JP2014087340A - Novel gene and protein of brachyspira hyodysenteriae and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel gene derived from B. hyodysenteriae and a protein coded by the gene, and to provide use of the gene and the protein coded by the gene for treatment and diagnosis of swine dysentery.SOLUTION: Provided are a novel gene of Brachyspira hyodysenteriae that is pathogenic bacteria of swine dysentery, a protein coded by the gene, use of the gene and the protein coded by the gene for diagnosis of B. hyodysenteriae-related diseases or vaccine against B. hyodysenteriae, and for screening of chemical compounds that sterilize B. hyodysenteriae or inhibit pathogenic effect of B. hyodysenteriae.

Description

  The present invention relates to a novel gene of Brachyspira hyodysenteria and a protein encoded thereby. The present invention further provides the B. of these novel genes and proteins. Diagnosis of hyodysenteriasis and B. B. Use for vaccines against hyodysenteriae; B. kill the hyodysenteria It also relates to the use in screening for compounds that inhibit the pathogenic effects of hyodysenteria. In addition, these sequences are also available in other Brachyspira species (B. suanatina, B. intermedia, B. alvinipulli, B. aalborgi, B. innocens, B. innocens). It may also be useful for diagnostic, therapeutic and / or prophylactic treatment of animal diseases caused by murdochii, B. pilosicoli, and the like.

  Swine dysentery is a serious endemic disease of pigs in Australia and around the world. Porcine dysentery is a contagious mucosal hemorrhagic diarrheal disease characterized by extensive inflammation and necrosis of the colon epithelial surface. Economic losses due to swine dysentery are mainly due to growth delays, treatment costs, and death. In 1971, an anaerobic spirochete (Treponema hyodysenteria) was first identified as the causative agent of swine dysentery. It was again assigned to the genus Brachyspira as a hyodysenteria. If swine dysentery develops in a pig farm, the range of disease can be mild, transient or obscure, severe, and even fatal. Clinical strategies may be hidden by treatment strategies in individual pig farms, and in some pig farms, swine dysentery may not be noticed or only suspected. An obvious disease is about to develop. Hyodysenteria can persist in infected pigs, other reservoir hosts (eg, rodents), or the environment. All these sources have the potential to transmit swine dysentery to uninfected herds. In addition, B. Although hyodysenteria can colonize, it is not clear how this usually occurs in field conditions.

  B. Since hyodicenteria colonization elicits a strong immunological response to spirochetes, indirect evidence of exposure to spirochetes can be obtained by measuring circulating antibody titers in infected animals. It has been reported that this antibody titer is maintained at a low level even in animals recovered from swine dysentery. Thus, serological tests for detecting antibodies can detect recovered carrier pigs with considerable potential and with asymptomatic infection and an undetectable number of spirochetes in the large intestine. Such a test would be particularly beneficial in an easy-to-use kit format (eg, an enzyme linked immunosorbent assay). B. Various techniques have been developed to demonstrate the presence of circulating antibodies against hyodysenteria, including indirect fluorescent antibody tests, hemagglutination tests, microtiter aggregation tests, complement binding tests, lipopolysaccharides or total An ELISA using a sonicated spirochete as an antigen can be mentioned. All these tests are problematic in terms of specificity as the relevant non-pathogenic intestinal spirochetes can induce cross-reactive antibodies. These tests are useful for detecting herds with obvious disease and high circulating antibody titers, but are problematic in identifying asymptomatic herds and individual infected pigs. Therefore, B.I. To date, no sufficiently sensitive and specific assay for detecting antibodies against hyodysenteria has been obtained. The lack of suitable diagnostic tests has hindered control of swine dysentery.

  Numerous methods are used to control swine dysentery, ranging from using antimicrobials prophylactically to reducing the number of infected herds and preventing the reentry of infected carrier pigs. It is. All of these options are expensive and, if they should be sufficiently effective, use advanced diagnostic tests to monitor their progress. Currently, the detection of swine dysentery in herd of asymptomatic infection and in individual healthy carrier animals remains a major problem and hinders effective control measures. For the definitive diagnosis of swine dysentery, B.D. Isolation and identification of hyodysenteria is necessary. The major problem with this is that such anaerobic bacteria are slow growing, have a biased nutritional requirement, and there are spirochetes that are morphologically similar to the resident flora of the intestines of pigs. Can be confusing. The diagnosis of individual affected pigs has been greatly improved by the development of polymerase chain reaction (PCR) assays to detect stool-derived spirochetes. Unfortunately, in practice, however, carrier animals with asymptomatic infection could not be detected due to the detection limit of PCR. Because of these diagnostic problems, B.B. There is clearly a need to develop a simple and effective diagnostic tool that can detect hyodysenteria infection.

  B. After colonization of hyodysenteria, a strong immunological response is induced against spirochetes, and pigs recovered from swine dysentery are protected from reinfection. Nonetheless, attempts to develop a vaccine to control swine dysentery have been largely unsuccessful because of the lack of protection on a herd basis or because it is too expensive and commercially viable. It is difficult to produce as possible. Although protected to some extent by bacterin vaccines, it is necessary to use multivalent bacterins because bacterin vaccines tend to be lipopolysaccharide serotype specific. Furthermore, large-scale production is difficult and expensive due to the spurious and anaerobic growth requirements of spirochetes.

  Several attempts have been made to develop live attenuated vaccines for swine dysentery. This approach is disadvantageous in that the immunostimulation is reduced in the case of attenuated strains due to reduced colony formation. In addition, in the case of live swine dysentery vaccine, there is a possibility of reversion to pathogenicity (particularly because little is known about genetic regulation and structure in B. hyodysenteria), producers and veterinarians He also hesitates to use it.

  The use of recombinant subunit vaccines is an attractive option because the product is well defined (essential for registration purposes) and may be relatively easy to produce on a large scale. To date, B. The first report using a recombinant protein from hyodysenteria as a vaccine candidate (38 kilodalton flagellar protein) failed to prevent colonization in pigs. This failure is specifically related to the specific recombinant protein used, as well as other downstream issues such as delivery system and route, administration rate, choice of adjuvant, etc. ( Non-patent document 1). Partially protected recombinant B. used for vaccination The first reported as a hyodysenteria protein was a 29.7 kDa outer membrane lipoprotein (Bhlp29.7, also referred to as “BmpB” or “BlpA”) that has homology to methionine-binding lipoproteins of various pathogens. It was. This his-tagged recombinant Bhlp29.7 protein was used for vaccination of pigs. Experimental challenge with hyodysenteria resulted in 50-70% of non-vaccinated control pigs versus 17-40% of vaccinated pigs Met. Since the incidence of Bhlp29.7 vaccinated pigs was significantly lower (P = 0.047) compared to control pigs, it was found that Bhlp29.7 might be a porcine dysentery vaccine component (Non-Patent Document 2) ). B. could be used as a recombinant vaccine component. Many other attempts have been made to identify coat proteins from hyodysenteria, but none of the vaccines have yet been successful. B. A more global approach is needed to identify potentially useful immunogenic recombinant proteins from hyodysenteria.

  To date, only one example of research using DNA for vaccination has been reported. In this study, the B. cerevisiae encoding putative ferritin. The hyodysenteria ftnA gene was cloned into an E. coli plasmid and the plasmid DNA was used to coat gold beads for ballistic vaccination. A mouse model of swine dysentery was used to confirm the protective properties of vaccination with DNA and / or recombinant protein. In the case of vaccination with recombinant protein, a good systemic response to ferritin was induced, whereas in the case of vaccination with DNA only a detectable systemic response was induced. When boosted with recombinant protein after vaccination with DNA, a systemic immune response to ferritin was induced only after boosting with protein. However, for any vaccination regimen tested, B. There was no protection against hyodysenteria colonization or related lesions. Interestingly, as a result of vaccination of mice with DNA alone, the disease was considerably exacerbated (Non-patent Document 3).

Gabe, JD, Chang, RJ, Slomiany, R, Andrews, WH and McCaman, MT (1995), of cytoplasmic proteins from Serpurina hyodysenteria B204 Isolation and molecular cloning of the flaB1 gene encoding a 38 kilodalton flagellar protein, Infection and Immunity 63: 142-148 La, T, Phillips, ND, Reichel, MP and Hampson, DJ (2004), recombinant BmpB (29.7 kDa outer membrane lipoprotein of Brachyspira hyodysenteria) Protection of swine from dysentery by vaccination used, Veterinary Microbiology 102: 97-109 Davis, A.D. J. et al. Smith, S .; C. And Moore, R .; J. et al. (2005), Brachyspira hyodysenteria ftnA gene: DNA vaccination and bacterial real-time PCR quantification in disease mouse model, Current Microbiology 50: 285-291

  The object of the present invention is to It is to provide a novel gene derived from hyodysenteria and a protein encoded by the gene. A further object of the present invention is that this novel gene and the protein encoded by the gene can be used for therapeutic and diagnostic purposes. B. In vaccines against hyodysenteria, B. The gene and / or the protein can be used in the diagnosis of a hyodysenteria infection.

  The object of the present invention is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65 having a nucleotide sequence contained in It is to provide a hyodysenteria gene. Further, the object of the present invention is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65 with% identity of at least 95%, 90%, 85%, 80%, 75% and It is to provide a nucleotide sequence that can be 70% (ie any integer from 100 to 70). The present invention also includes SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65 nucleotide sequences and at least 95%, 90%, 85%, 80%, 75% and 70% of said sequences ( That is, a DNA vaccine or DNA immunogenic composition comprising a sequence that is the same as any integer of 100 to 70) is also included. The present invention further includes SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43. 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65, and at least 95%, 90%, 85%, 80%, 75% and 70% of the sequence (ie , Any integer from 100 to 70) including diagnostic sequences comprising DNA having the same sequence.

  Further, the object of the present invention is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, Providing a plasmid comprising DNA having the sequence of 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65; and SEQ ID NOs: 1, 3, 5, 7, 9 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 Prokaryotic and / or eukaryotic expression vectors comprising DNA having the sequences of 61, 63 and 65, and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, To provide a cell comprising a plasmid containing a DNA having the sequence of 3,45,47,49,51,53,55,57,59,61,63 and 65.

  The object of the present invention is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66 have a novel amino acid sequence. It is to provide a hyodysenteria protein. Other objects of the invention are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66 and at least 95%, 90%, 85%, 80%, 75% and 70% (ie , Any integer from 100 to 70) to provide proteins that are homologous. In addition, the object of the present invention is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66, or SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66 The vaccine or immunogenic composition comprises a protein having an amino acid sequence that is at least 95%, 90%, 85%, 80%, 75% and 70% (ie, any integer from 100 to 70) homologous to the contained sequence. That is. Further embodiments of the present invention are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66, or SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 , 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66 To provide a diagnostic kit comprising one or more proteins having a sequence that is at least 95%, 90%, 85%, 80%, 75% and 70% homologous to the sequence.

  Other aspects of the invention include SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, Encodes a protein having the amino acid sequence contained in 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66; and SEQ ID NOs: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 , 62, 64 and 66, a nucleotide sequence encoding an amino acid sequence that is at least 95%, 90%, 85%, 80%, 75% and 70% homologous (ie any integer from 100 to 70). Is to provide. The present invention also includes SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, Encodes a protein having the amino acid sequence contained in 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66; and SEQ ID NOs: 2, 4, 6, 8, 10, 12 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 DNA having a sequence encoding an amino acid sequence that is at least 95%, 90%, 85%, 80%, 75% and 70% (ie any integer from 100 to 70) homologous to the sequences contained in Including plasmid, eukaryotic and prokaryotic expression Compactors, and also encompasses DNA vaccine. Cells containing such plasmids and expression vectors are also encompassed by the present invention.

  The present invention relates to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, Binds to proteins having amino acid sequences contained in 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66, or SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 And a monoclonal antibody that binds to a protein that is at least 95%, 90%, 85%, 80%, 75% and 70% (ie, any integer from 100 to 70) homologous to the sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66 bind to a protein having an amino acid sequence included in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 , 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66 Diagnostic kits comprising monoclonal antibodies that bind to proteins that are at least 95%, 90%, 85%, 80%, 75% and 70% (ie, any integer from 100 to 70) homologous to the sequences to be included The With such a diagnostic kit, B. The presence of hyodysenteria can be detected. As mammals, any mammal or bird is preferable, and chicken, goose, duck, turkey, parakeet, dog, cat, hamster, gerbil, rabbit, ferret, horse, cow, sheep, pig, monkey and human are more preferable.

  The present invention also includes SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65, or at least 95%, 90%, 85%, 80%, 75 with said sequence % And 70% (i.e. any integer from 100 to 70) identical to the B.V. Also contemplated are methods of preventing or treating a hyodysenteria infection. The present invention also includes SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66, or at least 95%, 90%, 85%, 80%, 75% and 70 with said sequence % (Ie any integer from 100 to 70) in animals by administering a vaccine comprising one or more proteins having a sequence that is homologous. Also included are methods of preventing or treating a hyodysenteria infection. As mammals, any mammal or bird is preferable, and chicken, goose, duck, turkey, parakeet, dog, cat, hamster, gerbil, rabbit, ferret, horse, cow, sheep, pig, monkey and human are more preferable.

  The present invention also includes SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65, or at least 95%, 90%, 85%, 80%, 75 with said sequence Also contemplated are methods of generating an immune response in an animal by administering to the animal an immunogenic composition comprising a sequence that is% and 70% (ie, any integer between 100 and 70) identical. The present invention also includes SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66, or at least 95%, 90%, 85%, 80%, 75% and 70 with said sequence Also encompassed are methods of generating an immune response in an animal by administering to the animal an immunogenic composition comprising one or more proteins having a sequence that is% (ie any integer between 100 and 70) homologous. As mammals, any mammal or bird is preferable, and chicken, goose, duck, turkey, parakeet, dog, cat, hamster, gerbil, rabbit, ferret, horse, cow, sheep, pig, monkey and human are more preferable.

  As used herein, the article “a” or “an” means that the article has one or more grammatical objects (ie, at least one). By way of example, “an element” means one element or more than one element.

