Vaccines and vaccine ingredients for microbial cell inhibition

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to a recovered polypeptide which is a biological target for methane-producing cell inhibition, as well as to a recovered polynucleotide which codes this polypeptide. There are disclosed expression vector and cloning vector containing this polynucleotide, and host cells containing the above expression vector. There are described conjugated molecules or fused molecule for methane-producing cell inhibition, as well as antibody or its functional fragment which binds to the above polypeptide. The invention also covers a pharmaceutical composition and methods for inhibiting and identifying the methane-producing cell with the use of the above conjugated molecule or fused molecule and the antibody or its fragment.

EFFECT: invention enables inhibiting the methane-producing cell effectively.

19 cl, 9 dwg, 6 ex

 

RELATED APPLICATIONS

This application claims the priority of applications for U.S. patent No. 60/975104 from 25 September 2007, No. 60/989840 from November 22, 2007, and No. 60/989841 from November 22, 2007, the contents of each of which is incorporated into this description by reference in full.

The technical FIELD TO WHICH the INVENTION RELATES

The invention relates to components of microbial cells that can be used to generate antibodies, including peptides, polypeptides comprising these peptides, polynucleotides that encode these peptides or polypeptides and antibodies directed to these peptides, polypeptides or polynucleotides. The invention also relates to expression vectors and cells-masters for the production of these peptides, polypeptides, polynucleotides and antibodies. The invention also relates to methods and compositions, particularly vaccine compositions for detection, targeting and suppression of microbial cells, especially cells methaneproducing, using one or more of the disclosed peptides, polypeptides, polynucleotides, antibodies, expression vectors and host cells.

The prior art TO WHICH the INVENTION RELATES

In New Zealand agricultural activities takes into account the emissions of most greenhouse gases. Thus, reducing the of agricultural greenhouse gas emissions is essential to the fulfillment of obligations to New Zealand under the Kyoto Protocol. The Protocol requires the reduction of greenhouse gas emissions to 1990 levels by the end of the first commitment period (2008-2012). This agricultural complex and the New Zealand government has established a scientific research Association of pastoral greenhouse gases (PGGRC), to find ways of reducing emissions of the New Zealand agricultural greenhouse gas emissions.

An important part of the activities of the PGGRC was research into reducing methane emissions from grazing ruminants in New Zealand. The reduction of methane emissions by ruminants is of commercial interest for two reasons. First, failure to comply with obligations under the Kyoto Protocol will force the government to buy additional amounts permitted carbon emissions. Currently, this cost is estimated at $ 350 million. Secondly, the production of methane leads to the loss of 8-12% of the total energy produced in the rumen. Instead, this energy can be used to improve the productivity of ruminants.

The methane produced in the rumen by bacteria called methaneproducing that are part of typeEuryarchaeotain the Kingdom ofArchaea. Most of methaneproducing grow on CO2and H2as the sole energy source, but some of them are for growth can be used AOC is tatie or methyl compounds. In the rumen, there are several different genera methaneproducing archaea, but the species of the genusMethanobrevibacterespeciallyM. ruminantiumandM. smithii,considered to be the predominant methaneproducing the ruminants in New Zealand.M. ruminantiumat the present time is the object of the project to sequence the genome funded PGGRC. This project is the first genome methaneproducing of the scar, and he aimed at creating a better understanding of the biologyMethanobrevibacter,to detect a target for suppression of methane formation.

To reduce methane production in the rumen requires suppression of methaneproducing or inactivation of their metabolic pathways of methanogenesis. Means for suppressing methane production is to identify specific molecules that inhibit cell-methaneproducing. This can be achieved, for example, by use of funds, targeting methaneproducing. In one of the approaches you can get vaccines targeting microbial cells. Thus, it would be useful to identify components, in particular components of the cell surfaces of microbial cells, including peptides and polypeptides, as well as the associated polynucleotide and antibodies that can be used for antimicrobial vaccines.

SUMMARY of the INVENTION

According to the SNO invention described selected peptides polypeptides and polynucleotideM. ruminantiumin particular components of the cell surfaceM. ruminantium, as well as expression vectors, cell-hosts and antibodies, and methods of their application as it is described in detail in the present description.

According to the invention specifically described, the selected peptide, including, for example, at least a fragment of one amino acid sequence selected from the group consisting of SEQ ID NO:1-702. In a particular aspect the peptide comprises at least a fragment of the amino acid sequence of any of SEQ ID NO:45-260 and 332-702. In another aspect the peptide comprises at least a fragment of the amino acid sequence of any of SEQ ID nos:10-17. In another aspect, the peptide is a fragment of, for example, containing at least one amino acid sequence comprising the extracellular domain of any of SEQ ID nos:10-17, 45-260 and 332-702.

According to the invention specifically described, the selected polypeptide, including, for example, at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702. In a specific aspect, the polypeptide includes the amino acid sequence of any of SEQ ID NO:45-260 and 332-702. In yet another aspect, the polypeptide includes the amino acid sequence of any of SEQ ID nos:10-17. In another aspect, the polypeptide is a fra is a COP, for example, containing at least one amino acid sequence comprising the extracellular domain of any of SEQ ID nos:10-17, 45-260 and 332-702.

