阅读说明：本技术 针对家禽坏死性肠炎的疫苗 (Vaccine against necrotic enteritis in poultry ) 是由 龚建华 D·莱普 于 2018-05-31 设计创作，主要内容包括：一种免疫原性多肽适用于制备供治疗或预防家禽小肠坏死用的疫苗,所述免疫原性多肽选自经分离的产气荚膜梭菌菌毛多肽、所述菌毛多肽的变异体；所述菌毛多肽的片段；和所述变异体的片段。所述经分离的产气荚膜梭菌菌毛多肽包括组装的菌毛或菌毛亚单元CnaA、FimA和/或FimB。(An immunogenic polypeptide selected from the group consisting of an isolated clostridium perfringens pilus polypeptide, a variant of said pilus polypeptide, a variant of a clostridium perfringens pilus polypeptide, suitable for use in the preparation of a vaccine for the treatment or prevention of small intestine necrosis in poultry; a fragment of said pilus polypeptide; and fragments of said variants. The isolated clostridium perfringens pilus polypeptides include assembled pili or pilus subunits CnaA, FimA, and/or FimB.)

1. An isolated clostridium perfringens pilus polypeptide.

2. The isolated clostridium perfringens pilus polypeptide of claim 1 wherein said pilus polypeptide is a CnaA polypeptide.

3. The isolated clostridium perfringens pilus polypeptide of claim 2 wherein said CnaA polypeptide is selected from the group consisting of: a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO 10 and SEQ ID NO 13; a polypeptide encoded by a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 4, and SEQ ID NO. 7; and a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 4, and SEQ ID NO. 7.

4. The isolated clostridium perfringens pilus polypeptide of claim 1 wherein said pilus polypeptide is a FimA polypeptide.

5. The isolated clostridium perfringens pilus polypeptide of claim 4, wherein said FimA polypeptide is selected from the group consisting of: a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO 11 and SEQ ID NO 14; a polypeptide encoded by a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 8; and a polypeptide encoded by a polynucleotide that hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 8.

6. The isolated clostridium perfringens pilus polypeptide of claim 1 wherein said pilus polypeptide is a FimB polypeptide.

7. The isolated clostridium perfringens pilus polypeptide of claim 6 wherein said FimB polypeptide is selected from the group consisting of: a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO 12 and SEQ ID NO 15; a polypeptide encoded by a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, and SEQ ID NO. 9; and a polypeptide encoded by a polynucleotide that hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 6, and SEQ ID NO. 9.

8. The isolated clostridium perfringens pilus polypeptide of claim 1, wherein said pilus polypeptide is an assembled pilus.

9. The isolated clostridium perfringens pilus polypeptide of claim 9 wherein the assembled pilus comprises one or more subunits each individually selected from the group consisting of a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide.

10. An immunogenic polypeptide selected from the group consisting of an isolated clostridium perfringens pilus polypeptide, a variant of said pilus polypeptide, according to any one of claims 1-9; a fragment of said pilus polypeptide; and a fragment of said variant, wherein said pilus polypeptide, said variant, a fragment of said polypeptide, and a fragment of said variant are immunogenic in poultry.

11. The immunogenic polypeptide of claim 10, wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% sequence identity to the pilus polypeptide.

12. A polynucleotide comprising a sequence encoding the immunogenic polypeptide of claim 10 or 11.

13. A vector comprising the polynucleotide of claim 12, wherein the vector is configured to express the immunogenic polypeptide in a host cell.

14. A vaccine for the treatment or prevention of necrotic enteritis in poultry, comprising at least one immunogenic polypeptide according to claim 10 or 11.

15. A vaccine for treating or preventing clostridium perfringens infection in poultry comprising at least one immunogenic polypeptide according to claim 10 or 11.

16. Use of an immunogenic polypeptide according to claim 10 or 11 for the preparation of a medicament for the treatment or prevention of necrotic enteritis in poultry.

17. Use of an immunogenic polypeptide according to claim 10 or 11 for the manufacture of a medicament for the treatment or prevention of clostridium perfringens infection in poultry.

18. A method of treating or preventing necrotic enteritis in poultry, the method comprising administering to the poultry an effective amount of the immunogenic polypeptide of claim 10 or 11 or an effective amount of the vaccine of claim 14.

19. A method of treating or preventing clostridium perfringens infection in poultry comprising administering to the poultry an effective amount of an immunogenic polypeptide according to claim 10 or 11 or an effective amount of a vaccine according to claim 15.

20. Use of the immunogenic polypeptide according to claim 10 or 11 as a vaccine for the treatment or prevention of necrotic enteritis in poultry.

21. Use of an immunogenic polypeptide according to claim 10 or 11 as a vaccine for the treatment or prevention of clostridium perfringens infection in poultry.

22. An antibody that selectively binds to the immunogenic polypeptide of claim 10 or 11.

23. A method of detecting clostridium perfringens infection in poultry by obtaining a biological sample from the poultry and detecting the presence of the antibody of claim 22 in the biological sample.

24. A method of detecting an immunogenic polypeptide according to claim 10 or 11, comprising exposing the immunogenic polypeptide to an antibody according to claim 22 and detecting binding of the immunogenic polypeptide to the antibody.

Background

The present application relates to polypeptides suitable for the preparation of a vaccine against necrotic enteritis in poultry. More particularly, the present application relates to a clostridium perfringens pilus polypeptide suitable for use in the manufacture of a vaccine against necrotic enteritis associated with clostridium perfringens infection of poultry.

Necrotic enteritis is a disease of the gut of poultry (e.g., broiler chicks) and it is estimated that it costs the poultry industry $ US 60 billion in 2015. Necrotic enteritis is mainly caused by certain type a strains of clostridium perfringens that produce NetB pore-forming toxins that overgrow and adhere to the intestinal mucosa, ultimately producing lesions characteristic of the disease. Clostridium perfringens is a normal inhabitant of the intestinal tract and typically only those strains carrying NetB toxin are able to cause necrotic enteritis. Necrotic enteritis is mainly controlled by administration of antibiotics in feed, which is becoming increasingly unpopular due to the potential spread of antimicrobial resistance, and some countries are currently gradually stopping production of antibiotics in feed. Therefore, from a financial and public health point of view it is important to find alternative methods of controlling necrotic enteritis, such as the development of vaccines.

Clostridium perfringens adherence locus (VR-10B) (lepp D), (Gong J), (Songer JG), (Bernin P), (Boerlin P), (Palea VR), (Parreira VR), Prescout JF (Prescott JF.), 2013, Identification of accessory genomic Regions in Isolates of Clostridium perfringens Carrying the netB plasmid (Identification of accession genes Regions in bacterial Clostridium perfringens BPlasis), Journal of science (Journal of Bacteriology)195: 2-. Of the 54 poultry isolates examined, the identified locus was found to be present in 87% of the netB positive isolates and 42% of the netB negative isolates. This locus (subsequently renamed The Collagen Adhesion (CA) locus) was later shown to be associated with collagen Binding and essential for The pathogenesis of necrotic enteritis (Wedd B (Wade B), Karbern AL (Keyburn AL), Harrey V (Haring V), Ford M (Ford M), LedeJI (Rood JI), Murr (Moore RJ) The adhesive capacity of Clostridium perfringens strains is critical for The pathogenesis of necrotic enteritis in poultry (The adhesive abilities of Clostridium perfringens strains criticism), veterinary microbiology (Vet biol)2016,197: 53-61; Wedd B, Karbern AL to, Shermann T (Seemanema T), Ledejir et AL, RJ: collagen Binding to collagen of chicken strains causing necrosis, veterinary microbiology 2015,180: 299-.

A variety of clostridium perfringens proteins have been previously evaluated as vaccine candidates. However, these proteins provide at most partial protection against necrotic enteritis. In addition, many of these proteins are not specific to the strain causing necrotic enteritis and are not known to contribute to the pathogenesis of necrotic enteritis. Accordingly, it would be desirable to identify alternative clostridium perfringens polypeptides that may be candidates for the production of vaccines against necrotic enteritis.

Disclosure of Invention

One aspect of the invention provides an isolated clostridium perfringens pilus polypeptide. In another aspect, the invention provides an immunogenic polypeptide selected from the group consisting of an isolated clostridium perfringens pilus polypeptide, a variant of a pilus polypeptide; a fragment of a pilus polypeptide; and fragments of said variants, wherein the pilus polypeptide, the variants, fragments of the polypeptide and fragments of the variants are immunogenic in poultry. In at least one embodiment, the pilus polypeptide is a CnaA polypeptide. In at least one embodiment, the pilus polypeptide is a FimA polypeptide. In at least one embodiment, the pilus polypeptide is a FimB polypeptide. In at least one embodiment, the pilus polypeptide is an assembled pilus.

Another aspect of the invention provides a polynucleotide comprising a sequence encoding an isolated clostridium perfringens pilus polypeptide or immunogenic polypeptide as described herein. In another aspect, the present application provides a vector comprising a polynucleotide having a sequence encoding an isolated clostridium perfringens pilus polypeptide or an immunogenic polypeptide as described herein, wherein the vector is configured to express the isolated clostridium perfringens pilus polypeptide or immunogenic polypeptide in a host cell.

In another aspect, the invention provides a vaccine for the treatment or prevention of necrotic enteritis in poultry, wherein the vaccine comprises an immunogenic polypeptide as described herein. In another aspect, the present application provides a vaccine for treating or preventing clostridium perfringens infection in poultry, wherein the vaccine comprises an immunogenic polypeptide as described herein.

In another aspect, the invention provides the use of an immunogenic polypeptide as described herein for the manufacture of a medicament for the treatment or prevention of necrotic enteritis in poultry or the treatment or prevention of clostridium perfringens infection in poultry.

In another aspect, the invention provides a method of treating or preventing necrotic enteritis in poultry or treating or preventing clostridium perfringens infection in poultry, the method comprising administering to the poultry an effective amount of an immunogenic polypeptide as described herein or a vaccine thereof.

In another aspect, the invention provides the use of an immunogenic polypeptide as described herein as a vaccine for the treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of clostridium perfringens infection in poultry.

Another aspect of the invention provides an antibody that selectively binds to an immunogenic polypeptide as described herein. In another aspect, the invention provides a method of detecting clostridium perfringens infection in poultry by obtaining a biological sample from the poultry and detecting in the biological sample the presence of an antibody that selectively binds to an immunogenic polypeptide as described herein. A further aspect of the invention provides a method of detecting an immunogenic polypeptide as described herein, comprising exposing the immunogenic polypeptide to an antibody that selectively binds to the immunogenic polypeptide and detecting binding of the immunogenic polypeptide to the antibody.

