggaat 1560accacaggat ttaaa atg agc gac aat gcc ttt ttt gtt ata gaa gaa tca 1611 Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser 1 5 10ggc aaa cgc tat att gaa aac ttt ggt att gaa cct ctt ggt aag caa 1659Gly Lys Arg Tyr Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln 15 20 25gaa gat ttt gat ttt gtc ggc ggc ttt tgg tct aac tta gtg aat cgt 1707Glu Asp Phe Asp Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg 30 35 40ggt ttg gaa agt att atc gac cca tcc ggt atc ggt gga acg gta aac 1755Gly Leu Glu Ser Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn 45 50 55 60ctt aac ttt acc ggc gag gtg gaa acc tac acg tta gac gaa aca agg 1803Leu Asn Phe Thr Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg 65 70 75ttt aaa gcg gaa gcg gcg aag aaa agc cat tgg agt tta gtg aat gcg 1851Phe Lys Ala Glu Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala 80 85 90gcg aaa gta tac ggc ggt tta gac caa att att aaa aaa cta tgg gac 1899Ala Lys Val Tyr Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp 95 100 105agt ggc tca att aag cat tta tat caa gat aaa gat acg ggc aaa tta 1947Ser Gly Ser Ile Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu 110 115 120aaa ccg att att tac ggc acg gcc ggc aac gac agt aag att gaa ggc 1995Lys Pro Ile Ile Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly125 130 135 140act aaa atc acc cgt agg att gcg ggt aaa gaa gtt acg ctt gat att 2043Thr Lys Ile Thr Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile 145 150 155gcc aat cag aaa att gaa aaa ggc gtg tta gag aaa ttg ggg ctg tct 2091Ala Asn Gln Lys Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser 160 165 170gtt agt ggt tcg gat atc att aaa ttg ttg ttt gga gca ttg act cca 2139Val Ser Gly Ser Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro 175 180 185act tta aat aga atg ttg cta tca caa ctt atc cag tct ttt tcc gat 2187Thr Leu Asn Arg Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp 190 195 200agc ttg gct aaa ctt gat aat ccc tta gcc cct tac act aaa aat ggc 2235Ser Leu Ala Lys Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly205 210 215 220gtg gtt tat gtc acc ggc aaa ggg aat gat gtg ctt aaa gga act gaa 2283Val Val Tyr Val Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu 225 230 235cat gag gat ttg ttt ctc ggt ggt gag ggg aat gat act tat tat gcg 2331His Glu Asp Leu Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala 240 245 250aga gta ggc gat aca att gaa gac gcc gac ggc aaa ggt aaa gtc tat 2379Arg Val Gly Asp Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr 255 260 265ttt gtg aga gaa aaa ggg gta cct aag gcg gat cct aag cgg gta gag 2427Phe Val Arg Glu Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu 270 275 280ttt agc gag tac ata acg aaa gaa gaa ata aaa gag gtt gaa aag ggg 2475Phe Ser Glu Tyr Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly285 290 295 300tta tta act tac gca gtt tta gaa aat tat aat tgg gaa gag aaa acg 2523Leu Leu Thr Tyr Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr 305 310 315gcg act ttc gct cat gcg act atg ctt aat gag ctt ttt act gat tat 2571Ala Thr Phe Ala His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr 320 325 330act aat tat cgt tat gaa gtt aaa gga cta aaa ttg ccc gcc gtt aaa 2619Thr Asn Tyr Arg Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys 335 340 345aag tta aaa agt ccg ttg gtg gag ttt aca gct gat tta tta act gtt 2667Lys Leu Lys Ser Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val 350 355 360acg cct att gac gaa aac gga aaa gca ctt agc gaa aaa agt att acg 2715Thr Pro Ile Asp Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr365 370 375 380gtt aaa aat ttt aaa aat ggt gat tta gga ata agg ttg ttg gat cct 2763Val Lys Asn Phe Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro 385 390 395aat agc tat tat tat ttc ctt gaa ggc caa gat acg ggt ttt tat ggt 2811Asn Ser Tyr Tyr Tyr Phe Leu Glu Gly Gln Asp Thr Gly Phe Tyr Gly 400 405 410cct gct ttt tat att gaa cga aaa aac ggt ggc ggc gct aaa aat aac 2859Pro Ala Phe Tyr Ile Glu Arg Lys Asn Gly Gly Gly Ala Lys Asn Asn 415 420 425tcg tcg gga gca gga aat agc aaa gat tgg ggc ggg aac ggg cat gga 2907Ser Ser Gly Ala Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly 430 435 440aat cac cga aat aat gcc tcc gac ctg aat aaa ccg gac gga aat aat 2955Asn His Arg Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn445 450 455 460ggg aat aac caa aat aac gga agc aat caa gat aat cat agc gat gtg 3003Gly Asn Asn Gln Asn Asn Gly Ser Asn Gln Asp Asn His Ser Asp Val 465 470 475aat gcg cca aat aac ccg gga cgt aac tat gat att tac gat cct tta 3051Asn Ala Pro Asn Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu 480 485 490gct tta gat tta gat gga gat ggg ctt gaa acc gtg tcg atg aac ggg 3099Ala Leu Asp Leu Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly 495 500 505cga caa ggc gcg tta ttc gat cat gaa gga aaa ggt att cgt acc gca 3147Arg Gln Gly Ala Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala 510 515 520acg ggc tgg ctc gct gcg gat gac ggt ttt tta gtg tta gat cgt aac 3195Thr Gly Trp Leu Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn525 530 535 540caa gac ggc att att aat gat ata agc gag tta ttt agt aat aaa aat 3243Gln Asp Gly Ile Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn 545 550 555caa ctt tcc gac ggc agt att tct gca cac ggt ttt gcg aca tta gcc 3291Gln Leu Ser Asp Gly Ser Ile Ser Ala His Gly Phe Ala Thr Leu Ala 560 565 570gat ttg gat aca aac caa gat cag cgt atc gac caa aat gat aag ctg 3339Asp Leu Asp Thr Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu 575 580 585ttt tct aaa ctc caa att tgg cgg gat tta aat caa aac ggt ttt agt 3387Phe Ser Lys Leu Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser 590 595 600gaa gcg aat gag ctg ttt agc tta gaa agt ttg aat att aaa tct tta 3435Glu Ala Asn Glu Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu605 610 615 620cat acc gcc tat gaa gag cgt aat gat ttt cta gcg ggc aat aat atc 3483His Thr Ala Tyr Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile 625 630 635ctt gct cag ctt ggg aag tat gaa aaa acg gac ggt act ttt gca caa 3531Leu Ala Gln Leu Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Ala Gln 640 645 650atg ggc gat tta aat ttc agt ttt aac ccg ttt tat agc cga ttt acc 3579Met Gly Asp Leu Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr 655 660 665gaa gcg tta aat tta acc gag caa caa cgt cgc aca att aat cta acc 3627Glu Ala Leu Asn Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr 670 675 680ggc acc ggt cgg gtt cgg gat ttg cgt gaa gcc gcc gca ctt tct gag 3675Gly Thr Gly Arg Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu685 690 695 700gag ttg gct gct tta tta caa cag tac act aag gcc tcc gat ttt cag 3723Glu Leu Ala Ala Leu Leu Gln Gln Tyr Thr Lys Ala Ser Asp Phe Gln 705 710 715gca caa cga gaa tta ttg cct gcc att tta gat aaa tgg gcg gca acg 3771Ala Gln Arg Glu Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr 720 725 730gat tta cag tat caa cat tat gat aaa aca tta ctt aaa acg gta gaa 3819Asp Leu Gln Tyr Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Val Glu 735 740 745agt acc gat agt agt gct tct gtc gtt aga gtc acg cct tct caa tta 3867Ser Thr Asp Ser Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu 750 755 760agt agt ata cgc aat gca aag cat gat cct acc gtt atg caa aac ttt 3915Ser Ser Ile Arg Asn Ala Lys His Asp Pro Thr Val Met Gln Asn Phe765 770 775 780gaa cag agt aag gca aaa att gcg act tta aat tcg ctc tac ggg tta 3963Glu Gln Ser Lys Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu 785 790 795aat atc gat caa ctt tat tac acg acg gat aaa gac att cgc tat att 4011Asn Ile Asp Gln Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile 800 805 810act gat aaa gtg aat aat atg tat caa aca acc gta gaa ctt gcc tac 4059Thr Asp Lys Val Asn Asn Met Tyr Gln Thr Thr Val Glu Leu Ala Tyr 815 820 825cgt tct tta ctt tta caa acg cgt ttg aag aaa tat gtt tat agc gtt 4107Arg Ser Leu Leu Leu Gln Thr Arg Leu Lys Lys Tyr Val Tyr Ser Val 830 835 840aat gcg aaa caa ttc gaa ggg aaa tgg gta acc gat tat tct cgt act 4155Asn Ala Lys Gln Phe Glu Gly Lys Trp Val Thr Asp Tyr Ser Arg Thr845 850 855 860gaa gcc tta ttt aac tct act ttt aaa caa tcg cct gaa aat gca tta 4203Glu Ala Leu Phe Asn Ser Thr Phe Lys Gln Ser Pro Glu Asn Ala Leu 865 870 875tat gat tta agc gaa tac ctt tct ttc ttt aac gat cct acg gaa tgg 4251Tyr Asp Leu Ser Glu Tyr Leu Ser Phe Phe Asn Asp Pro Thr Glu Trp 880 885 890aaa gaa ggg cta tta ctg tta agc cgt tat ata gat tat gct aaa gca 4299Lys Glu Gly Leu Leu Leu Leu Ser Arg Tyr Ile Asp Tyr Ala Lys Ala 895 900 905caa gga ttt tat gaa aac tgg gcg gct act tct aac tta act att gcc 4347Gln Gly Phe Tyr Glu Asn Trp Ala Ala Thr Ser Asn Leu Thr Ile Ala 910 915 920cgt tta aga gag gct gga gta att ttt gca gaa tcg acg gat tta aaa 4395Arg Leu Arg Glu Ala Gly Val Ile Phe Ala Glu Ser Thr Asp Leu Lys925 930 935 940ggc gat gaa aaa aat aat att ttg tta ggt agc caa aaa gat aat aac 4443Gly Asp Glu Lys Asn Asn Ile Leu Leu Gly Ser Gln Lys Asp Asn Asn 945 950 955tta tcg ggt agt gca ggt gat gat cta ctt atc ggc gga gag ggt aat 4491Leu Ser Gly Ser Ala Gly Asp Asp Leu Leu Ile Gly Gly Glu Gly Asn 960 965 970gat acg tta aaa ggc agc tac ggt gca gac acc tat atc ttt agc aaa 4539Asp Thr Leu Lys Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys 975 980 985gga cac gga cag gat atc gtt tat gaa gat acc aat aat gat aac cgc 4587Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg 990 995 1000gca aga gat atc gac acc tta aaa ttt acc gat gtg aat tat gcg gaa 4635Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu1005 1010 1015 1020gtg aag ttt cga cga gta gat aat gac tta atg tta ttc ggt tat cat 4683Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His 1025 1030 1035gat acg gat tcg gtc acg gta aaa tcc