  “Amino acid” is meant to encompass all molecules, both natural and synthetic, that have both amino and acid functionalities and that can be included in polymers of naturally occurring amino acids. Examples of amino acids include naturally occurring amino acids, analogs and derivatives thereof, congeners, amino acid analogs having various side chains, and any of the above stereoisomers.

  The animal may be any mammal or bird. Examples of mammals include dogs, cats, hamsters, gerbils, rabbits, ferrets, horses, cows, sheep, pigs, monkeys, and humans. Examples of birds include chickens, geese, ducks, turkeys and parakeets.

  “Conserved residue” means an amino acid that is a member of a group of amino acids having certain common properties. By “conservative amino acid substitution” is meant a substitution (conceptually or otherwise) of one group of amino acids with another amino acid of the same group. A practical way to define common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of the same organism (Schulz, GE and RH). Schinner, Principle of protein structure, Springer-Verlag). According to such an analysis, a group of amino acids can be defined when the amino acids in a group are preferentially exchanged with each other and are therefore most similar to each other in their influence on the overall protein structure (Schulz ), GE and RH Schinner, Principle of Protein Structure, Springer-Verlag). Examples of amino acid groups thus defined include (i) a positively charged group including Lys, Arg and His, (ii) a negatively charged group including Glu and Asp, and (iii) an aromatic group including Phe, Tyr and Trp. (Vi) a nitrogen ring group comprising His and Trp; (v) a large aliphatic nonpolar group comprising Val, Leu and De; (vi) a weakly polar group comprising Met and Cys; (vii) Ser, Thr, Asp , Asn, Gly, Ala, Glu, Gln and Pro, a small residue group comprising (viii) an aliphatic group comprising Val, Leu, De, Met and Cys, and (ix) a small hydroxyl group comprising Ser and Thr. Can be mentioned.

  “Fusion protein” or “fusion polypeptide” means a chimeric protein as is known in the art, but can be constructed using methods known in the art. In many instances of fusion proteins, there are two different polypeptide sequences, but in some cases, there may be more polypeptide sequences. The polynucleotide sequence encoding the fusion protein can be operably linked in frame so that the fusion protein can be correctly translated. The fusion protein may comprise polypeptide sequences from the same species or from different species. In various embodiments, a fusion polypeptide can contain one or more amino acid sequences linked to a first polypeptide. Where more than one amino acid sequence is fused to the first polypeptide, the fusion sequence may be multiple copies of the same sequence or may be different amino acid sequences. The fusion polypeptide can be fused to the N-terminus, C-terminus, or N- and C-termini of the first polypeptide. Examples of fusion proteins include a polypeptide containing a glutathione S-transferase tag (GST-tag), a histidine tag (His-tag), an immunoglobulin domain or an immunoglobulin binding domain.

  By “isolated polypeptide” is meant, in one embodiment, a polypeptide prepared from recombinant DNA or RNA, a polypeptide of synthetic or natural origin, or some combination of said polypeptides. Whether the polypeptide is (1) unrelated to the protein normally found in nature, (2) separated from the cells in which it normally resides, or (3) free of other proteins from the same cell source (4) expressed by cells from different species, or (5) non-naturally occurring. An isolated polypeptide can exist but is not considered a purified polypeptide.

  “Isolated nucleic acid” and “isolated polynucleotide” are synthetically derived, whether they are polynucleotides derived from genomic DNA, cDNA, mRNA, tRNA, rRNA, iRNA or cellular organelles (eg, mitochondria and chloroplasts) A polynucleotide that is operatively linked to a polynucleotide that is (1) unrelated to cells in which the “isolated nucleic acid” is not found in nature, or (2) is not linked in nature. means. An isolated polynucleotide can exist but is not considered a purified polynucleotide.

  "Nucleic acid" and "polynucleotide" refer to a polymeric form of nucleotides and refer to ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide species. These terms include, as equivalents, analogs of RNA or DNA obtained from nucleotide analogs, and are applicable to the embodiments described herein as single stranded (eg, sense or It should also be understood to encompass antisense) polynucleotides and double stranded polynucleotides.

  “Nucleic acid of the present invention” and “polynucleotide of the present invention” mean a nucleic acid encoding the polypeptide of the present invention. The polynucleotide of the present invention comprises a nucleic acid sequence of the present invention; at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the nucleic acid sequence of the present invention (ie, 60-100). Nucleotide sequences that are identical; nucleotide sequences that hybridize with the nucleic acid sequences of the invention under stringent conditions; nucleotide sequences that encode polypeptides functionally equivalent to the polypeptides of the invention; amino acid sequences of the invention Nucleotides encoding polypeptides that are homologous or identical to at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% (ie any integer between 60 and 100) Sequence; having the activity of a polypeptide of the present invention and at least about 60%, 70%, 80%, 85%, 90% to the amino acid sequence of the present invention , 95%, 98%, 99% or more (ie any integer from 60 to 100) homologous or identity-encoding nucleotide sequence; 1 to about 2, 3, 5, 7, 10, 15 A nucleotide sequence (eg, allelic variant) that differs from the nucleic acid sequence of the present invention by more than 20, 30, 50, 75 or more nucleotide substitutions, additions or deletions; nucleic acids derived from the nucleic acid sequence of the present invention and evolutionarily related; and All of the above and other nucleic acids of the present invention may include all or part of the nucleotide sequence resulting from the degeneracy of the genetic code and its complement. The nucleic acid of the present invention also includes homologues (for example, orthologs and paralogs) of the nucleic acid sequence of the present invention, and the present invention is codon-optimized for expression in a specific living body (for example, host cell). Also encompassed are variants of the nucleic acid sequence.

  “Operationally linked” when describing the relationship between two nucleic acid regions means juxtaposition where the regions are in a relationship that allows them to function as intended. For example, when an appropriate molecule (eg, an inducer or polymerase) is conjugated to a control sequence “operably linked” to a coding sequence, the coding sequence is expressed under conditions that are compatible with the control sequence. To a regulatory (or regulatory) sequence.

  “Polypeptide”, “protein” and “peptide” are used interchangeably herein and refer to a polymer of amino acids. Examples of polypeptides include gene products and naturally occurring proteins, homologues of the above, orthologs, paralogs, fragments, other equivalents, variants and analogs.

  A “polypeptide fragment” or “fragment”, when used for a reference polypeptide, lacks amino acid residues compared to the reference polypeptide itself, but the remaining amino acid sequence is usually Means a polypeptide that is identical to the corresponding position. Such deletions can occur at the amino terminus or the carboxy terminus of the reference polypeptide, or both. Fragments are usually at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 amino acids long It is. Fragments can retain one or more of the biological activities of the reference polypeptide. In certain embodiments, the fragment may comprise a domain having the desired biological activity, optionally including additional amino acids on one or both sides of the domain, the number of additional amino acids being 5, It can be 10, 15, 20, 30, 40, 50 or up to 100 or more residues. Furthermore, a fragment may contain a sub-fragment of a specific region, but this sub-fragment retains the function of the region of origin. In other embodiments, the fragment may have immunogenic properties.

  The “polypeptide of the present invention” means a polypeptide containing the amino acid sequence of the present invention or an equivalent or fragment thereof. The polypeptide of the present invention includes a polypeptide comprising all or part of the amino acid sequence of the present invention; the polypeptide of the present invention includes the amino acid sequence of the present invention; 1 to about 2, 3, 5, 7, 10, An amino acid sequence of the present invention having 15, 20, 30, 50, 75 or more conservative amino acid substitutions; an amino acid sequence of the present invention and at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% (ie any integer between 60 and 100) homologous amino acid sequences; and functional fragments thereof. The polypeptide of the present invention also includes homologues of the amino acid sequence of the present invention (for example, orthologs and paralogs).

  In addition, the structure of the polypeptide of the present invention can be modified for the purpose of enhancing therapeutic or prophylactic efficacy and stability (for example, ex vivo effective period and in vivo proteolytic resistance). Such modified polypeptides are considered “functional equivalents” of the polypeptides described in more detail herein if they are designed to retain at least one activity of the naturally occurring form of the protein. It is done. Such modified polypeptides can be produced, for example, by amino acid substitutions, deletions or additions, which substitutions can be composed in whole or in part by conservative amino acid substitutions.

  For example, isolated conservative amino acid substitutions (eg, replacement of leucine with isoleucine or valine, replacement of aspartic acid with glutamic acid, replacement of threonine with serine) would not significantly affect the biological activity of the resulting molecule. It makes sense to expect. Whether the amino acid sequence of the polypeptide is changed to obtain a functional homolog can be easily confirmed by evaluating that the mutant polypeptide can produce a response similar to the wild-type protein. Polypeptides with more than one substitution can be readily tested in the same manner.

  “Purify” means obtaining a target species that is the predominant species present (ie, species that are abundant (on a molar basis) compared to any other species in the composition). A “purified fraction” is a composition that comprises at least about 50 percent (on a molar basis) of a target species of all species present. When checking the purity or species in a solution or dispersion, the solvent or matrix in which the species is dissolved or dispersed is usually not used for such confirmation; only the species in which the species is dissolved or dispersed (the target Including). In general, a purified composition has more than about 80% of all species present in the composition, or one that accounts for more than about 85%, 90%, 95%, 99% or more of all species present. The target species can be purified until it is essentially homogeneous (ie, contaminant species cannot be detected in the composition by conventional detection methods), but the composition is essentially a single species. . One of ordinary skill in the art, in light of the teachings herein, can purify the polypeptides of the present invention using standard techniques for protein purification. The purity of the polypeptide can be determined by a number of methods known to those skilled in the art, and examples of the method include amino terminal amino acid sequence analysis, gel electrophoresis, mass spectrometry, and the methods described herein.

  “Recombinant protein” or “recombinant polypeptide” means a polypeptide produced by recombinant DNA techniques. As an example of such a technique, a DNA or a polypeptide encoded by the DNA may be produced by inserting a DNA encoding the expressed protein into a suitable expression vector and using it to transform a host cell. Can be mentioned.

  “Regulatory sequence” is a general term used throughout this specification, and means a polynucleotide sequence such as an initiation signal, enhancer, regulator, promoter, etc., and a coding sequence or non-coding sequence to which they are operatively linked. Necessary or desirable to affect the expression of Examples of regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990), eg, SV40 early and late promoters, adenoviruses or sites Megalovirus early promoter, lac system, trp system, TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, major operator and promoter region of phage λ, regulatory region for fd coat protein, 3-phospho Promoter for glycerate kinase or other glycolytic enzymes, promoter of acid phosphatase (eg Pho5), promoter of yeast alpha mating factor, gene expression of prokaryotic cell or eukaryotic cell or its virus Polyhedron promoter of the baculovirus system and other sequences known to control the, and their various combinations. The nature and use of such control sequences can vary depending on the host organism. In prokaryotes, such regulatory sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence. “Regulatory sequence” means at least including components whose presence may affect expression, and may also include additional components whose presence is advantageous (eg, leader sequences and fusion partner sequences). In certain embodiments, transcription of the polynucleotide sequence is under the control of a promoter sequence (or other regulatory sequence) that controls the expression of the polynucleotide in the cell type intended for expression. It will also be appreciated that the polynucleotide may be under the control of regulatory sequences that are the same or different from the regulatory sequences that control the expression of naturally occurring polynucleotide forms.

  “Sequence homology” means the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. Where sequence homology is expressed as a percentage (eg, 50%), the percentage indicates the percentage of matches over the sequence length obtained by comparing the desired sequence with other sequences. Gap (in one of the two sequences) is allowed to maximize matching, usually a gap length of 15 bases or less is used, more frequently a gap length of 6 bases or less is used, and Frequently gap lengths of 2 bases or less are used. “Sequence identity” means that the sequences are identical over the window of comparison (ie, nucleotide-nucleotide basis for nucleic acids, or amino acid-amino acid basis for polypeptides). “Percentage of sequence identity” is a matched position by comparing two optimally aligned sequences over a window of comparison and confirming the number of positions where identical amino acids or nucleotides are present in both sequences. Is calculated by dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result obtained by 100 to obtain the percentage of sequence identity. Methods for calculating sequence identity are known to those skilled in the art and are described in more detail below.

  As used herein, “soluble” as used for a polypeptide or other protein of the present invention means that at the time of expression in cell culture, at least a portion of the expressed polypeptide or protein remains in the cytoplasmic fraction of the cell, and the lysate It means not fractionating with cell debris during lysis or centrifugation. Polypeptide solubility can be determined by various methods known in the art, such as fusion with heterologous amino acid sequences, deletion of amino acid residues, amino acid substitution (eg, amino acid residues having hydrophilic side chains in the sequence). Can be improved by chemical modification (eg, addition of hydrophilic groups).

  Polypeptide solubility can be determined by various techniques known in the art, such as dynamic light scattering to confirm aggregation, UV absorption, centrifugation to separate aggregates from non-aggregates, SDS gels. It can be measured by electrophoresis (eg, comparing the amount of protein in the soluble fraction to the amount of protein in the mixture of soluble and insoluble fractions). When expressed in a host cell, the polypeptides of the present invention are at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more is soluble (eg, at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% of the total amount of protein expressed in the cell) 80%, more than 90% are found in the cytoplasmic fraction). In certain embodiments, at least about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 milligrams by 1 L culture of cells expressing a polypeptide of the invention. These soluble proteins are produced. In an exemplary embodiment, the polypeptides of the invention are at least about 10% soluble and produce at least about 1 milligram of protein from a 1 L cell culture.

  “Specifically hybridize” means detectable and specific nucleic acid binding. The polynucleotides, oligonucleotides and nucleic acids of the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize a significant amount of detectable binding to non-specific nucleic acids. Stringent conditions can be used to obtain selective hybridization conditions known in the art and described herein. In general, the nucleic acid sequence identity between the polynucleotides, oligonucleotides and nucleic acids of the invention and the nucleic acid sequence of interest is at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% or more (ie any integer from 30 to 100). In certain instances, the hybridization and washing conditions are performed under stringent conditions in accordance with conventional hybridization procedures and further description herein.