According to the invention additionally describes the selected polynucleotide comprising the coding sequence of at least one peptide. In one aspect of polynucleotide contains the coding sequence of at least a fragment of amino acid sequence selected from the group consisting of SEQ ID NO:1-702. In a particular aspect of polynucleotide contains the coding sequence of at least a fragment of any of SEQ ID NO:45-260 and 332-702. In another aspect of polynucleotide contains the coding sequence of at least a fragment of any of SEQ ID nos:10-17. In another aspect of polynucleotide contains a fragment of a sequence that encodes, for example, at least one amino acid sequence comprising the extracellular domain of any of SEQ ID nos:10-17, 45-260 and 332-702.

According to the invention additionally describes the selected polynucleotide comprising the coding sequence of at least one polypeptide. In one aspect of polynucleotide contains a sequence encoding at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702. In a particular aspect of polynucleotide contains the encoding of th is sequence of any of SEQ ID NO:45-260 and 332-702. In another aspect of polynucleotide contains the coding sequence of any of SEQ ID nos:10-17. In another aspect of polynucleotide contains a fragment of a sequence that encodes, for example, at least one amino acid sequence comprising the extracellular domain of any of SEQ ID nos:10-17, 45-260 and 332-702.

In an additional aspect according to the invention described selected polynucleotide, including, for example, a nucleic acid sequence selected from the group consisting of SEQ ID NO:703-1395. In a particular aspect of polynucleotide contains a nucleic acid sequence with SEQ ID NO:703-710. In another aspect of polynucleotide is a fragment or oligonucleotide comprising, for example, a nucleic acid sequence comprising the extracellular domain encoded by any of SEQ ID NO:703-710, 737-931 and 1003-1395. In addition, the invention includes a dedicated polynucleotide or its fragment, hybridity with any of these sequences of nucleic acids from SEQ ID NO:703-1395. The invention additionally includes a dedicated polynucleotide, including complement, reverse complement, reverse sequence, or fragments of any of these nucleic acids sequences.

According to the invention described expression vector comprising polynucleotide according to the invention. In one aspect, the vector expr the Russia contains the sequence encoding at least a fragment of the amino acid sequence selected from the group consisting of SEQ ID NO:1-702. In a particular aspect, the expression vector contains the coding sequence of at least a fragment of at least one of SEQ ID NO:45-260 and 332-702. In yet another aspect, the expression vector contains a sequence encoding at least one amino acid sequence of at least one of SEQ ID nos:10-17. In another aspect, the expression vector contains a sequence encoding at least one amino acid sequence comprising the extracellular domain of any of SEQ ID nos:10-17, 45-260 and 332-702.

According to the invention also describes a host cell, such as microbial a host cell comprising at least one expression vector.

According to the invention specifically described antibody directed to a peptide, polypeptide or polynucleotide, as indicated in the present description. In some aspects, the antibody is directed to an amino acid sequence selected from the group consisting of SEQ ID NO:1-702. In alternative aspects, the antibody is directed to at least the fragment of a polypeptide sequence selected from the group consisting of SEQ ID NO:10-17, 45-260 and 332-702. In a particular aspect, the antibody binds at least one fragment of the peptide sequence of any of the C SEQ ID NO:10-17. In yet another aspect, the antibody binds at least one fragment of a polypeptide sequence of any of SEQ ID NO:45-260 and 332-702. In an alternative aspect, the antibody binds to at least a fragment of a peptide or polypeptide comprising the extracellular domain of any of SEQ ID nos:10-17, 45-260 and 332-702. In another aspect, the antibody comprises one or more of chimeras or conjugates at least one cell inhibitor, for example, compounds that prevent methanogenesis (for example, bromoethanesulfonate), with antibodies and fragments of antibodies to lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, as described in detail in the present description.

According to the invention is additionally described modified peptides or polypeptides, for example at least one of SEQ ID NO:1-702, including biologically active modifications, fragments, variants and derivatives described in the present description. Also described polynucleotide encoding these modified peptides or polypeptides, as well as modifications, fragments, variants and derivatives disclosed polynucleotides; antibodies obtained using these modified peptides, polypeptides or polynucleotides; expression vectors comprising these polynucleotides, and cell-hosts, including these ve the Torah. The following describes the modified antibodies, including biologically active modifications, fragments, variants and derivatives described in the present description. In some aspects, the compositions and methods of the invention use these modified peptides, polypeptides, polynucleotides, antibodies or the corresponding expression vectors or cells of hosts.

According to the invention described composition comprising the selected peptide or polypeptide, such as at least one of SEQ ID NO:1-702. Also described composition, including a dedicated polynucleotide, for example at least one of SEQ ID NO:703-1395. According to the invention is additionally described composition comprising the antibody, for example, aimed to sequence peptides, polypeptides or polynucleotides disclosed in the present description. The following describes the composition, which includes the expression vector or the cell host comprising the expression vector, in accordance with the invention. This composition may include any of the biologically active modifications, fragments, variants and derivatives described in the present description. The composition may include at least one cell inhibitor (such as chimeras or conjugate) and may be composed, for example, in the form of pharmaceutical compositions, in particular in the form of compositions vaccines.

According to the invention also describes a method of obtaining a peptide or polypeptide, such as at least a fragment of any of SEQ ID NO:1-702, comprising: a) culturing the expression vector or host cell comprising the expression vector, which contains at least part of the sequence that encodes at least one peptide or polypeptide, under conditions suitable for the expression of this peptide or polypeptide; and b) removing the peptide or polypeptide from the culture. In specific aspects, the peptide or polypeptide contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or modified sequences.