Drawings

Other features of the present invention will become apparent from the following written description and the accompanying drawings, in which:

FIG. 1 is a schematic representation of the 5.2 kilobase pair Clostridium perfringens VR-10B chromosomal locus;

FIG. 2A is a photograph showing the separation of histidine-tagged recombinant pilus subunit polypeptide CnaA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as visualized by Coomassie staining;

FIG. 2B is a photograph showing the separation of the recombinant FimA subunit polypeptide FimA by SDS-PAGE as detected by Coomassie staining;

FIG. 2C is a photograph showing the separation of the recombinant fimbrial subunit polypeptide FimB by SDS-PAGE as detected by Coomassie staining;

FIG. 2D is a photograph showing the mixed fractions of CnaA, FimA, and FimB visually after concentration and desalting by SDS-PAGE and Coomassie staining;

figure 3A is a graph showing serum IgY antibody responses (absorbance at 405 nm) against CnaA recombinant polypeptides in first vaccination trials in chickens immunized with adjuvant alone or with CnaA. Each point represents a single individual and the horizontal line represents the mean. Comparison of Individual Means (comparative experimental Means in the Analysis of Variance) "by Variance Analysis, as measured by the dukay test (Tukey's test) (Tukey J. (Tukey, J)," biological measurements (1949)5(2):99-114), indicates a significant difference in the pre-immune samples (d8) in each group at a level of p < 0.05; indicates significant differences at p <0.01 levels; and represents a significant difference when p <0.001 level;

FIG. 3B is a graph showing the serum IgY antibody response (absorbance at 405 nm) against the FimA recombinant polypeptide in birds immunized with adjuvant alone or with FimA in the assay of FIG. 3A. Each point represents a single individual and the horizontal line represents the mean. Represents a significant difference in the preimmune samples (d8) in each group at p <0.05 level, as measured by dukeley test; indicates significant differences at p <0.01 levels; and represents a significant difference when p <0.001 level;

FIG. 4 is a graph showing the Necrotic Enteritis (NE) damage score in groups of chickens immunized with adjuvant alone or with CnaA or FimA followed by challenge with C.perfringens strain CP1 in feed in the experiment of FIG. 3A. Each dot represents a single individual and the horizontal line represents the mean necrotic enteritis lesion;

fig. 5A is a graph showing serum IgY antibody responses (absorbance at 405 nm) against CnaA recombinant polypeptides in chickens immunized with adjuvant alone, CnaA, or CnaA, FimA in combination with FimB (Comb) in a second vaccination experiment. Each point represents a single individual and the horizontal line represents the mean. Represents a significant difference in the preimmune samples (d7) in each group at p <0.05 level, as measured by dukeley test; indicates significant differences at p <0.01 levels; and represents a significant difference when p <0.001 level;

FIG. 5B is a graph showing serum IgY antibody responses (absorbance at 405 nm) against the FimA recombinant polypeptide in the assay of FIG. 5A in chickens immunized with adjuvant alone or with a combination of CnaA, FimA and FimB (Comb). Each point represents a single individual and the horizontal line represents the mean. Represents a significant difference in the preimmune samples (d7) in each group at p <0.05 level, as measured by dukeley test; indicates significant differences at p <0.01 levels; and represents a significant difference when p <0.001 level;

FIG. 5C is a graph showing the serum IgY antibody response (absorbance at 405 nm) against the fimB recombinant polypeptide in the assay of FIG. 5A in chickens immunized with adjuvant alone, fimB alone or with a combination of CnaA, FimA and fimB (Comb). Each point represents a single individual and the horizontal line represents the mean. Represents a significant difference in the preimmune samples (d7) in each group at p <0.05 level, as measured by dukeley test; indicates significant differences at p <0.01 levels; and represents a significant difference when p <0.001 level;

FIG. 6 is a graph showing the Necrotic Enteritis (NE) damage score in groups of chickens immunized with adjuvant alone, with CnaA, FimB or combination of CnaA, FimA and FimB (Comb), followed by challenge with C.perfringens CP1 in feed in the experiment of FIG. 5A. Each dot represents a single individual and the horizontal line represents the mean necrotic enteritis lesion. The letters (a, b) represent groups with significant differences (DuKay; p < 0.01);

FIG. 7 is a graph showing the Necrotic Enteritis (NE) damage score after challenge of groups of chickens with C.perfringens in feed, strain CP1 or CP1 knockout mutants (CP 1. delta. fimA and CP 1. delta. fimB) containing pilus subunit genes fimA and fimB. Lines represent the mean necrotic enteritis lesion score;

FIG. 8A is a photograph showing the separation of surface polypeptides extracted from Clostridium perfringens strain CP1 or CP1 knock-out mutants (CP 1. DELTA. cnaA, CP 1. DELTA. fimA, and CP 1. DELTA. fimB) containing the genes for each of the pilus subunits cnaA, fimA, and fimB by SDS-PAGE with Coomassie stain;

FIG. 8B is a photograph showing Western blot analysis of surface polypeptides separated by SDS-PAGE, which were extracted from Clostridium perfringens strain CP1 or CP1 knockout mutants (CP 1. delta. cnaA, CP 1. delta. fimA, and CP 1. delta. fimB) containing the genes for each of the pilus subunits cnaA, fimA, and fimB, detected using anti-FimA antibodies obtained from chicken serum as primary antibodies;

FIG. 8C is a photograph showing Western blot analysis of surface polypeptides separated by SDS-PAGE, which were extracted from Clostridium perfringens strain CP1 or CP1 knockout mutants (CP 1. delta. cnaA, CP 1. delta. fimA, and CP 1. delta. fimB) containing the genes for each of the pilus subunits cnaA, fimA, and fimB, detected using rabbit-produced anti-FimA antibody as a primary antibody;

FIG. 9A is a photograph showing SDS-PAGE separation of surface polypeptides extracted from various Clostridium perfringens strains by visual inspection with Coomassie stain;

FIG. 9B is a photograph showing Western blot analysis of surface polypeptides separated by SDS-PAGE, visually confirmed by SDS-PAGE of surface polypeptides extracted from various Clostridium perfringens strains using anti-FimA antibody obtained from chicken serum as a primary antibody; and is

FIG. 10 is a series of photographs of Clostridium perfringens strain CP1 or CP1 knockout mutant CP 1. delta. fimA and gold particle labeled CP 1. delta. fimB cells obtained by transmission electron microscopy using rabbit anti-FimA antibody as the primary antibody and 6nm immunogold labeled goat anti-rabbit IgG as the secondary antibody.

Detailed Description

The applicant has found that the VR-10B locus identified in Clostridium perfringens strains associated with necrotic enteritis in poultry (Lepu D et al, Journal of Bacteriology (2013)195:1152-1166) contains six putative genes found to encode adherent fimbriae: three genes encoding the structural pilus subunits (cnaA, fimA and fimB), and genes encoding two transpeptidases and one signal peptidase that may be involved in pilus assembly. A schematic representation of the VR-10B locus is shown in FIG. 1.

Pili are hair-like structures that are present on the surface of many bacteria and often involve toxicity. Pili of this type are composed of covalently linked major and minor polypeptide subunits that form cell surface structures of about 1 μm in length. Pili of Gram-negative bacteria (Gram-negative bacteria) have been extensively studied, but recently several Gram-positive species, including Corynebacterium diphtheriae (Corynebacterium diphtheriae), Streptococcus pneumoniae (Streptococcus pneumoniae), and Streptococcus pyogenes (Streptococcus pyogenes), have been shown to produce specific types of pili that are assembled by transpeptidases. This type of adherent gram-positive pilus is assembled at the cell surface from pilin subunits by housekeeping and pilin-specific transpeptidases, by covalent bonds, and finally covalently linked to cell wall peptidoglycans to form assembled pili.

Without being bound by theory, it is expected that the clostridium perfringens pilus polypeptides described herein may be a viable and promising target for developing vaccines against necrotic enteritis from multiple causes. The locus is predominantly present in clostridium perfringens strains that cause necrotic enteritis. Thus, the immune response induced by the immunogenic pilin is expected to specifically target the clostridium perfringens strain that causes the disease. Furthermore, pili are present on the surface of bacterial cells and are often associated with attachment to the host during the pathogenic phase of a bacterial infection, which can expose the pili to the host immune system. In addition, pili have been successfully used to develop vaccines against a variety of other infectious diseases, possibly due to their role in the disease and their location on the bacterial cell surface.

Accordingly, one aspect of the present application provides an immunogenic polypeptide selected from the group consisting of an isolated clostridium perfringens pilus polypeptide, a variant of a pilus polypeptide; a fragment of a pilus polypeptide; and fragments of said variants, wherein the pilus polypeptide, the variants, fragments of the polypeptide and fragments of the variants are immunogenic in poultry.

As used herein, the term "poultry" is used to refer to poultry birds or poultry species that are agriculturally raised for produce, including (but not limited to) meat, eggs, and feathers. Poultry includes, but is not limited to, chickens, turkeys, ducks, geese, quail, ostriches, pheasants, and other agriculturally related avians or flying birds. Especially included are poultry susceptible to necrotic enteritis caused by infection with clostridium perfringens. In at least one embodiment, the poultry is broiler or chicken raised for meat production.

As used herein, the term "polypeptide" means a compound containing two or more amino acid residues joined together by peptide bonds. Polypeptides include, but are not limited to, oligopeptides or polypeptides in which two or more amino acid residues are sequentially joined together by covalent peptide bonds to form a single polypeptide chain; and proteins comprising two or more polypeptide chains that are non-covalently associated with each other or linked to each other by covalent bonds other than peptide bonds, including (but not limited to) disulfide bonds and isopeptide bonds. As used herein, the term "isopeptide bond" means an amide bond formed between an amino group of one amino acid and a carboxyl group of a second amino acid, wherein at least one of the amino and carboxyl groups is located in the side chain of the corresponding amino acid.

As used herein, the term "clostridium perfringens pilus polypeptide" means a polypeptide that has the function of a pilus or pilus subunit and is encoded by one or more genes found in clostridium perfringens strains associated with necrotic enteritis in poultry. In at least one embodiment, the gene is the cnaA gene, fimA gene or fimB gene found in the VR-10B locus identified in the Journal of bacteriology (2013)195:1152-1166, by Dimethoprim et al, as shown diagrammatically in FIG. 1.

As used herein, the term "variant" when used in connection with a polypeptide means a polypeptide whose amino acid sequence differs from the sequence of a reference polypeptide to which the variant is being compared for one or more amino acid residues. Differences between the variant and reference polypeptide sequences may include substitution of one or more amino acid residues with a different amino acid residue, insertion of an additional amino acid residue, or deletion of an amino acid residue. In certain embodiments, a variant can differ from a reference polypeptide by conservative substitutions of one or more amino acid residues with a replacement amino acid residue, which may have similar properties including, but not limited to, charge, size, and hydrophilicity as the amino acid residue replaced by the new residue. In certain embodiments, a variant may retain, in whole or in part, one or more biological functions of a reference polypeptide, including (but not limited to) immunogenicity. In at least one embodiment, the reference polypeptide is an isolated clostridium perfringens pilus polypeptide as described herein.

In at least one embodiment, the sequence of the variant can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% identity to the sequence of the reference polypeptide. As used herein, the term "percent identity" or "percent identity" when used in connection with a sequence of a polypeptide or polynucleotide means the percentage of the total number of amino acid or nucleotide residues in the sequence that are identical to the amino acid or nucleotide residue at the corresponding position in the reference polypeptide or polynucleotide sequence, respectively. In at least one embodiment, when the length of the variant sequence does not correspond to the length of the reference sequence, the percent identity can be calculated based on the total number of residues in the variant sequence or based on the total number of residues in the reference sequence. Percent identity can be measured by a variety of local or global sequence alignment algorithms well known in the art, including, but not limited to, the Smith-Waterman algorithm (Smith-Waterman algorithm) and the niedeman-wunschel algorithm (Needleman-wunschel algorithm). Tools for using these or other suitable algorithms include, but are not limited to, BLAST (basic local alignment search tool) and other such tools well known in the art.

As used herein, the term "fragment," when in reference to a polypeptide or variant, means a smaller polypeptide containing fewer amino acid residues than the polypeptide or variant and having a sequence identical to a portion of the sequence of the polypeptide or variant. In at least one embodiment, the fragment retains one or more biological activities of the polypeptide or variant, including (but not limited to) immunogenicity. In at least one embodiment, a fragment comprises an epitope of a polypeptide or variant. In at least one embodiment, a fragment has a length of at least 6 amino acids or a length of at least 7 amino acids, or a length of at least 8 amino acids or a length of at least 9 amino acids or a length of at least 10 amino acids.

As used herein, the term "immunogenic" means that an agent (including, but not limited to, a polypeptide or polynucleotide or fragment thereof) is capable of inducing an immunoprotective response in a subject to which the immunogenic agent is administered. As used herein, the term "immune protective response" means an immune response that prevents, reduces or eliminates one or more symptoms of a disease in an infected subject.