  “Stringent conditions” or “stringent hybridization conditions” means conditions that promote specific hybridization between two complementary polynucleotide strands so as to form a double strand. . Stringent conditions can be selected to be about 5 ° C. lower than the melting point (Tm) of a given polynucleotide duplex at a given ionic strength and pH. The length of the complementary polynucleotide strand and its GC amount determine the Tm of the double strand, and thus the hybridization conditions necessary to obtain the desired hybridization specificity. Tm is a temperature (under a predetermined ionic strength and pH) at which 50% of the polynucleotide sequence hybridizes with a complementary strand that perfectly matches. In some cases, it may be desirable to increase the stringency of the hybridization conditions to be approximately equal to the Tm of a particular double strand.

  Various techniques can be used to estimate Tm. Normally, it is estimated that the GC base pair in the duplex contributes about 3 ° C. to Tm up to the theoretical maximum of about 80-100 ° C., whereas the AT base pair contributes about 2 ° C. It is estimated that.

  However, a more advanced Tm model can be used that takes into account GC stacking interactions, solvent effects, desired assay temperature, and the like. For example, the following formula: Td = (((3 × # GC) + (2 × # AT)) × 37) −562) / # bp) −5 (where #GC, #AT and #bp are respectively The number of guanine-cytosine base pairs involved in duplex formation, the number of adenine-thymine base pairs and the number of total base pairs), and the dissociation temperature (Td) is about 60 ° C. Can be designed.

  Hybridization should be performed in 5 × SSC, 4 × SSC, 3 × SSC, 2 × SSC, 1 × SSC or 0.2 × SSC for at least about 1 hour, 2 hours, 5 hours, 12 hours or 24 hours. Can do. The stringency of the reaction can be adjusted, for example, by raising the hybridization temperature from about 25 ° C. (room temperature) to about 45 ° C., 50 ° C., 55 ° C., 60 ° C. or 65 ° C. The hybridization reaction can also include other agents that affect stringency. For example, when hybridization is performed in the presence of 50% formamide, the stringency of hybridization at a predetermined temperature increases.

  Subsequent to the hybridization reaction, a single washing step or two or more washing steps can be performed, which can be performed at the same or different salinity and temperature. For example, stringency can be adjusted by increasing the temperature of the wash from about 25 ° C. (room temperature) to about 45 ° C., 50 ° C., 55 ° C., 60 ° C., 65 ° C. or higher. The washing step can be performed in the presence of a detergent (eg, 0.1 or 0.2% SDS). For example, following the hybridization, two washing steps (each step in 2 × SSC, 0.1% SDS for about 20 minutes) can be performed at 65 ° C., and if necessary, two further washing steps ( Each step can be performed at 65 ° C. in 0.2 × SSC, 0.1% SDS for about 20 minutes).

  Examples of stringent hybridization conditions include 50% formamide, 10 × Denhart solution (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin) and 200 μg / mL denatured carrier DNA ( For example, after overnight hybridization at 65 ° C. in a solution containing sheared salmon sperm DNA), two washing steps (each step is 2 × SSC, 0.1% SDS for about 20 minutes) It may be performed at 65 ° C., and further two washing steps (each step in 0.2 × SSC, 0.1% SDS for about 20 minutes) may be performed at 65 ° C.

  In hybridization, two kinds of nucleic acids in a solution may be hybridized, or nucleic acids in a solution may be hybridized with a nucleic acid attached to a solid support (for example, a filter). If there is one nucleic acid on the solid support, a prehybridization step can be performed prior to hybridization. Prehybridization can be performed at least about 1 hour, 3 hours or 10 hours in the same solution and temperature as the hybridization solution (in the absence of complementary polynucleotide strands).

  Suitable stringency conditions are known to those skilled in the art or can be determined experimentally by those skilled in the art. See, for example, current protocols in molecular biology, John Wiley & Sons, N.M. Y. (1989), 6.3.1-12.3.6; Sambrook et al., 1989, Molecular Cloning, Laboratory Manual, Cold Spring Harbor Press, N .; Y. S .; Edited by Agrawal, Methods in Molecular Biology, Volume 20; Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization Using Nucleic Acid Probes (eg, Part I Chapter 2 “ Overview of hybridization principles and strategies for nucleic acid probe assays ", Elsevier, New York; Tibanyenda, N. et al., Eur. J. Biochem. 139: 19 (1984) and Ebel, S. et al., Biochem. 31: 12083 (1992).

  “Vector” means a nucleic acid capable of transporting linked nucleic acids. One type of vector that can be used in the present invention is an episome (ie, a nucleic acid that enables extrachromosomal replication). Other vectors include vectors that allow self-replication and expression of linked nucleic acids. A vector capable of directing the expression of an operatively linked gene is referred to herein as an “expression vector”. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids, but “plasmid” means a circular double-stranded DNA molecule that is not linked to a chromosome in vector form. In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to encompass other forms of expression vectors that have equivalent functions and then become known in the art.

  Using the nucleic acid of the present invention as a diagnostic reagent, the presence or absence of a target DNA or RNA sequence to which the nucleic acid specifically binds can be detected, and for example, the expression level of the nucleic acid of the present invention can be determined. In one aspect, the invention is a method for detecting the presence of a nucleic acid of the invention or part thereof in a sample, comprising: (a) at least 8 complementary to a part of the nucleic acid of the invention A step of preparing an oligonucleotide having a nucleotide length; and (b) a sample containing at least one nucleic acid in the oligonucleotide under conditions that allow hybridization between the oligonucleotide and the nucleic acid of the present invention or a part thereof. And (c) detecting the hybridization between the oligonucleotide and the nucleic acid in the sample, and detecting the presence of the nucleic acid of the present invention or a part thereof in the sample. Intended. In another aspect, the invention provides a method for detecting the presence of a nucleic acid of the invention or a part thereof in a sample, wherein (a) each is complementary to the sequence of the nucleic acid of the invention. Providing a pair of single-stranded oligonucleotides having an oligonucleotide length of at least 8 nucleotides, the sequence complementary to the oligonucleotide being at least 10 nucleotides away from each other; and (b) the oligonucleotide under hybridization conditions Contacting the nucleotide with a sample containing at least one nucleic acid; (c) amplifying the nucleotide sequence between the two oligonucleotide primers; and (d) detecting the presence of the amplified sequence; A method performed by detecting the presence of the nucleic acid of the present invention or a part thereof in a sample is contemplated.

  In another aspect of the invention, the polynucleotide of the invention is inserted into an expression vector that includes a nucleotide sequence that encodes a polypeptide of the invention and is operably linked to at least one regulatory sequence. Of course, the design of an expression vector can depend on factors such as the choice of host cells to transform and / or the type of protein desired to be expressed. It is necessary to consider the copy number of the vector, the ability to control the copy number, and the expression of any other protein encoded by the vector (eg, an antimicrobial marker).

  B. using an expression vector containing the polynucleotide of the present invention as a drug; Animals infected with hyodysenteria can be treated, or used as a vaccine (and also as a drug), It can be prevented from infection with hyodysenteria, and when an animal is infected, the symptoms and progression of the disease can be suppressed. One method of using an expression vector as a drug is to administer a nucleic acid vaccine to an animal at risk of infection or an infected animal. Nucleic acid vaccine technology is well described in the art. Some of the descriptions include US Pat. No. 6,562,376 (Hooper et al.), US Pat. No. 5,589,466 (Felgner et al.), US Pat. No. 6,673,776 ( Felgner et al.) And US Pat. No. 6,710,035 (Felgner et al.). Nucleic acid vaccines can be injected intramuscularly or intradermally, and animal electroporation (see WO01 / 23537 (King et al., And WO01 / 68889 (Malone et al.)) Is a lipid composition. (See US Pat. No. 5,703,055 (Felgner et al.)) Or by other mechanisms known in the art.

  Alternatively, an expression vector can be transfected into a bacterium and the bacterium can be administered to a target animal to induce an immune response against the protein encoded by the nucleotide of the present invention contained in the expression vector. Expression vectors can contain eukaryotic expression sequences so that the nucleotides of the invention are transcribed and translated in the host animal. Alternatively, the expression vector can be transcribed in bacteria and then translated in the host animal. Bacteria used as carriers of expression vectors need to be invasive even when attenuated. To name a few, Shigella species, Salmonella species, Escherichia coli species, and Aeromonas species that are still invasive when attenuated can be used. Examples of such methods are US Pat. No. 5,824,538 (Branstrom et al.), US Pat. No. 5,877,159 (Powell et al.), US Pat. No. 6,150, 170 (Powell et al.), US Pat. No. 6,500,419 (Hone et al.), And US Pat. No. 6,682,729 (Powell et al.).

  Alternatively, the polynucleotides of the present invention can be inserted into certain viruses that act as vectors. Viral vectors can express the protein of the invention on the surface of the virus or transport the polynucleotide to animal cells where the polynucleotide of the invention is transcribed and translated into protein. Animals infected with a viral vector can exhibit an immune response against the protein encoded by the polynucleotide of the invention. Thus, in animals infected with viral vectors, Infectious diseases caused by hyodysenteria can be reduced or prevented. Examples of viral vectors include US Pat. No. 5,283,191 (Morgan et al.), US Pat. No. 5,554,525 (Sondermeijer et al.), And US Pat. No. 5,712,118. (Murphy) can be found.

  The polynucleotide of the present invention can be used to cause expression and overexpression of the polypeptide of the present invention in cells grown in culture (for example, production of proteins and polypeptides such as fusion proteins and polypeptides). Can be made).

  The present invention relates to a host cell transfected with a recombinant gene to express the polypeptide of the present invention. The host cell can be any prokaryotic or eukaryotic cell. For example, the polypeptides of the present invention can be expressed in bacterial cells (eg, E. coli), insect cells (baculovirus), yeast, plant cells or mammalian cells. In such instances, when the host cell is a human cell, it may or may not be present in a live subject. Other suitable host cells are known to those skilled in the art. In addition, tRNA molecules not normally found in the host can be added to the host cell to optimize polypeptide expression. Alternatively, the nucleotide sequence can be altered to optimize expression in the host cell, but the protein produced will still have a high homology to the originally encoded protein. Other methods suitable for maximizing polypeptide expression will be known to those skilled in the art.

  The invention further relates to a method for producing the polypeptides of the invention. For example, host cells transfected with an expression vector encoding a polypeptide of the invention can be cultured under appropriate conditions to cause expression of the polypeptide. The polypeptide can be secreted and isolated from a mixture of media and cells containing the polypeptide. Alternatively, the polypeptide can be retained in the cytoplasm, the cells can be recovered, lysed, and the protein isolated.

  Cell culture media includes host cells, media and other by-products. Suitable media for cell culture are well known in the art. Protein purification techniques known in the art (for example, ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using an antibody specific for a specific epitope of the polypeptide of the present invention, etc. ) Can be used to isolate a polypeptide from cell culture media or host cells, or both.

  Thus, recombinant proteins can be produced by microbial or eukaryotic processes using nucleotide sequences encoding all or selected portions of the polypeptides of the invention. Ligating sequences into polynucleotide constructs (eg, expression vectors) and transforming or transfecting hosts (eukaryotes (yeasts, birds, insects or mammals) or prokaryotes (bacterial cells)). Is a standard procedure. Similar procedures or variations thereof can be used to prepare the recombinant polypeptides of the invention by microbial means or tissue culture techniques.

  Vectors suitable for expression of the polypeptides of the present invention include the following types of plasmids: pTrcHis-derived plasmids for expression in prokaryotic cells (eg, E. coli), pET-derived plasmids, pBR322-derived plasmids, pEMBL-derived plasmids, Examples include pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids. Various methods used for plasmid preparation and host organism transformation are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, and general recombination procedures, see Molecular Cloning, Laboratory Manual, Second Edition, Sambrook, Fritsch and Maniatis. (Maniatis) (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17.

  The coding sequence of the polypeptide of interest can be incorporated as part of a fusion gene comprising a nucleotide sequence that encodes a different polypeptide. The invention comprises a nucleic acid of the invention and at least one heterologous sequence encoding a heterologous peptide linked in-frame with the nucleotide sequence of the nucleic acid of the invention to encode a fusion protein comprising the heterologous polypeptide. Is intended to be an isolated polynucleotide. The heterologous polypeptide can be fused to (a) the C-terminus of the polypeptide of the invention, (b) the N-terminus of the polypeptide of the invention, or (c) the C-terminus and N-terminus of the polypeptide of the invention. . In certain instances, the heterologous sequence encodes a polypeptide that allows detection, isolation, solubilization and / or stabilization of the polypeptide to be fused. In yet another embodiment, the heterologous sequence is a poly His tag, myc, HA, GST, protein A, protein G, calmodulin binding peptide, thioredoxin, maltose binding protein, polyarginine, poly His-Asp, FLAG, immunoglobulin. It encodes a part of a protein, a polypeptide such as a transcellular transport peptide.

  Fusion expression systems can be useful when production of immunogenic fragments of the polypeptides of the invention is desired. For example, rotavirus VP6 capsid protein can be used as an immunological carrier protein for a polypeptide moiety in monomeric or viral particle form. A nucleic acid sequence corresponding to a polypeptide portion of the invention that results in the production of an antibody is incorporated into a fusion gene construct comprising a coding sequence for a late vaccinia virus structural protein to produce a fusion protein comprising a portion of the protein as part of a virion. A set of recombinant viruses can be produced that are expressed. Hepatitis B surface antigen can also be used for this role. Similarly, a chimeric construct encoding a fusion protein comprising a portion of a polypeptide of the invention and a poliovirus capsid protein can be produced to enhance immunogenicity (eg, EP Publication No. 0259149; Evans). (1989) Nature 339: 385; Huang et al. (1988) J. Virol. 62: 3855; and Schlienger et al. (1992) J. Virol. 66: 2).

Fusion proteins can facilitate protein expression and / or purification. For example, the polypeptide of the present invention can be produced as a glutathione S-transferase (GST) fusion protein. Such GST fusion proteins can be used to simplify the purification of the polypeptides of the invention, for example by using a glutathione derivatized matrix (eg, current protocols in molecular biology, Ausubel et al. Ed. (N.Y .: John Wiley & Sons, 1991). In other embodiments, a fusion gene encoding a purified leader sequence, such as a poly (His) / enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, used a Ni ²⁺ metal resin. Affinity chromatography may allow purification of the expressed fusion protein. The purified leader sequence can then be removed by treatment with enterokinase to obtain a purified protein (eg, Hochuli et al., (1987) J. Chromatography 411: 177; and Janknecht et al., PNAS USA 88: 8972).