According to the invention also describes a method of obtaining antibodies, for example, directed at least one fragment of any of SEQ ID NO:1-702, comprising: a) culturing the expression vector or host cell comprising the expression vector, which contains at least part of the sequence coding for less is th least one antibody or antibody fragment, under conditions suitable for expression of the antibody or antibody fragment; and (b) removing this amino acid sequences of the culture. In particular aspects, the antibody or antibody fragment directed to at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or modified sequences. In an alternative aspect, the antibody is produced by immunizing an animal host, as described in detail in the present description.

According to the invention is additionally described is a method of obtaining antibodies, for example, directed at least one fragment of any of SEQ ID NO:1-702, which includes the Chimera or conjugate with at least one cell inhibitor. This method comprises: a) culturing the expression vector or host cell comprising an expression vector that contains a sequence encoding at least one antibody or fragment of the antibody, under conditions suitable for expression of the antibody or antibody fragment; (b) obtaining chimeras or conjugate with the antibody or antibody fragment (e.g., through the expression of the fusion sequence or chemical conjugation with cellular data inhibitor); and c) removing chimeras or conjugate.

In particular aspects, the antibody is directed to at least one frag is into any of SEQ ID NO:1-702 or modified sequences. In further aspects, the inhibitor is selected from compounds that prevent methanogenesis (e.g., bromoethanesulfonate), antibodies and fragments of antibodies, lytic enzymes, peptide-nucleic acids, antimicrobial peptides and other antibiotics, as described in detail in the present description. In an alternative aspect, the antibody is produced by immunizing an animal host, and then by conjugation, as described in the present description.

In addition, according to the invention described by way of suppression (e.g., inhibiting growth or replication) of microbial cells, in particular cells of methaneproducing, including: the introduction of the cells into contact with the antibody or antibody fragment, for example, directed at least one fragment of any of SEQ ID NO:1-702, either Chimera or conjugate antibodies or with any modified antibody.

As another method, the cell was suppressed by the introduction of a vaccine composition as described in detail in the present description.

According to the invention is further described by way of suppression (e.g., inhibiting growth or replication) of microbial cells, in particular cells of methaneproducing, comprising: a) optionally, obtaining or allocating at least one antibody as described in the present description; and b) introducing cells into contact with the antibody. In particular TSA which the antibody is directed at least to a fragment of any of SEQ ID NO:1-702 or modified sequences. In some aspects, the antibody further includes at least one cell inhibitor, is attached, for example, in the form of a Chimera or conjugate. In other aspects, the antibody is administered to the patient in the form of a composition, for example in the form of a composition of the vaccine.

In addition, according to the invention described by way of suppression (e.g., inhibiting growth or replication) of microbial cells, in particular cells of methaneproducing, comprising: a) optionally, obtaining or allocating at least one peptide or polypeptide, as described in the present description; and b) introducing a peptide or polypeptide to a patient to induce them immune response. In a particular aspect, the peptide or polypeptide contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or modified sequences. In other aspects, the peptide or polypeptide is administered to the patient in the form of a composition, for example in the form of a composition of the vaccine.

According to the invention, furthermore, the described method of detecting and/or measuring the concentrations of polypeptides, in particular polypeptide on the cell surface, or the corresponding peptides or polynucleotides, including: 1) introduction of the sample from the patient into contact with an antibody directed to this polypeptide (for example, at least one fragment of any of SEQ ID NO:1-702 or the x modified sequences), or with the corresponding peptide or polynucleotide (for example, at least one fragment of SEQ ID NO:703-1395 or modified sequences); and 2) determining the presence or concentration of the formed complex of the antibody with the corresponding polypeptide, peptide or polynucleotide in the sample. Such methods can also be used for detection and/or measurement of titers of microbial cells, in particular cells methaneproducing.

According to the invention also provides a method of detecting and/or measuring the concentrations of polynucleotides, in particular polynucleotide encoding a component of the cell surface, including: 1) introduction of the sample from the patient into contact with complementary polynucleotides (for example, with a sequence complementary to at least a fragment of any of SEQ ID NO:703-1395 or modified sequences); and 2) determining the presence or concentration of the hybridization complex formed with polynucleotides in the sample. Such methods can also be used for detection and/or measurement of titers of microbial cells, in particular cells methaneproducing.

In specific aspects of the methods according to the invention using the components of thein vivoorin vitroexpression. In other aspects, these methods use peptides, polypeptides, polynucleotide or as is tetela, produced by recombinant, synthetic or semi-synthetic means or endogenous means.

Other aspects and embodiments of the invention described below.

BRIEF DESCRIPTION of DRAWINGS

This invention is described with reference to specific embodiments of such, and with reference to the figures.

FIGURES 1A-1C. The comparison of the genomes ofMethanobacteriales(Fig. 1A); the statistics of the genomeM. ruminantium(Fig. 1B); the genes presumably involved in methanogenesis species ofMethanobacteriales(Fig. 1C).

FIGURE 2. The vaccination scheme.

FIGURE 3. Humoral immune responses of sheep to the vaccine preparations of the cell wallM. ruminantiumand peptides designed againstM. ruminantiummtr,as well as proteins on the cell surface.

FIGURE 4. The sequence of the peptides used for the production of antibodies.

FIGURES 5A-5B. Open reading frames selected for the production of antibodies: the nucleotide sequence (Fig. 5A); the amino acid sequence (Fig. 5B).