The immunogenic polypeptides of the invention (including the isolated clostridium perfringens pilus polypeptides, variants of pilus polypeptides, fragments of pilus polypeptides, and fragments of variants of the invention) are immunogenic in poultry. Thus, in at least one embodiment, the elicitation by poultry immunized with an isolated clostridium perfringens pilus polypeptide, pilus polypeptide variant, pilus polypeptide fragment, and variant fragment of the present invention, will exhibit an immunoprotective response to one or more of the following: a clostridium perfringens cell, an assembled clostridium perfringens pilus, a clostridium perfringens pilus polypeptide, a fragment of a clostridium perfringens pilus polypeptide, or a portion of a clostridium perfringens cell, including but not limited to a cell membrane or a portion thereof or a cell wall or a portion thereof carrying one or more of an assembled clostridium perfringens pilus, a clostridium perfringens pilus polypeptide, or a clostridium perfringens pilus polypeptide fragment.

Another aspect of the application provides a polynucleotide comprising a sequence encoding an isolated clostridium perfringens pilus polypeptide or an immunogenic polypeptide as described herein. In at least one embodiment, the polynucleotide is messenger rna (mrna) having a sequence that is capable of being translated to produce an isolated clostridium perfringens pilus polypeptide or an immunogenic polypeptide. In at least one embodiment, the polynucleotide is DNA, at least one strand of which is capable of being transcribed to produce mRNA, which in turn can be translated to produce an isolated clostridium perfringens pilus polypeptide or immunogenic polypeptide. In at least one embodiment, the DNA is capable of being expressed by a biochemical system, including but not limited to a cell, to produce an isolated clostridium perfringens pilus polypeptide or immunogenic polypeptide. In at least one such embodiment, the DNA can be incorporated into a vector configured to express the DNA in a host cell, as is well known in the art.

In at least one embodiment, the polynucleotide can include a variant polynucleotide sequence that hybridizes under at least moderately stringent conditions to a polynucleotide comprising a sequence encoding an isolated clostridium perfringens pilus polypeptide or immunogenic polypeptide as described herein. "at least moderately stringent hybridization conditions" means conditions selected to promote the hybridization of two complementary nucleic acids in solutionConditions under which selective hybridization occurs between molecules. Hybridization can occur over all or a portion of a nucleic acid sequence molecule. The hybridizing portion typically has a length of at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides. Those skilled in the art will recognize that the stability of nucleic acid duplexes or hybrids is based on the melting temperature (T;)_m) It was confirmed that the melting temperature was the sodium ion concentration ([ Na ] in the sodium-containing buffer⁺]) And function of temperature (T)_m＝81.5℃-16.6(Log₁₀[Na⁺]) +0.41 (% (G + C) -600/l, where% G + C is the percentage of cytosine and guanine nucleotides in a nucleic acid and l is the length of the nucleic acid in a base pair, or similar equation). Accordingly, the wash condition parameters that determine the stability of the hybrids are sodium ion concentration and temperature. To identify molecules that are similar to, but not identical to, known nucleic acid molecules, it can be assumed that a 1% mismatch causes T_mA decrease of about 1 ℃ occurs. For example, if seeking to have>Nucleic acid molecules of 95% identity, the final wash temperature can be reduced by about 5 ℃. Based on these considerations, one skilled in the art will be readily able to select appropriate hybridization conditions.

In some embodiments, stringent hybridization conditions are selected. By way of example, the following conditions may be used to achieve stringent hybridization: based on the above equation, at T_mHybridization was performed in 5 Xsodium chloride/sodium citrate (SSC)/5 Xdanhart's solution/1.0% Sodium Dodecyl Sulfate (SDS) at-5 ℃; followed by washing with 0.2 XSSC/0.1% SDS at 60 ℃. Moderately stringent hybridization conditions include a wash step in 3 XSSC at 42 ℃. However, it is understood that equivalent stringencies may be achieved using alternative buffers, salts, and temperatures. Other guidelines for hybridization conditions can be found in: modern Molecular Biology techniques (Current Protocols in Molecular Biology), John Wiley father, Inc. (John Wiley)&Sons), new york (n.y.), 2002; and sabeluk (Sambrook) et al, molecular cloning: a Laboratory Manual (Molecular Cloning: a Laboratory Manual), Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), 2001.

In at least one embodiment, the isolated clostridium perfringens pilus polypeptide is a CnaA polypeptide. In at least one embodiment, the CnaA polypeptide has an amino acid sequence selected from SEQ ID NO 10 and SEQ ID NO 13. In at least one embodiment, the CnaA polypeptide is encoded by a polynucleotide having a sequence selected from SEQ ID No. 1, SEQ ID No. 4, and SEQ ID No. 7. In at least one embodiment, the CnaA polypeptide is encoded by a polynucleotide that hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 4, and SEQ ID NO. 7. In at least one embodiment, when the pilus polypeptide is a CnaA polypeptide, the variant has at least 75%, 80%, 85%, 90%, 95%, 99% or 99.9% sequence identity to an amino acid sequence selected from SEQ ID No. 10 and SEQ ID No. 13.

In at least one embodiment, the isolated clostridium perfringens pilus polypeptide is a FimA polypeptide. In at least one embodiment, the FimA polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO: 14. In at least one embodiment, the FimA polypeptide is encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 8. In at least one embodiment, the FimA polypeptide is encoded by a polynucleotide that hybridizes under at least moderately stringent conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 8. In at least one embodiment, when the pilus polypeptide is a FimA polypeptide, the variant has at least 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to an amino acid sequence selected from SEQ ID No. 11 and SEQ ID No. 14.

In at least one embodiment, the isolated clostridium perfringens pilus polypeptide is a FimB polypeptide. In at least one embodiment, the FimB polypeptide has an amino acid sequence selected from SEQ ID No. 12 and SEQ ID No. 15. In at least one embodiment, the FimB polypeptide is encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 6, and SEQ ID No. 9. In at least one embodiment, the FimB polypeptide is encoded by a polynucleotide that hybridizes under at least medium stringency conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 6, and SEQ ID No. 9. In at least one embodiment, when the pilus polypeptide is a FimB polypeptide, the variant has at least 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% sequence identity to an amino acid sequence selected from SEQ ID No. 12 and SEQ ID No. 15.

In at least one embodiment, the isolated clostridium perfringens pilus polypeptide is an assembled pilus. In at least one embodiment, the assembled pilus comprises one or more subunits, each subunit independently selected from the group consisting of a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide. In at least one embodiment, one or more of the subunits are covalently bonded to each other. In at least one embodiment, the assembled pilus is a pilus isolated from a clostridium perfringens cell or a portion thereof, including (but not limited to) a cell membrane or a portion thereof, or a cell wall or a portion thereof. In at least one embodiment, the assembled pilus is a pilus fragment isolated from a clostridium perfringens cell or a portion thereof, including but not limited to a cell membrane or a portion thereof, or a cell wall or a portion thereof, wherein the fragment comprises one or more subunits each independently selected from a CnaA polypeptide, a FimA polypeptide, and a FimB polypeptide.

In at least one embodiment, the isolated clostridium perfringens polypeptide can be isolated from a culture of clostridium perfringens. Thus, in at least one embodiment, an isolated clostridium perfringens pilus polypeptide can be part of a formulation containing one or more portions of a clostridium perfringens cell, including (but not limited to) a cell membrane or a portion thereof, or a cell wall or a portion thereof, carrying a pilus polypeptide or fragment thereof as described herein. In at least one embodiment, an isolated clostridium perfringens pilus polypeptide can be produced recombinantly by expressing a vector comprising a polynucleotide having a sequence encoding a pilus polypeptide in a suitable host cell. In at least one embodiment, when the pilus polypeptide is an assembled pilus, the assembled pilus can be produced recombinantly by expressing a vector comprising a polynucleotide having a sequence encoding a gene and other nucleotide sequences necessary for assembly of the assembled pilus in a suitable host cell. Furthermore, the isolated clostridium perfringens pilus polypeptide can be at least partially purified after isolation or recombinant production. Suitable vectors and host cells, including but not limited to prokaryotic and eukaryotic host cells suitable for the production of recombinant polypeptides, and methods for the isolation or recombinant production of such polypeptides, including methods for at least partially purifying such polypeptides, are well known in the art, or can be readily identified and selected by the skilled artisan through only routine experimental work.

In another aspect, the present application provides a vaccine for treating or preventing necrotic enteritis in poultry or for treating or preventing clostridium perfringens infection in poultry, wherein the vaccine comprises at least one immunogenic polypeptide as described herein. As used herein, the term "vaccine" means an immunogenic formulation for preventing, treating or reducing the infectious effects of clostridium perfringens. Vaccine formulations typically contain an immunologically effective amount of an immunogenic agent, and may or may not also contain an adjuvant. In the case of the vaccine of the invention, the immunogenic agent may be an immunogenic polypeptide as described herein.

As used herein, the term "adjuvant" means an agent effective to enhance the immune response against an immunogenic agent in a subject vaccinated with a vaccine comprising the immunogenic agent. Suitable adjuvants are well known in the art and include, but are not limited to, inorganic compounds including, but not limited to, alum, aluminum hydroxide, and other aluminum-containing compounds; saponins including, but not limited to, Quil-A^TM(ii) a Freund's complete and incomplete adjuvant; adjuvants containing lipids or mineral oils, including (but not limited to) oil-in-water emulsions; a polysaccharide adjuvant; a protein adjuvant; an immunomodulator; an adjuvant obtained from killed or attenuated bacterial cells; and other suitable adjuvants known in the art.

The vaccine can be formulated in one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable" means molecular entities and compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to an animal or human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals or humans. The term "carrier" as used herein means a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Suitable carriers are well known in the art and, in at least one embodiment, are described in e.w. martin, Remington's pharmaceutical Sciences, 18 th edition, or other versions.

The vaccine can be formulated for administration by any convenient route known in the art, including, but not limited to, oral, rectal, nasal, transmucosal, transdermal, parenteral, intravenous, intramuscular, subcutaneous, in ovo, or other known routes. In at least one embodiment, it is contemplated that the vaccine can be administered orally. Without being bound by theory, it is contemplated that oral vaccination can directly target gut-associated lymphoid tissue, the site of infection by necrotic enteritis-associated clostridium perfringens strains. In at least one embodiment, it is contemplated that the offspring can be immunized by maternal vaccination and subsequent transfer of maternal immunity, including (but not limited to) transfer of maternal antibodies to offspring.

In another aspect, the invention provides the use of an immunogenic polypeptide as described herein for the manufacture of a medicament for the treatment or prevention of necrotic enteritis in poultry or the treatment or prevention of clostridium perfringens infection in poultry. The medicament may be a vaccine as described herein.

In another aspect, the invention provides a method of treating or preventing necrotic enteritis in poultry or treating or preventing clostridium perfringens infection in poultry, the method comprising administering to the poultry an effective amount of an immunogenic polypeptide or vaccine as described herein. Administration can be by routes well known in the art, including, but not limited to, oral, rectal, nasal, parenteral, intravenous, intramuscular, subcutaneous, or other routes. In at least one embodiment, administration can be by subcutaneous injection. In at least one embodiment, the administration can be oral. In at least one embodiment, the vaccine can be administered to the poultry more than once to provide an initial immunization, followed by one or more booster immunizations, as understood in the art. In at least one embodiment, one or more of the prime immunization and the one or more boost immunizations are administered to the poultry after maternal antibodies in the poultry have disappeared. In at least one such embodiment, the poultry are administered one or more of an initial immunization and one or more booster immunizations no earlier than about 10 days after hatch.

In another aspect, the invention provides the use of an immunogenic polypeptide as described herein as a vaccine for the treatment or prevention of necrotic enteritis in poultry or for the treatment or prevention of clostridium perfringens infection in poultry.