  Techniques for producing fusion genes are well known. In essence, ligation of various DNA fragments encoding various polypeptide sequences consists of blunt or twisted ends for ligation, restriction enzyme digestion to provide appropriate ends, and filling-in as necessary. ), Alkaline phosphatase treatment to avoid undesired ligation, and enzymatic ligation. In other embodiments, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that cause complementary overhangs between two consecutive gene fragments, followed by annealing the consecutive gene fragments to produce a chimeric gene sequence. (See, eg, current protocols in molecular biology, edited by Ausubel et al., John Wiley & Sons: 1992).

  In other embodiments, the present invention provides solid surfaces (eg, plates, microtiter plates, slides, beads, particles, spheres, films, strands, precipitates, gels, sheets, tubes, containers, capillaries, pads, slices. Etc.) The nucleic acid of the present invention immobilized thereon is provided. The nucleic acid of the present invention can be immobilized on a chip as part of the array. The array can contain one or more of the polynucleotides of the invention described herein. In one embodiment, the chip includes one or more polynucleotides of the invention as part of an array of polynucleotide sequences from the same pathogenic species as the polynucleotide of the invention.

  In a preferred form of the invention, the isolated B.M. In addition to providing a hyodysenteria polypeptide, a polynucleotide sequence encoding the polypeptide is also provided. B. More preferably, the hyodysenteria polypeptide is provided in a substantially purified form.

  Preferred polypeptides of the present invention have one or more biological properties (eg, in vivo, in vitro or immunological properties) of natural full-length polypeptides. Non-functional polypeptides are also encompassed within the scope of the invention because they can be useful, for example, as antagonists of functional polypeptides. The biological properties of analogs, fragments or derivatives relative to the wild type can be confirmed, for example, by biological assays.

  Polypeptides (including analogs, fragments and derivatives) can be prepared synthetically (eg, using well-known techniques of solid phase or liquid phase peptide synthesis). It is preferred to use solid phase synthesis techniques. Alternatively, the polypeptides of the present invention can be prepared using well-known genetic engineering techniques described below. In still other embodiments, the polypeptide may be a biological fluid (eg, but not limited to animal-derived plasma, stool, serum, urine. Examples of animals include pigs, chickens, geese, etc.) , Ducks, turkeys, parakeets, humans, monkeys, dogs, cats, horses, hamsters, gerbils, rabbits, ferrets, horses, cows, sheep, etc. can do. The animal may be any mammal or bird.

  B. Hyodysenteria polypeptide analogs include polypeptides having an amino acid sequence in which one or more amino acids are replaced by other amino acids, but the biological activity of the molecule is not substantially altered by the substitution. It is done.

  According to the present invention, an antibody that recognizes the polypeptide using the polypeptide of the present invention produced by recombinant or chemical synthesis, and fragments or other derivatives or analogs thereof (such as fusion proteins) as an immunogen. Can be manufactured.

  A molecule is “antigenic” if it can specifically interact with an antigen recognition molecule of the immune system (eg, an immunoglobulin (antibody) or a T cell antigen receptor). The antigen amino acid sequence includes at least about 5, preferably at least about 10 amino acids. The antigenic part of the molecule can be an immunodominant part for antibody or T cell receptor recognition, or the part used to produce an antibody against the molecule by conjugating the antigenic part to an immunization carrier molecule It can be. Antigenic molecules need not be immunogenic per se (ie, capable of eliciting an immune response without a carrier).

By “antibody” is meant any immunoglobulin (including antibodies and fragments thereof) that binds to a specific epitope. “Antibodies” include polyclonal and monoclonal antibodies, chimeric antibodies, those further described in US Pat. Nos. 4,816,397 and 4,816,567, and antigen binding portions of antibodies (eg, Fab And F (ab ′) ₂ , F (v) (including single chain antibodies)). Thus, an “antibody molecule” as used herein in various grammatical forms is intended to be an intact immunoglobulin molecule as well as an immunologically active portion of an immunoglobulin molecule that includes an antibody binding site. An “antibody binding site” is a structural part of an antibody molecule that specifically binds to an antigen and consists of a heavy chain variable region, a light chain variable region and a hypervariable region.

Examples of antibody molecules include intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and portions of immunoglobulin molecules containing paratopes (eg, Fab, Fab ′, F (ab ′) ₂ , F (v ), Which are known in the art, such a portion is preferably used in the therapeutic methods described herein.

The Fab and F (ab ′) ₂ portions of an antibody molecule are each prepared by the proteolytic reaction of papain and pepsin on a substantially intact antibody molecule by well-known methods. See, for example, US Pat. No. 4,342,566 (Theofilopolous et al.). Fab ′ antibody molecules are also well known. After production from the F (ab ′) ₂ moiety, the disulfide bond linking the two heavy chain moieties is reduced with mercaptoethanol, and the resulting protein mercaptan is then obtained. Is alkylated with a reagent such as iodoacetamide. In the present invention, an antibody containing an intact antibody molecule is preferred.

  “Monoclonal antibody” means an antibody having only one kind of antibody-binding site capable of immunoreaction with a specific antigen, although it may be described in a grammatically different form of expression. Thus, a monoclonal antibody usually displays a single binding affinity for any antigen with which it immunoreacts. Thus, a monoclonal antibody may contain antibody molecules (eg, bispecific (chimeric) monoclonal antibodies) having multiple antibody binding sites, each immunospecific for a different antigen.

  “Adjuvant” means a compound or mixture that improves the immune response to an antigen. Adjuvants can function as tissue depots that gradually release antigen and can also act as lymphatic activators that non-specifically enhance immune responses [Hood et al., Immunology, page 384, 2nd edition. Benjamin / Cummings, Menlo Park, Calif. (1984)]. In many cases, a primary challenge with an antigen alone in the absence of an adjuvant will not elicit a humoral or cellular immune response. Adjuvants include complete Freund's and incomplete Freund's adjuvants, mineral gels such as saponin and aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions or hydrocarbon emulsions, keyhole phosphorus Examples include, but are not limited to, pet hemocyanin, dinitrophenol, and potentially useful human adjuvants (such as BCG (Bacille Calmette Guerin) and Corynebacterium parvum). The adjuvant is preferably pharmaceutically acceptable.

  Polyclonal antibodies against the polypeptides of the present invention can be produced using various procedures known in the art. In the production of antibodies, various host animals can be immunized with the polypeptides of the invention by injection. Host animals include, but are not limited to, rabbits, mice, rats, sheep, goats and the like. In one embodiment, the polypeptides of the invention can be conjugated to an immunogenic carrier such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Depending on the host species, various adjuvants can be used to improve the immunological response. Adjuvants include Freund's adjuvant (complete and incomplete adjuvants), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin , Dinitrophenol, and potentially useful human adjuvants such as, but not limited to, BCG (Bacille Calmette Guerin) and Corynebacterium parvum.

  In preparing monoclonal antibodies against the polypeptides of the present invention, any technique for producing antibody molecules by continuous cell systems in culture can be used. Such techniques include the hybridoma technique originally developed by Kohler et al. (Kohler et al., (1975) Nature, 256: 495-497), the trioma technique, the human B cell hybridoma technique [Kozbor ) Et al. (1983) Immunology Today 4:72] and the EBV-hybridoma technique for producing human monoclonal antibodies [Cole et al. (1985) Monoclonal antibodies and cancer therapy, pages 77-96, Alan. R. Liss, Inc. However, it is not limited to these. Immortal antibody-producing cell lines can be produced by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. For example, U.S. Pat. Nos. 4,341,761, 4,399,121, 4,427,783, 4,444,877, 4,451,570, 4,466,917. No. 4,472,500, 4,491,632 and 4,493,890.

  In a further embodiment of the invention, monoclonal antibodies can be produced in sterile animals using modern techniques. According to the invention, chicken or pig antibodies can be used, which can be obtained by using chicken or pig hybridomas or by transforming B cells with EBV virus in vitro. In fact, according to the present invention, a gene derived from a mouse antibody molecule specific for the polypeptide of the present invention is spliced together with a gene derived from an antibody molecule of appropriate biological activity to produce a “chimeric antibody”. Techniques [Morrison et al. (1984) J. MoI. Bacteriol. 159-870; Neuberger et al., (1984) Nature, 312: 604-608; Takeda et al., (1985) Nature, 314: 452-454]. Is within the range. Such chimeric antibodies are much less likely to induce their own immune response (especially allergic responses) than cross-species antibodies, and therefore are used for the treatment of intestinal diseases and disorders (discussed below). Is preferred.

  In accordance with the present invention, techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for the polypeptides of the present invention. In a further embodiment of the present invention, the techniques described for the construction of Fab expression libraries [Huse et al., (1989) Science, 246: 1275-1281] are used for the polypeptides of the present invention. Monoclonal Fab fragments having the desired specificity can be identified quickly and easily.

Antibody fragments that contain the idiotype of the antibody molecule can be produced by known techniques. Such fragments include, for example, F (ab ′) ₂ fragments that can be produced by pepsin digestion of antibody molecules, Fab ′ fragments that can be produced by reducing disulfide bridges of F (ab ′) ₂ fragments, antibody molecules Fab fragments that can be generated by treating with papain and a reducing agent include, but are not limited to.

  In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art such as radioimmunoassay, ELISA, “sandwich” immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay. (Eg, using colloidal gold, enzyme or radioisotope label), Western blot, precipitation reaction, agglutination assay (eg, gel aggregation assay or hemagglutination assay), complement fixation assay, immunofluorescence assay, protein A assay, immunization It can be performed by electrophoresis assay or the like. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In other embodiments, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and such means are within the scope of the present invention. For example, to select an antibody that recognizes a specific epitope of a polypeptide of the present invention, the produced hybridoma is assayed to determine the presence of a product that binds to a fragment of the polypeptide of the present invention containing the epitope. can do.

  In addition, the present invention can be performed using the polypeptide of the present invention and / or an antibody that binds to the polypeptide of the present invention. A diagnostic and prognostic method for detecting the presence of hyodysenteriae; Also included are kits useful for diagnosis and prognostic observation of hyodysenteria infections.

  Diagnosis and prognostic methods are generally performed using biological samples obtained from animals such as chickens and pigs. “Sample” means a sample of animal tissue or fluid suspected of containing a Brachyspira species (eg, B. hyodysenteriae) or a polynucleotide or polypeptide thereof. Examples of such tissues and fluids include, but are not limited to, plasma, serum, feces, urine, lung, heart, skeletal muscle, stomach, intestine, and in vitro cell culture components.

  The invention is a method for detecting the presence of a polypeptide of the invention in a sample comprising the following steps: (a) an antibody (preferably a solid support) that specifically binds to the polypeptide of the invention. Contacting the antibody with a sample suspected of containing the polypeptide of the present invention under conditions capable of forming a reaction complex comprising the polypeptide of the present invention) and (b) Detecting the formation of a reaction complex comprising the antibody in the sample and the polypeptide of the invention, wherein the detection of the formation of the reaction complex indicates the presence of the polypeptide of the invention in the sample. provide.

The antibody used in this method is preferably derived from an affinity purified polyclonal antibody, more preferably from a monoclonal antibody. Furthermore, the antibody molecules used in the present invention are preferably in the form of Fab, Fab ′, F (ab ′) ₂ or F (v) moieties or complete antibody molecules.

  B. Based on the method described above. Particularly preferred methods for detecting hyodysenteria include enzyme-linked immunosorbent assay, radioimmunoassay, immunoradiometric assay, immunoenzyme assay (sandwich assay using monoclonal antibody and / or polyclonal antibody, etc.).

Among these procedures, three procedures that are particularly useful include polypeptides of the invention (or fragments thereof) labeled with a detectable label, antibody Ab ₁ labeled with a detectable label, or labeled with a detectable label. the antibody Ab ₂ is used. Such a procedure is summarized by the following formula (wherein the asterisk indicates that the particle is labeled and “AA” represents the polypeptide of the invention):
A. AA ^* + Ab ₁ = AA ^* Ab ₁
B. AA + Ab ₁ ^* = AA Ab ₁ ^*
C. AA + Ab ₁ + Ab ₂ ^* = Ab ₁ AA Ab ₂ ^*

  Those skilled in the art are familiar with such procedures and their applications and can therefore be used within the scope of the present invention. Procedure A, a “competition” procedure, is described in US Pat. Nos. 3,654,090 and 3,850,752. Procedure B is typical of well-known competitive assay techniques. Procedure C is a “sandwich” procedure and is described in US Pat. Nos. RE 31,006 and 4,016,043. Still other procedures such as the “double antibody” procedure and the “DASP” procedure are known.

  In any instance, the polypeptides of the invention form a complex with one or more antibodies or binding partners, and one component of the complex is labeled with a detectable label. The fact that a complex has been formed and, if necessary, the amount of complex can be confirmed by known methods applicable to label detection.

From the above, it will be appreciated that the property of Ab ₂ is that it reacts with Ab ₁ . The reason is that Ab ₁ generated in one mammalian species is used as an antigen to generate antibody Ab ₂ in other species. For example, a rabbit antibody can be used as an antigen to generate Ab ₂ in a goat body. Thus, Ab ₂ will be a anti-rabbit antibody raised in goats. In the present specification and claims, Ab ₁ is referred to as the primary antibody and Ab ₂ is referred to as the secondary antibody or anti-Ab ₁ antibody.