FIGURES 6A-6C. Open reading frames encoding the enzymes of the metabolic pathway of methanogenesis identified by theM. ruminantium: annotations (Fig. 6A); the nucleotide sequence (Fig. 6B); the amino acid sequence (Fig. 6C).

FIGURES 7A-7C. Open reading frames for proteins on the cell surface, Eden is oficerowie the M. ruminantium: annotations (Fig. 7A); the nucleotide sequence (Fig. 7B); the amino acid sequence (Fig. 7C).

FIGURES 8A-8C. Open reading frames encoding the biosynthesis of exopolysaccharides identified by theM. ruminantium: annotations (Fig. 8A); the nucleotide sequence (Fig. 8B); the amino acid sequence (Fig. 8C).

FIGURES 9A-9C. Open reading frames, including transmembrane domains identified by theM. ruminantium: annotations (Fig. 9A); the nucleotide sequence (Fig. 9B); the amino acid sequence (Fig. 9C).

DETAILED description of the INVENTION

Definition

The term "antibody" should be understood in its broadest sense, and he presumably includes the intact monoclonal antibodies and polyclonal antibodies. He presumably also encompasses fragments and derivatives of antibodies to the length, yet they still exhibit the desired biological activity. Antibodies include immunoglobulin molecules and immunologically active molecules of immunoglobulins (Ig), i.e. molecules containing antigennegative site which specifically binds (immunochemical reacts with the antigen. They include, but are not limited to the above, polyclonal, monoclonal, chimeric, single-chain, Fc, Fab, Fab' and Fab2fragments, and is also a library of expressed Fab.

Molecules antibodies belong to any of the classes: IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chain present in the molecule. They include subclasses such as IgG1, IgG2, and others. The light chain can be Kappa-chain or lambda chain. Reference in the present description antibody includes a reference to all the classes, subclasses and types. Also includes chimeric antibodies, such as monoclonal antibodies or fragments thereof that are specific for more than one source, such as one or more sequences of mouse, human or ruminant animals. Added "camel" antibodies (single-stranded Manantial family of camelids) or nanotesla. It is clear that each reference to an "antibody" or any similar term in the present description includes intact antibodies, and any fragments, modifications, derivatives, or variants.

"Modified" nucleic acid sequences encoding peptides, polypeptides or antibodies used in the present description, include these sequences with deletions, insertions, or substitutions of different nucleotides, resulting in polynucleotide that encodes the same or a functionally equivalent sequence. The encoded peptide, polypeptide or antibody may also be "altered" and in order to win such deletions, insertion or replacement of amino acid residues that cause silent change and result in a functionally equivalent sequence. Deliberate amino acid substitutions may be done on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of the residues as long as there remains biological activity (for example, the Association of cells, the Association of membranes) or immunogenic/immunological activity. For example, negatively charged amino acids may include aspartic and glutamic acids, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar terminal groups having similar hydrophilicity values may include leucine, isoleucine and valine, glycine and alanine, asparagine and glutamine, serine and threonine, phenylalanine and tyrosine.

"Amino acid sequence", as used in the present description, is a sequence of oligopeptides, peptide, polypeptide, protein, or antibody and its fragment, and any molecules that have a natural, recombinant, or synthetic or semi-synthetic origin. Sequence according to the invention contain at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250 amino acids, preferably by ENISA least from 5 to 10, from 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 100, from 100 to 150, from 150 to 200, or 200 to 250 amino acids. Sequence retain biological activity (e.g., effects on cell growth and/or proliferation) or immunogenic/immunological activity of amino acid sequence. "Amino acid sequence" and any similar terms are not limited to only the complete, native amino acid sequence associated with a full-sized molecules, but also include any fragments thereof, modifications, derivatives and options.

"Amplification"as used in this description, called the production of additional copies of a nucleic acid sequence and is generally carried out with the help of technical means of polymerase chain reaction (PCR), are well known in the art (Dieffenbach, C.W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY).

The terms "biologically active" or "functional"as used in this description, called a peptide or polypeptide that retains one or more structural, immunogenic or biochemical functions (for example, the Association of cells, the Association of membranes) sequence of natural origin.

The terms "cellular inhibitor" or "inhibitor", as used in the present description, referred to as funds, to the which reduce or inhibit the growth or replication of microbial cells, especially cells methaneproducing. Cellular inhibitor may act to reduce or block, for example, cell division. The inhibitor can reduce or block, for example, DNA synthesis, RNA synthesis, protein synthesis or post-translational modification. The inhibitor can also reduce or block the activity of enzymes involved in metabolic pathways of methanogenesis. The inhibitor may also target cell recognition components of the immune system. Suppression of cell also includes murder and cell death, for example, in the lysis, apoptosis, necrosis, etc. are Useful inhibitors include, but are not limited to the above, compounds that prevent methanogenesis (e.g., bromoethanesulfonate), antibodies and antibody fragments, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, as described in detail in the present description.

The terms "complementary" or "complementarity"as used in this description, called the normal linking polynucleotides in gentle salt and temperature conditions by mating grounds. For the sequence A-G-T complementary sequence is T-C-A, reverse complement represents A-C-T, and the reverse sequence is T-G-A. Complem nternet between two single-stranded molecules may be partial, when contacted, only some of the nucleic acids, or it may be complete when there is full complementarity between the single-stranded molecules. The degree of complementarity between the chains of nucleic acids has a significant impact on the efficiency and strength of hybridization between the chains of nucleic acids. This is of particular importance for amplification reactions, which depend upon binding between the chains of nucleic acids, as well as for the development and application of molecules, peptide nucleic acid (PNA).