ttc tac agc cat gta gat tat 4731Asp Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr 1040 1045 1050caa ttt gac aaa ttg gag ttt gct gac cgc agt ata act cgc gat gaa 4779Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu 1055 1060 1065ctg att aaa gca ggg ctt cat cta tac ggc acc gat ggc aat gat gat 4827Leu Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp 1070 1075 1080ata aag gat cat gcg gat tgg gac agc att ttg gaa ggc ggc aaa ggc 4875Ile Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly1085 1090 1095 1100aac gat att cta aga ggt ggc tac ggt gcg gac acc tat atc ttt agc 4923Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser 1105 1110 1115aaa gga cac gga cag gat atc gtt tat gaa gat acc aat aat gat aac 4971Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn 1120 1125 1130cgc gca aga gat atc gac acc tta aaa ttt act gat gtg aat tat gcg 5019Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala 1135 1140 1145gaa gtg aaa ttc cga cga gta gat aat gac tta atg tta ttc ggt tat 5067Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr 1150 1155 1160cat gat acg gat tcg gtc acg ata aaa tcc ttc tac aac cat gta gat 5115His Asp Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp1165 1170 1175 1180tat caa ttt gac aaa ttg gaa ttt gct gac cgc agt ata act cgt gat 5163Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp 1185 1190 1195gaa cta ggt aaa caa ggt atg gca tta ttt ggc act gac ggt gat gat 5211Glu Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp 1200 1205 1210aat atc aac gac tgg gga cgt aac tcg gtg att gat gcc ggt gcg ggt 5259Asn Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly 1215 1220 1225aat gat acg gtt aat ggc ggt aat ggc gat gac acc ctc atc ggc ggc 5307Asn Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly 1230 1235 1240aaa ggt aat gat att cta aga ggt ggc tac ggt gcg gac acc tat atc 5355Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile1245 1250 1255 1260ttt agc aaa gga cac gga cag gat atc gtt tat gaa gat acc aat aat 5403Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn 1265 1270 1275gat aac cgc gca aga gat atc gac acc tta aaa ttt acc gat gtg aat 5451Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn 1280 1285 1290tat gcg gaa gtg aaa ttc cga cga gta gat aat gac tta atg tta ttc 5499Tyr Ala Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe 1295 1300 1305ggt tat cat gat acg gat tcg gtc acg gta aaa tcc ttc tac agc cat 5547Gly Tyr His Asp Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His 1310 1315 1320gta gat tat caa ttt gac aaa ttg gag ttt gct gac cgc agt ata act 5595Val Asp Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr1325 1330 1335 1340cgc gat gaa ctg att aaa gca ggg ctt cat cta tac ggc acc gat ggc 5643Arg Asp Glu Leu Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly 1345 1350 1355aat gat gat ata aag gat cat gcg gat tgg gac agc att ttg gaa ggc 5691Asn Asp Asp Ile Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly 1360 1365 1370ggc aaa ggc aac gat att cta aga ggt ggc tac ggt gcg gac acc tat 5739Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr 1375 1380 1385atc ttt agc aaa gga cac gga cag gat atc gtt tat gaa gat acc aat 5787Ile Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn 1390 1395 1400aat gat aac cga gca aga gat atc gac acc tta aaa ttt act gat gtg 5835Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val1405 1410 1415 1420aat tat gcg gaa gtg aaa ttc cga cga gta gat aat gac tta atg tta 5883Asn Tyr Ala Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu 1425 1430 1435ttc ggt tat cat gat acg gat tcg gtc acg ata aaa tcc ttc tac aac 5931Phe Gly Tyr His Asp Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn 1440 1445 1450cat gta gat tat caa ttt gac aaa ttg gaa ttt gct gac cgc agt ata 5979His Val Asp Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile 1455 1460 1465act cgt gat gaa cta ggt aaa caa ggt atg gca tta ttt ggc act gac 6027Thr Arg Asp Glu Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp 1470 1475 1480ggt gat gat aat atc aac gac tgg gga cgt aac tcg gtg att gat gcc 6075Gly Asp Asp Asn Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala1485 1490 1495 1500ggt gcg ggt aat gat acg gtt aat ggc ggt aat ggc gat gac acc ctc 6123Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu 1505 1510 1515atc ggc ggc aaa ggt aat gat att cta aga ggt ggc tac ggt gcg gac 6171Ile Gly Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp 1520 1525 1530acc tat atc ttt agc aaa gga cac gga cag gat atc gtt tat gaa gat 6219Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp 1535 1540 1545acc aat aat gat aac cgc gca aga gat atc gac acc tta aaa ttt act 6267Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr 1550 1555 1560gat att aat tta tcc gaa ctt tgg ttt agc cga gaa aat aac gat ttg 6315Asp Ile Asn Leu Ser Glu Leu Trp Phe Ser Arg Glu Asn Asn Asp Leu1565 1570 1575 1580att att aaa tca tta tta agt gag gat aaa gtc acg gtt caa aat tgg 6363Ile Ile Lys Ser Leu Leu Ser Glu Asp Lys Val Thr Val Gln Asn Trp 1585 1590 1595tat tca cac caa gat cat aaa ata gaa aat att cgt tta tcg aat gag 6411Tyr Ser His Gln Asp His Lys Ile Glu Asn Ile Arg Leu Ser Asn Glu 1600 1605 1610caa acg ttg gtg agc act cag gtg gag aag atg gtt gag tcg atg gcc 6459Gln Thr Leu Val Ser Thr Gln Val Glu Lys Met Val Glu Ser Met Ala 1615 1620 1625ggc ttt gct cag aag cac gga gga gag ata tct ctt gtg tcg ctt gaa 6507Gly Phe Ala Gln Lys His Gly Gly Glu Ile Ser Leu Val Ser Leu Glu 1630 1635 1640gag gta aaa caa tat atc aat agc tta aca gct gct tta taa 6549Glu Val Lys Gln Tyr Ile Asn Ser Leu Thr Ala Ala Leu1645 1650 1655catacgaaag aaatcggcac agtttttttg aactgtgccg atttgatttt agtgtaagaa 6609tatagcctga ttttaagaaa tttactcttg gctaataact atttcccatt ttataagtta 6669ttgacggatg gttttatcaa atatgagatc aaatcttatt ttaaattcgc tttccattaa 6729gcgatat 6736 2 1657 PRT Actinobacillus pleuropneumoniae 2Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser Gly Lys Arg Tyr 1 5 10 15Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln Glu Asp Phe Asp 20 25 30Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg Gly Leu Glu Ser 35 40 45Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn Leu Asn Phe Thr 50 55 60Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg Phe Lys Ala Glu 65 70 75 80Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala Ala Lys Val Tyr 85 90 95Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp Ser Gly Ser Ile 100 105 110Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu Lys Pro Ile Ile 115 120 125Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly Thr Lys Ile Thr 130 135 140Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile Ala Asn Gln Lys145 150 155 160Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser Val Ser Gly Ser 165 170 175Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro Thr Leu Asn Arg 180 185 190Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp Ser Leu Ala Lys 195 200 205Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly Val Val Tyr Val 210 215 220Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu His Glu Asp Leu225 230 235 240Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala Arg Val Gly Asp 245 250 255Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr Phe Val Arg Glu 260 265 270Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu Phe Ser Glu Tyr 275 280 285Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly Leu Leu Thr Tyr 290 295 300Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr Ala Thr Phe Ala305 310 315 320His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr Thr Asn Tyr Arg 325 330 335Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys Lys Leu Lys Ser 340 345 350Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val Thr Pro Ile Asp 355 360 365Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr Val Lys Asn Phe 370 375 380Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro Asn Ser Tyr Tyr385 390 395 400Tyr Phe Leu Glu Gly Gln Asp Thr Gly Phe Tyr Gly Pro Ala Phe Tyr 405 410 415Ile Glu Arg Lys Asn Gly Gly Gly Ala Lys Asn Asn Ser Ser Gly Ala 420 425 430Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly Asn His Arg Asn 435 440 445Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn Asn Gln 450 455 460Asn Asn Gly Ser Asn Gln Asp Asn His Ser Asp Val Asn Ala Pro Asn465 470 475 480Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu Ala Leu Asp Leu 485 490 495Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly Arg Gln Gly Ala 500 505 510Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala Thr Gly Trp Leu 515 520 525Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn Gln Asp Gly Ile 530 535 540Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn Gln Leu Ser Asp545 550 555 560Gly Ser Ile Ser Ala His Gly Phe Ala Thr Leu Ala Asp Leu Asp Thr 565 570 575Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu Phe Ser Lys Leu 580 585 590Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser Glu Ala Asn Glu 595 600 605Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu His Thr Ala Tyr 610 615 620Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile Leu Ala Gln Leu625 630 635 640Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Ala Gln Met Gly Asp Leu 645 650 655Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr Glu Ala Leu Asn 660 665 670Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr Gly Thr Gly Arg 675 680 685Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu Glu Leu Ala Ala 690 695 700Leu Leu Gln Gln Tyr Thr Lys Ala Ser Asp Phe Gln Ala Gln Arg Glu705 710 715 720Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr Asp Leu Gln Tyr 725 730 735Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Val