Another aspect of the invention provides an antibody that selectively binds to an immunogenic polypeptide as described herein. In at least one embodiment, the antibody is a poultry antibody. In at least one embodiment, the antibody can be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or an antibody fragment that retains the property of selectively binding to an immunogenic polypeptide as described herein. As used herein, the term "antibody fragment" is intended to include, but is not limited to, Fab ', F (ab')₂scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimeric and bispecific antibody fragments thereof. Antibodies can be fragmented using conventional techniques. For example, F (ab')₂And (3) fragment. Can process the obtained F (ab')₂The fragments are reduced to disulfide bridges, thereby producing Fab' fragments. Papain digestion can cause Fab fragment formation. Fab, Fab 'and F (ab')₂scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

Methods of making and characterizing such antibodies and fragments thereof are well known in the art and can be readily performed by the skilled artisan without undue effort. For example, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal (e.g., a mouse, hamster, or rabbit), avian bird (e.g., poultry), or other animal can be immunized with an immunogenic form of an immunogenic polypeptide of the invention that induces an antibody response in the mammal. Techniques for conferring immunogenicity to a peptide include conjugation to a carrier or other techniques well known in the art. For example, the peptide can be administered in the presence of an adjuvant. The progress of the immunity can be monitored by detecting antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with immunogenic agents as antigens to assess antibody levels. After immunization, antisera can be obtained and, if necessary, polyclonal antibodies isolated from the serum.

To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from the immunized animal and fused with myeloma cells by standard somatic cell fusion procedures, thereby immortalizing these cells and producing hybridoma cells. Such techniques are well known in the art. The hybridoma cells can be screened immunochemically to produce antibodies specifically reactive with the immunogenic polypeptides as described herein and to enable isolation of monoclonal antibodies. Accordingly, the disclosure also encompasses hybridoma cells that secrete monoclonal antibodies specific for the immunogenic polypeptides as described herein.

Specific antibodies or antibody fragments reactive with an immunogenic polypeptide as described herein can also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria containing peptides produced by nucleic acid molecules as described herein. For example, the complete Fab fragment, VH region, and FV region can be expressed in bacteria using a phage expression library.

In another aspect, the invention provides a method of detecting infection of poultry by a clostridium perfringens strain associated with necrotic enteritis, wherein the method comprises obtaining a biological sample from the poultry and detecting the presence of an antibody in the biological sample that selectively binds to an immunogenic polypeptide as described herein. In at least one embodiment, the biological sample is a blood sample. In at least one embodiment, the sample is a stool sample. In at least one embodiment, detecting comprises measuring the amount or concentration of antibody present in the biological sample using methods well known to those skilled in the art.

A further aspect of the invention provides a method of detecting an immunogenic polypeptide as described herein, comprising exposing the immunogenic polypeptide to an antibody that selectively binds to the immunogenic polypeptide and detecting binding of the immunogenic polypeptide to the antibody. In at least one embodiment, the immunogenic polypeptide can be an isolated clostridium perfringens pilus polypeptide as described herein. In at least one embodiment, the immunogenic polypeptide can be an assembled pilus attached to the surface of a clostridium perfringens bacterial cell. Such embodiments of the methods can be useful for identifying and detecting strains of clostridium perfringens that are capable of causing necrotic enteritis in poultry.

As used herein, the term "about" or "approximately" when applied to a numerical value or range of values means that the value can vary within an acceptable degree of error of the measured quantity given the nature or accuracy of the measurement, such that the deviation is deemed to be equivalent in the art to the value and provides the same function or result. For example, as understood in the art, the degree of error can be represented by the number of significant figures specified for measurement, and includes, but is not limited to, a deviation of ± 1 from the most precise significant figure reported for measurement. Exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5%, of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may index values within an order of magnitude, preferably within 5 times a given value, and more preferably within 2 times a given value. Unless otherwise indicated, the numerical quantities set forth herein are approximations that mean that the term "about" or "approximately" can be inferred when not expressly stated.

As used herein, the term "substantially" refers to the complete or nearly complete range or degree of an action, feature, characteristic, state, structure, clause, or result. For example, two substances having "substantially" the same property will have exactly the same property or will have properties that are so nearly exactly the same that the difference is not measurable or significant. The degree of permissible accuracy of the deviation from absolute integrity may in some cases depend on the particular situation. However, in general, proximity to completeness will have the same overall result as if absolute and overall completeness were obtained. The use of "substantially" is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, feature, characteristic, state, structure, item, or result.

Examples of the invention

Other features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.

Example 1: production of purified recombinant pilus-related polypeptides from clostridium perfringens

The coding regions of the three pilus subunits (cnaA, fimA and fimB) were codon optimized and truncated to exclude the predicted N-terminal signal peptide and C-terminal cell wall classification signal LPXTG transmembrane domain. The C-terminal domain is a hydrophobic region predicted to be removed during pilus assembly. Synthesis of a truncated codon-optimized coding region (Integrated DNA technologies, Coralville, USA) using In-Fusion^TMCloning, cloning into the pET28a expression vector (millipore-sigma (millipore sigma), the green grain region of ceramic, Canada, Ontario, Canada) according to the manufacturer's instructions (american Bio engineering corporation (Takara USA), Mountain city, California, USA), sequence verification, and then transformation into escherichia coli BL21 cells. Transformed colonies were grown in 1L LB medium supplemented with 50. mu.g/ml kanamycin (kanamycin) and 1mM IPTG at 37 ℃ for 18 hours with shaking. The culture was centrifuged and resuspended in 20ml binding buffer (20mM NaPO)₄0.5M NaCl, 30mM imidazole) and dissolved by sonication on ice for 10 minutes (10 second pulse, 20 second pause). Cell lysate in

HisTrap on prime plus System using a gradient of 50 to 500mM imidazole^TMFF crude column (GE Healthcare), Montreal, Canada (R) ((R))Montreal, Canada)) under natural conditions. Fractions of 1ml were collected and fractions exhibiting a peak at 280nm were mixed and Pierce was used^TMProtein concentrator (9K molecular weight cut-off) (Fisher Scientific, Unionville, Ontario, Canada) concentrates and uses Zeba^TMSpin 7K molecular weight cut-off desalting column (fisher technologies) for desalting. Quantification of the purified protein was performed using BCA (bisquinolinecarboxylic acid) protein assay kit (feishale) according to the manufacturer's instructions. The polypeptides were visualized by SDS-PAGE and Coomassie staining.

The truncation sequence resulted in high level expression of histidine (His) -tagged polypeptides as demonstrated by the SDS-PAGE gels shown in fig. 2a (CnaA), 2b (FimA), 2c (FimB), and 2D (mixed fractions of CnaA, FimA, and FimB after concentration and desalting). These enhanced expression levels can be attributed to enhanced solubility of the resulting polypeptide.

Table 1 shows the sequences of the full-length, codon-optimized, and truncated gene sequences, as well as the sequences of the full-length subunit polypeptide and the expressed truncated and His-tagged subunit polypeptide.

Table 1: sequence of pilus subunit genes and polypeptides

Example 2: preparation of clostridium perfringens strain CP1 pilus subunit knockout mutant

The pathogenesis of necrotic enteritis in poultry by Clostridium capsular, 2017, requires the induction of an Agr-like quorum sensing system (agar-like consistency strain for experimental recovery), Lepp D (Lepp, D.), meidizaded gaharley I (Mehdizadeh Gohari, I.), Wu T (Wu, T.), Zhou H (Zhou, H.), Yin X (Yin, X.), Yu H (Yu, H.), purce co-kott J.F (prescot, J.F), nie s s.p. (Nie, s.p.), Xie M.Y (Xie, M.Y.), Gong J (Gong, J.), 2017, Clostridium capsular, the pathogenesis of necrotic enteritis in poultry by mor-like quorum sensing system (induction of necrosis of the same strain for experimental recovery, western strain, T. knot, n. T.) (loop, r-happy, r-like), and T. loop, r. T. c.),85, 646:165-182), each of the three pilus subunit genes (cnaA, fimA and fimB) in the toxic clostridium perfringens strain CP1 was insertionally inactivated to generate CP1 knockout mutants (CP1 Δ cnaA, CP1 Δ fimA and CP1 Δ fimB) of the respective pilus subunit genes. Briefly, regions targeting the closttron intron were designed using the pelutcard algorithm (Perutka algorithm) implemented at www.clostron.com, inserting the following gene positions: base pair (bp)183 for the cnasense strand, bp 231 for the fimA sense strand, and bp 273 for the fimB sense strand. Regions of the targeting intron were synthesized and cloned into the ClosTron plasmid pMTL007C-E2 by DNA 2.0 (Menlopak, Calif. (Menlopak, Calif., USA.) As described previously (with slight modifications) (Gilas Cuwa A, Wettak L, Ferviririe J, Lung T, Brown P., 2005, Clostridium perfringens electroporation Rapid Generation (Rapidrotocol)for electrophoresis of Clostridium perfringens, journal of microbiological Methods (JMicrobiol Methods)62:125-127), the resulting plasmids were electroporated into CP1, briefly, CP1 was subcultured in 50ml of TGY and grown to exponential phase (600nm optical density [ OD 600. sup. tm. [ OD 600. sup. f.) after overnight anaerobic culture in 5ml of TGY broth (3% tryptone, 2% glucose, 1% yeast extract) at 37 ℃ and overnight],0.8). Cells were harvested by centrifugation at 6,000g for 10 min at 20 ℃ and electroporated with 10ml Sucrose Electroporation Buffer (SEB) (272mM sucrose, 1mM MgCl)₂、5mMNa₂HPO₄pH 7.4) and then resuspended in 5ml of SEB. Aliquots (0.2ml) were mixed with 2 μ g of concentrated plasmid DNA in pre-cooled cuvettes (0.2cm spacing) and the plasmid DNA was introduced into the cells by electroporation (1,000V, 25F) using a GenePulser Xcell apparatus from Bole (Bio-Rad) (Burle, Hercules, Calif.). Immediately after transformation, the mixture was transferred to 1ml of TGY medium and incubated anaerobically at 37 ℃ for 3 hours, followed by anaerobic inoculation at 37 ℃ onto TGY agar containing 15. mu.g/ml thiamphenicol (thiamphenicol) overnight to select transformants. The resulting colonies were subcultured on TGY agar containing 10. mu.g/ml erythromycin to select integrants, and then passaged for 10 consecutive days to solidify the shuttle vector. Those clone lines were selected for further analysis that were resistant to erythromycin (erythromycin) but sensitive to thiamphenicol.

Example 3: animal testing

Two vaccination trials were performed to evaluate the ability of the three purified His-tagged recombinant pilus subunits to prevent Necrotic Enteritis (NE) in the chicken challenge model. Commercial one day male White rock broiler chicks (White ply chicken chicks) were randomly assigned to experimental groups (n-15-17) and housed in isolation units. A summary of the experimental design is shown in table 2. In addition, toxicity of CP1 Δ fimA and CP1 Δ fimB mutants in the same model was evaluated.

Table 2: overview of the design of vaccination trials

Test 1:

the first experiment included three groups of 18 birds vaccinated with adjuvant control, CnaA or FimA alone. Each bird was injected intramuscularly (i.m.) in the pectoral muscle with 200. mu.l of Quil-A containing solution on days 8 and 20^TMAdjuvant (50 μ g) and recombinant pilus polypeptide (50 μ g) in Phosphate Buffered Saline (PBS), and birds were euthanized on day 31.