The term "derivative", as used in the present description, is called the chemical modification of a nucleic acid that encodes a peptide, polypeptide or antibody, or a nucleic acid complementary to it. Such modifications include, for example, substitution of a hydrogen on alkyl, acyl, or amino group. In preferred aspects derived nucleic acids encode a peptide, polypeptide or antibody that retain the biological or immunogenic/immunological activity of natural molecules. Derivative peptide, polypeptide, or antibody are what is modified by glycosylation, paglierani or any similar process that retains one or more biological functions (for example, the Association of cells, the Association of membranes) or monogenous/immunological activity sequence, from which they are derived.

The term "homology", as used in the present description, is called the degree of complementarity. There is partial homology (i.e. less than 100% identity or homology (that is, 100% identity). A partially complementary sequence that at least partially retains an identical sequence from hybridizing with the target nucleic acid, called functional term "virtually homologous". The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (e.g., southern blotting or Northern blotting, hybridization in solution and the like) under conditions of low stringency. Almost homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of low stringency. This does not mean that the conditions of low stringency are that Pets nonspecific binding; conditions of low stringency require that the binding of two sequences to each other was specific (i.e., selective) interaction.

The term "hybridization"as used in this description, is any process SIP is utilized which chain of nucleic acid binds with a complementary chain by mating grounds.

"Insertion" or "addition"is used in the present description, is called the change of amino acid or nucleotide sequence by adding one or more amino acid residues or nucleotides, respectively, compared to a molecule of natural origin.

"Methaneproducing"used in this description are called microbes that produce gaseous methane, includingMethanobrevibacter,Methanothermobacter, Methanomicrobium, MethanobacteriumandMethanosarcina. Specific methaneproducing include, but are not limited to the above,Methanobrevibacterruminantium(i.e. strain M1 or strain DSM1093),Methanobrevibactersmithii,Methanobrevibacteracididurans,Methanobrevibacterthaueri, Methanobacterium bryantii, Methanobacterium formicicum, Methanothermobacter marburgensis, Methanothermvobacter wolfeii, Methanosphaera stadtmanae, Methanomicrobium mobile, Methanosarcina barkeri, Methanosarcina mazei, Methanococcoides burtoniiandMethanolobus taylorii. All species and genera of methaneproducing covered by this term.

"Microbial cells used in the present description, are called cells of microorganisms of natural origin or genetically modified, including archaebacteria, such as methaneproducing, halophila and thermoacidophile, and eubacteria, such as cyanobacteria, spirochaetes, proteobacteria and gram-positive and gram-negative bacteria.

The term "modified" on yaytsa modified sequences and fragments of the sequences, options and derivatives, as described in the present description.

"The sequence of the nucleic acid" or "nucleotide sequence", as used in the present description, is a sequence of polynucleotide, the oligonucleotide or its fragments, and the DNA or RNA of natural, recombinant, or synthetic or semi-synthetic origin which may be single - or double-stranded and may represent the sense or antisense chain, coding and non-coding region. Sequence according to the invention preferably include at least 12, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 300, 450, 600, 750 nucleotides, preferably at least 15 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, from 150 to 300 300 to 450 450 to 600, or 600 to 750 nucleotides, or at least 1000 nucleotides, or at least 1500 nucleotides. It is clear that each reference to "the sequence of the nucleic acid" or "nucleotide sequence" in the present description will include native full-length sequence, and any complements, fragments, modifications, derivatives, or variants.

The term "oligonucleotide" is a nucleic acid sequence with a length of at least 6, 8, 10, 12, 15, 18, 21, 25, 27, 30, or 36 nucleotides, or at least from 12 to 36 nucleotides, or the least about 15 to 30 nucleotides, which can be used in PCR amplification, sequencing or hybridization assays. Used in the present description, the term "oligonucleotide" is virtually equivalent to the terms "amplier", "primers", "oligomers" and "probes", as usually formulated in this field.

The term "polynucleotide", whether you used it in the singular or the plural, usually called any sequence of nucleic acids, for example any polyribonucleotide or polyethoxylated, which may be unmodified RNA or DNA or modified RNA or DNA. This sequence includes, without limitation, single - and double-stranded DNA, DNA, including single - and double-stranded region, the single - and double-stranded RNA, and RNA including single - and double-stranded region, the hybrid molecules of DNA and RNA that may be single-stranded or, more typically, double-stranded or include single - and double-stranded region. Also includes triplex region comprising RNA or DNA or both DNA and RNA. Specifically included are mRNA, cDNA, genomic DNA, or any fragments thereof. The term includes DNA and RNA, which contain one or more modified bases, such as tritium-labeled bases or minor reasons, such casinosin. These polynucleotide according to the invention may include the coding or noncoding sequences, sense or antisense sequence, or interfering RNA, such as small interfering RNA. It is clear that each reference to "polynucleotide" or a similar term in this description will include the full sequence, as well as any accessories, parts, modifications, derivatives, or variants.