The most commonly used labels in such studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and the like. Examples of fluorescent substances that can be used as labels include fluorescein, rhodamine, and auramine. Specific detection substances include anti-rabbit antibodies prepared in the body of goats and conjugated with fluorescein by isothiocyanate. Examples of preferable isotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re. Radioactive labels can be detected by any currently available measurement procedure. Although many enzymes can be used, examples of preferable enzymes include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, a combination of glucose oxidase and peroxidase, and alkaline phosphatase. Enzymes are bound to selected particles by reaction with crosslinking molecules such as carbodiimide, diisocyanate, glutaraldehyde and the like. Enzyme labels can be detected by any of the currently used colorimetric, spectrophotometric, fluorescent spectrophotometric, amperometric or gas measuring methods. Examples of alternative labeling materials and methods disclosed include US Pat. Nos. 3,654,090, 3,850,752, and 4,016,043.

  The present invention also relates to a method for detecting an antibody against a polypeptide of the present invention in a biological sample, comprising: (a) preparing the polypeptide of the present invention or a fragment thereof; and (b) biological Incubating the sample with the polypeptide of the present invention under conditions capable of forming an antibody-antigen complex, and (c) confirming whether an antibody-antigen complex having the polypeptide of the present invention has been formed. A method of using is also provided.

  In another embodiment of the invention, an in vitro method for assessing the level of an antibody against a polypeptide of the invention in a biological sample, comprising: (a) a reaction in the biological sample according to the method described above Providing a method comprising detecting the formation of a complex and (b) assessing the amount of reaction complex formed that corresponds to the level of a polypeptide of the invention in the biological sample. .

  In addition, B. in animal hosts. An in vitro method for monitoring a therapeutic treatment of a disease associated with hyodysenteria, comprising the steps of the present invention in a series of biological samples obtained at various time points from an animal host undergoing the therapeutic treatment. Methods are provided for assessing and monitoring the level of antibodies to a polypeptide as described above.

  The present invention further provides B. in biological samples. A method for detecting the presence or absence of hyodysenteria, comprising: (a) contacting a biological sample with a polynucleotide probe or primer of a polynucleotide of the invention under suitable hybridization conditions; (b) the probe Or by detecting the duplex formed between the primer and the nucleic acid in the sample. A method for detecting the presence or absence of hyodysenteria is provided.

  According to one embodiment of the present invention, B.I. Detection of hyodysenteria can be performed by directly amplifying the polynucleotide sequence from the biological sample using known techniques and then detecting the presence of the polynucleotide sequence of the present invention.

  In one form of the invention, the target nucleic acid sequence is amplified by PCR and then detected using any of the specific methods described above. Other useful diagnostic techniques for detecting the presence of polynucleotide sequences include (1) allele-specific PCR, (2) single-stranded conformation analysis, (3) denaturing gradient gel electrophoresis, (4 ) RNase protection assay, (5) use of proteins that recognize nucleotide mismatches such as E. coli mutS protein, (6) allele-specific oligonucleotides, (7) fluorescent in situ hybridization, but not limited to .

  In addition to the methods described above, polynucleotide sequences can be detected by conventional probe techniques. When detecting the presence of a desired polynucleotide sequence using a probe, a biological sample (blood, serum, etc.) to be analyzed can be processed as necessary to extract nucleic acids. Sample polynucleotide sequences can be prepared by various methods (eg, denaturation, restriction digestion, electrophoresis, dot blotting) to facilitate detection of the target sequence. The target region of the sample polynucleotide sequence usually needs to be at least partially single stranded to hybridize with the target sequence of the probe. No denaturation is necessary if the sequence is naturally single stranded. However, if the sequence is double stranded, it may be necessary to denature the sequence. Denaturation can be performed by various techniques known in the art.

  The sample polynucleotide sequence and probe are incubated under conditions that promote stable hybridization of the target sequence in the probe with the desired putative polynucleotide sequence in the sample. In order to prevent false positives, it is preferable to use high stringency conditions.

  Detection of the obtained hybrid is usually performed using a labeled probe. Alternatively, the probe can be detected directly or indirectly by specific binding to the labeled ligand without labeling. Suitable labels and methods for labeling probes and ligands are known in the art, eg, radioactive labels or biotin that can be incorporated by known methods (eg, nick translation, random priming, kinase treatment), Examples include fluorescent groups, chemiluminescent groups (for example, dioxetane, particularly induced dioxetane), enzymes, antibodies, and the like. This change in the basic scheme is known in the art, and examples thereof include a change that facilitates separation of the hybrid to be detected from the foreign substance and / or a change that amplifies the signal from the labeling moiety.

  It is also contemplated within the scope of the present invention that a nucleic acid probe cocktail capable of detecting the desired polynucleotide sequence of the present invention can be used in the nucleic acid probe assay of the present invention. Thus, in one example, more than one probe complementary to a polynucleotide sequence is used to detect the presence of a polynucleotide sequence of the invention in a cell sample, but in particular, the number of different probes is 2, 3 or 5 Different types of nucleic acid probe sequences.

  In addition, a polynucleotide sequence described herein (preferably in the form of a probe) is immobilized on a solid support, and Brachyspira species (for example, B. hyodysenteria, B. intermedia, B. albinipri, B. Aalborghi, B. innocence, B. murdoquii, B. pilosicoli, but not limited thereto) can also be detected. Alternatively, the polynucleotide sequences described herein are B. It may form part of a DNA molecule library that can be used to simultaneously detect multiple different genes from Brachyspira species such as hyodysenteria. In yet another aspect of the invention, the polynucleotide sequences described herein are immobilized to a solid support along with other polynucleotide sequences (eg, derived from other bacteria or viruses) and bound to the solid support. B. The presence of Brachyspira species such as hyodysenteria and / or other polynucleotide sequences can be identified.

  Techniques for producing a fixed library of DNA molecules have been described in the art. In general, many prior art methods describe the synthesis of single stranded nucleic acid molecule libraries using, for example, masking techniques that form permutations of various sequences at various discrete locations on a solid substrate. U.S. Pat. No. 5,837,832 describes an improved method for producing a DNA array immobilized on a silicon substrate based on a very large scale integration technique. In particular, US Pat. No. 5,837,832 describes a strategy called “tiling” for synthesizing specific probe sets at spatially defined locations on a substrate. Is a technique that can be used to produce the fixed DNA library of the present invention. US Pat. No. 5,837,832 also provides references for earlier techniques that can also be used. Thus, the polynucleotide sequence probe can be synthesized in situ on the surface of the substrate.

  Alternatively, single-stranded molecules can be synthesized away from the solid substrate, and each of the pre-formed sequences can be attached to separate locations on the solid substrate. For example, a polynucleotide device can be printed directly on a substrate using a robotic device with pins or piezoelectric devices.

  The library sequence is typically immobilized on or within a separate area of the solid substrate. The substrate may be porous, allowing it to be immobilized within the substrate, or it may be substantially non-porous, in which case the library sequence is typically immobilized on the surface of the substrate. The solid substrate may be formed of any material to which the polypeptide can bind directly or indirectly. Examples of suitable solid substrates include organic polymers such as flat glass, silicon wafers, mica, ceramics, and plastics (eg, polystyrene and polymethacrylate). It is also possible to use widely available semipermeable membranes such as nitrocellulose membranes and nylon membranes. The semipermeable membrane can be attached to a stronger solid surface such as glass. If necessary, the surface can be coated with a metal layer such as gold, platinum, or other transition metals.

The solid substrate is generally preferably a material having a rigid or semi-rigid surface. In a preferred embodiment, at least one surface of the substrate is substantially flat, but in some embodiments it is desirable to physically separate the synthetic regions of different polymers, for example by raised regions or etched grooves. It can happen. In addition, it is preferable that the solid substrate is suitable for high-density adhesion of DNA sequences in a discontinuous region of usually 50 to 100 μm. In this case, the density is 10,000 to 40,000 dots / cm ⁻² .

  It is convenient to divide the solid substrate into sections. This can be done by techniques such as photo-etching or by applying a hydrophobic ink (eg, Teflon-based ink (Cel-line, USA)).

  The separate locations at which each of the different components of the library are provided may have any suitable shape, for example circular, rectangular, elliptical, wedge shaped, etc.

  Covalent or non-covalent means can be used to attach the polynucleotide sequence to the substrate. The polynucleotide sequence can be attached to the substrate through a layer of molecules to which the library sequence is bound. For example, the polynucleotide sequence can be labeled with biotin and the substrate can be coated with avidin and / or streptavidin. The convenience of using a biotinylated polynucleotide sequence is that the binding efficiency to a solid substrate can be easily confirmed. Since polynucleotide sequences can be difficult to bind to certain solid substrates, it is often necessary to provide a chemical interface between the solid substrate (eg, glass) and the nucleic acid sequence. An example of a suitable chemical interface is hexaethylene glycol. Another example is the use of glass coated with polylysine, in which case polylysine is chemically modified by standard procedures to introduce an affinity ligand. Other methods for attaching molecules to the surface of a solid substrate using a coupling agent are known in the art (see, for example, WO 98/49557).

  The complementary polynucleotide sequence bound to the fixed nucleic acid library means that various means such as a change in the optical property value of the bound polynucleotide sequence (ie, use of ethidium bromide), a polypeptide labeled with a fluorophore, etc. This can be confirmed by using a labeled nucleic acid. Other detection techniques that do not require the use of labels include optical techniques such as acousto-optic methods, reflectometry, ellipsometry, surface plasmon resonance (see WO97 / 49989), and the like.

  Therefore, the present invention provides a solid substrate on which at least one polynucleotide of the present invention (preferably, two or more different polynucleotide sequences of the present invention) are immobilized.

  The present invention can also be used as a prophylactic or therapeutic agent, which stimulates a humoral response or a cell-mediated response in animals such as chickens and pigs, so that Brachyspira species (for example, B. hyodysenteria and B. Suanatina, B. Intermedia, B. Albinipuri, B. Aalborghi, B. Innocence, B. Murdokii, B. Pyrochicory, etc., to protect against colonization Can do. B. Natural infection with Brachyspira species such as hyodysenteria induces circulating antibody titers against the proteins described herein. Therefore, the amino acid sequence described in the present specification or a part thereof has a possibility of constituting a main component of a preventive or therapeutic agent administered systemically or orally for protection against intestinal spirocheta disease.

  Accordingly, in one embodiment, the present invention provides an amino acid sequence or fragment thereof as described herein mixed in a therapeutically effective amount with a pharmaceutically acceptable carrier, diluent or excipient, An antibody that binds, or a polynucleotide sequence described herein is provided.

  As used herein, a “therapeutically effective amount” means at least about 15%, preferably at least 50%, more preferably at least 90%, most preferably a clinically significant defect in the activity, function and response of an animal host. Means an amount sufficient to prevent such defects. Alternatively, a therapeutically effective amount means an amount sufficient to ameliorate a clinically significant condition in the animal host.

  “Pharmaceutically acceptable” refers to a molecule or composition that is physiologically acceptable and usually does not cause allergic reactions or similar adverse reactions (eg, stomach upset, etc.) when administered to animals. Used to indicate. “Carrier” means a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Examples of such pharmaceutical carriers include sterilized liquids such as water, oils derived from petroleum, animals, plants, or synthetics, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water, physiological saline solution, dextrose aqueous solution and glycerol aqueous solution are particularly preferably used as carriers for injection solutions. Suitable pharmaceutical carriers are Martin, Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. Easton, Pennsylvania (1990).

  In a more specific form of the invention, a therapeutically effective amount of an amino acid sequence described herein or an analog, fragment or derivative thereof, or an antibody thereto is pharmaceutically acceptable diluent, preservative. , Pharmaceutical compositions comprising solubilizers, emulsifiers, adjuvants and / or carriers are provided. Such compositions include various buffers (eg, Tris-HCl, acetate, phosphate), pH and ionic strength diluents, detergents and solubilizers (eg, Tween 80 and polysorbate 80), antioxidants (eg, , Ascorbic acid and sodium metabisulfite), preservatives (eg, thimerosol and benzyl alcohol) and other additives and bulking substances (eg, lactose and mannitol). The material may be incorporated into a particulate preparation of a polymer compound such as polylactic acid or polyglycolic acid, or incorporated into a liposome. Hyaluronic acid can also be used. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the invention. See, for example, Martin, Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton, PA 18042), pages 1435-1712 (this document is incorporated herein by reference) Incorporated herein). The composition may be prepared in a liquid form or as a dry powder such as a lyophilized form.

  Alternatively, the polynucleotides of the invention can be optimized for expression in plants (eg, corn). Plants can be transformed with plasmids containing optimized polynucleotides. The plant is then grown and the protein of the invention expressed in the plant or expressed optimized in the plant. Thereafter, the plant is recovered, and the plant part containing the protein of the present invention is processed into animal feed. When an animal eats this animal feed, Immunity to hyodysenteria is conferred. Prior art examples detailing such methods include US Pat. No. 5,914,123 (Arntzen et al.), US Pat. No. 6,034,298 (Lam et al.) And US Pat. No. 6,136,320 (Arntzen et al.).

  It will be appreciated that the pharmaceutical compositions provided by the present invention may be administered by any means known in the art. The pharmaceutical composition for administration is preferably administered by injection, oral, pulmonary route or nasal route. More preferably, the amino acid sequences described herein or antibodies derived from the sequences are delivered by intravenous, intraarterial, intraperitoneal, intramuscular or subcutaneous routes of administration. Alternatively, an appropriately formulated amino acid sequence described herein or an antibody derived therefrom can be administered by nasal or oral administration.

  Further, the present invention relates to B.I. Use of a polynucleotide sequence of the present invention for the manufacture of a medicament for modulating a disease associated with hyodysenteria, and antisense capable of hybridizing to a polynucleotide sequence encoding the amino acid sequence of the present invention for its manufacture Also included is the use of sequences and ribozyme polynucleotide sequences.

  Desirably, an antisense construct or polynucleotide sequence encoding a ribozyme for use in a therapeutic method is administered directly as an uncoated nucleic acid construct. The uptake of uncoated nucleic acid constructs by bacterial cells is enhanced by several known transfection techniques, for example techniques involving the use of transfection agents. Examples of such agents include cationic agents (eg, calcium phosphate or DEAE-dextran) and lipofectants. Usually, a nucleic acid construct is mixed with a transfection agent to produce a composition.