Glu Ser Thr Asp Ser 740 745 750Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu Ser Ser Ile Arg 755 760 765Asn Ala Lys His Asp Pro Thr Val Met Gln Asn Phe Glu Gln Ser Lys 770 775 780Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu Asn Ile Asp Gln785 790 795 800Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile Thr Asp Lys Val 805 810 815Asn Asn Met Tyr Gln Thr Thr Val Glu Leu Ala Tyr Arg Ser Leu Leu 820 825 830Leu Gln Thr Arg Leu Lys Lys Tyr Val Tyr Ser Val Asn Ala Lys Gln 835 840 845Phe Glu Gly Lys Trp Val Thr Asp Tyr Ser Arg Thr Glu Ala Leu Phe 850 855 860Asn Ser Thr Phe Lys Gln Ser Pro Glu Asn Ala Leu Tyr Asp Leu Ser865 870 875 880Glu Tyr Leu Ser Phe Phe Asn Asp Pro Thr Glu Trp Lys Glu Gly Leu 885 890 895Leu Leu Leu Ser Arg Tyr Ile Asp Tyr Ala Lys Ala Gln Gly Phe Tyr 900 905 910Glu Asn Trp Ala Ala Thr Ser Asn Leu Thr Ile Ala Arg Leu Arg Glu 915 920 925Ala Gly Val Ile Phe Ala Glu Ser Thr Asp Leu Lys Gly Asp Glu Lys 930 935 940Asn Asn Ile Leu Leu Gly Ser Gln Lys Asp Asn Asn Leu Ser Gly Ser945 950 955 960Ala Gly Asp Asp Leu Leu Ile Gly Gly Glu Gly Asn Asp Thr Leu Lys 965 970 975Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln 980 985 990Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile 995 1000 1005Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys Phe Arg 1010 1015 1020Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp Ser1025 1030 1035 1040Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr Gln Phe Asp Lys 1045 1050 1055Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Ile Lys Ala 1060 1065 1070Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp Ile Lys Asp His 1075 1080 1085Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly Asn Asp Ile Leu 1090 1095 1100Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly1105 1110 1115 1120Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Asp 1125 1130 1135Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys Phe 1140 1145 1150Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp 1155 1160 1165Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp Tyr Gln Phe Asp 1170 1175 1180Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Gly Lys1185 1190 1195 1200Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp Asn Ile Asn Asp 1205 1210 1215Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val 1220 1225 1230Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp 1235 1240 1245Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly 1250 1255 1260His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala1265 1270 1275 1280Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val 1285 1290 1295Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp 1300 1305 1310Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr Gln 1315 1320 1325Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu 1330 1335 1340Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp Ile1345 1350 1355 1360Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly Asn 1365 1370 1375Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys 1380 1385 1390Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg 1395 1400 1405Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu 1410 1415 1420Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His1425 1430 1435 1440Asp Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp Tyr 1445 1450 1455Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu 1460 1465 1470Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp Asn 1475 1480 1485Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly Asn 1490 1495 1500Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly Lys1505 1510 1515 1520Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe 1525 1530 1535Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp 1540 1545 1550Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Ile Asn Leu 1555 1560 1565Ser Glu Leu Trp Phe Ser Arg Glu Asn Asn Asp Leu Ile Ile Lys Ser 1570 1575 1580Leu Leu Ser Glu Asp Lys Val Thr Val Gln Asn Trp Tyr Ser His Gln1585 1590 1595 1600Asp His Lys Ile Glu Asn Ile Arg Leu Ser Asn Glu Gln Thr Leu Val 1605 1610 1615Ser Thr Gln Val Glu Lys Met Val Glu Ser Met Ala Gly Phe Ala Gln 1620 1625 1630Lys His Gly Gly Glu Ile Ser Leu Val Ser Leu Glu Glu Val Lys Gln 1635 1640 1645Tyr Ile Asn Ser Leu Thr Ala Ala Leu 1650 1655 3 7004 DNA Actinobacillus pleuropneumoniae CDS 1566..5714 3atcgatatgc cgccgggtac gggcgatatc caacttactc tttcgcaaca aattccggtt 60accggtgcgg tagtggtaac tactccgcaa gatattgcgt tattagatgc ggtgaaaggt 120atttcaatgt tccaaaaagt gtcggtaccg gtcttaggta tcattgaaaa tatgagcgtg 180catatctgcc aaaattgcgg tcaccacgaa gatattttcg gcaccggcgg tgcggagaaa 240gtggcgaaga aatacggtac taaagtatta ggacaaatgc cgttgcatat tcgcttacgt 300caagatttgg atgccggcac accgaccgtc gttgcggcac cggaacacga caccagcaga 360gcctatattg aattagcggc aaaagtcgct tcggaattat actggcaagg ttcggttatc 420ccgtctgaaa ttatgattcg tgaagtaaaa taagcctaca taaccacgga ataccagata 480acacagaagg aaaacaagcg gtagaatttg cagaaaaagt tgcaaattct accgcttttt 540tattagtacg attcgctgtt ggactgccat ttgatttggt ttgtcaggat attatgttat 600tgtaatgaaa tgttagtgaa ttatttttat taatttgaaa ggagacaaaa tgaaaataaa 660aaaacgttac attgcgctgc tagctttagg cagtgttatt ggctatgcct ggtatcaaaa 720ttatcaatgg gaacagttga tgttaagtgg ctattgtgaa aaggacggaa gctattgtga 780tgataggcat acgaagcagg aactgattga tagggcaatt aactatgtgc tggaaaatca 840aattcaacag acatatgaag gtgatgacct tgtggatata aaacaatatt caacaataga 900ggaatttaaa aaactaaatc cgaattgttg taaggtagat tcttggccgg atgatgctgt 960tcgtgaggat gctgatttac agcgagaggg caaagcgtat aaatacgtaa aagtcaaata 1020tttaagaacc tatttagcga atagagaacc tgaacaatgg gaaaattaca tagtatttga 1080taattgcagt ggaattaaag aaagacacca actgtattaa aaatagatta gatggagaca 1140acacgatgac aaaactaact atccaagatg tgactaattt atatttatat aagcaaagaa 1200ctttacctac ggataggtta gatgattcgc ttattagcaa aacaggaaaa ggggaaaata 1260ttgataaaaa ggaatttatg gcggggccgg gacgttttgt gacggccgat aattttagtg 1320ttgtaaaaga cttttttact gcaaaggatt cattaataaa cctaagcttg cagactcgta 1380tattagcgaa tttaaagccg ggcaaatatt ccaaagcgca gatattagaa atgttgggct 1440atacgaaaaa tggagaaaag gtagatggca tgtttaccgg tgaagtccag acattaggct 1500tttatgacga tggcaaaggg gatttactcg aacgcgccta tatctgaaat accacaggat 1560ttaaa atg agc gac aat gcc ttt ttt gtt ata gaa gaa tca ggc aaa 1607 Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser Gly Lys 1 5 10cgc tat att gaa aac ttt ggt att gaa cct ctt ggt aag caa gaa gat 1655Arg Tyr Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln Glu Asp 15 20 25 30ttt gat ttt gtc ggc ggc ttt tgg tct aac tta gtg aat cgt ggt ttg 1703Phe Asp Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg Gly Leu 35 40 45gaa agt att atc gac cca tcc ggt atc ggt gga acg gta aac ctt aac 1751Glu Ser Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn Leu Asn 50 55 60ttt acc ggc gag gtg gaa acc tac acg tta gac gaa aca agg ttt aaa 1799Phe Thr Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg Phe Lys 65 70 75gcg gaa gcg gcg aag aaa agc cat tgg agt tta gtg aat gcg gcg aaa 1847Ala Glu Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala Ala Lys 80 85 90gta tac ggc ggt tta gac caa att att aaa aaa cta tgg gac agt ggc 1895Val Tyr Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp Ser Gly 95 100 105 110tca att aag cat tta tat caa gat aaa gat acg ggc aaa tta aaa ccg 1943Ser Ile Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu Lys Pro 115 120 125att att tac ggc acg gcc ggc aac gac agt aag att gaa ggc act aaa 1991Ile Ile Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly Thr Lys 130 135 140atc acc cgt agg att gcg ggt aaa gaa gtt acg ctt gat att gcc aat 2039Ile Thr Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile Ala Asn 145 150 155cag aaa att gaa aaa ggc gtg tta gag aaa ttg ggg ctg tct gtt agt 2087Gln Lys Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser Val Ser 160 165 170ggt tcg gat atc att aaa ttg ttg ttt gga gca ttg act cca act tta 2135Gly Ser Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro Thr Leu175 180 185 190aat aga atg ttg cta tca caa ctt atc cag tct ttt tcc gat agc ttg 2183Asn Arg Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp Ser Leu 195 200 205gct aaa ctt gat aat ccc tta gcc cct tac act aaa aat ggc gtg gtt 2231Ala Lys Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly Val Val 210 215 220tat gtc acc ggc aaa ggg aat gat gtg ctt aaa gga act gaa cat gag 2279Tyr Val Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu His Glu 225 230 235gat ttg ttt ctc ggt ggt gag ggg aat gat act tat tat gcg aga gta 2327Asp Leu Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala Arg Val 240 245 250ggc gat aca att gaa gac gcc gac ggc aaa ggt aaa gtc tat ttt gtg 2375Gly Asp Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr Phe Val255 260 265 270aga gaa aaa ggg gta cct aag gcg gat cct aag cgg gta gag ttt agc 2423Arg Glu Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu Phe Ser 275 280 285gag tac ata acg aaa gaa gaa ata aaa gag gtt gaa aag ggg tta tta 2471Glu Tyr Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly Leu Leu 290 295 300act tac gca gtt tta gaa aat tat aat tgg gaa gag aaa acg gcg act 2519Thr Tyr Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr Ala Thr 305 310 315ttc gct cat gcg act atg ctt aat gag ctt ttt act gat tat act aat 2567Phe Ala His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr Thr Asn 320 325 330tat cgt tat gaa gtt aaa gga cta aaa ttg ccc gcc gtt aaa aag tta 2615Tyr Arg Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys Lys Leu335 340 345 350aaa agt ccg ttg gtg gag ttt aca gct gat tta tta act gtt acg cct 2663Lys Ser Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val Thr Pro 355 360 365att gac gaa aac gga aaa gca ctt agc gaa