"Peptide" and "polypeptide", as used in the present description, called the selected peptides or polypeptides according to the invention, obtained from any species, preferably microorganisms, from any source, whether natural, synthetic, semi-synthetic or recombinant origin. In particular, the peptide or polypeptide according to the invention can be obtained from cells of methaneproducing, such as cellsMethanobrevibacterin particularM. ruminantiumor cellsM. smithii. For recombinant production of peptide or polypeptide according to the invention can be derived from microbial or eukaryotic cells, such asEscherichia, Streptomyces, Bacillus, Salmonella, yeast, insect cells such as Drosophila, animal cells such as COS cells and Cho, or plant cells. It is clear that each reference to "a peptide" or "polypeptide" in the present description will be on the th full sequence, as well as any fragments, modifications, derivatives, or variants.

"Peptide nucleic acid" or "PNA"used in this description, called antisense molecule or antigenic agent, which include the Foundation associated through peptide backbone.

The term "ruminant"used in this description are called animals that have the scar as a special kind of digestive organ. Ruminants include, but are not limited to the above, cattle, sheep, goats, Buffalo, elk, antelope, caribou and deer.

The terms "stringent conditions" or "stringency"as used in the present description, are conditions for hybridization due to nucleic acid, salt and temperature. These conditions are well known in this field and can be modified in order to identify and detect identical or related polynucleotide sequences. See, for example, Sambrook, J.et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY and Ausubel, F. M.et al.(1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. Numerous relevant conditions, covering either the low or high severity, depend on such factors as the length and feature sequence (DNA, RNA, nucleotide composition), the nature of the target molecules (DNA, RNA, nucleotide composition), environmental conditions (the solution or immobilized on a solid substrate), the concentration of salts and other components (for example, formamide, dextran sulfate and/or polyethylene glycol) and reaction temperature (for example, in the range from about 5°C below the melting temperature of the probe to about 20°C-25°C below the melting temperature). One or more factors can be modified to generate conditions of either low or high stringency than, but equivalent to the above conditions.

The term "subject" includes humans and non-human animals. Non-human animals include, but are not limited to the above, birds and mammals, such as ruminants, and in particular, mice, rabbits, cats, dogs, pigs, sheep, goats, cows and horses.

The terms "virtually purified" or "isolated"as used in this description, called the sequence of nucleic acids or amino acids that are extracted from his cell, recombinant or synthetic environments and at least 60% free, preferably 75%and most preferably at least 90% free, or at least 99% free from other components with which they are associated in their environment. "Selected" polynucleotide and polypeptides have been identified and separated from at least one impurity molecules with which they are associated in their natural state. Accordingly, onate, selected polynucleotide and polypeptides are in a form which differs from the form and context in which they are found in nature. In addition, it should be understood that "isolated" does not necessarily reflect an accurate measure (for example, a specific percentage)to which this sequence was cleared.

"Transformation", as used in the present description, describes the process by which exogenous DNA enters and changes a recipient cell. This may occur under natural or artificial conditions using various methods, well known in this field. The transformation can be based on any known method for the insertion of foreign sequences of nucleic acids in prokaryotic and eukaryotic cells-owners. This method is selected depending on the type of host cell, subject to transformation, and may include, but are not limited to the above, viral infection, electroporation, heat shock, lipofection and the bombing of the microparticles. Such "transformed" cells are composed of stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the chromosome of the host. They also include cells that are temporarily Express the inserted DNA or R Is It for a limited period of time.

"Vaccine"as used in this description, includes all components and compositions for stimulating an immune response in the subject. Particularly useful in this respect are subunit vaccines, including peptide vaccines, and vector vaccines, vaccines based on nucleic acids, as well as edible vaccines. Vaccines can be used to create or enhance the immune response to an antigen, in particular microbial antigen. In specific aspects vaccines contain antigens that elicit protective immunological reactions such as the formation of antibodies, T-helper and T-cell responses. The vaccine may also include antibodies, for example, for passive immunization.

"Variant" of a peptide, polypeptide, or antibody used in the present description, is called an amino acid sequence that is altered by one or more amino acids. Variant of polynucleotide modified by one or more nucleotides. Option may be the "conservative substitutions", when substituted amino acid has similar structural or chemical properties, for example the substitution of leucine for isoleucine. Rarely, a variant may be the result of "non-conservative substitutions, such as replacing glycine to tryptophan. Similar minor modifications can also include amino acid is major deletions, or insertions, or both at the same time. Methodological principles to determine which amino acid residues can be substituted, inserted or deleterow without destroying the biological or immunogenic/immunological activity can be found using computer programs well known in this field, for example the software package LASERGENE (DNASTAR).

The invention also includes variants that retain at least one biological activity (for example, the Association of cells, the Association of membranes) or immunogenic/immunological activity. The preferred option is the one that has almost the same or a functionally equivalent sequence, for example having a sequence at least 80%, and more preferably at least 90% identical disclosed in the present description of the sequence. The most preferred option is the one that has at least 95%, at least 97%, at least 98% or at least 99% identity disclosed in the present description of the sequence. The percentage identity is determined by aligning the two sequences, which should be compared, as described below, determining the number of identical residues in the aligned parts, dividing that number by the total number of residues in the patent (search) sequence and is the Nozhai the result by 100. Useful equalization program is AlignX (Vector NTI).