  Alternatively, the antisense construct or ribozyme can be mixed with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include, for example, isotonic saline solutions such as phosphate buffered saline. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration. The routes of administration described herein are merely guidelines, and those skilled in the art will be able to easily determine the optimal route of administration and dose for any particular animal and condition.

  Further, the present invention relates to B.I. A kit for screening an animal suspected of being infected with a Brachyspira species such as hyodysenteria or Also included is a kit for confirming infection with a Brachyspira species such as hyodysenteria. In a further embodiment of the present invention, a kit suitable for use by an expert is prepared and Brachyspira species (eg, B. hyodysenteriae, B. suanatina, B. interna) in animals suspected of being infected. Check for the presence of media, B. albinipri, B. aalborghi, B. innocence, B. muldkii, B. pilosicoli, or Brachyspira species (e.g., B. hyodycentria) And B. Suanatina, B. Intermedia, B. Albinipuri, B. Aalborghi, and B. pirocolic) can be quantitatively measured. According to the testing techniques described above, such a kit comprises at least one labeled with one of the amino acid sequences described herein or a binding partner thereof (eg, an antibody specific for the amino acid sequence). And may contain instructions depending on the method selected (eg, “competition”, “sandwich”, “DASP”, etc.). Alternatively, such a kit can contain at least a polynucleotide sequence that is complementary to a portion of one of the polynucleotide sequences described herein and can contain instructions for its use. Such kits may also contain peripheral reagents such as buffers and stabilizers.

Accordingly, Brachyspira species (for example, but not limited to, B. hyodysenteria, B. suanatina, B. intermedia, B. albinipri, B. aalborghi, B. innocence, B. muldkii, B. pirochicory) The following components may be included in a test kit for demonstrating the presence of:
(A) a predetermined amount of at least one labeled immunity obtained by directly or indirectly attaching one of the amino acid sequences described herein or a specific binding partner thereof to a detectable label; Chemically reactive components;
(B) Other reagents;
(C) Instructions for using the kit

More specifically, the diagnostic test kit may contain the following.
(A) a known amount of one of the amino acid sequences described herein (or its binding partner), generally bound to a solid phase to form an immunosorbent, or bound to a suitable tag, or Multiple such final products, etc. (b) Other reagents as required (c) Instructions for use of the test kit

In a further variation, the test kit includes
(A) a labeling component obtained by binding one of the amino acid sequences described herein to a detectable label; (b) one or more additional immunochemical reagents, at least one of which is ,
(I) a ligand capable of binding to the labeling component (a),
(Ii) a ligand capable of binding to the binding partner of the labeling component (a),
A ligand selected from the group consisting of (iii) a ligand capable of binding to at least one of the components to be measured, and (iv) a ligand capable of binding to at least one binding partner of at least one of the components to be measured, or An additional immunochemical reagent that is a fixed ligand;
(C) instructions for carrying out a protocol for detecting and / or measuring one or more components of an immunochemical reaction between one of the amino acid sequences described herein and its specific binding partner; Can be included.

Preparation of genomic library A genomic library is prepared using a swine isolate of hyodysenteria (WA1 strain). This strain has been well characterized and found to become pathogenic after an experimental challenge in pigs. The cetyltrimethylammonium bromide (CTAB) method is used to prepare high quality chromosomal DNA suitable for the preparation of genomic DNA libraries. B. The hyodysenteria is grown in 100 mL of anaerobic trypticase soy broth medium to a cell density of 10 ⁹ cells / mL. Cells are harvested at 4000 × g for 10 minutes and the cell pellet is resuspended in 9.5 mL TE buffer. SDS is added to a final concentration of 0.5% (w / v) and cells are lysed with 100 μg proteinase K at 37 ° C. for 1 hour. NaCl is added to a final concentration of 0.7 M, and 1.5 mL of CTAB / NaCl solution (10% w / v CTAB, 0.7 M NaCl) is added, then the solution is incubated at 65 ° C. for 20 minutes. The lysate is extracted with an equal volume of chloroform / isoamyl alcohol and the tube is centrifuged at 6000 × g for 10 minutes for phase separation. Transfer the aqueous phase to a new tube and add 0.6 volumes of isopropanol to precipitate high molecular weight DNA. Precipitated DNA is recovered using a glass rod and transferred to a tube containing 1 mL of 70% (v / v) ethanol. The tube is centrifuged at 10,000 × g and the pelleted DNA is redissolved overnight in 4 mL TE buffer. A cesium chloride gradient containing 1.05 g / mL CsCl and 0.5 mg / mL ethidium bromide is prepared using the redissolved DNA solution. The gradient is transferred to a 4 mL sealable centrifuge tube and centrifuged at 70000 × g overnight at 15 ° C. The separated DNA is visualized under ultraviolet light and high molecular weight DNA is recovered from the gradient using a 15 g needle. Ethidium bromide is removed from the DNA by sequential extraction with CsCl saturated isopropanol. The purified chromosomal DNA is dialyzed against 2 L TE buffer and precipitated with isopropanol. The resuspended genomic DNA is sheared using GeneMachines Hydroshear (Genomic Solutions, Ann Arbor, Mich.) And the sheared DNA is filled using Klenow DNA polymerase to produce blunt-ended fragments. Using CloneSmart DNA ligase, 100 ng of blunt-ended DNA fragment is ligated to 25 ng of pSMART VC vector (Lucigen, Middleton, Wis.). The ligated DNA is then electroporated into E. coli electrocompetent cells. A small insertion (2 to 3 kb) library and a medium insertion (3 to 10 kb) library are constructed and used as a low copy pSMART VC vector.

Genomic sequencing After obtaining the genomic library, individual E. coli clones containing the pSMART VC vector are selected. Plasmid DNA is purified and sequenced. The purified plasmid is subjected to automated direct sequencing of the pSMART VC vector using forward and reverse primers specific for the pSMART VC vector. Each sequencing reaction is performed in a volume of 10 μL consisting of 200 ng plasmid DNA, 2 pmol primer and 4 μL ABI PRISM ^™ BigDye Terminator Cycle Sequencing Ready Reaction Mix (PE Applied Biosystems, Foster City, Calif.). Cycling conditions include a denaturation step at 96 ° C. for 2 minutes, followed by 25 cycles of denaturation at 96 ° C. for 10 seconds, and a combined primer annealing and extension step at 60 ° C. for 4 minutes. Residual dye terminators are removed from the sequencing products by precipitation with 95% (v / v) ethanol containing 85 mM sodium acetate (pH 5.2) and 3 mM EDTA (pH 8) and vacuum dried. Plasmid sequencing is performed in duplicate using each primer. Sequencing products are analyzed using an ABI 373A DNA sequencer (PE Applied Biosystems).

Annotation B. Using various publicly available bioinformatics tools. Construct and annotate the partial genomic sequence of the hyodysenteria and analyze and reanalyze the sequence as part of the quality assurance procedure for data analysis. The open reading frame (ORF) is predicted using various programs such as GeneMark, GLIMMER, ORPHEUUS, SELFID, and GetORF. The estimated ORF is examined for homology (DNA and protein) with existing international databases using a search such as BLAST or FASTA. Using a program such as PSI-BLAST, FASTA, MOTIFS, FINDPATTTERNS, PHD, SIGNALP, PSORT, etc., all the predicted ORFs are analyzed to confirm their cell localization. Using databases such as Interpro, Prosite, ProDom, Pfam and Blocks, surface related proteins such as transmembrane domains, leader peptides, homology to known surface proteins, lipoprotein signatures, outer membrane anchor motifs, host cell binding domains, etc. Predict. Phylogenetic and other molecular evolution analyzes are performed using identified genes and other species to assist in assigning functions. In silico analysis of both subsequenced genomes gives a comprehensive list of all predicted ORFs present in the available sequence data. Each ORF can be examined for descriptive information such as predicted molecular weight, isoelectric point, hydrophobicity, subcellular localization, and related in vitro properties of the natural gene product. Predictive genes encoding proteins similar to surface localization components and / or virulence factors in other pathogens are selected as potential vaccine targets.

Bioinformatics results Shotgun sequencing of the hyodysenteria genome determines 94.6% (3028.6 kb of the predicted 3200 kb) of the genome. B. The hyodysenteria sequence consists of 294 contigs (average contig size is 10.3 kb). B. In the case of a hyodysenteria, 2593 open reading frames (ORF) are predicted from 294 contigs. By comparing the predicted ORF with the genes present in the nucleic acid and protein databases, it can be seen that about 70% of the ORFs are homologous to the genes contained in the public database. The remaining 30% of the predicted ORF has no known identity.

Vaccine candidates ORFs that exhibit reasonable homology (E value less than e- ¹⁵ ) to outer surface proteins, secreted proteins, and to reduce the number of ORFs that will be tested as vaccine candidates Select possible virulence factors present in public databases as potential vaccine candidates. Many of the 2593 ORFs obtained by genomic shotgun sequencing pass this test, but the results for 33 of them are presented herein. Tables 1-1 and 1-2 show 33 genes selected as potential vaccine targets, and the similarity between the genes and other known amino acid sequences obtained from the SWISS-PROT database. . Of note, the% identity (% homology) of amino acids remains less than 71% while the% identity of amino acids does not exceed 58%, which makes these ORFs unique. I understand that.

  The DNA and amino acid sequences of NAV-H54 are found in SEQ ID NOs: 1 and 2, respectively. The DNA and amino acid sequences of NAV-H55 are found in SEQ ID NOs: 3 and 4, respectively. The DNA and amino acid sequences of NAV-H56 are found in SEQ ID NOs: 5 and 6, respectively. The DNA and amino acid sequences of NAV-H57 are found in SEQ ID NOs: 7 and 8, respectively. The DNA and amino acid sequences of NAV-H58 are found in SEQ ID NOs: 9 and 10, respectively. The DNA and amino acid sequences of NAV-H59 are found in SEQ ID NOs: 11 and 12, respectively. The DNA and amino acid sequences of NAV-H60 are found in SEQ ID NOs: 13 and 14, respectively. The DNA and amino acid sequences of NAV-H61 are found in SEQ ID NOs: 15 and 16, respectively. The DNA and amino acid sequences of NAV-H62 are found in SEQ ID NOs: 17 and 18, respectively. The DNA and amino acid sequences of NAV-H63 are found in SEQ ID NOs: 19 and 20, respectively. The DNA and amino acid sequences of NAV-H64 are found in SEQ ID NOs: 21 and 22, respectively. The DNA and amino acid sequences of NAV-H65 are found in SEQ ID NOs: 23 and 24, respectively. The DNA and amino acid sequences of NAV-H66 are found in SEQ ID NOs: 25 and 26, respectively. The DNA and amino acid sequences of NAV-H67 are found in SEQ ID NOs: 27 and 28, respectively. The DNA and amino acid sequences of NAV-H68 are found in SEQ ID NOs: 29 and 30, respectively. The DNA and amino acid sequences of NAV-H69 are found in SEQ ID NOs: 31 and 32, respectively. The DNA and amino acid sequences of NAV-H70 are found in SEQ ID NOs: 33 and 34, respectively. The DNA and amino acid sequences of NAV-H71 are found in SEQ ID NOs: 35 and 36, respectively. The DNA and amino acid sequences of NAV-H72 are found in SEQ ID NOs: 37 and 38, respectively. The DNA and amino acid sequences of NAV-H73 are found in SEQ ID NOs: 39 and 40, respectively. The DNA and amino acid sequences of NAV-H74 are found in SEQ ID NOs: 41 and 42, respectively.

  The DNA and amino acid sequences of NAV-H22 are found in SEQ ID NOs: 43 and 44, respectively. The DNA and amino acid sequences of NAV-H23 are found in SEQ ID NOs: 45 and 46, respectively. The DNA and amino acid sequences of NAV-H24 are found in SEQ ID NOs: 47 and 48, respectively. The DNA and amino acid sequences of NAV-H30 are found in SEQ ID NOs: 49 and 50, respectively. The DNA and amino acid sequences of NAV-H32 are found in SEQ ID NOs: 51 and 52, respectively. The DNA and amino acid sequences of NAV-H33 are found in SEQ ID NOs: 53 and 54, respectively. The DNA and amino acid sequences of NAV-H37 are found in SEQ ID NOs: 55 and 56, respectively. The DNA and amino acid sequences of NAV-H40 are found in SEQ ID NOs: 57 and 58, respectively. The DNA and amino acid sequences of NAV-H41 are found in SEQ ID NOs: 59 and 60, respectively. The DNA and amino acid sequences of NAV-H43 are found in SEQ ID NOs: 61 and 62, respectively. The DNA and amino acid sequences of NAV-H44 are found in SEQ ID NOs: 63 and 64, respectively. The DNA and amino acid sequences of NAV-H45 are found in SEQ ID NOs: 65 and 66, respectively.

  To further reduce the number of ORFs that will be tested as vaccine candidates, abandon gene products that are predicted to be localized in the cytoplasm and inner membrane of spirochetes by in silico analysis. As a result, 21 of the 33 genes shown in Table 1 are further analyzed. These 21 genes include NAV-H58, NAV-H60, NAV-H62, NAV-H64, NAV-H66, NAV-H67, NAV-H69, NAV-H71, NAV-H73, NAV-H22, NAV- H23, NAV-H24, NAV-H30, NAV-H32, NAV-H33, NAV-H37, NAV-H40, NAV-H41, NAV-H43, NAV-H44 and NAV-H45.

Analysis of gene distribution using polymerase chain reaction (PCR) One or two primer pairs that anneal to different regions of the target gene coding region are designed and optimized for PCR detection. Individual primers are designed using Oligo Explorer 1.2 and a primer set with a melting temperature estimated at about 55-60 ° C. is selected. These primer sets are also selected to produce PCR products exceeding 200 bp. Select a moderate stringency primer annealing temperature (50 ° C.) and perform distribution analysis PCR. Due to this moderate stringency condition, a small number of potential mismatch sequences (due to strain differences) occurring at the primer binding site will not affect primer binding. 21 B.B. The distribution analysis of the hyodysenteria target gene is described in B.C. For 23 strains of hyodysenteria (including 2 strains known to be non-pathogenic). PCR analysis is performed in a total volume of 25 μL using Taq DNA polymerase (Biotech International, Thurmont, MD). The amplification mixture consists of 1 × PCR buffer (containing 1.5 mM MgCl ₂ ), 1 U Taq DNA polymerase, 0.2 mM each dNTP (Amersham Pharmacia Biotech, Piscataway, NJ), 0.5 μM primer pair, and Consists of 1 μL of purified chromosomal template DNA. Cycling conditions include an initial template denaturation step at 94 ° C. for 5 minutes, followed by 35 cycles of denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 15 seconds, and primer extension at 68 ° C. for 4 minutes. PCR products were electrophoresed on 1% (w / v) agarose gels in 1 × TAE buffer (40 mM Tris-acetate, 1 mM EDTA), stained with 1 μg / mL ethidium bromide solution, UV Observe with light.