aaa agt att acg gtt aaa 2711Ile Asp Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr Val Lys 370 375 380aat ttt aaa aat ggt gat tta gga ata agg ttg ttg gat cct aat agc 2759Asn Phe Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro Asn Ser 385 390 395tat tat tat ttc ctt gaa ggc caa gat acg ggt ttt tat ggt cct gct 2807Tyr Tyr Tyr Phe Leu Glu Gly Gln Asp Thr Gly Phe Tyr Gly Pro Ala 400 405 410ttt tat att gaa cga aaa aac ggt gga ggc tct aaa aat aac tcg tcg 2855Phe Tyr Ile Glu Arg Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser415 420 425 430gga gca gga aat agc aaa gat tgg ggc ggg aac ggg cat gga aat cac 2903Gly Ala Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly Asn His 435 440 445cga aat aat gcc tcc gac ctg aat aaa ccg gac gga aat aat ggg aat 2951Arg Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn 450 455 460aac caa aat aac gga agc aat caa gat aat cat agc gat gtg aat gcg 2999Asn Gln Asn Asn Gly Ser Asn Gln Asp Asn His Ser Asp Val Asn Ala 465 470 475cca aat aac ccg gga cgt aac tat gat att tac gat cct tta gct tta 3047Pro Asn Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu Ala Leu 480 485 490gat tta gat gga gat ggg ctt gaa acc gtg tcg atg aac ggg cga caa 3095Asp Leu Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly Arg Gln495 500 505 510ggc gcg tta ttc gat cat gaa gga aaa ggt att cgt acc gca acg ggc 3143Gly Ala Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala Thr Gly 515 520 525tgg ctc gct gcg gat gac ggt ttt tta gtg tta gat cgt aac caa gac 3191Trp Leu Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn Gln Asp 530 535 540ggc att att aat gat ata agc gag tta ttt agt aat aaa aat caa ctt 3239Gly Ile Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn Gln Leu 545 550 555tcc gac ggg agt att tct gca cac ggt ttt gcg aca tta gcc gat ttg 3287Ser Asp Gly Ser Ile Ser Ala His Gly Phe Ala Thr Leu Ala Asp Leu 560 565 570gat aca aac caa gat cag cgt atc gac caa aat gat aag ctg ttt tct 3335Asp Thr Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu Phe Ser575 580 585 590aaa ctc caa att tgg cgg gat tta aat caa aac ggt ttt agt gaa gcg 3383Lys Leu Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser Glu Ala 595 600 605aat gag ctg ttt agc tta gaa agt ttg aat att aaa tct tta cat acc 3431Asn Glu Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu His Thr 610 615 620gcc tat gaa gag cgt aat gat ttt cta gcg ggc aat aat atc ctt gct 3479Ala Tyr Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile Leu Ala 625 630 635cag ctt ggg aag tat gaa aaa acg gac ggt act ttt gga caa atg ggc 3527Gln Leu Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Gly Gln Met Gly 640 645 650gat tta aat ttc agt ttt aac ccg ttt tat agc cga ttt acc gaa gcg 3575Asp Leu Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr Glu Ala655 660 665 670tta aat tta acc gag caa caa cgt cgc aca att aat cta acc ggc acc 3623Leu Asn Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr Gly Thr 675 680 685ggt cgg gtt cgg gat ttg cgt gaa gcc gcc gca ctt tct gag gag ttg 3671Gly Arg Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu Glu Leu 690 695 700gct gct tta tta caa cag tac act aag ggc tcc gat ttt cag gca caa 3719Ala Ala Leu Leu Gln Gln Tyr Thr Lys Gly Ser Asp Phe Gln Ala Gln 705 710 715cga gaa tta ttg cct gcc att tta gat aaa tgg gcg gca acg gat tta 3767Arg Glu Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr Asp Leu 720 725 730cag tat caa cat tat gat aaa aca tta ctt aaa acg gta gaa agt acc 3815Gln Tyr Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Val Glu Ser Thr735 740 745 750gat agt agt gct tct gtc gtt aga gtc acg cct tct caa tta agt agt 3863Asp Ser Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu Ser Ser 755 760 765ata cgc aat gta aag cat gat cct acc gtt atg caa aac tgt gaa caa 3911Ile Arg Asn Val Lys His Asp Pro Thr Val Met Gln Asn Cys Glu Gln 770 775 780agt aag gca aaa att gcg act tta aat tcg ctc tac ggg tta aat att 3959Ser Lys Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu Asn Ile 785 790 795gat caa ctt tat tat acg acg gat aaa gac att cgt tat att act gac 4007Asp Gln Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile Thr Asp 800 805 810aaa gtg aat aat atg tat caa aca acc gga gaa ctc ggc tat cgt tct 4055Lys Val Asn Asn Met Tyr Gln Thr Thr Gly Glu Leu Gly Tyr Arg Ser815 820 825 830tta ctt tta caa acg cgt gtg aag aaa tat gtt tat agc gtt aat gcg 4103Leu Leu Leu Gln Thr Arg Val Lys Lys Tyr Val Tyr Ser Val Asn Ala 835 840 845aaa caa ttc gaa ggg aaa tgg gta gcc gat tat tct cgt act gaa gcc 4151Lys Gln Phe Glu Gly Lys Trp Val Ala Asp Tyr Ser Arg Thr Glu Ala 850 855 860tta ttt aac tct act tat aaa caa tcg ccc gaa aat gta tta tat gat 4199Leu Phe Asn Ser Thr Tyr Lys Gln Ser Pro Glu Asn Val Leu Tyr Asp 865 870 875tta cgc gaa tac ctt tct ttc tat aac gac cct acg gaa tgg aaa gaa 4247Leu Arg Glu Tyr Leu Ser Phe Tyr Asn Asp Pro Thr Glu Trp Lys Glu 880 885 890ggg cta tta ctg tta agc cgt tat ata gat tat gct aaa gca caa gga 4295Gly Leu Leu Leu Leu Ser Arg Tyr Ile Asp Tyr Ala Lys Ala Gln Gly895 900 905 910ttt tat gaa aac tgg gcg gct act tct aac tta act att gcc cgt tta 4343Phe Tyr Glu Asn Trp Ala Ala Thr Ser Asn Leu Thr Ile Ala Arg Leu 915 920 925aga gag gct gga gta att tgt gca gaa tcg acg gat tta aaa ggc gat 4391Arg Glu Ala Gly Val Ile Cys Ala Glu Ser Thr Asp Leu Lys Gly Asp 930 935 940gaa aaa aat aat att gtg tta ggt agc caa aaa gat aat aac tta tcg 4439Glu Lys Asn Asn Ile Val Leu Gly Ser Gln Lys Asp Asn Asn Leu Ser 945 950 955ggt agt gca ggt gat gat cta ctt atc ggc gga gag ggt aat gat acg 4487Gly Ser Ala Gly Asp Asp Leu Leu Ile Gly Gly Glu Gly Asn Asp Thr 960 965 970tta aaa ggc agc tac ggt gca gac acc tat atc ttt agc aaa ggg cat 4535Leu Lys Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His975 980 985 990gga caa gat gta att tat gaa tat tcc gac agt gca aac tct aaa aaa 4583Gly Gln Asp Val Ile Tyr Glu Tyr Ser Asp Ser Ala Asn Ser Lys Lys 995 1000 1005gat att gat acc tta aaa ttt acc gat gtg aat tat gcg gaa gtg aag 4631Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys 1010 1015 1020ttt cga cga gta gat aat gac tta atg tta ttc ggt tat cat gat acg 4679Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr 1025 1030 1035gat tcg gtc acg gta aaa tcc ttc tac agc cat gta gat tat caa ttt 4727Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr Gln Phe 1040 1045 1050gac aaa ttg gag ttt gct gac cgc agt ata act cgc gat gaa ctg att 4775Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Ile1055 1060 1065 1070aaa gca ggg ctt cat cta tac ggc acc gat ggc aat gat gat ata aag 4823Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp Ile Lys 1075 1080 1085gat cat gcg gat tgg gac agc att gtg gaa ggc ggc aaa ggc aac gat 4871Asp His Ala Asp Trp Asp Ser Ile Val Glu Gly Gly Lys Gly Asn Asp 1090 1095 1100att cta aga ggt ggc tac ggt gcg gac acc tat atc ttt agc aaa gga 4919Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly 1105 1110 1115cac gga cag gat atc gtt tat gaa gat acc aat aat gat aac cga gca 4967His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala 1120 1125 1130aga gat atc gac acc tta aca ttt act gat gtg aat tat gcg gaa gtg 5015Arg Asp Ile Asp Thr Leu Thr Phe Thr Asp Val Asn Tyr Ala Glu Val1135 1140 1145 1150aaa ttc cga cga gta gat aat gac tta atg tta ttc ggt tat cat gat 5063Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp 1155 1160 1165acg gat tcg gtc acg ata aaa tcc ttc tac aac cat gta gat tat caa 5111Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp Tyr Gln 1170 1175 1180tgt gac aaa ttg gac ttt gct gac cgc agt ata act cgt gat gaa cta 5159Cys Asp Lys Leu Asp Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu 1185 1190 1195ggt aaa caa ggt atg gca tta ttt ggc act gac ggc gat gat aat atc 5207Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp Asn Ile 1200 1205 1210aac gac tgg gga cgt aac tcg gtg att gat gcc ggt gcg ggt aat gat 5255Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly Asn Asp1215 1220 1225 1230acg gtt aat ggc ggt aat ggc gat gac acc ctc atc ggc ggc aaa ggt 5303Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly Lys Gly 1235 1240 1245aat gat att cta aga ggt ggc tac ggt gcg gac acc tat atc ttt agc 5351Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser 1250 1255 1260aaa gga cac gga cag gat atc gtt tat gaa gat acc aat aat gat aac 5399Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn 1265 1270 1275cgc gca aga gat atc gac acc tta aaa ttt act gat att aat tta tcc 5447Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Ile Asn Leu Ser 1280 1285 1290gaa ctt tgg ttt agc cga gaa aat aac gat ttg att att aaa tca tta 5495Glu Leu Trp Phe Ser Arg Glu Asn Asn Asp Leu Ile Ile Lys Ser Leu1295 1300 1305 1310tta agt gag gat aaa gtc acg gtt caa aat tgg tat tca cac caa gat 5543Leu Ser Glu Asp Lys Val Thr Val Gln Asn Trp Tyr Ser His Gln Asp 1315 1320 1325cat aaa ata gaa aat att cgt tta tcg aat gag caa atg ttg gtg agc 5591His Lys Ile Glu Asn Ile Arg Leu Ser Asn Glu Gln Met Leu Val Ser 1330 1335 1340act cag gtg gag aag atg gtt gag tcg atg gcc ggc ttt gct cag aag 5639Thr Gln Val Glu Lys Met Val Glu Ser Met Ala Gly Phe Ala Gln Lys 1345 1350 1355cac gga gga gag ata tct ctt ctg tcg cct gaa gag gta aaa caa tat 5687His Gly Gly Glu Ile Ser Leu Leu Ser Pro Glu Glu Val Lys Gln Tyr 1360 1365 1370atc aat agc tta aca gct gct tta taa catacgaaag aaatcggcac 5734Ile Asn Ser Leu Thr Ala Ala Leu1375 1380agtttttgtg aactgtgccg atttgatttt agtgtaagaa tatagcctga ttttaagaaa 5794tttactcttg gctaataact atttcccatt ttataagtta ttgacggatg gttttatcaa 5854atatgagatc aaatcttatt ttaaattcgc tttccattaa gcgatattga tcttttaagt 5914ttggggccgc atgagtttgg aaccgatacc actcattgtg ggaatcaata cacaatacgc 5974tgtaatcgga ctcttgcagt tcataataat gctttctctc cgttaattct tcttgcgtat 6034atggcgagag