Description of the invention

Methane is formed by methaneproducing in the anterior intestine of ruminants, which act as the final reducing carbon in the system of the scar. Multi-step metabolic pathway of methanogenesis well elucidated mainly from the study "nerovnovych" methaneproducing, but adaptations that allow methaneproducing to grow and survive in the rumen is not fully understood.Methanobrevibacter ruminantiumis known methaneproducing the ruminants in New Zealand. As indicated in the present description, geneM. ruminantiumwas sequenced, and shown that its size is approximately 3,0 MPa with GC content at the level 33,68%. Identified all the components of the metabolic pathway of methanogenesis, and compared these sequences of genes with similar ofMethanobacterium thermoautotrophicumandMethanosphaera stadtmanaeindicates that the gene organization of methanogenesis fromMethanobacterialesconservative (Fig. 1C). This genome contains many large surface proteins with characteristics that indicate that they mediate the Association with other microbes of the rumen. In various aspects of the invention identified polynucleotide and polypeptides can be used as a means to suppress methaneproducing and/or methanogenesis in ru is CE, and further elucidate the role ofM. ruminantiumin the formation of methane. Especially useful are disclosed polynucleotide and polypeptides identified as components involved in methanogenesis (Fig. 6A-6C), components of the cell surface (Fig. 7A-7C), the components involved in the biosynthesis of exopolysaccharides (Fig. 8A-8C), components with transmembrane domains (Fig. 9A-9C), as well as polynucleotide and polypeptides for antibody production (Fig. 5A-5B).

Peptides, polypeptides and polynucleotide

The invention includes peptides and polypeptides, including at least one of SEQ ID NO:1-702, as well as fragments, variants and derivatives thereof. The peptides and polypeptides of the present invention can be expressed and used in various assays to determine their biological activity. The peptides and polypeptides can be used for large-scale synthesis and allocation patterns, for example for commercial production. Such peptides and polypeptides can be used to build antibodies to select the appropriate amino acid sequence, as well as to quantify the concentration of these amino acid sequences. The peptides and polypeptides can be used in vaccines for targeting and suppression of microbial cells, especially glue is OK-methaneproducing. The peptides and polypeptides can also be used for the production of antibodies that inhibit the growth and replication of these cells. The peptides and polypeptides of the present invention can also be used as compositions, such as pharmaceutical compositions, particularly vaccine compositions. In particular aspects can be used vnutrichasovye of a slow-release device in combination with the peptides, polypeptides, antibodies, and compositions (such as pharmaceutical compositions, particularly vaccine compositions) according to the invention.

The peptides of the present invention contain at least one sequence selected from the group consisting of: (a) peptides comprising at least a fragment of one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or fragments, variants, or derivatives thereof; (b) peptides comprising the functional domain of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702, and fragments and variants; and (c) peptides comprising at least a specified number of contiguous residues in least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or its variants or derivatives thereof. In one embodiment, the invention includes : the Lenna peptide, comprising the amino acid sequence of at least one of SEQ ID nos:1-9. All these sequences are collectively referred to in this description as the peptides according to the invention.

The polypeptides of the present invention contain at least one sequence selected from the group consisting of: (a) polypeptides comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or its fragments, variants, or derivatives; (b) polypeptides comprising a functional domain of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702, and fragments and variants; and (c) polypeptides comprising at least a specified number of contiguous residues at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or its variants or derivatives thereof. In one embodiment, the invention includes the selected polypeptide comprising the amino acid sequence of at least one of SEQ ID nos:1-9. All these sequences are collectively referred to in this description as the polypeptides according to the invention.

The invention also includes a dedicated polynucleotide that encodes a peptide or polypeptide with SEQ ID NO:1-702. Selected polynucleotide on this image is meniu have practical significance in genome mapping, in physical mapping and cloning of genes more or less related components of the cell surface. The probes developed using polynucleotides of the present invention, can be used for detection of the presence and study the expression patterns of genes in any organism whose cells sufficiently homologous sequences of DNA and RNA, using methods that are well known in the field, methods such as slot blot or analysis using microarrays. The probes developed using polynucleotides of the present invention, can be used for sequencing and PCR amplification. Polynucleotide according to the invention can be used for the manufacture of expression vectors and cell-hosts for vaccines with the aim of targeting and suppression of microbial cells, especially cells methaneproducing. The invention further includes the use of polynucleotides for the production of antibodies that inhibit the growth and replication of these cells. Polynucleotide of the present invention can also be used as compositions, such as pharmaceutical compositions, particularly vaccine compositions. In particular aspects can be used vnutrichasovye of a slow-release device in combination with data from polynucleotides is, vectors, cells masters and compositions (such as pharmaceutical compositions, particularly vaccine compositions) according to the invention.

Polynucleotide of the present invention contain at least one sequence selected from the group consisting of: (a) sequences comprising a sequence encoding at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or its fragments or variants; (b) complements, reverse sequences, or reverse complements sequence that encodes at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or its fragments, and variants; (c) open reading frames containing the sequence coding for least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702, and fragments and variants; (d) functional domains of the sequence that encodes at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702, and fragments and variants; and (e) sequences comprising at least a specified number of contiguous residues of a sequence that encodes at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-702 or its variants; and (f) sequences comprising at least a specified number of contiguous nucleotides of any of SEQ ID NO:703-1395. Also provides oligonucleotide probes and primers, as well as their options. All these polynucleotide and oligonucleotide probes and primers in the remainder of this description are referred to collectively as polynucleotide according to the invention.