Sera were collected from five birds per group on day 8 (before immunization), and on days 20 and 31 (after immunization). Serum IgY titers against CnaA and FimA were determined by ELISA (enzyme linked immunosorbent assay). Clostridium perfringens recombinant pili polypeptide is diluted to 10 μ g/ml with 50mM carbonate/bicarbonate coating buffer pH 9.6 and 100 μ l to 96 well MaxiSorp is added^TMIn each well of an immuno-culture plate (Feishell technologies). Each well was plated at 37 ℃ for 1 hour, then at 4 ℃ overnight, washed three times with wash buffer (PBS containing 0.05% Tween 20), and then blocked with wash buffer containing 1% Bovine Serum Albumin (BSA) (Sigma)) for 2 hours at 37 ℃. Two-fold serial dilutions (1/64 to 1/65,536) of each serum sample diluted in wash buffer containing 1% BSA were incubated in individual wells at 37 ℃ for 2 hours, followed by three washes with wash buffer. Each well was incubated with goat anti-chicken IgY horseradish peroxidase (HRP) conjugated polyclonal antibody, diluted 1:5,000 in wash buffer for 1 hour at room temperature, and then washed three times with wash buffer. To each well was added a substrate solution (0.2mg/ml2,2' -nitrilo-bis (3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) (sigma) in 1X ABTS buffer (sigma)) and incubated at room temperature for 30 minutes after stopping the reaction with 0.5% Sodium Dodecyl Sulfate (SDS), BioTek was used^TMThe disc reader measures absorbance at 405 nm. The titer is log as the lowest serum dilution at absorbance greater than twice that of the background wells₂Values were calculated using PBS with 1% BSA instead of serum. The statistical difference between pre-and post-immune titers for each antigen was determined by one-way ANOVA followed by dukih's post-hoc test in different vaccination groups.

The results are shown in fig. 3A and B, respectively. The mean serum response to CnaA in the CnaA immunized group at d31 was significantly higher than that of preimmunized birds (d8), however the overall increase was smaller. The mean response to FimA after immunization in the FimA immunization group did not increase significantly. However, two birds truncated to d31 did exhibit higher titers.

Antibiotic-free pup diets containing 20% protein were fed to birds until experimental induction of Necrotic Enteritis (NE). On day 27, birds fasted for 24 hours and then switched to antibiotic-free turkey diets (28% protein) containing clostridium perfringens CP1 culture on days 28 and 29, followed by euthanasia on day 31. Daily infected diets were prepared by mixing with clostridium perfringens CP1 culture at a 2:1(v/w) ratio at 37 ℃ for 15 hours or 24 hours in liquid thioglycolate (FTG) (Difco) medium, respectively, each morning and afternoon. Following euthanasia, birds were examined generally for their small intestine (duodenum to ileum) and using the system described by keberen et al, blindly scored as follows from 1 to 6 (keberen al (keyburn al), boyes jd (boyce jd), watts p (vaz p), banland tl (bannamtl), ford me (ford me), park d (parker d), diru a (di rubo a), lud ji (rod ji), moore rj), 2008, NetB: a neotoxin associated with necrotic enteritis in birds caused by clostridium perfringens (NetB, a new toxin which is associated with necrotic enteritis in birds with avian experimental use of byClostridium perfringens), public museum books of science books fringens (PLoS pathalog) 4: 26:

0: no eye damage exists;

1: thin or brittle walls;

2: focal necrosis or ulceration (1-5 lesions);

3: focal necrosis or ulceration (6-15 lesions);

4: focal necrosis or ulceration (16 or more lesions);

5: necrotic plaques 2-3cm in length;

6: diffuse necrosis characteristic of the field condition.

Statistical differences between Necrotic Enteritis (NE) scores for each group were determined by one-way ANOVA (analysis of variance), followed by dukes' post hoc test. The results shown in fig. 4 indicate that all groups have the same high mean lesion score. The mean necrotic enteritis scores of the adjuvant alone control, CnaA immunized, and FimA immunized groups were 3.1, 3.0, and 3.3, respectively.

Without being bound by theory, it is expected that day 8 immunization may have been disturbed by maternal antibodies, and that day 20 immunization may not have had time to elicit a sufficient immune response before challenge with clostridium perfringens CP 1. Thus, a second vaccination trial was performed, including additional immunizations prior to clostridium perfringens CP1 challenge.

Test 2:

the second test consisted of: four groups of 18 birds were inoculated subcutaneously (s.c.) with adjuvant controls, CnaA, FimB, alone, or in combination with CnaA, FimA, and FimB. In this experiment, each bird was immunized subcutaneously with 50. mu.g of recombinant polypeptide and 50. mu.g of Quil-A on days 7, 14 and 19^TMCombinations of adjuvants, and sera were collected on days 7, 19 and 29 to measure antibody titers. Birds were challenged with c.perfringens strain CP1 in feed on days 26 and 27 as described in trial 1; and on day 29, birds were euthanized and bowel injury was scored.

Compared to the pre-immune control group, significant (p <0.001) serum antibody (IgY) responses were observed on day 19 and day 29 in all immune groups (except the group immunized with FimB on day 19) and the magnitude of the response was also much greater than in test 1. The results are shown in fig. 5A (anti-CnaA serum response), 5B (anti-FimA serum response) and 5C (anti-FimB serum response).

In addition, as seen in figure 6, both the CnaA and FimB immunised groups had significantly lower necrotic enteritis scores (2 and 2.06 respectively) when measured and scored as in test 1 compared to the adjuvant control group (3.75), indicating that these antigens provided at least partial protection against necrotic enteritis. For FimB antigen, the number of birds with severe disease (necrotic enteritis score >2) accounted for 33.3%, compared to 93.7% in the control group. The combination of vaccination subunits did not appear to reduce the severity of the disease (mean necrotic enteritis score of 3.7), but elicited strong serum responses against all three subunits, as seen in fig. 5A-C.

Clostridium perfringens strain CP1 pilus subunit knockout mutant for stimulating chicken

Three groups of 18 birds in trial 2, which had not been immunized, were challenged twice daily on days 26 and 27 with CP1, CP1 Δ fimA or CP1 Δ fimB prepared as described in example 2 in the feed. On day 29, birds were euthanized and necrotic enteritis lesions were scored as described in example 3. As can be seen from the results presented in fig. 7, neither CP1 Δ fimA nor CP1 Δ fimB mutant strains caused disease in the challenged avian, suggesting that functional fimbriae appear to be essential for the pathogenesis of necrotic enteritis.

Example 4: characterization of Clostridium perfringens pilus surface polypeptides

Clostridium perfringens strains CP1 and CP1 pilus subunit mutants:

surface polypeptides were extracted from clostridium perfringens strain CP1 and pilus subunit mutants CP1 Δ cnaA, CP1 Δ fimA and CP1 Δ fimB described in example 3 using methods in the following references: usual C. (Chang, C.), yellow i. -H. (Huang, i. -H.), hendelick a.p.a. (Hendrickx, a.p.a.), Ton-leiter H. (Ton-That, H), 2013, Visualization of Gram-positive Bacterial Pili (Visualization of Gram-positive Bacterial Pili) in: delcour H.A (Delcour, H.A.) (eds), bacterial cell surface: methods and Protocols (Bacterial Cell Surfaces: methods and Protocols), Horizontan Press (Humana Press), Tetorwa, N.J., 77-95. The strains were grown anaerobically in TGY medium (3% tryptone, 2% glucose, 1% yeast extract) at 37 ℃ overnight, subcultured 1:100 in 10ml TGY medium and grown to OD₆₀₀About 1. Cells were centrifuged at 6,000x g for 5 minutes and at SMM buffer pH 6.8(0.5M sucrose, 10mM MgCl)₂10mM maleate). The bacterial pellet was resuspended in 1ml of SMM buffer and 60. mu.l of muramidase buffer (2mM acetic acid, 48mM sodium acetate) containing 5U/. mu.l mutanolysin (Sigma) and 10. mu.l of 0.1M phenylmethylsulfonyl fluoride (PMSF) (Sigma) were added thereto.After incubation at 37 ℃ for at least 4 hours at constant optical rotation, the protoplasts were centrifuged at 20,000x g for 5 minutes and the supernatant fractions containing the cell wall proteins were removed. Proteins were precipitated by adding 81 μ l 100% (w/v) trichloroacetic acid (TCA) (sigma) per ml and incubating at 4 ℃ overnight. After centrifugation at 20,000x g for 20 minutes at 4 ℃, the protein pellet was washed with acetone and slowly resuspended in 50 μ l of sample-loaded buffer (62.5mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 4% β -mercaptoethanol, 3M urea, 0.01% bromophenol blue) for at least 15 minutes at room temperature.

Surface protein extract (5. mu.l) was loaded on Novex^TMNuPAGE^TM3-8% Tris-acetate gel (Feishale technologies) and electrophoresed at 150V for 1 hour. Bio-Safe for gel^TMCoomassie stain (burle) or transferred to polyvinylidene fluoride (PVDF) membranes at 350V over 1 hour in 1X transfer buffer (48mM Tris, 39mM glycine, 20% methanol, 0.1% SDS). Using Westernbreeze^TMChemiluminescent assay kit (life technologies company) chemiluminescent detection was performed using chicken anti-FimA serum (1:200) as primary antibody and goat anti-chicken IgY Alkaline Phosphatase (AP) conjugated secondary antibody (1:2,000) according to the manufacturer's instructions. The FimA immunized chickens from experiment 1 were sacrificed and sera were obtained for use as primary abs (example 3) which subsequently exhibited higher anti-FimA titers or produced polyclonal antibodies in rabbits against the recombinant pilus polypeptide described in example 1. The results are shown in FIGS. 8A-C.

Western blot analysis of the SDS-PAGE separated transpeptidase dependent pili is known to produce High Molecular Weight (HMW) ladder-like patterns reflecting the different polymer lengths, reflecting the mechanism by which the pili assemble at the cell surface. The pilin subunits are covalently linked by housekeeping and pilin-specific transpeptidases, causing growth of the hetero-polymer structure, which is ultimately covalently linked to the cell wall peptidoglycan. The termination of assembly and hence polymer length is variable, resulting in a characteristic high molecular weight ladder-like pattern when these pili are visualized by western blotting. As seen in fig. 8B and C, ladder-like patterns showing pilus structures were observed in western blots of surface polypeptides extracted from clostridium perfringens strain CP1, but not in corresponding western blots of surface polypeptides extracted from mutant strains, whether visualized using antibodies obtained from chicken serum or antibodies produced in rabbits.

Various clostridium perfringens strains:

surface polypeptides were extracted from five clostridium perfringens isolates derived from avian (CP1, JGS4141 and JGS4120) or non-avian (strain 13, ATCC13124) sources as described above. Surface protein extracts (5. mu.l) were loaded on two Novex plates^TMNuPAGE^TM3-8% Tris-acetate gel (Feishale technologies) and electrophoresed at 150V for 1 hour. One gel for Bio-Safe^TMCoomassie stain (burle) and the second gel was transferred to polyvinylidene fluoride (PVDF) membrane at 350V over 1 hour in 1X transfer buffer (48mM tris, 39mM glycine, 20% methanol, 0.1% SDS). Using Westernbreeze^TMChemiluminescent assay kit (life technologies company) chemiluminescent detection was performed using chicken anti-FimA serum (1:200) as primary antibody and goat anti-chicken IgY Alkaline Phosphatase (AP) conjugated secondary antibody (1:2,000) according to the manufacturer's instructions. FimA immunized chickens were sacrificed and serum obtained for use as the primary Ab, which subsequently exhibited higher anti-FimA titers.

The results are shown in FIGS. 9A-B. The presence (+) or absence (-) of loci (VR-10B (CA) loci) of pilus subunit genes cnaA, fimA, and fimB in various Clostridium perfringens strains has been previously determined by microarray analysis and Polymerase Chain Reaction (PCR) methods (Lepu D (Lepp D), et al, Journal of bacteriology (Journal of bacteriology) (2013)195: 1152-. As seen in fig. 9A-B, when western blots were visualized by chicken anti-FimA antibodies, the pilus structure of the extracted surface polypeptides of the strains carrying the pilus locus in the genome (JGS4141 and CP1) displayed a characteristic ladder-like pattern (shown in fig. 9B by the vertical right-hand gel image), whereas other strains not carrying a pilus locus in the genome did not display this pattern. The visual observation of the smaller molecular weight bands in the extracts may be due to the presence of irrelevant antibodies in the crude chicken serum. None of the extracts displayed a band corresponding to the FimA polypeptide itself, the expected position of which is indicated in fig. 9B by the arrow on the right side of the gel image. This is not surprising, as no surface-associated proteins are expected to include the FimA monomer, which is found only intracellularly.