attaaagctg aatggctggt tcgcactaac aaacaggttc tccgatttca 6094gatattcaca accgtaatgg ctaccggttt cctgcggttt tacataattg gtatgatttt 6154gtttagctgt tatacggtag atgcctaatt gtggtaaatt gcgtgtgtca atatagcttt 6214cttgttctcc gtaaccgaaa tactcaatgg cgttttctgt tttagctaag aagaaacgta 6274agccgaagcg gggtaaatac ggtaattcga tcgggcgaat agcgttaatt tcaaccgaaa 6334gttgtccgtc attgaagata cgataacgaa tatccagtgt taaaatgcga ccgcgagaaa 6394ttgacacaat tgcagatttt actgaaaatt cgaccgcttg ttcgctttgc tgccactgaa 6454tttcatacgc tctggtatag gctttatcgt agccggcatt ttggcacgcc tcacgaatga 6514ggcgatcatt gtcggttggc gcacgccaaa tattaaaatc taacgattgt tggataatcg 6574ctttaccggc tttttcaata cgggtgaaaa tccctttctg tttatctaat tgataactaa 6634attgaccgtt gtgtacgtta atgtggaagc gatcttcttg tacttcaaat gcactgttct 6694caattgtgaa ttgtggtaat actaatttat tttcgctaaa taaattgagc tgctcgaagc 6754caagtgaatg tgcttcgtct aataattcga ccgcggtatt taagcgataa tttaaattca 6814gtagccataa atgcccgtta ttttttggta actcaatcgg taatactacg ctgccgtgcg 6874gttggcaaga aacggataaa ttcccaccgc ttgtcaccac gccgttttcg acaaattcgt 6934aatcaatcgt taaataatcg gcaagatcag tgaaatccaa gtagttgtgg atcacaattt 6994ggttatcgat 7004 4 1382 PRT Actinobacillus pleuropneumoniae 4Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser Gly Lys Arg Tyr 1 5 10 15Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln Glu Asp Phe Asp 20 25 30Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg Gly Leu Glu Ser 35 40 45Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn Leu Asn Phe Thr 50 55 60Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg Phe Lys Ala Glu 65 70 75 80Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala Ala Lys Val Tyr 85 90 95Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp Ser Gly Ser Ile 100 105 110Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu Lys Pro Ile Ile 115 120 125Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly Thr Lys Ile Thr 130 135 140Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile Ala Asn Gln Lys145 150 155 160Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser Val Ser Gly Ser 165 170 175Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro Thr Leu Asn Arg 180 185 190Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp Ser Leu Ala Lys 195 200 205Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly Val Val Tyr Val 210 215 220Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu His Glu Asp Leu225 230 235 240Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala Arg Val Gly Asp 245 250 255Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr Phe Val Arg Glu 260 265 270Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu Phe Ser Glu Tyr 275 280 285Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly Leu Leu Thr Tyr 290 295 300Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr Ala Thr Phe Ala305 310 315 320His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr Thr Asn Tyr Arg 325 330 335Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys Lys Leu Lys Ser 340 345 350Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val Thr Pro Ile Asp 355 360 365Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr Val Lys Asn Phe 370 375 380Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro Asn Ser Tyr Tyr385 390 395 400Tyr Phe Leu Glu Gly Gln Asp Thr Gly Phe Tyr Gly Pro Ala Phe Tyr 405 410 415Ile Glu Arg Lys Asn Gly Gly Gly Ser Lys Asn Asn Ser Ser Gly Ala 420 425 430Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly Asn His Arg Asn 435 440 445Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn Asn Gln 450 455 460Asn Asn Gly Ser Asn Gln Asp Asn His Ser Asp Val Asn Ala Pro Asn465 470 475 480Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu Ala Leu Asp Leu 485 490 495Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly Arg Gln Gly Ala 500 505 510Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala Thr Gly Trp Leu 515 520 525Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn Gln Asp Gly Ile 530 535 540Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn Gln Leu Ser Asp545 550 555 560Gly Ser Ile Ser Ala His Gly Phe Ala Thr Leu Ala Asp Leu Asp Thr 565 570 575Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu Phe Ser Lys Leu 580 585 590Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser Glu Ala Asn Glu 595 600 605Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu His Thr Ala Tyr 610 615 620Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile Leu Ala Gln Leu625 630 635 640Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Gly Gln Met Gly Asp Leu 645 650 655Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr Glu Ala Leu Asn 660 665 670Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr Gly Thr Gly Arg 675 680 685Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu Glu Leu Ala Ala 690 695 700Leu Leu Gln Gln Tyr Thr Lys Gly Ser Asp Phe Gln Ala Gln Arg Glu705 710 715 720Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr Asp Leu Gln Tyr 725 730 735Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Val Glu Ser Thr Asp Ser 740 745 750Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu Ser Ser Ile Arg 755 760 765Asn Val Lys His Asp Pro Thr Val Met Gln Asn Cys Glu Gln Ser Lys 770 775 780Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu Asn Ile Asp Gln785 790 795 800Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile Thr Asp Lys Val 805 810 815Asn Asn Met Tyr Gln Thr Thr Gly Glu Leu Gly Tyr Arg Ser Leu Leu 820 825 830Leu Gln Thr Arg Val Lys Lys Tyr Val Tyr Ser Val Asn Ala Lys Gln 835 840 845Phe Glu Gly Lys Trp Val Ala Asp Tyr Ser Arg Thr Glu Ala Leu Phe 850 855 860Asn Ser Thr Tyr Lys Gln Ser Pro Glu Asn Val Leu Tyr Asp Leu Arg865 870 875 880Glu Tyr Leu Ser Phe Tyr Asn Asp Pro Thr Glu Trp Lys Glu Gly Leu 885 890 895Leu Leu Leu Ser Arg Tyr Ile Asp Tyr Ala Lys Ala Gln Gly Phe Tyr 900 905 910Glu Asn Trp Ala Ala Thr Ser Asn Leu Thr Ile Ala Arg Leu Arg Glu 915 920 925Ala Gly Val Ile Cys Ala Glu Ser Thr Asp Leu Lys Gly Asp Glu Lys 930 935 940Asn Asn Ile Val Leu Gly Ser Gln Lys Asp Asn Asn Leu Ser Gly Ser945 950 955 960Ala Gly Asp Asp Leu Leu Ile Gly Gly Glu Gly Asn Asp Thr Leu Lys 965 970 975Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly Gln 980 985 990Asp Val Ile Tyr Glu Tyr Ser Asp Ser Ala Asn Ser Lys Lys Asp Ile 995 1000 1005Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys Phe Arg 1010 1015 1020Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp Ser1025 1030 1035 1040Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr Gln Phe Asp Lys 1045 1050 1055Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Ile Lys Ala 1060 1065 1070Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp Ile Lys Asp His 1075 1080 1085Ala Asp Trp Asp Ser Ile Val Glu Gly Gly Lys Gly Asn Asp Ile Leu 1090 1095 1100Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly1105 1110 1115 1120Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Asp 1125 1130 1135Ile Asp Thr Leu Thr Phe Thr Asp Val Asn Tyr Ala Glu Val Lys Phe 1140 1145 1150Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp 1155 1160 1165Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp Tyr Gln Cys Asp 1170 1175 1180Lys Leu Asp Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Gly Lys1185 1190 1195 1200Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp Asn Ile Asn Asp 1205 1210 1215Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly Asn Asp Thr Val 1220 1225 1230Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn Asp 1235 1240 1245Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly 1250 1255 1260His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala1265 1270 1275 1280Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Ile Asn Leu Ser Glu Leu 1285 1290 1295Trp Phe Ser Arg Glu Asn Asn Asp Leu Ile Ile Lys Ser Leu Leu Ser 1300 1305 1310Glu Asp Lys Val Thr Val Gln Asn Trp Tyr Ser His Gln Asp His Lys 1315 1320 1325Ile Glu Asn Ile Arg Leu Ser Asn Glu Gln Met Leu Val Ser Thr Gln 1330 1335 1340Val Glu Lys Met Val Glu Ser Met Ala Gly Phe Ala Gln Lys His Gly1345 1350 1355 1360Gly Glu Ile Ser Leu Leu Ser Pro Glu Glu Val Lys Gln Tyr Ile Asn 1365 1370 1375Ser Leu Thr Ala Ala Leu 1380 5 6736 DNA Actinobacillus pleuropneumoniae CDS 1..453 CDS 1132..6549 -10_signal 617..623 -35_signal 594..599 promoter 454..1131 5atc gat atg ccg ccg ggt acg ggc gat atc caa ctt act ctt tcg caa 48Ile Asp Met Pro Pro Gly Thr Gly Asp Ile Gln Leu Thr Leu Ser Gln 1 5 10 15caa att ccg gtt acc ggt gcg gtg gtg gta acc act ccg caa gat att 96Gln Ile Pro Val Thr Gly Ala Val Val Val Thr Thr Pro Gln Asp Ile 20 25 30gcg tta tta gat gcg gtg aaa ggt att tca atg ttc caa aaa gtg tcg 144Ala Leu Leu Asp Ala Val Lys Gly Ile Ser Met Phe Gln Lys Val Ser 35 40 45gta ccg gtc tta ggt atc att gaa aat atg agc gta cat atc tgc caa 192Val Pro Val Leu Gly Ile Ile Glu Asn Met Ser Val His Ile Cys Gln 50 55 60aat tgc ggt cac cac gaa gat att ttc ggc acc ggc ggt gcg gag aaa 240Asn Cys Gly His His Glu Asp Ile Phe Gly Thr Gly Gly Ala Glu Lys 65 70 75 80gtg gcg aag aaa tac ggt act aaa gta tta gga caa atg ccg ttg cat 288Val Ala Lys Lys Tyr Gly Thr Lys Val Leu Gly Gln Met Pro Leu His 85 90 95att cgc tta cgt caa gat ttg gat gcc ggc aca ccg acc gtc gtt gcg 336Ile Arg Leu Arg Gln Asp Leu Asp Ala Gly Thr Pro Thr Val Val Ala 100 105 110gca ccg gaa cac gaa acc agc cga gcc tat att gaa tta gcg gca aaa 384Ala Pro Glu His Glu Thr Ser Arg Ala Tyr Ile Glu Leu Ala Ala Lys 115 120 125gtc gct tcg gaa tta tac tgg caa ggt tcg gtt atc ccg tct gaa att 432Val Ala Ser Glu Leu Tyr Trp Gln Gly Ser Val Ile Pro Ser Glu Ile 130 135 140atg att cgt gaa gta aaa taa gttttaataa ccacgaaaac acaaagaaca 483Met Ile Arg Glu Val Lys145 150caagcggtag aatttgcaga aaaatttgca aatcctaccg