  The primers used for 18 genes (out of 21) are shown in Table 2. Of these 18 genes, 3 (NAV-H23, NAV-H41 and NAV-H71) were tested. Present in 83% of the hyodysenteria strains, 3 (NAV-H24, NAV-H30 and NAV-H73) are present in 87% of the tested strains and 7 (NAV-H22, NAV-H32, NAV- H33, NAV-H37, NAV-H43, NAV-H64 and NAV-H69) are present in 91% of the tested strains, 3 (NAV-H40, NAV-H44 and NAV-H45) are 100% of the tested strains. % Present. The remaining three genes were tested for B. cerevisiae. It is present in less than 80% of the hyodysenteria strains. These genes are not very useful as vaccine subunits due to their low distribution. For this reason, further analysis of these genes was abandoned.

Extraction of pTrcHis Plasmid E. coli JM109 clone carrying the pTrcHis plasmid (Invitrogen, Carlsbad, Calif.) is streaked from a glycerol stock onto Luria-Bertani (LB) agar plates supplemented with 100 mg / L ampicillin and incubated at 37 ° C. for 16 hours. To do. Inoculate a single colony into 10 mL LB broth supplemented with 100 mg / L ampicillin and incubate the broth medium at 37 ° C. for 12 hours with shaking. The whole amount of the overnight culture is centrifuged at 5000 × g for 10 minutes, and the plasmid contained in the cells is extracted using QIAprep Spin Miniprep Kit (Qiagen, Doncaster, Victoria). The pelleted cells are resuspended with 250 μL of cell resuspension buffer P1, and then 250 μL of cell lysis buffer P2 is added to lyse. Lysed cells are neutralized with 350 μL of neutralization buffer N3 and pelleted cell debris is pelleted by centrifugation (20000 × g, 10 minutes). The supernatant is transferred to a spin column and centrifuged at 10000 × g for 1 minute. After discarding the flow-through, 500 μL of wash buffer PE is added to the column and centrifuged as described above. Discard the flow-through and dry the column by centrifugation at 20000 xg for 3 minutes. Elute plasmid DNA from the column using 100 μL of elution buffer EB. The purified plasmid is quantified using a Dynaquant DNA fluorometer (Hoefer, San Francisco, Calif.) And diluted with TE buffer to adjust the DNA concentration to 100 μg / mL. The purified pTrcHis plasmid is stored at -20 ° C.

Preparation of vector 2 μg of purification in a total volume of 50 μL containing 5 U of two restriction enzymes in 100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT and 100 μg / mL BSA The pTrcHis plasmid is digested at 37 ° C. for 1-4 hours. The particular restriction enzyme pair used depends on the sequence of the primer and the sequence of the ORF (the goal is to use a primer that does not cleave the ORF). Confirmation of the restriction vector is performed by electrophoresis of 1 μL of the digested reaction product at 90 V for 1 hour on a 1% (w / v) agarose gel in 1 × TAE buffer. The electrophoresed DNA is stained with 1 μg / mL ethidium bromide and observed using ultraviolet (UV) light.

  The linearized pTrcHis vector is purified using an UltraClean PCR Clean-up Kit (Mo Bio Laboratories, Carlsbad, CA). That is, the restriction reaction (50 μL) is mixed with 250 μL of SpinBind buffer B1, and the entire amount is added to the spin column. After centrifuging at 8000 × g for 1 minute, discard the flow-through and add 300 μL SpinClean buffer B2 to the column. After centrifuging the column as described above and discarding the flow-through, the column is dried at 20000 × g for 3 minutes. The purified vector is eluted from the column using 50 μL of TE buffer. The purified linearized vector is quantified using a fluorimeter and diluted with TE buffer to adjust the DNA concentration to 50 μg / mL. The purified restriction vector is stored at -20 ° C.

Primer design for insert preparation Primer pairs are designed so as to amplify the coding region of the target gene as much as possible using genomic DNA as a starting point. All primer sequences contain a terminal restriction enzyme site that allows for cohesive end ligation of the resulting amplicon to the linearized pTrcHis vector. The primers are tested using Amplify 1.2 (University of Wisconsin, Madison, Wis.) And the theoretical amplicon sequence is inserted at the appropriate position in the pTrcHis vector sequence. Putative translation of the chimeric pTrcHis expression cassette is performed using Vector NTI version 6 (Informax) to confirm that the gene insert is in the correct reading frame. Table 3 shows gene size, protein size, predicted molecular weight of natural protein (unit: dalton), and predicted pI of protein. It is noteworthy that the histidine fusion of the recombinant protein adds about 4 kDa to the predicted molecular weight of the natural protein.

Amplification of gene inserts Genomic DNA is used to amplify all target gene inserts by PCR in a total volume of 100 [mu] L using Taq DNA polymerase (Biotech International) and Pfu DNA polymerase (Promega, Madison, Wis.). Amplification mixture consists of 1 × PCR buffer (containing 1.5 mM MgCl ₂ ), 1 U Taq DNA polymerase, 0.01 U Pfu DNA polymerase, 0.2 mM each dNTP (Amersham Pharmacia Biotech), 0.5 μM Consists of appropriate primer pairs and 1 μL of purified chromosomal DNA. Chromosomal DNA is the same as that used for genomic sequencing. Prepare from a Hyodycentria strain. Cycling conditions include an initial template denaturation step at 94 ° C. for 5 minutes, followed by 35 cycles of denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 15 seconds, and primer extension at 68 ° C. for 4 minutes. PCR products are electrophoresed on a 1% (w / v) agarose gel in 1 × TAE buffer, stained with 1 μg / mL ethidium bromide solution, and observed using UV light. After confirming the presence of the correct size PCR product, the PCR reaction is purified using an UltraClean PCR Clean-up Kit (Mo Bio Laboratories, Carlsbad, CA). The PCR reaction (100 μL) is mixed with 500 μL SpinBind buffer B1, and the entire amount is added to the spin column. After centrifuging at 8000 × g for 1 minute, discard the flow-through and add 300 μL SpinClean buffer B2 to the column. After centrifuging the column as described above and discarding the flow-through, the column is dried at 20000 × g for 3 minutes. The purified vector is eluted from the column using 100 μL TE buffer.

Restriction enzyme digestion of gene inserts 30 μL of purified PCR product is digested in a total volume of 50 μL using each restriction enzyme (1 U) compatible with the terminal restriction endonuclease recognition site identified by the cloning oligonucleotide primer. The restriction reaction is performed at 37 ° C. for 1 to 4 hours using 100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT, 100 μg / mL BSA and 1 U of each restriction enzyme. . The digested insert DNA is purified using an UltraClean PCR Clean-up Kit (see above). The purified digested insert DNA is quantified with a fluorometer and diluted with TE buffer to adjust the DNA concentration to 20 μg / mL. The purified restriction insert DNA is used immediately for vector ligation.

Ligation of gene insert to pTrcHis vector All ligation reactions are performed in a total volume of 20 [ mu] L. 100 ng linearized pTrcHis with 20 ng restriction insert in 30 mM Tris-HCl (pH 7.8), 10 mM MgCl ₂ , 10 mM DTT and 1 mM ATP containing 1 U T4 DNA ligase (Promega) at 16 ° C. Incubate for 16 hours. The same ligation reaction without insert DNA is also performed as a negative control for vector recirculation. Appropriate restriction enzymes are used for each reaction.

Transformation of pTrcHis ligation into E. coli cells Competent E. coli JM109 (Promega) cells are thawed on ice from -80 <0> C storage and contain 5 [mu] L overnight ligation reaction (equivalent to 25 ng of pTrcHis vector) Transfer 50 μL of the cells into an ice-cold 1.5 mL microtube. Gently tap the bottom of each tube on the bench to mix and leave on ice for 30 minutes. The tube is then placed in a 42 ° C. water bath for 45 seconds to heat shock the cells and then the tube is returned to ice for 2 minutes. Transformed cells are collected in 1 mL LB broth for 1 hour at 37 ° C. with gentle mixing. Harvested cells are collected at 2500 × g for 5 minutes and resuspended in 50 μL fresh LB broth. A total volume of 50 μL of resuspended cells is evenly spread on a LB agar plate containing 100 mg / L ampicillin using a sterile glass rod. Plates are incubated for 16 hours at 37 ° C.

Detection of recombinant pTrcHis construct in E. coli by PCR Streak 12 single transformant colonies of each construct onto a new LB agar plate containing 100 mg / L ampicillin and incubate at 37 ° C. for 16 hours. Single colonies from each transformation event are resuspended in 50 μL TE buffer and boiled for 1 minute. 2 μL of boiled cells are used as PCR template. The amplification mixture is 1 × PCR buffer (containing 1.5 mM MgCl ₂ ), 1 U Taq DNA polymerase, 0.2 mM each dNTP, 0.5 μM pTrcHis-F primer (5′-CAATTTTCAGACAATCTTGTG-3 ′: SEQ ID NO: 107) and 0.5 μM pTrcHis-R primer (5′-TGCCTGGCAGTTCCCTACTCTCG-3 ′: SEQ ID NO: 108). Cycling conditions include an initial template denaturation step at 94 ° C. for 5 minutes followed by 35 cycles of denaturation at 94 ° C. for 30 seconds, annealing at 60 ° C. for 15 seconds, and primer extension at 72 ° C. for 1 minute. PCR products are electrophoresed on a 1% (w / v) agarose gel in 1 × TAE buffer, stained with 1 μg / mL ethidium bromide solution, and observed using UV light. Cloning of various inserts into the pTrcHis expression vector produces recombinant constructs of various sizes.

Pilot expression of recombinant His-tagged protein 5-10 isolated colonies of recombinant pTrcHis construct in E. coli JM109 were transferred to 3 mL LB in a 5 mL tube containing 100 mg / L ampicillin and 1 mM IPTG. Inoculate broth and incubate for 16 hours at 37 ° C. with shaking. Centrifugation is performed at 5000 × g for 10 minutes at 4 ° C. to recover the cells. The supernatant is discarded and each pellet is resuspended in 10 μL Ni-NTA denaturing lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8 M urea, pH 8.0). After vortexing the tube for 1 minute, the cell debris is pelleted by centrifugation (10000 × g, 10 minutes, 4 ° C.). The supernatant is transferred to a new tube and stored at −20 ° C. until analysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE analysis of proteins is performed using a discontinuous Tris-glycine buffer system. 30 μL of protein sample was added to 10 μL of 4 × sample treatment buffer (250 mM Tris-HCl (pH 6.0), 8% (w / v) SDS, 200 mM DTT, 40% (v / v) glycerol and 0. 04% (w / v) bromophenol blue). Immediately after boiling the sample for 5 minutes, 10 μL of sample is added to the wells in the gel. The gel is a gel for concentration (125 mM Tris-HCl (pH 6.8), 4% w / v acrylamide, 0.15% w / v bisacrylamide and 0.1% w / v SDS) and a separation gel. (375 mM Tris-HCl (pH 8.8), 12% w / v acrylamide, 0.31% w / v bisacrylamide and 0.1% w / v SDS). These gels were polymerized with the addition of 0.1% (v / v) TEMED and 0.05% (w / v) freshly prepared ammonium sulfate solution to produce a mini-PROTEAN double slab cell (Bio-Rad, CA). Pour into Hercules). Samples are processed at room temperature (RT), 150 V, until the bromophenol blue dye reaches the bottom of the gel. In order to obtain an estimate of molecular weight, a pre-stained molecular weight standard is electrophoresed in parallel with the sample. Immediately after electrophoresis, the gel is stained with Coomassie Brilliant Blue G250 (Bio-Rad) or electrotransferred to a nitrocellulose membrane for Western blotting.

Western blot analysis Using a Towbin transfer buffer system, the proteins separated from the SDS-PAGE gel are electrophoresed on a nitrocellulose membrane. After electrophoresis, the gel is equilibrated for 15 minutes in transfer buffer (25 mM Tris, 192 mM glycine, 20% v / v methanol, pH 8.3). Proteins in the gel are electrotransferred to a nitrocellulose membrane (Protran, Schleicher and Schuell BioScience, Keene, NH) overnight at 30 V, 4 ° C. using a mini-protean transblot instrument (Bio-Rad). The freshly transferred nitrocellulose membrane containing the separated protein is blocked for 1 hour at room temperature with 10 mL Tris-buffered saline (TBS) containing 5% (w / v) nonfat dry milk. The membrane is washed with 0.1% (v / v) Tween 20-containing TBS (TBST) and then incubated with 10 mL of mouse anti-his antibody (5000-fold diluted with TBST) for 1 hour at room temperature. After washing 3 times with TBST for 5 minutes, the membrane is incubated with 10 mL of goat anti-mouse IgG (all molecules) -AP diluted 1: 5000 with TBST for 1 hour at RT. Color the membrane using an alkaline phosphatase substrate kit (Bio-Rad). The color development reaction is stopped by washing the membrane with distilled water. The membrane is then dried and scanned for presentation.