Immunogold labeling of clostridium perfringens strains CP1 and CP1 pilus subunit mutants:

the cells of Clostridium perfringens strain CP1 or CP1 knockout mutants CP1 Δ fimA and CP1 Δ fimB are labeled with gold particles by using an immunogold technique, and comprise rabbit anti-FimA as a primary antibody and 6nm colloidal gold-Affinipure^TMGoat anti-rabbit IgG (H + L) (min X Hu, Ms, Rat Sr Prot) (Cedarlane) as a secondary antibody and examined by transmission electron microscopy, substantially as described previously (often C. (Chang, C.), yellow i. -H. (Huang, i. -H.), hendrick a.p.a. (Hendrickx, a.p.a.), Ton leite H. (Ton-That), 2013, Visualization of Gram-positive Bacterial Pili (Visualization of Gram-positive Bacterial Pili), in dale H.A. (Delcour, H.A.) (compiled), Bacterial Cell surface: Methods and Protocols (Bacterial Cell Surfaces: Methods and Protocols), humann Press (Humana Press), new western tile (towarwa), towarwa, tokyo 95-wawa), tokay. As seen in fig. 10, cells of the native CP1 strain displayed the presence of pilus structures on the cell surface, whereas cells of the CP1 Δ fimA and CP1 Δ fimB mutants lacked such structures.

The examples described herein are intended to illustrate the compositions and methods of this invention and are not intended to limit the scope of the invention. It is intended to include modifications and variations consistent with the entire specification and which are apparent to those skilled in the art. The following claims should not be limited by the specific embodiments described in the examples, but should be given the broadest interpretation consistent with the entire specification.

Sequence listing

<110> the kawang (Her Majesty the) represented by the canadian agricultural and agricultural product ministry

Queen in Right of Canada, as represented by the Minister of

Agriculture and Agri-Food)