cttttttatt agtacgattc 543gctgttggac tgctatttga tttggtttgt caggatatta tgttattgta atgaaatgtt 603agtgaattat ttttattaat ttgaaaggaa acaaaatgaa aataaaaaaa cgttacattg 663cgctgttggt cttaggtgtc gttatcagct atgcctggta tcaaaattat caatgggaac 723agctgatgtt aagcggttat tgtgaaaagg acggaagtta ttttgatgat aggcatacga 783agcaagaact gattgatagg gcaattaact atatgctgga gcatcaatct aaaaaaacat 843acgatgctta tactgatgaa cctttagaaa taaaaccata tttaacaata gaggaattta 903aaaaactcaa tccaaattgt tgtgaaatta cctcatggcc agcagatgca gttccacaag 963attgggatgt tcgtgtggaa ggtaaggcat ataggtatgt aatcgtaaaa tatttaagaa 1023ccttagcaaa tagagaacct gaacgatggg aaactagtat tgtttttgat aattgcggca 1083atcctaaaag agcaagctac ttatattatt taaagagaga aatttatt atg aca aaa 1140 Met Thr Lys 1tta act atg caa gat gtg acc aat tta tat tta tat aaa acg aaa act 1188Leu Thr Met Gln Asp Val Thr Asn Leu Tyr Leu Tyr Lys Thr Lys Thr 5 10 15cta cct aaa gat aga ttg gat gat tca ctt att tct gaa ata gga aaa 1236Leu Pro Lys Asp Arg Leu Asp Asp Ser Leu Ile Ser Glu Ile Gly Lys 20 25 30 35gga gat gat gat att gat aga aaa gaa ttt atg gtg ggg ccg gga cgt 1284Gly Asp Asp Asp Ile Asp Arg Lys Glu Phe Met Val Gly Pro Gly Arg 40 45 50ttt gtg acc gct gat aac ttt agc gtt gta aga gat ttt ttt aat gct 1332Phe Val Thr Ala Asp Asn Phe Ser Val Val Arg Asp Phe Phe Asn Ala 55 60 65ggg aaa tca cgc att att gcg ccg caa gtc ccg cct att cgt tca cag 1380Gly Lys Ser Arg Ile Ile Ala Pro Gln Val Pro Pro Ile Arg Ser Gln 70 75 80cag gaa aaa atc ttg gtc ggt tta aaa ccg ggc aaa tat tcc aaa gcg 1428Gln Glu Lys Ile Leu Val Gly Leu Lys Pro Gly Lys Tyr Ser Lys Ala 85 90 95cag ata ttg gaa atg ctg ggt tat acg aaa ggc gga gaa gtg gta aat 1476Gln Ile Leu Glu Met Leu Gly Tyr Thr Lys Gly Gly Glu Val Val Asn100 105 110 115ggc atg ttt gcc ggt gaa gtc cag aca tta ggc ttt tat gac gat ggc 1524Gly Met Phe Ala Gly Glu Val Gln Thr Leu Gly Phe Tyr Asp Asp Gly 120 125 130aaa ggg gat tta ctc gaa cgc gcc tat atc tgg aat acc aca gga ttt 1572Lys Gly Asp Leu Leu Glu Arg Ala Tyr Ile Trp Asn Thr Thr Gly Phe 135 140 145aaa atg agc gac aat gcc ttt ttt gtt ata gaa gaa tca ggc aaa cgc 1620Lys Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser Gly Lys Arg 150 155 160tat att gaa aac ttt ggt att gaa cct ctt ggt aag caa gaa gat ttt 1668Tyr Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln Glu Asp Phe 165 170 175gat ttt gtc ggc ggc ttt tgg tct aac tta gtg aat cgt ggt ttg gaa 1716Asp Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg Gly Leu Glu180 185 190 195agt att atc gac cca tcc ggt atc ggt gga acg gta aac ctt aac ttt 1764Ser Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn Leu Asn Phe 200 205 210acc ggc gag gtg gaa acc tac acg tta gac gaa aca agg ttt aaa gcg 1812Thr Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg Phe Lys Ala 215 220 225gaa gcg gcg aag aaa agc cat tgg agt tta gtg aat gcg gcg aaa gta 1860Glu Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala Ala Lys Val 230 235 240tac ggc ggt tta gac caa att att aaa aaa cta tgg gac agt ggc tca 1908Tyr Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp Ser Gly Ser 245 250 255att aag cat tta tat caa gat aaa gat acg ggc aaa tta aaa ccg att 1956Ile Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu Lys Pro Ile260 265 270 275att tac ggc acg gcc ggc aac gac agt aag att gaa ggc act aaa atc 2004Ile Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly Thr Lys Ile 280 285 290acc cgt agg att gcg ggt aaa gaa gtt acg ctt gat att gcc aat cag 2052Thr Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile Ala Asn Gln 295 300 305aaa att gaa aaa ggc gtg tta gag aaa ttg ggg ctg tct gtt agt ggt 2100Lys Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser Val Ser Gly 310 315 320tcg gat atc att aaa ttg ttg ttt gga gca ttg act cca act tta aat 2148Ser Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro Thr Leu Asn 325 330 335aga atg ttg cta tca caa ctt atc cag tct ttt tcc gat agc ttg gct 2196Arg Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp Ser Leu Ala340 345 350 355aaa ctt gat aat ccc tta gcc cct tac act aaa aat ggc gtg gtt tat 2244Lys Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly Val Val Tyr 360 365 370gtc acc ggc aaa ggg aat gat gtg ctt aaa gga act gaa cat gag gat 2292Val Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu His Glu Asp 375 380 385ttg ttt ctc ggt ggt gag ggg aat gat act tat tat gcg aga gta ggc 2340Leu Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala Arg Val Gly 390 395 400gat aca att gaa gac gcc gac ggc aaa ggt aaa gtc tat ttt gtg aga 2388Asp Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr Phe Val Arg 405 410 415gaa aaa ggg gta cct aag gcg gat cct aag cgg gta gag ttt agc gag 2436Glu Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu Phe Ser Glu420 425 430 435tac ata acg aaa gaa gaa ata aaa gag gtt gaa aag ggg tta tta act 2484Tyr Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly Leu Leu Thr 440 445 450tac gca gtt tta gaa aat tat aat tgg gaa gag aaa acg gcg act ttc 2532Tyr Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr Ala Thr Phe 455 460 465gct cat gcg act atg ctt aat gag ctt ttt act gat tat act aat tat 2580Ala His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr Thr Asn Tyr 470 475 480cgt tat gaa gtt aaa gga cta aaa ttg ccc gcc gtt aaa aag tta aaa 2628Arg Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys Lys Leu Lys 485 490 495agt ccg ttg gtg gag ttt aca gct gat tta tta act gtt acg cct att 2676Ser Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val Thr Pro Ile500 505 510 515gac gaa aac gga aaa gca ctt agc gaa aaa agt att acg gtt aaa aat 2724Asp Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr Val Lys Asn 520 525 530ttt aaa aat ggt gat tta gga ata agg ttg ttg gat cct aat agc tat 2772Phe Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro Asn Ser Tyr 535 540 545tat tat ttc ctt gaa ggc caa gat acg ggt ttt tat ggt cct gct ttt 2820Tyr Tyr Phe Leu Glu Gly Gln Asp Thr Gly Phe Tyr Gly Pro Ala Phe 550 555 560tat att gaa cga aaa aac ggt ggc ggc gct aaa aat aac tcg tcg gga 2868Tyr Ile Glu Arg Lys Asn Gly Gly Gly Ala Lys Asn Asn Ser Ser Gly 565 570 575gca gga aat agc aaa gat tgg ggc ggg aac ggg cat gga aat cac cga 2916Ala Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly Asn His Arg580 585 590 595aat aat gcc tcc gac ctg aat aaa ccg gac gga aat aat ggg aat aac 2964Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn Gly Asn Asn 600 605 610caa aat aac gga agc aat caa gat aat cat agc gat gtg aat gcg cca 3012Gln Asn Asn Gly Ser Asn Gln Asp Asn His Ser Asp Val Asn Ala Pro 615 620 625aat aac ccg gga cgt aac tat gat att tac gat cct tta gct tta gat 3060Asn Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu Ala Leu Asp 630 635 640tta gat gga gat ggg ctt gaa acc gtg tcg atg aac ggg cga caa ggc 3108Leu Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly Arg Gln Gly 645 650 655gcg tta ttc gat cat gaa gga aaa ggt att cgt acc gca acg ggc tgg 3156Ala Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala Thr Gly Trp660 665 670 675ctc gct gcg gat gac ggt ttt tta gtg tta gat cgt aac caa gac ggc 3204Leu Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn Gln Asp Gly 680 685 690att att aat gat ata agc gag tta ttt agt aat aaa aat caa ctt tcc 3252Ile Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn Gln Leu Ser 695 700 705gac ggc agt att tct gca cac ggt ttt gcg aca tta gcc gat ttg gat 3300Asp Gly Ser Ile Ser Ala His Gly Phe Ala Thr Leu Ala Asp Leu Asp 710 715 720aca aac caa gat cag cgt atc gac caa aat gat aag ctg ttt tct aaa 3348Thr Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu Phe Ser Lys 725 730 735ctc caa att tgg cgg gat tta aat caa aac ggt ttt agt gaa gcg aat 3396Leu Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser Glu Ala Asn740 745 750 755gag ctg ttt agc tta gaa agt ttg aat att aaa tct tta cat acc gcc 3444Glu Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu His Thr Ala 760 765 770tat gaa gag cgt aat gat ttt cta gcg ggc aat aat atc ctt gct cag 3492Tyr Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile Leu Ala Gln 775 780 785ctt ggg aag tat gaa aaa acg gac ggt act ttt gca caa atg ggc gat 3540Leu Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Ala Gln Met Gly Asp 790 795 800tta aat ttc agt ttt aac ccg ttt tat agc cga ttt acc gaa gcg tta 3588Leu Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr Glu Ala Leu 805 810 815aat tta acc gag caa caa cgt cgc aca att aat cta acc ggc acc ggt 3636Asn Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr Gly Thr Gly820 825 830 835cgg gtt cgg gat ttg cgt gaa gcc gcc gca ctt tct gag gag ttg gct 3684Arg Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu Glu Leu Ala 840 845 850gct tta tta caa cag tac act aag gcc tcc gat ttt cag gca caa cga 3732Ala Leu Leu Gln Gln Tyr Thr Lys Ala Ser Asp Phe Gln Ala Gln Arg 855 860 865gaa tta ttg cct gcc att tta gat aaa tgg gcg gca acg gat tta cag 3780Glu Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr Asp Leu Gln 870 875 880tat caa cat tat gat aaa aca tta ctt aaa acg gta gaa agt acc gat 3828Tyr Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Val Glu Ser Thr Asp 885 890 895agt agt gct tct gtc gtt aga gtc acg cct tct caa tta agt agt ata 3876Ser Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu Ser Ser Ile900 905 910 915cgc aat gca aag cat gat cct acc gtt atg caa aac ttt gaa cag agt 3924Arg Asn Ala Lys His Asp Pro Thr Val Met Gln Asn Phe Glu Gln Ser 920 925 930aag gca aaa att gcg act tta aat tcg ctc tac ggg tta aat atc gat 3972Lys Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu Asn Ile Asp 935 940 945caa ctt tat tac acg acg gat aaa gac att cgc tat att act gat aaa 4020Gln Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile Thr Asp Lys 950 955 960gtg aat