Verification of the reading frame of the recombinant pTrcHis construct by direct sequence analysis. Two transformant clones of each construct that produced the correct size PCR product were inoculated into 10 mL LB broth containing 100 mg / L ampicillin. And incubate at 37 ° C. for 12 hours with shaking. The entire overnight culture is centrifuged at 5000 × g for 10 minutes and the plasmid contained in the cells is extracted using the QIAprep Spin Miniprep Kit as described above. The purified plasmid is quantified with a fluorometer. Both purified plasmids are subjected to automated direct sequencing of the pTrcHis expression cassette using the pTrcHis-F primer and the pTrcHis-R primer. Each sequencing reaction is performed in a volume of 10 μL consisting of 200 ng plasmid DNA, 2 pmol primer and 4 μL ABI PRISM ^™ BigDye Terminator Cycle Sequencing Ready Reaction Mix (PE Applied Biosystems, Foster City, Calif.). Cycling conditions include a denaturation step at 96 ° C. for 2 minutes, followed by 25 cycles of denaturation at 96 ° C. for 10 seconds and a combined primer annealing and extension step at 60 ° C. for 4 minutes. Residual dye terminator is removed from the sequencing product by precipitation with 95% (v / v) ethanol containing 85 mM sodium acetate (pH 5.2) and 3 mM EDTA (pH 8) and dried in vacuo. Plasmid sequencing is performed in duplicate using each primer. Sequencing products are analyzed using an ABI 373A DNA sequencer (PE Applied Biosystems). Nucleotide sequencing of pTrcHis is performed to confirm that the expression cassette is in the correct reading frame for each construct.

Expression and purification of recombinant His-tagged protein A single colony of recombinant pTrcHis construct in E. coli JM109 is inoculated into 50 mL LB broth containing 100 mg / L ampicillin in a 250 mL conical flask and shaken. Incubate for 16 hours at 37 ° C. Inoculate a 2 L conical flask containing 1 L LB broth supplemented with 100 mg / L ampicillin with 10 mL overnight culture at 37 ° C. until the optical density of the cells is 0.5 at 600 nm (approximately 3-4 Time) incubate. IPTG is then added to a final concentration of 1 mM to induce culture and the cells are returned to 37 ° C. with shaking. After 5 hours induction, the culture is transferred to a 250 mL centrifuge bottle and the bottle is centrifuged at 5000 × g for 20 minutes at 4 ° C. The supernatant is discarded and each pellet is resuspended in 8 mL Ni-NTA denaturing lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCI, 8 M urea, pH 8.0). Store the resuspended cells at −20 ° C. overnight.

The cell suspension is removed from storage at −20 ° C. and thawed on ice. The cell lysate is then sonicated 3 times on ice for 30 seconds, with a 1 minute incubation on ice between each sonication. Lysed cells are clarified by centrifugation at 20000 × g for 10 minutes at 4 ° C., and the supernatant is transferred to a 15 mL column containing a total volume of 0.5 mL Ni-NTA agarose resin (Qiagen). Recombinant His ₆ tag protein is allowed to bind to the resin at 4 ° C. for 1 hour with rotary mixing. The resin was then washed with 30 mL of Ni-NTA denaturing wash buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8 M urea, pH 6.3), and then 12 ml of Ni-NTA denaturing elution buffer ( Elute with 100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8M urea, pH 4.5). Four fractions (3 mL) of the eluate are collected and stored at 4 ° C. Treat 30 μL of each eluate with 10 μL of 4 × sample treatment buffer and boil for 5 minutes. Samples are subjected to SDS-PAGE and stained with Coomassie Brilliant Blue G250 (Bio-Rad). The dyed gel is equilibrated with distilled water for 1 hour, sandwiched between two cellulose sheets and dried overnight at RT.

  Expression of the selected recombinant E. coli clone is performed on a medium scale to produce a recombinant protein sufficient for vaccination of mice (see below).

Dialysis and lyophilization of purified recombinant His-tagged protein Pool the eluted protein and transfer to a 3500 Da molecular weight cut-off (MWCO) hydration dialysis tube (Spectrum Laboratories, Los Angeles, CA). An aliquot (200 μL) of the pooled eluate is taken and quantified using a commercially available protein assay (Bio-Rad). The protein is dialyzed against 2 L of distilled water with stirring at 4 ° C. Change dialysis buffer 8 times at 12 hour intervals. The dialyzed protein is transferred from the dialysis tube to a 50 mL centrifuge tube (maximum volume: 40 mL) and the tube is placed at −80 ° C. overnight. The tube is placed in a MAXI lyophilizer (Hero-Holten, Allerod, Denmark), lyophilized to dryness. The lyophilized protein is then rehydrated with PBS to an estimated concentration of 2 mg / mL and stored at −20 ° C.
Subsequent dialysis, lyophilization, and stable recombinant antigens were successfully produced.

  Eight of the 18 genes have been successfully cloned into the E. coli expression system and recombinant proteins can be stably expressed from these clones.

Serology with Purified Recombinant Protein 20 μg of purified recombinant protein is added to a 7 cm IEF well, electrophoresed on a 10% (w / v) SDS-PAGE gel and electrotransferred to a nitrocellulose membrane. The membrane is blocked with TBS-skim milk (5% w / v) and incorporated into a multi-screen device (Bio-Rad). The wells are incubated with 100 μL of diluted porcine serum (100 ×) for 1 hour at room temperature. Porcine serum recovered from high health pigs (n = 3), experimentally challenged clinical SD (n = 5), naturally infected seroconverted pigs (n = 5), and spontaneous infection Collect from pigs (n = 4). The membrane is then removed from the device, washed 3 times with TBST (0.1% v / v) and then incubated with 10 mL of goat anti-pig IgG (all molecules) -AP (5000 ×) for 1 hour at RT. The membrane is washed 3 times with TBST and then developed using an alkaline phosphatase substrate kit (Bio-Rad). When sufficient color develops, the membrane is washed with tap water, dried, and scanned for presentations.

  The reactivity of porcine serum collected from pigs with different health conditions is shown in the table below. All proteins are recognized in a 100% serum panel, indicating that the gene is expressed in vivo and can induce a systemic immune response after exposure to spirochetes.

Vaccination of mice with purified recombinant his-tag protein For each purified recombinant his-tag protein, 10 mice are immunized systemically or orally, and the recombinant protein is immunogenic. Check if it exists. The recombinant protein is emulsified with 30% (v / v) water-in-oil adjuvant and injected intramuscularly into the quadriceps of 10 mice (Balb / cJ: 5 weeks old, male). All mice receive 100 μg protein (in a total volume of 100 μL). Three weeks after the first vaccination, all mice receive the same second intramuscular vaccination as the first vaccination. All mice are killed 2 weeks after the second vaccination. Serum was collected from the heart after death and analyzed by Western blot analysis. Test for antibodies against cell extracts of hyodysenteriae.

Western blot analysis 20 μg of purified recombinant protein is added to a 7 cm IEF well, electrophoresed on a 10% (w / v) SDS-PAGE gel and electrophoresed to a nitrocellulose membrane. The membrane is blocked with TBS-skim milk (5% w / v) and incorporated into a multi-screen device (Bio-Rad). The wells are incubated with 100 μL of diluted mouse serum (100 ×) for 1 hour at room temperature. The membrane is removed from the device, washed 3 times with TBST (0.1% v / v) and then incubated with 10 mL of goat anti-mouse IgG (all molecules) -AP (5000 ×) for 1 hour at room temperature. The membrane is washed 3 times with TBST and then developed using an alkaline phosphatase substrate kit (Bio-Rad). When sufficient color develops, the membrane is washed with tap water, dried, and scanned for presentations.

  Western blot analysis shows that antibody reactivity to recombinant vaccine antigen is significantly increased in mice after vaccination. All mice recognize a recombinant protein with a molecular weight equivalent to the purified recombinant protein stained with Coomassie blue. These experiments provide evidence that recombinant proteins are immunogenic when used for vaccination of mice and that specific circulating antibody titers against the antigen can be induced by the vaccination protocol used. From this result, B.I. It can be seen that the recombinant protein may be useful in an effective vaccine against animal species colonized by hyodysenteriae.

Pig vaccination with purified recombinant his-tag protein For each purified recombinant his-tag protein, 10 seronegative pigs were muscled with 1 mg of specific antigen (in 1 mL of vaccine volume). Inject. Antigen is emulsified with an equal volume of water-in-oil adjuvant. Pigs are vaccinated at 3 weeks of age and again at 6 weeks of age. A second group of 10 seronegative pigs is used as a negative control and left unvaccinated. All pigs had 100 mL of active B.B. Challenge with hyodysenteria cultures (-10 ⁹ cells / mL) and observe clinical signs of swine dysentery during the experiment (up to 6 weeks after challenge) and postmortem examination.

Diagnostic Kit B. Pigs in pig farms with known infection with hyodysenteria Pigs known not to be infected with hyodysenteria, and B. Serum is collected from pigs in pig farms where infection with hyodysenteria is unknown. 20 μg of purified recombinant protein is added to a 7 cm IEF well, electrophoresed on a 10% (w / v) SDS-PAGE gel and electrophoresed to a nitrocellulose membrane. The membrane is blocked with TBS-skim milk (5% w / v) and incorporated into a multi-screen device (Bio-Rad). The wells are incubated with 100 μL of diluted porcine serum (100 ×) for 1 hour at room temperature. The membrane is then removed from the device, washed 3 times with TBST (0.1% v / v) and incubated with 10 mL of goat anti-pig IgG (all molecules) -AP (5000 ×) for 1 hour at room temperature. The membrane is washed 3 times with TBST and then developed using an alkaline phosphatase substrate kit (Bio-Rad). When sufficient color develops, the membrane is washed with tap water, dried, and scanned for presentations. By comparing the results obtained with positive and negative controls, pigs You can check if you are infected with hyodysenteriae.

  Although the invention has been described with reference to specific embodiments, variations of the methods and compositions described above can be used, and the method can be different from those specifically described herein. It will be apparent to those skilled in the art that the invention is intended to be practiced. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims.

Claims (44)

  A polynucleotide comprising the sequence of SEQ ID NO: 59.   A plasmid comprising the polynucleotide according to claim 1.   The plasmid according to claim 2, which is an expression vector.   A cell comprising the plasmid according to claim 2.   A cell comprising the plasmid according to claim 3.   An immunogenic composition comprising the plasmid of claim 3.   A vaccine composition for the treatment or prevention of Brachyspira hyodysenteria comprising the plasmid according to claim 3.   A vaccine composition for the treatment or prevention of Brachyspira hyodysenteria comprising the cell according to claim 4.   A DNA molecule comprising a sequence that is at least 70% identical to the polynucleotide of claim 1.   A plasmid comprising the DNA molecule according to claim 9.   10. A DNA molecule according to claim 9 which is at least 80% identical to the polynucleotide of claim 1.   A plasmid comprising the DNA molecule according to claim 11.   10. A DNA molecule according to claim 9 which is at least 90% identical to the polynucleotide of claim 1.   A plasmid comprising the DNA molecule of claim 13.   A polypeptide comprising the sequence of SEQ ID NO: 60.   A polynucleotide comprising a sequence encoding the polypeptide of claim 15.   A plasmid comprising the polynucleotide according to claim 16.   The plasmid according to claim 17, which is an expression vector.   A cell comprising the plasmid according to claim 17.   20. An immunogenic composition comprising the cell of claim 19.   A protein comprising a sequence that is at least 70% identical to the polypeptide of claim 15.   24. A protein according to claim 21 which is at least 80% identical to the polypeptide of claim 15.   23. A protein according to claim 22 which is at least 90% identical to the polypeptide of claim 15.   24. An immunogenic composition comprising the protein of claim 23.   An immunogenic composition comprising the polypeptide of claim 15.   A vaccine composition for the treatment or prevention of Brachyspira hyodysenteria comprising the polypeptide of claim 15.   A monoclonal antibody that binds to the polypeptide of claim 15.   A kit for diagnosing the presence of Brachyspira hyodysenteria in an animal comprising the monoclonal antibody of claim 27.   A kit for diagnosing the presence of Brachyspira hyodysenteria in an animal comprising the polypeptide of claim 15.   A kit for diagnosing the presence of Brachyspira hyodysenteria in an animal comprising the polynucleotide of claim 1.   25. A method of generating an immune response against Brachyspira hyodysenteria in an animal (excluding humans), comprising administering to said animal an immunogenic composition according to claim 24.   26. A method of generating an immune response against Brachyspira hyodysenteria in an animal (excluding humans), comprising administering to said animal an immunogenic composition according to claim 25.   7. A method of generating an immune response against Brachyspira hyodysenteria in an animal (excluding humans), comprising administering to said animal an immunogenic composition according to claim 6.   21. A method of generating an immune response against Brachyspira hyodysenteria in an animal (excluding humans), comprising administering to said animal an immunogenic composition according to claim 20.   A method for treating or preventing a disease caused by Brachyspira hyodysenteriae in an animal (excluding humans) in need of the treatment, wherein the therapeutically effective amount of the vaccine composition according to claim 7 is applied to the animal. A method comprising administering.   A method for treating or preventing a disease caused by Brachyspira hyodysenteriae in an animal (excluding humans) in need of the treatment, wherein the animal is treated with a therapeutically effective amount of the vaccine composition according to claim 8. A method comprising administering.   27. A method of treating or preventing a disease caused by Brachyspira hyodysenteriae in an animal (excluding humans) in need of the treatment, wherein the animal is treated with a therapeutically effective amount of the vaccine composition of claim 26. A method comprising administering.   24. Use of the protein of claim 23 for the preparation of a medicament for generating an immune response against Brachyspira hyodysenteria in an animal.   16. Use of the polypeptide according to claim 15 for the preparation of a medicament for generating an immune response against Brachyspira hyodysenteria in an animal.   Use of the plasmid according to claim 3 for the preparation of a medicament for generating an immune response against Brachyspira hyodysenteria in an animal.   20. Use of a cell according to claim 19 for the preparation of a medicament for generating an immune response against Brachyspira hyodysenteria in an animal.   Use of the plasmid according to claim 3 for the preparation of a medicament for the treatment or prevention of diseases caused by Brachyspira hyodysenteria in animals in need of said treatment.   Use of a cell according to claim 4 for the preparation of a medicament for the treatment or prevention of a disease caused by Brachyspira hyodysenteria in an animal in need of said treatment.   Use of a polypeptide according to claim 15 for the preparation of a medicament for the treatment or prevention of a disease caused by Brachyspira hyodysenteria in an animal in need of said treatment.