<120> vaccine against necrotic enteritis in poultry

<130>3688-157

<150>US 62/513,001

<151>2017-05-31

<160>15

<170> PatentIn 3.5 edition

<210>1

<211>2091

<212>DNA

<213> Clostridium perfringens bacterium

<400>1

atgaaaataa ataaaaaaat ttttagcatg ctatttatgg ttattgtact ttttacatgc 60

atatcatcaa atttttctgt ttctgcttct tctattcaaa gaggaagaga tatcagtaat 120

gaggtagtta caagcctagt ggctactcca aatagtataa atgatggtgg aaacgttcag 180

gttcgtttgg aatttaaaga aaatcatcaa agaaatatac aaagtggaga tactataact 240

gtcaaatgga caaattcagg ggaagtattt tttgaaggat atgaaaaaac aattccactt 300

tatataaaag accaaaatgt tggtcaagca gtaatagaga aaacaggtgc aacacttaca 360

tttaatgata aaatagataa attagatgat gttggtggat gggcaacatt tactttgcaa 420

ggaagaaaca ttacctcagg taatcatgaa cacacaggaa tagcatatat tatatctggt 480

tcaaagcggg cagatgtaaa tataaccaaa ccagaatcag gtacaactag tgtattctat 540

tataaaacag gtagtatgta taccaatgat acaaatcatg tcaattggtg gttactggtg 600

aatccaagca aggtatattc tgaaaaaaac gtttatattc aagatgaaat ccaaggcgga 660

caaacattag aacctgattc ttttgaaata gtagtaactt ggtatgatgg ttatgtagaa 720

aagtttaaag gaaaagaagc gataagggaa ttccataata aatatccaaa ttcaaatata 780

tcggtatcag aaaataaaat aacagtaaac atttcacaag aggattccac acaaaagttt 840

attaatattt tttataaaac taagattaca aatccgaaac aaaaagaatt cgttaataat 900

acaaaagcat ggtttaaaga gtataataag ccagctgtaa atggagaatc ctttaaccat 960

agcgtacaaa atattaatgc agatgctgga gttaatggaa ctgtaaaagg cgaattaaaa 1020

atcataaaaa cattaaaaga taaaagtatt ccaattaaag atgttcagtt taagatgaga 1080

agagttgata atacagttat caaagatggt aaaaaagaat tattactaac aactgatgat 1140

aaaggtattg caaatgtaaa aggtcttcct gtaggaaaat atgaagtaaa agagatttca 1200

gctccagaat ggattgcttt taatcctctt attgcaccaa aattggaatt cacaatatca 1260

gatcaggaca cagaaggcaa attgtgggct gttgaaaatg aattaaagac aatttcaatt 1320

ccggttgaaa aggtctgggt aggacaaact agtgaacgag cagaaatcaa gctttttgca 1380

gatggtattg aagtagacaa agtgatttta aatgcagata acaattggaa acacacattt 1440

gaaaataaac ctgaatataa ttcagaaaca aaacagaaaa tcaattattc tgtgtcagag 1500

acaactattt ctggatatga aagcaatatc acaggcgatg ctaagaatgg ttttattgta 1560

accaatacag aacttcctga tttgactatt ggtaaagaag ttataggaga attgggtgac 1620

aagacgaagg tatttaactt tgagcttaca ttaaagcaag cagatggaaa gcctatcaat 1680

ggtaaattta attacattgg tagtgtagat gacaggtaca aaaaagaaag cataaagcct 1740

tctgatggtg agattacttt tatagaagga aaagcaacta taactttatc acatggacaa 1800

gagattacaa tcaaggattt accatatggg gttacatata aagttatgga aaaagaagct 1860

aatgaaaatg gctatttaac tacctataat ggaaataacg aagtcacaac aggtgaattg 1920

aaacaggata caaaagtaca ggtagttaac aacaaagagt ttgttccaac aactggtata 1980

tcaaccacaa cagagcaagg tacaatggtt ggaatggtga ttttttctat aggaatactt 2040

atggtcatga ttgtagttct tttacaattg aataaaggac tgaaaagatg a 2091

<210>2

<211>1074

<212>DNA

<213> Clostridium perfringens bacterium

<400>2

atgataaaca agaaaaaatt aagtgcatta ttattaagtg gagcaatgtt tatgagtatg 60

aatacaaatg tattcgcatc aaatttacct tctggagggg tagaaggtac agaacagaat 120

cctgcaaaag caacaattac aaagaatttt gaatttccag aaggtattaa tacacctagt 180

gcaacattca agtttacagc agaaaaaata actaatgatg cgccagatgc aacaattgga 240

gatattaatt atacacaagg ggataatgga actttatcaa atggaaaata tagtgtaaag 300

aaaacaactg aaattacttt tggaaatttc ccacatgcag gagaatatga ttataatgta 360

aaagaaacga atgagggagt aggtggtatt acatatgata caaaagaata caaagttcat 420

gtgtatgttg caaatagtaa cgctatggat ggaaaaactt atgtaaaagc cattacatca 480

gaaaatggag gtgaaaaagc tccaattgag tttgttaata catataaaaa ggacacttct 540

ttacttatag aaaagaatgt aataggagat ttagctgact taacaaaaca gtttgagttt 600

cagattaatt taaaaaaatc agcaacatct gacataacaa aattcgaagg aaatattatt 660

agaaaagatg gtaaaataga gcctgtaaca tatacagctg aaaatacaga aacttttaaa 720

ttagcaaatg gagataaact taagtttgaa agtattccag caggaacaaa atatgaagta 780

aaagagatag gtgctagtga tggatataca ccttctataa cagtaattga aaatggaaat 840

gagacttcta ataatcgtac ggtagctgaa aaagatggta tatcatctaa gtcaaattct 900

aatgataact taattggtga aggtgaaaac aaagtaacat ttacaaacac atataatgac 960

aaacctatca caggtattgt tatgaataat attccattta ttctaatgat tagttttgct 1020

gtccttggat ttggtgcttt agctattata aaaagacgta aaactataag ataa 1074

<210>3

<211>660

<212>DNA

<213> Clostridium perfringens bacterium

<400>3

atggaaacaa agaaaataag aaacaaaatc cttatggcta tcgtagcatt gagctttata 60

ttgcttccaa acactagagt atatgctact gaaaatacag caaatattcc tttgatagtt 120

agacaggaat ttaatgtata tacgaaagat tcaaaagcaa tagacatgat tggaaaatac 180

gagctaaagg caataagtga aaatgcccct atgccagaag aaagtaaaaa tggaagtttt 240

atctttaata tagatggaaa tgataagcag tttactattc cattagctta tacacatggt 300

ggtgtgtata tctatcaaat tcaacagata acgcaatcta aagataatta catatatgat 360

aaaaatagct ataagataac tgtatatgta aaaaatgcag aaaataatca tttaatacca 420

caaattattg tgaaaaatga aaataatgaa aaatgtgaag aaatatgttt ttataacatt 480

tacaaacaaa aaaataaaat taatgagatt tctaaaacac catataagcc aaatggaata 540

aatgttccta aaacaggcga taccacaaac attggatttt atattgtaat acttataatt 600

tcacttggat tacttgtggt attgaaatgg aaagaatata aaaagagaaa aaaagaataa 660

<210>4

<211>2088

<212>DNA

<213> Artificial sequence

<220>

<223> codon optimized cnaA

<400>4

atgaagatca acaagaagat cttcagcatg ttatttatgg tcattgtgct gttcacctgt 60

atcagctcta acttcagtgt gagcgcgtca agcatccagc gcggccggga catcagcaac 120

gaggtggtga catcgctcgt agctaccccg aatagcatca acgatggtgg taacgtccaa 180

gtgcgtctgg aatttaaaga gaatcaccag cggaacattc agtccggcga cacgattacg 240

gtcaaatgga ctaactcagg tgaggtcttt tttgaaggct acgaaaaaac catcccgctg 300

tatatcaagg atcagaacgt tggccaggcg gttattgaaa aaaccggtgc aacattaaca 360

ttcaacgata agatcgacaa attagatgat gtcggcggct gggccacatt cacgctccag 420

ggtcgcaata ttacttcagg aaatcatgag catactggta ttgcgtacat tatctcgggt 480

agcaaacgtg cggacgttaa catcacaaaa cctgaatccg gaacaacgtc tgtgttttac 540

tacaagacgg gttcgatgta caccaatgac acaaatcatg tgaattggtg gctgctggtt 600

aacccgagca aagtatactc tgagaaaaat gtctatattc aggatgaaat tcaaggcggt 660

cagaccctgg agccggacag ttttgaaatc gtcgttacat ggtacgatgg ttatgtggaa 720

aaatttaaag gtaaagaagc gatccgggag ttccacaata aatatccgaa tagtaatatc 780

tcggtcagtg aaaataaaat cacggtaaat atttcgcaag aagattccac ccaaaaattc 840

attaacatct tttacaagac taaaatcacc aacccgaagc agaaagaatt tgtaaacaac 900

accaaagcct ggttcaaaga gtacaataag ccggcggtta acggtgaaag ttttaatcac 960

agtgtgcaga atatcaacgc agatgccggg gtaaatggta ctgttaaagg tgaattgaaa 1020

attatcaaaa ccctgaaaga taaaagtatt ccgatcaagg atgtgcagtt taagatgcgc 1080

cgcgtggata ataccgttat taaagacggc aagaaagagc tgctgttgac cacagatgat 1140

aaagggattg caaacgtgaa aggtctgcca gtcgggaaat acgaagtcaa agaaatcagt 1200

gcgcctgagt ggatcgcctt caatccactg attgcgccca aacttgaatt tacgatcagc 1260

gatcaagaca cagaggggaa attatgggca gtggaaaacg aactcaaaac catctcgatt 1320

ccggtcgaaa aagtctgggt aggtcagacg agtgaacggg cggagatcaa actgtttgcg 1380

gatggaattg aagttgataa ggtgatcctg aacgcggata ataattggaa gcacaccttt 1440

gagaataaac ccgaatataa ctccgagact aaacaaaaaa tcaactatag tgtgagcgaa 1500

actaccatca gtggctatga atcaaatatt actggcgatg cgaaaaacgg atttattgtc 1560

accaacacag aactgcctga tttgacgatc gggaaagagg taatcggcga actcggcgat 1620

aaaaccaagg tattcaactt tgaactgaca cttaagcagg ctgacggaaa gcccattaac 1680

gggaaattta actatattgg ttcggtggat gatcgttata agaaggaatc gattaagcct 1740

agcgatgggg aaattacgtt catcgaggga aaagcaacga ttaccctctc ccacggacaa 1800

gagatcacca ttaaggacct tccgtatggt gtgacctata aagtcatgga aaaagaagcc 1860

aacgagaatg gatatttaac cacttacaac ggaaataacg aagtcaccac cggggagttg 1920

aaacaggata cgaaagtaca agtggttaat aataaagaat tcgtcccgac aaccgggatc 1980

agcaccacca ccgaacaggg aaccatggtc gggatggtga tctttagcat cggtattctc 2040

atggtaatga ttgtcgttct gctgcagctg aataaaggac tgaaacgc 2088

<210>5

<211>1086

<212>DNA

<213> Artificial sequence

<220>

<223> codon optimized fimA

<400>5

atgattaata aaaagaaact gtcggcgctg ctcttaagcg gggccatgtt tatgagcatg 60

aacacgaatg tgttcgcgtc taacctccca tcgggtggtg tggagggcac cgaacaaaac 120

ccagcgaaag cgacaatcac gaaaaacttc gagtttccgg aaggtattaa tacacccagc 180

gcgacattca aatttaccgc cgaaaaaatt accaacgatg cgccggatgc tactattggc 240

gacatcaatt atacccaagg tgataatggg acgttaagca atggcaaata cagtgtgaaa 300

aagactaccg agattacctt cgggaacttc ccgcatgctg gtgagtatga ttataacgtc 360

aaagaaacca atgaaggcgt gggtggcatt acttacgata cgaaagaata taaagttcat 420

gtgtatgtgg ccaactcaaa tgcgatggac ggtaagacat atgttaaagc gattactagc 480

gaaaatggcg gggaaaaagc accgatcgaa ttcgttaaca cctataaaaa agatacgtcg 540

ttactgattg aaaaaaatgt aattggcgat ctggcagacc tcaccaaaca gtttgagttt 600

caaatcaact tgaaaaagag cgcgactagt gatattacca agtttgaagg taacattatt 660

cgcaaagacg gtaagattga acccgtgacc tataccgcgg aaaataccga gacctttaag 720

ttagccaacg gagacaagtt aaaattcgag tccatccccg ccggtacaaa atatgaagtc 780

aaggaaatcg gggcgagcga tgggtacacg ccctcaatca ccgttatcga aaatggcaac 840

gaaacctcaa ataaccgcac tgtagccgaa aaagatggaa tctctagcaa aagcaactcg 900

aacgacaatt taatcggcga aggcgaaaat aaagtgacct ttaccaatac gtacaacgat 960

aaaccaatca cgggaatcgt aatgaataat attccgttca ttcttatgat tagctttgcc 1020

gttcttggct tcggtgcatt agcgatcatt aaacgccgca aaaccatccg ccccatcgat 1080

acgcgt 1086

<210>6

<211>624

<212>DNA

<213> Artificial sequence

<220>

<223> codon optimized fimB

<400>6

atggctattg ttgctttgtc atttatcctg ctcccgaata cccgggtcta tgcgacggag 60

aacaccgcta atatcccgtt aattgtacgc caagaattta atgtttacac taaagattct 120

aaagccattg acatgatcgg aaaatatgaa ttaaaagcca tttctgagaa cgctcccatg 180

ccggaggaat caaaaaatgg tagctttatt tttaacatcg acggtaatga taaacagttt 240

actattccgc tggcgtacac tcacggtggc gtctacatct atcaaatcca gcaaattacc 300

cagagcaagg ataactacat ctacgataaa aacagctata aaatcacggt atatgtcaag 360

aacgcagaaa acaatcatct gatcccgcag attattgtaa aaaatgagaa caatgaaaaa 420

tgtgaagaaa tctgcttcta caatatctac aaacagaaaa acaagatcaa tgagatctct 480

aaaaccccct ataagccgaa tggtattaat gtcccgaaaa cgggtgatac cacgaacatc 540

ggattctaca ttgtgatctt gattatttcc ctgggcctgc tggtggtctt gaagtggaaa 600

gaatataaaa aacgtaagaa ggaa 624

<210>7

<211>1887

<212>DNA

<213> Artificial sequence

<220>

<223> truncated cnaA

<400>7

tcaagcatcc agcgcggccg ggacatcagc aacgaggtgg tgacatcgct cgtagctacc 60

ccgaatagca tcaacgatgg tggtaacgtc caagtgcgtc tggaatttaa agagaatcac 120

cagcggaaca ttcagtccgg cgacacgatt acggtcaaat ggactaactc aggtgaggtc 180

ttttttgaag gctacgaaaa aaccatcccg ctgtatatca aggatcagaa cgttggccag 240

gcggttattg aaaaaaccgg tgcaacatta acattcaacg ataagatcga caaattagat 300

gatgtcggcg gctgggccac attcacgctc cagggtcgca atattacttc aggaaatcat 360

gagcatactg gtattgcgta cattatctcg ggtagcaaac gtgcggacgt taacatcaca 420

aaacctgaat ccggaacaac gtctgtgttt tactacaaga cgggttcgat gtacaccaat 480

gacacaaatc atgtgaattg gtggctgctg gttaacccga gcaaagtata ctctgagaaa 540

aatgtctata ttcaggatga aattcaaggc ggtcagaccc tggagccgga cagttttgaa 600

atcgtcgtta catggtacga tggttatgtg gaaaaattta aaggtaaaga agcgatccgg 660

gagttccaca ataaatatcc gaatagtaat atctcggtca gtgaaaataa aatcacggta 720

aatatttcgc aagaagattc cacccaaaaa ttcattaaca tcttttacaa gactaaaatc 780

accaacccga agcagaaaga atttgtaaac aacaccaaag cctggttcaa agagtacaat 840

aagccggcgg ttaacggtga aagttttaat cacagtgtgc agaatatcaa cgcagatgcc 900

ggggtaaatg gtactgttaa aggtgaattg aaaattatca aaaccctgaa agataaaagt 960

attccgatca aggatgtgca gtttaagatg cgccgcgtgg ataataccgt tattaaagac 1020

ggcaagaaag agctgctgtt gaccacagat gataaaggga ttgcaaacgt gaaaggtctg 1080

ccagtcggga aatacgaagt caaagaaatc agtgcgcctg agtggatcgc cttcaatcca 1140

ctgattgcgc ccaaacttga atttacgatc agcgatcaag acacagaggg gaaattatgg 1200

gcagtggaaa acgaactcaa aaccatctcg attccggtcg aaaaagtctg ggtaggtcag 1260

acgagtgaac gggcggagat caaactgttt gcggatggaa ttgaagttga taaggtgatc 1320

ctgaacgcgg ataataattg gaagcacacc tttgagaata aacccgaata taactccgag 1380

actaaacaaa aaatcaacta tagtgtgagc gaaactacca tcagtggcta tgaatcaaat 1440

attactggcg atgcgaaaaa cggatttatt gtcaccaaca cagaactgcc tgatttgacg 1500

atcgggaaag aggtaatcgg cgaactcggc gataaaacca aggtattcaa ctttgaactg 1560

acacttaagc aggctgacgg aaagcccatt aacgggaaat ttaactatat tggttcggtg 1620

gatgatcgtt ataagaagga atcgattaag cctagcgatg gggaaattac gttcatcgag 1680

ggaaaagcaa cgattaccct ctcccacgga caagagatca ccattaagga ccttccgtat 1740

ggtgtgacct ataaagtcat ggaaaaagaa gccaacgaga atggatattt aaccacttac 1800

aacggaaata acgaagtcac caccggggag ttgaaacagg atacgaaagt acaagtggtt 1860

aataataaag aattcgtccc gacaacc 1887

<210>8

<211>894

<212>DNA

<213> Artificial sequence

<220>

<223> truncated fimA

<400>8

tctaacctcc catcgggtgg tgtggagggc accgaacaaa acccagcgaa agcgacaatc 60