aat atg tat caa aca acc gta gaa ctt gcc tac cgt tct tta 4068Val Asn Asn Met Tyr Gln Thr Thr Val Glu Leu Ala Tyr Arg Ser Leu 965 970 975ctt tta caa acg cgt ttg aag aaa tat gtt tat agc gtt aat gcg aaa 4116Leu Leu Gln Thr Arg Leu Lys Lys Tyr Val Tyr Ser Val Asn Ala Lys980 985 990 995caa ttc gaa ggg aaa tgg gta acc gat tat tct cgt act gaa gcc tta 4164Gln Phe Glu Gly Lys Trp Val Thr Asp Tyr Ser Arg Thr Glu Ala Leu 1000 1005 1010ttt aac tct act ttt aaa caa tcg cct gaa aat gca tta tat gat tta 4212Phe Asn Ser Thr Phe Lys Gln Ser Pro Glu Asn Ala Leu Tyr Asp Leu 1015 1020 1025agc gaa tac ctt tct ttc ttt aac gat cct acg gaa tgg aaa gaa ggg 4260Ser Glu Tyr Leu Ser Phe Phe Asn Asp Pro Thr Glu Trp Lys Glu Gly 1030 1035 1040cta tta ctg tta agc cgt tat ata gat tat gct aaa gca caa gga ttt 4308Leu Leu Leu Leu Ser Arg Tyr Ile Asp Tyr Ala Lys Ala Gln Gly Phe 1045 1050 1055tat gaa aac tgg gcg gct act tct aac tta act att gcc cgt tta aga 4356Tyr Glu Asn Trp Ala Ala Thr Ser Asn Leu Thr Ile Ala Arg Leu Arg1060 1065 1070 1075gag gct gga gta att ttt gca gaa tcg acg gat tta aaa ggc gat gaa 4404Glu Ala Gly Val Ile Phe Ala Glu Ser Thr Asp Leu Lys Gly Asp Glu 1080 1085 1090aaa aat aat att ttg tta ggt agc caa aaa gat aat aac tta tcg ggt 4452Lys Asn Asn Ile Leu Leu Gly Ser Gln Lys Asp Asn Asn Leu Ser Gly 1095 1100 1105agt gca ggt gat gat cta ctt atc ggc gga gag ggt aat gat acg tta 4500Ser Ala Gly Asp Asp Leu Leu Ile Gly Gly Glu Gly Asn Asp Thr Leu 1110 1115 1120aaa ggc agc tac ggt gca gac acc tat atc ttt agc aaa gga cac gga 4548Lys Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His Gly 1125 1130 1135cag gat atc gtt tat gaa gat acc aat aat gat aac cgc gca aga gat 4596Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg Asp1140 1145 1150 1155atc gac acc tta aaa ttt acc gat gtg aat tat gcg gaa gtg aag ttt 4644Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys Phe 1160 1165 1170cga cga gta gat aat gac tta atg tta ttc ggt tat cat gat acg gat 4692Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr Asp 1175 1180 1185tcg gtc acg gta aaa tcc ttc tac agc cat gta gat tat caa ttt gac 4740Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr Gln Phe Asp 1190 1195 1200aaa ttg gag ttt gct gac cgc agt ata act cgc gat gaa ctg att aaa 4788Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Ile Lys 1205 1210 1215gca ggg ctt cat cta tac ggc acc gat ggc aat gat gat ata aag gat 4836Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp Ile Lys Asp1220 1225 1230 1235cat gcg gat tgg gac agc att ttg gaa ggc ggc aaa ggc aac gat att 4884His Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly Asn Asp Ile 1240 1245 1250cta aga ggt ggc tac ggt gcg gac acc tat atc ttt agc aaa gga cac 4932Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys Gly His 1255 1260 1265gga cag gat atc gtt tat gaa gat acc aat aat gat aac cgc gca aga 4980Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg Ala Arg 1270 1275 1280gat atc gac acc tta aaa ttt act gat gtg aat tat gcg gaa gtg aaa 5028Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu Val Lys 1285 1290 1295ttc cga cga gta gat aat gac tta atg tta ttc ggt tat cat gat acg 5076Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His Asp Thr1300 1305 1310 1315gat tcg gtc acg ata aaa tcc ttc tac aac cat gta gat tat caa ttt 5124Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp Tyr Gln Phe 1320 1325 1330gac aaa ttg gaa ttt gct gac cgc agt ata act cgt gat gaa cta ggt 5172Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu Leu Gly 1335 1340 1345aaa caa ggt atg gca tta ttt ggc act gac ggt gat gat aat atc aac 5220Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp Asn Ile Asn 1350 1355 1360gac tgg gga cgt aac tcg gtg att gat gcc ggt gcg ggt aat gat acg 5268Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly Asn Asp Thr 1365 1370 1375gtt aat ggc ggt aat ggc gat gac acc ctc atc ggc ggc aaa ggt aat 5316Val Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly Lys Gly Asn1380 1385 1390 1395gat att cta aga ggt ggc tac ggt gcg gac acc tat atc ttt agc aaa 5364Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys 1400 1405 1410gga cac gga cag gat atc gtt tat gaa gat acc aat aat gat aac cgc 5412Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg 1415 1420 1425gca aga gat atc gac acc tta aaa ttt acc gat gtg aat tat gcg gaa 5460Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu 1430 1435 1440gtg aaa ttc cga cga gta gat aat gac tta atg tta ttc ggt tat cat 5508Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His 1445 1450 1455gat acg gat tcg gtc acg gta aaa tcc ttc tac agc cat gta gat tat 5556Asp Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr1460 1465 1470 1475caa ttt gac aaa ttg gag ttt gct gac cgc agt ata act cgc gat gaa 5604Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu 1480 1485 1490ctg att aaa gca ggg ctt cat cta tac ggc acc gat ggc aat gat gat 5652Leu Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp 1495 1500 1505ata aag gat cat gcg gat tgg gac agc att ttg gaa ggc ggc aaa ggc 5700Ile Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly 1510 1515 1520aac gat att cta aga ggt ggc tac ggt gcg gac acc tat atc ttt agc 5748Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser 1525 1530 1535aaa gga cac gga cag gat atc gtt tat gaa gat acc aat aat gat aac 5796Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn1540 1545 1550 1555cga gca aga gat atc gac acc tta aaa ttt act gat gtg aat tat gcg 5844Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala 1560 1565 1570gaa gtg aaa ttc cga cga gta gat aat gac tta atg tta ttc ggt tat 5892Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr 1575 1580 1585cat gat acg gat tcg gtc acg ata aaa tcc ttc tac aac cat gta gat 5940His Asp Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp 1590 1595 1600tat caa ttt gac aaa ttg gaa ttt gct gac cgc agt ata act cgt gat 5988Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp 1605 1610 1615gaa cta ggt aaa caa ggt atg gca tta ttt ggc act gac ggt gat gat 6036Glu Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp1620 1625 1630 1635aat atc aac gac tgg gga cgt aac tcg gtg att gat gcc ggt gcg ggt 6084Asn Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly 1640 1645 1650aat gat acg gtt aat ggc ggt aat ggc gat gac acc ctc atc ggc ggc 6132Asn Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly 1655 1660 1665aaa ggt aat gat att cta aga ggt ggc tac ggt gcg gac acc tat atc 6180Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile 1670 1675 1680ttt agc aaa gga cac gga cag gat atc gtt tat gaa gat acc aat aat 6228Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn 1685 1690 1695gat aac cgc gca aga gat atc gac acc tta aaa ttt act gat att aat 6276Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Ile Asn1700 1705 1710 1715tta tcc gaa ctt tgg ttt agc cga gaa aat aac gat ttg att att aaa 6324Leu Ser Glu Leu Trp Phe Ser Arg Glu Asn Asn Asp Leu Ile Ile Lys 1720 1725 1730tca tta tta agt gag gat aaa gtc acg gtt caa aat tgg tat tca cac 6372Ser Leu Leu Ser Glu Asp Lys Val Thr Val Gln Asn Trp Tyr Ser His 1735 1740 1745caa gat cat aaa ata gaa aat att cgt tta tcg aat gag caa acg ttg 6420Gln Asp His Lys Ile Glu Asn Ile Arg Leu Ser Asn Glu Gln Thr Leu 1750 1755 1760gtg agc act cag gtg gag aag atg gtt gag tcg atg gcc ggc ttt gct 6468Val Ser Thr Gln Val Glu Lys Met Val Glu Ser Met Ala Gly Phe Ala 1765 1770 1775gag aag cac gga gga gag ata tct ctt gtg tcg ctt gaa gag gta aaa 6516Gln Lys His Gly Gly Glu Ile Ser Leu Val Ser Leu Glu Glu Val Lys1780 1785 1790 1795caa tat atc aat agc tta aca gct gct tta taa catacgaaag aaatcggcac 6569Gln Tyr Ile Asn Ser Leu Thr Ala Ala Leu 1800 1805agtttttttg aactgtgccg atttgatttt agtgtaagaa tatagcctga ttttaagaaa 6629tttactcttg gctaataact atttcccatt ttataagtta ttgacggatg gttttatcaa 6689atatgagatc aaatcttatt ttaaattcgc tttccattaa gcgatat 6736 6 150 PRT Actinobacillus pleuropneumoniae 6Ile Asp Met Pro Pro Gly Thr Gly Asp Ile Gln Leu Thr Leu Ser Gln 1 5 10 15Gln Ile Pro Val Thr Gly Ala Val Val Val Thr Thr Pro Gln Asp Ile 20 25 30Ala Leu Leu Asp Ala Val Lys Gly Ile Ser Met Phe Gln Lys Val Ser 35 40 45Val Pro Val Leu Gly Ile Ile Glu Asn Met Ser Val His Ile Cys Gln 50 55 60Asn Cys Gly His His Glu Asp Ile Phe Gly Thr Gly Gly Ala Glu Lys 65 70 75 80Val Ala Lys Lys Tyr Gly Thr Lys Val Leu Gly Gln Met Pro Leu His 85 90 95Ile Arg Leu Arg Gln Asp Leu Asp Ala Gly Thr Pro Thr Val Val Ala 100 105 110Ala Pro Glu His Glu Thr Ser Arg Ala Tyr Ile Glu Leu Ala Ala Lys 115 120 125Val Ala Ser Glu Leu Tyr Trp Gln Gly Ser Val Ile Pro Ser Glu Ile 130 135 140Met Ile Arg Glu Val Lys145 150 7 1805 PRT Actinobacillus pleuropneumoniae 7Met Thr Lys Leu Thr Met Gln Asp Val Thr Asn Leu Tyr Leu Tyr Lys 1 5 10 15Thr Lys Thr Leu Pro Lys Asp Arg Leu Asp Asp Ser Leu Ile Ser Glu 20 25 30Ile Gly Lys Gly Asp Asp Asp Ile Asp Arg Lys Glu Phe Met Val Gly 35 40 45Pro Gly Arg Phe Val Thr Ala Asp Asn Phe Ser Val Val Arg Asp Phe 50 55 60Phe Asn Ala Gly Lys Ser Arg Ile Ile Ala Pro Gln Val Pro Pro Ile 65 70 75 80Arg Ser Gln Gln Glu Lys Ile Leu Val Gly Leu Lys Pro Gly Lys Tyr 85 90 95Ser Lys Ala Gln Ile Leu Glu Met Leu Gly Tyr Thr Lys Gly Gly Glu 100 105 110Val Val Asn Gly Met Phe Ala Gly Glu Val Gln Thr Leu Gly Phe Tyr 115 120 125Asp Asp Gly Lys Gly Asp Leu Leu Glu Arg Ala Tyr Ile Trp Asn Thr 130 135 140Thr Gly Phe Lys Met Ser Asp Asn Ala Phe Phe Val Ile Glu Glu Ser145 150 155 160Gly Lys Arg Tyr Ile Glu Asn Phe Gly Ile Glu Pro Leu Gly Lys Gln 165 170 175Glu Asp Phe Asp Phe Val Gly Gly Phe Trp Ser Asn Leu Val Asn Arg 180 185 190Gly Leu Glu Ser Ile Ile Asp Pro Ser Gly Ile Gly Gly Thr Val Asn 195 200 205Leu Asn Phe Thr Gly Glu Val Glu Thr Tyr Thr Leu Asp Glu Thr Arg 210 215 220Phe Lys Ala Glu Ala Ala Lys Lys Ser His Trp Ser Leu Val Asn Ala225 230 235 240Ala Lys Val Tyr Gly Gly Leu Asp Gln Ile Ile Lys Lys Leu Trp Asp 245 250 255Ser Gly Ser Ile Lys His Leu Tyr Gln Asp Lys Asp Thr Gly Lys Leu 260 265 270Lys Pro Ile Ile Tyr Gly Thr Ala Gly Asn Asp Ser Lys Ile Glu Gly 275 280 285Thr Lys Ile Thr Arg Arg Ile Ala Gly Lys Glu Val Thr Leu Asp Ile 290 295 300Ala Asn Gln Lys Ile Glu Lys Gly Val Leu Glu Lys Leu Gly Leu Ser305 310 315 320Val Ser Gly Ser Asp Ile Ile Lys Leu Leu Phe Gly Ala Leu Thr Pro 325 330 335Thr Leu Asn Arg Met Leu Leu Ser Gln Leu Ile Gln Ser Phe Ser Asp 340 345 350Ser Leu Ala Lys Leu Asp Asn Pro Leu Ala Pro Tyr Thr Lys Asn Gly 355 360 365Val Val Tyr Val Thr Gly Lys Gly Asn Asp Val Leu Lys Gly Thr Glu 370 375 380His Glu Asp Leu Phe Leu Gly Gly Glu Gly Asn Asp Thr Tyr Tyr Ala385 390 395 400Arg Val Gly Asp Thr Ile Glu Asp Ala Asp Gly Lys Gly Lys Val Tyr 405 410 415Phe Val Arg Glu Lys Gly Val Pro Lys Ala Asp Pro Lys Arg Val Glu 420 425 430Phe Ser Glu Tyr Ile Thr Lys Glu Glu Ile Lys Glu Val Glu Lys Gly 435 440 445Leu Leu Thr Tyr Ala Val Leu Glu Asn Tyr Asn Trp Glu Glu Lys Thr 450 455 460Ala Thr Phe Ala His Ala Thr Met Leu Asn Glu Leu Phe Thr Asp Tyr465 470 475 480Thr Asn Tyr Arg Tyr Glu Val Lys Gly Leu Lys Leu Pro Ala Val Lys 485 490 495Lys Leu Lys Ser Pro Leu Val Glu Phe Thr Ala Asp Leu Leu Thr Val 500 505 510Thr Pro Ile Asp Glu Asn Gly Lys Ala Leu Ser Glu Lys Ser Ile Thr 515 520 525Val Lys Asn Phe Lys Asn Gly Asp Leu Gly Ile Arg Leu Leu Asp Pro 530 535 540Asn Ser Tyr Tyr Tyr Phe Leu Glu Gly Gln Asp Thr Gly Phe Tyr Gly545 550 555 560Pro Ala Phe Tyr Ile Glu Arg Lys Asn Gly Gly Gly Ala Lys Asn Asn 565 570 575Ser Ser Gly Ala Gly Asn Ser Lys Asp Trp Gly Gly Asn Gly His Gly 580 585 590Asn His Arg Asn Asn Ala Ser Asp Leu Asn Lys Pro Asp Gly Asn Asn 595 600 605Gly Asn Asn Gln Asn Asn Gly Ser Asn Gln Asp Asn His Ser Asp Val 610 615 620Asn Ala Pro Asn Asn Pro Gly Arg Asn Tyr Asp Ile Tyr Asp Pro Leu625 630 635 640Ala Leu Asp Leu Asp Gly Asp Gly Leu Glu Thr Val Ser Met Asn Gly 645 650 655Arg Gln Gly Ala Leu Phe Asp His Glu Gly Lys Gly Ile Arg Thr Ala 660 665 670Thr Gly Trp Leu Ala Ala Asp Asp Gly Phe Leu Val Leu Asp Arg Asn 675 680 685Gln Asp Gly Ile Ile Asn Asp Ile Ser Glu Leu Phe Ser Asn Lys Asn 690 695 700Gln Leu Ser Asp Gly Ser Ile Ser Ala His Gly Phe Ala Thr Leu Ala705 710 715 720Asp Leu Asp Thr Asn Gln Asp Gln Arg Ile Asp Gln Asn Asp Lys Leu 725 730 735Phe Ser Lys Leu Gln Ile Trp Arg Asp Leu Asn Gln Asn Gly Phe Ser 740 745 750Glu Ala Asn Glu Leu Phe Ser Leu Glu Ser Leu Asn Ile Lys Ser Leu 755 760 765His Thr Ala Tyr Glu Glu Arg Asn Asp Phe Leu Ala Gly Asn Asn Ile 770 775 780Leu Ala Gln Leu Gly Lys Tyr Glu Lys Thr Asp Gly Thr Phe Ala Gln785 790 795 800Met Gly Asp Leu Asn Phe Ser Phe Asn Pro Phe Tyr Ser Arg Phe Thr 805 810 815Glu Ala Leu Asn Leu Thr Glu Gln Gln Arg Arg Thr Ile Asn Leu Thr 820 825 830Gly Thr Gly Arg Val Arg Asp Leu Arg Glu Ala Ala Ala Leu Ser Glu 835 840 845Glu Leu Ala Ala Leu Leu Gln Gln Tyr Thr Lys Ala Ser Asp Phe Gln 850 855 860Ala Gln Arg Glu Leu Leu Pro Ala Ile Leu Asp Lys Trp Ala Ala Thr865 870 875 880Asp Leu Gln Tyr Gln His Tyr Asp Lys Thr Leu Leu Lys Thr Val Glu 885 890 895Ser Thr Asp Ser Ser Ala Ser Val Val Arg Val Thr Pro Ser Gln Leu 900 905 910Ser Ser Ile Arg Asn Ala Lys His Asp Pro Thr Val Met Gln Asn Phe 915 920 925Glu Gln Ser Lys Ala Lys Ile Ala Thr Leu Asn Ser Leu Tyr Gly Leu 930 935 940Asn Ile Asp Gln Leu Tyr Tyr Thr Thr Asp Lys Asp Ile Arg Tyr Ile945 950 955 960Thr Asp Lys Val Asn Asn Met Tyr Gln Thr Thr Val Glu Leu Ala Tyr 965 970 975Arg Ser Leu Leu Leu Gln Thr Arg Leu Lys Lys Tyr Val Tyr Ser Val 980 985 990Asn Ala Lys Gln Phe Glu Gly Lys Trp Val Thr Asp Tyr Ser Arg Thr 995 1000 1005Glu Ala Leu Phe Asn Ser Thr Phe Lys Gln Ser Pro Glu Asn Ala Leu 1010 1015 1020Tyr Asp Leu Ser Glu Tyr Leu Ser Phe Phe Asn Asp Pro Thr Glu Trp1025 1030 1035 1040Lys Glu Gly Leu Leu Leu Leu Ser Arg Tyr Ile Asp Tyr Ala Lys Ala 1045 1050 1055Gln Gly Phe Tyr Glu Asn Trp Ala Ala Thr Ser Asn Leu Thr Ile Ala 1060 1065 1070Arg Leu Arg Glu Ala Gly Val Ile Phe Ala Glu Ser Thr Asp Leu Lys 1075 1080 1085Gly Asp Glu Lys Asn Asn Ile Leu Leu Gly Ser Gln Lys Asp Asn Asn 1090 1095 1100Leu Ser Gly Ser Ala Gly Asp Asp Leu Leu Ile Gly Gly Glu Gly Asn1105 1110 1115 1120Asp Thr Leu Lys Gly Ser Tyr Gly Ala Asp Thr Tyr Ile Phe Ser Lys 1125 1130 1135Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn Arg 1140 1145 1150Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala Glu 1155 1160 1165Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr His 1170 1175 1180Asp Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His Val Asp Tyr1185 1190 1195 1200Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp Glu 1205 1210 1215Leu Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly Asn Asp Asp 1220 1225 1230Ile Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly Gly Lys Gly 1235 1240 1245Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile Phe Ser 1250 1255 1260Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn Asp Asn1265 1270 1275 1280Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn Tyr Ala 1285 1290 1295Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe Gly Tyr 1300 1305 1310His Asp Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn His Val Asp 1315 1320 1325Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr Arg Asp 1330 1335 1340Glu Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp Gly Asp Asp1345 1350 1355 1360Asn Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala Gly Ala Gly 1365 1370 1375Asn Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu Ile Gly Gly 1380 1385 1390Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr Ile 1395 1400 1405Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn Asn 1410 1415 1420Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val Asn1425 1430 1435 1440Tyr Ala Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu Phe 1445 1450 1455Gly Tyr His Asp Thr Asp Ser Val Thr Val Lys Ser Phe Tyr Ser His 1460 1465 1470Val Asp Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile Thr 1475 1480 1485Arg Asp Glu Leu Ile Lys Ala Gly Leu His Leu Tyr Gly Thr Asp Gly 1490 1495 1500Asn Asp Asp Ile Lys Asp His Ala Asp Trp Asp Ser Ile Leu Glu Gly1505 1510 1515 1520Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp Thr Tyr 1525 1530 1535Ile Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp Thr Asn 1540 1545 1550Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr Asp Val 1555 1560 1565Asn Tyr Ala Glu Val Lys Phe Arg Arg Val Asp Asn Asp Leu Met Leu 1570 1575 1580Phe Gly Tyr His Asp Thr Asp Ser Val Thr Ile Lys Ser Phe Tyr Asn1585 1590 1595 1600His Val Asp Tyr Gln Phe Asp Lys Leu Glu Phe Ala Asp Arg Ser Ile 1605 1610 1615Thr Arg Asp Glu Leu Gly Lys Gln Gly Met Ala Leu Phe Gly Thr Asp 1620 1625 1630Gly Asp Asp Asn Ile Asn Asp Trp Gly Arg Asn Ser Val Ile Asp Ala 1635 1640 1645Gly Ala Gly Asn Asp Thr Val Asn Gly Gly Asn Gly Asp Asp Thr Leu 1650 1655 1660Ile Gly Gly Lys Gly Asn Asp Ile Leu Arg Gly Gly Tyr Gly Ala Asp1665 1670 1675 1680Thr Tyr Ile Phe Ser Lys Gly His Gly Gln Asp Ile Val Tyr Glu Asp 1685 1690 1695Thr Asn Asn Asp Asn Arg Ala Arg Asp Ile Asp Thr Leu Lys Phe Thr 1700 1705 1710Asp Ile Asn Leu Ser Glu Leu Trp Phe Ser Arg Glu Asn Asn Asp Leu 1715 1720 1725Ile Ile Lys Ser Leu Leu Ser Glu Asp Lys Val Thr Val Gln Asn Trp 1730 1735 1740Tyr Ser His Gln Asp His Lys Ile Glu Asn Ile Arg Leu Ser Asn Glu1745 1750 1755 1760Gln Thr Leu Val Ser Thr Gln Val Glu Lys Met Val Glu Ser Met Ala 1765 1770 1775Gly Phe Ala Gln Lys His Gly Gly Glu Ile Ser Leu Val Ser Leu Glu 1780 1785 1790Glu Val Lys Gln Tyr Ile Asn Ser Leu Thr Ala Ala Leu 1795 1800 1805 8 18 DNA Actinobacillus pleuropneumoniae 8tggcactgac ggtgatga 18 9 18 DNA Actinobacillus pleuropneumoniae 9ggccatcgac tcaaccat 18 10 26 DNA Actinobacillus pleuropneumoniae 10agccatatgg gcgatttaaa tttcag 26 11 24 DNA Actinobacillus pleuropneumoniae 11tatggatcct ccgtgcttct gagc 24 12 20 DNA Actinobacillus pleuropneumoniae 12gggacagtgg ctcaattaag 20 13 20 DNA Actinobacillus pleuropneumoniae 13agctgtaaac tccaccaacg 20 14 28 DNA Actinobacillus pleuropneumoniae 14cgccatatga caaaattaac tatgcaag 28 15 26 DNA Actinobacillus pleuropneumoniae 15cgcgaattca gcgacacaag agatat 26 16 9 PRT Actinobacillus pleuropneumoniae 16Tyr Ser Asp Ser Ala Asn Ser Lys Lys1 5

Claims

1. A subunit vaccine for the protection of animals against infection with a bacterium of the species Actinobacillus pleuropneumoniae, comprising purified ApxIV toxin and a pharmaceutically acceptable carrier.

2. The vaccine according to claim 1, further comprising an adjuvant.

3. The vaccine according to claim 1, which is in a freeze-dried form.

4. The vaccine according to claim 1, further comprising one or more antigens from pig-pathogenic microorganisms or viruses.

5. The vaccine according to claim 4, wherein said one or more antigens are from microorganisms or viruses selected from the group consisting of Porcine Reproductive Respiratory Syndrome (PRRS) virus, Pseudorabies virus, Porcine Influenza virus, Porcine Parvovirus, Transmissible Gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Streptococcus suis.

6. A method for the protection of a susceptible animal against Actinobacillus pleuropneumoniae infection, comprising administering a vaccine according to claim 1 to the animal.

7. A method for the preparation of a vaccine according to claim 1, comprising admixing purified ApxIV toxin with a pharmaceutically acceptable carrier.

8. A subunit vaccine for the protection of animals against infection with a bacterium of the species Actinobacillus pleuropneumoniae, comprising purified ApxIV toxin and an adjuvant.