acgaaaaact tcgagtttcc ggaaggtatt aatacaccca gcgcgacatt caaatttacc 120

gccgaaaaaa ttaccaacga tgcgccggat gctactattg gcgacatcaa ttatacccaa 180

ggtgataatg ggacgttaag caatggcaaa tacagtgtga aaaagactac cgagattacc 240

ttcgggaact tcccgcatgc tggtgagtat gattataacg tcaaagaaac caatgaaggc 300

gtgggtggca ttacttacga tacgaaagaa tataaagttc atgtgtatgt ggccaactca 360

aatgcgatgg acggtaagac atatgttaaa gcgattacta gcgaaaatgg cggggaaaaa 420

gcaccgatcg aattcgttaa cacctataaa aaagatacgt cgttactgat tgaaaaaaat 480

gtaattggcg atctggcaga cctcaccaaa cagtttgagt ttcaaatcaa cttgaaaaag 540

agcgcgacta gtgatattac caagtttgaa ggtaacatta ttcgcaaaga cggtaagatt 600

gaacccgtga cctataccgc ggaaaatacc gagaccttta agttagccaa cggagacaag 660

ttaaaattcg agtccatccc cgccggtaca aaatatgaag tcaaggaaat cggggcgagc 720

gatgggtaca cgccctcaat caccgttatc gaaaatggca acgaaacctc aaataaccgc 780

actgtagccg aaaaagatgg aatctctagc aaaagcaact cgaacgacaa tttaatcggc 840

gaaggcgaaa ataaagtgac ctttaccaat acgtacaacg ataaaccaat cacg 894

<210>9

<211>468

<212>DNA

<213> Artificial sequence

<220>

<223> truncated fimB

<400>9

acggagaaca ccgctaatat cccgttaatt gtacgccaag aatttaatgt ttacactaaa 60

gattctaaag ccattgacat gatcggaaaa tatgaattaa aagccatttc tgagaacgct 120

cccatgccgg aggaatcaaa aaatggtagc tttattttta acatcgacgg taatgataaa 180

cagtttacta ttccgctggc gtacactcac ggtggcgtct acatctatca aatccagcaa 240

attacccaga gcaaggataa ctacatctac gataaaaaca gctataaaat cacggtatat 300

gtcaagaacg cagaaaacaa tcatctgatc ccgcagatta ttgtaaaaaa tgagaacaat 360

gaaaaatgtg aagaaatctg cttctacaat atctacaaac agaaaaacaa gatcaatgag 420

atctctaaaa ccccctataa gccgaatggt attaatgtcc cgaaaacg 468

<210>10

<211>696

<212>PRT

<213> Clostridium perfringens bacterium

<400>10

Met Lys Ile Asn Lys Lys Ile Phe Ser Met Leu Phe Met Val Ile Val

1 5 10 15

Leu Phe Thr Cys Ile Ser Ser Asn Phe Ser Val Ser Ala Ser Ser Ile

20 25 30

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

35 40 45

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

50 55 60

Phe Lys Glu Asn His Gln Arg Asn Ile Gln Ser Gly Asp Thr Ile Thr

65 70 75 80

Val Lys Trp Thr Asn Ser Gly Glu Val Phe Phe Glu Gly Tyr Glu Lys

85 90 95

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

100 105 110

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

115 120 125

Asp Asp Val Gly Gly Trp Ala Thr Phe Thr Leu Gln Gly Arg Asn Ile

130 135 140

Thr Ser Gly Asn His Glu His Thr Gly Ile Ala Tyr Ile Ile Ser Gly

145 150 155 160

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

165 170 175

Ser Val Phe Tyr Tyr Lys Thr Gly Ser Met Tyr Thr Asn Asp Thr Asn

180 185 190

His Val Asn Trp Trp Leu Leu Val Asn Pro Ser Lys Val Tyr Ser Glu

195 200 205

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

210 215 220

Pro Asp Ser Phe Glu Ile Val Val Thr Trp Tyr Asp Gly Tyr Val Glu

225 230 235 240

Lys Phe Lys Gly Lys Glu Ala Ile Arg Glu Phe His Asn Lys Tyr Pro

245 250 255

Asn Ser Asn Ile Ser Val Ser Glu Asn Lys Ile Thr Val Asn Ile Ser

260 265 270

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

275 280 285

Ile Thr Asn Pro Lys Gln Lys Glu Phe Val Asn Asn Thr Lys Ala Trp

290 295 300

Phe Lys Glu Tyr Asn Lys Pro Ala Val Asn Gly Glu Ser Phe Asn His

305 310 315 320

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

325 330 335

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

340 345 350

Lys Asp Val Gln Phe Lys Met Arg Arg Val Asp Asn Thr Val Ile Lys

355 360 365

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

370 375 380

Asn Val Lys Gly Leu Pro Val Gly Lys Tyr Glu Val Lys Glu Ile Ser

385 390 395 400

Ala Pro Glu Trp Ile Ala Phe Asn Pro Leu Ile Ala Pro Lys Leu Glu

405 410 415

Phe Thr Ile Ser Asp Gln Asp Thr Glu Gly Lys Leu Trp Ala Val Glu

420 425 430

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

435 440 445

Gln Thr Ser Glu Arg Ala Glu Ile Lys Leu Phe Ala Asp Gly Ile Glu

450 455 460

Val Asp Lys Val Ile Leu Asn Ala Asp Asn Asn Trp Lys His Thr Phe

465 470 475 480

Glu Asn Lys Pro Glu Tyr Asn Ser Glu Thr Lys Gln Lys Ile Asn Tyr

485 490 495

Ser Val Ser Glu Thr Thr Ile Ser Gly Tyr Glu Ser Asn Ile Thr Gly

500 505 510

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

515 520 525

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

530 535 540

Phe Asn Phe Glu Leu Thr Leu Lys Gln Ala Asp Gly Lys Pro Ile Asn

545 550 555 560

Gly Lys Phe Asn Tyr Ile Gly Ser Val Asp Asp Arg Tyr Lys Lys Glu

565 570 575

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

580 585 590

Thr Ile Thr Leu Ser His Gly Gln Glu Ile Thr Ile Lys Asp Leu Pro

595 600 605

Tyr Gly Val Thr Tyr Lys Val Met Glu Lys Glu Ala Asn Glu Asn Gly

610 615 620

Tyr Leu Thr Thr Tyr Asn Gly Asn Asn Glu Val Thr Thr Gly Glu Leu

625 630 635 640

Lys Gln Asp Thr Lys Val Gln Val Val Asn Asn Lys Glu Phe Val Pro

645 650 655

Thr Thr Gly Ile Ser Thr Thr Thr Glu Gln Gly Thr Met Val Gly Met

660 665 670

Val Ile Phe Ser Ile Gly Ile Leu Met Val Met Ile Val Val Leu Leu

675 680 685

Gln Leu Asn Lys Gly Leu Lys Arg

690 695

<210>11

<211>357

<212>PRT

<213> Clostridium perfringens bacterium

<400>11

Met Ile Asn Lys Lys Lys Leu Ser Ala Leu Leu Leu Ser Gly Ala Met

1 5 10 15

Phe Met Ser Met Asn Thr Asn Val Phe Ala Ser Asn Leu Pro Ser Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Ser Val Lys Lys Thr Thr Glu Ile Thr Phe Gly Asn Phe Pro His

100 105 110

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

115 120 125

Gly Ile Thr Tyr Asp Thr Lys Glu Tyr Lys Val His Val Tyr Val Ala

130 135 140

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

145 150 155 160

Glu Asn Gly Gly Glu Lys Ala Pro Ile Glu Phe Val Asn Thr Tyr Lys

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Lys Ile Glu Pro Val Thr Tyr Thr Ala Glu Asn Thr Glu Thr Phe Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Lys Pro Ile Thr Gly Ile Val Met Asn Asn Ile Pro Phe Ile Leu Met

325 330 335

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

340 345 350

Arg Lys Thr Ile Arg

355

<210>12

<211>219

<212>PRT

<213> Clostridium perfringens bacterium

<400>12

Met Glu Thr Lys Lys Ile Arg Asn Lys Ile Leu Met Ala Ile Val Ala

1 5 10 15

Leu Ser Phe Ile Leu Leu Pro Asn Thr Arg Val Tyr Ala Thr Glu Asn

20 25 30

Thr Ala Asn Ile Pro Leu Ile Val Arg Gln Glu Phe Asn Val Tyr Thr

35 40 45

Lys Asp Ser Lys Ala Ile Asp Met Ile Gly Lys Tyr Glu Leu Lys Ala

50 55 60

Ile Ser Glu Asn Ala Pro Met Pro Glu Glu Ser Lys Asn Gly Ser Phe

65 70 75 80

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

85 90 95

Tyr Thr His Gly Gly Val Tyr Ile Tyr Gln Ile Gln Gln Ile Thr Gln

100 105 110

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

115 120 125

Tyr Val Lys Asn Ala Glu Asn Asn His Leu Ile Pro Gln Ile Ile Val

130 135 140

Lys Asn Glu Asn Asn Glu Lys Cys Glu Glu Ile Cys Phe Tyr Asn Ile

145 150 155 160

Tyr Lys Gln Lys Asn Lys Ile Asn Glu Ile Ser Lys Thr Pro Tyr Lys

165 170 175

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

180 185 190

Phe Tyr Ile Val Ile Leu Ile Ile Ser Leu Gly Leu Leu Val Val Leu

195 200 205

Lys Trp Lys Glu Tyr Lys Lys Arg Lys Lys Glu

210 215

<210>13

<211>680

<212>PRT

<213> Artificial sequence

<220>

<223> His-tagged expressed cnaA

<400>13

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser HisMet Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Ser Ser Ile Gln Arg Gly Arg Asp Ile Ser Asn Glu

35 40 45

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

50 55 60

Asn Val Gln Val Arg Leu Glu Phe Lys Glu Asn His Gln Arg Asn Ile

65 70 75 80

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

85 90 95

Phe Phe Glu Gly Tyr Glu Lys Thr Ile Pro Leu Tyr Ile Lys Asp Gln

100 105 110

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

115 120 125

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

130 135 140

Thr Leu Gln Gly Arg Asn Ile Thr Ser Gly Asn His Glu His Thr Gly

145 150 155 160

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

165 170 175

Lys Pro Glu Ser Gly Thr Thr Ser Val Phe Tyr Tyr Lys Thr Gly Ser

180 185 190

Met Tyr Thr Asn Asp Thr Asn His Val Asn Trp Trp Leu Leu Val Asn

195 200 205

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

210 215 220

Gln Gly Gly Gln Thr Leu Glu Pro Asp Ser Phe Glu Ile Val Val Thr

225 230 235 240

Trp Tyr Asp Gly Tyr Val Glu Lys Phe Lys Gly Lys Glu Ala Ile Arg

245 250 255

Glu Phe His Asn Lys Tyr Pro Asn Ser Asn Ile Ser Val Ser Glu Asn

260 265 270

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

275 280 285

Asn Ile Phe Tyr Lys Thr Lys Ile Thr Asn Pro Lys Gln Lys Glu Phe

290 295 300

Val Asn Asn Thr Lys Ala Trp Phe Lys Glu Tyr Asn Lys Pro Ala Val

305 310 315 320

Asn Gly Glu Ser Phe Asn His Ser Val Gln Asn Ile Asn Ala Asp Ala

325 330 335

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

340 345 350

Lys Asp Lys Ser Ile Pro Ile Lys Asp Val Gln Phe Lys Met Arg Arg

355 360 365

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

370 375 380

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

385 390 395 400

Tyr Glu Val Lys Glu Ile Ser Ala Pro Glu Trp Ile Ala Phe Asn Pro

405 410 415

Leu Ile Ala Pro Lys Leu Glu Phe Thr Ile Ser Asp Gln Asp Thr Glu

420 425 430

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

435 440 445

Val Glu Lys Val Trp Val Gly Gln Thr Ser Glu Arg Ala Glu Ile Lys

450 455 460

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

465 470 475 480

Asn Asn Trp Lys His Thr Phe Glu Asn Lys Pro Glu Tyr Asn Ser Glu

485 490 495

Thr Lys Gln Lys Ile Asn Tyr Ser Val Ser Glu Thr Thr Ile Ser Gly

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Asp Asp Arg Tyr Lys Lys Glu Ser Ile Lys Pro Ser Asp Gly Glu Ile

580 585 590

Thr Phe Ile Glu Gly Lys Ala Thr Ile Thr Leu Ser His Gly Gln Glu

595 600 605

Ile Thr Ile Lys Asp Leu Pro Tyr Gly Val Thr Tyr Lys Val Met Glu

610 615 620

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

625 630 635 640

Glu Val Thr Thr Gly Glu Leu Lys Gln Asp Thr Lys Val Gln Val Val

645 650 655

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

660 665 670

Leu Glu His His His His His His

675 680

<210>14

<211>349

<212>PRT

<213> Artificial sequence

<220>

<223> His-tagged expressed fimA

<400>14

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Ser Asn Leu Pro Ser Gly Gly Val Glu Gly Thr Glu

35 40 45

Gln Asn Pro Ala Lys Ala Thr Ile Thr Lys Asn Phe Glu Phe Pro Glu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Glu Ile Thr Phe Gly Asn Phe Pro His Ala Gly Glu Tyr Asp Tyr

115 120 125

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

130 135 140

Lys Glu Tyr Lys Val His Val Tyr Val Ala Asn Ser Asn Ala Met Asp

145 150 155 160

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

165 170 175

Ala Pro Ile Glu Phe Val Asn Thr Tyr Lys Lys Asp Thr Ser Leu Leu

180 185 190

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

195 200 205

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

210 215 220

Phe Glu Gly Asn Ile Ile Arg Lys Asp Gly Lys Ile Glu Pro Val Thr

225 230 235 240

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

245 250 255

Leu Lys Phe Glu Ser Ile Pro Ala Gly Thr Lys Tyr Glu Val Lys Glu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Lys Val Thr Phe Thr Asn Thr Tyr Asn Asp Lys Pro Ile Thr Val Asp

325 330 335

Lys Leu Ala Ala Ala Leu Glu His His His His His His

340 345

<210>15

<211>207

<212>PRT

<213> Artificial sequence

<220>

<223> His-tagged expressed fimB

<400>15

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Thr Glu Asn Thr Ala Asn Ile Pro Leu Ile Val Arg

35 40 45

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

50 55 60

Gly Lys Tyr Glu Leu Lys Ala Ile Ser Glu Asn Ala Pro Met Pro Glu

65 70 75 80

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

85 90 95

Gln Phe Thr Ile Pro Leu Ala Tyr Thr His Gly Gly Val Tyr Ile Tyr

100 105 110

Gln Ile Gln Gln Ile Thr Gln Ser Lys Asp Asn Tyr Ile Tyr Asp Lys

115 120 125

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

130 135 140

Leu Ile Pro Gln Ile Ile Val Lys Asn Glu Asn Asn Glu Lys Cys Glu

145 150 155 160

Glu Ile Cys Phe Tyr Asn Ile Tyr Lys Gln Lys Asn Lys Ile Asn Glu

165 170 175

Ile Ser Lys Thr Pro Tyr Lys Pro Asn Gly Ile Asn Val Pro Lys Thr

180 185 190

Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His

195200 205