Specialists in this field it is clear that as a result of the degeneracy of the genetic code can be lots of nucleotide sequences encoding peptides or polypeptides according to the invention, some of which have minimal homology to the nucleotide sequences of any known gene of natural origin. Thus, the invention considers without exception, all possible variants of the nucleotide sequence, which can be made by selecting combinations based on possible codon. These combinations are made in accordance with the standard triplet genetic code as applied to amino acid sequences of natural origin, and all such variations should be considered specifically disclosed.

The nucleotide sequence encoding the peptides and polypeptides or fragments or variants are preferably capable of g is britishly with nucleotide sequence of natural origin when selected properly the parameters of the peptide or severity of the conditions. However, it may be advantageous to produce nucleotide sequences encoding a peptide or polypeptide, or a fragment, or derivative, with significantly different frequency of use of the codon. Codons can be selected to increase the speed with which the expression of the peptide or polypeptide in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for significant modification of the nucleotide sequence encoding the peptides or polypeptides and their derivatives without altering the encoded amino acid sequences include obtaining RNA transcripts with more desirable properties, for example with an extended half-life, than transcripts derived from the sequence of natural origin.

The invention also includes obtaining DNA sequences or fragments which encode peptides or polypeptides or fragments or variants, using synthetic chemistry. After receiving the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the field. In addition, synthetic x is MIA can be used to introduce mutations into a sequence, encoding the peptide or polypeptide or any of their variants or fragments. Also included in the invention sequence polynucleotide capable of hybridizing to the claimed nucleotide sequences, and, in particular, with those specified in SEQ ID NO:703-1395, and varying severity, as discussed in Wahl, G.M. and Berger, S.L. (1987; Methods Enzymol. 152:399-407), and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).

Methods for determining DNA sequences are well known and widely available in this area and can be used in the practice of any of embodiments according to the invention. When these methods can be applied such enzymes as the fragment maple DNA polymerase I, sequenase (SEQUENASE) (U.S. Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase Amersham Pharmacia Biotech (Piscataway, NJ), or combinations of polymerases and "editorial" nucleases, as found in the amplification system, elongase (ELONGASE Amplification System)sold on the market by Life Technologies (Gaithersburg, MD). Preferably, this process can be automated by using an instrument such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA), and the ABI Catalyst and 373 and 377 DNA sequencers (Perkin Elmer) or Genome Sequencer 20™ (Roche Diagnostics).

Nucleic acid sequences encoding peptides or polypeptides, may be extended utilizing a partial nucleotide placentas is required and applying different methods, known in this area for the detection of 3'-5'-directional sequences, such as promoters and regulatory elements. For example, one method that can be used site-restriction" PCR, uses universal primers to recover the unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA is first amplified in the presence of primer to the linker sequence and a primer specific to the known region. Amplificatoare sequences are then subjected to a second round of PCR with the same linker primer and other specific primers internal to the first. The products of each round of PCR transcribers using the appropriate RNA polymerase and is sequenced using reverse transcriptase.

Commercially available capillary electrophoresis system can be used to analyze the size or confirm the nucleotide sequence of the products of sequencing or PCR. In particular, capillary sequencing can use flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide), activated by the laser, and the detection of the emitted wavelengths by a camera on charge-coupled devices. The intensity of ihoda/light can be converted to electrical signal using appropriate software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer)and the entire process from loading of samples to computer analysis and display of electronic data, can be controlled by the computer. Capillary electrophoresis is particularly preferred for the sequencing of small DNA fragments that may be present in limited amounts in a particular sample.

In another embodiment, the invention polynucleotide or fragments thereof, encoding the peptides or polypeptides may be used in recombinant DNA molecules to direct expression of these peptides or polypeptides or fragments or variants in the appropriate cell hosts. Due to the natural degeneracy of the genetic code can be obtained from other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence, and these sequences can be used for cloning and expression of peptides or polypeptides. The nucleotide sequence of the present invention can be engineered using methods well-known in this field, with the aim of modifying the sequences encoding amino acids for a variety of reasons, including, not limited to the above, changes that modify the cloning, processing and/or expression of the gene products is impressive. DNA shuffling by random fragmentation and secondary assemblies using PCR gene fragments and synthetic oligonucleotides may be used to design the nucleotide sequences. For example, can be used site-directed mutagenesis to insert new restriction sites, modification models of glycosylation changes of preference codons, the introduction of mutations and so on.

In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding the peptides or polypeptides can be legirovanyh with a heterologous sequence to encode a chimeric protein. For example, it may be useful to encode a chimeric sequence recognized commercially available antibodies. The chimeric protein may also be engineered to contain a cleavage site located between the peptide or polypeptide according to the invention and the heterologous protein sequence, so that the peptide or polypeptide can be cleaved and purified away from the heterologous balance.

In another embodiment, sequences encoding the peptides or polypeptides may be synthesized in whole or in part, using chemical methods well known in the art (see Caruthers, .H. et al.(1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T.et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the polypeptide may be produced using chemical methods to synthesize the amino acid sequence or its fragment. For example, the fusion polypeptide can be performed using various solid-phase techniques (Roberge, J.Y.et al.(1995) Science 269:202-204; Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154)and automated synthesis may be achieved, for example, by using a peptide synthesizer ABI 431A Peptide Synthesizer (Perkin Elmer). Various fragments of the peptides or polypeptides can be chemically synthesized separately and combined using chemical methods to obtain the full-length molecule.

The newly synthesized peptide or polypeptide can be isolated using preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins Structures and Molecular Principles, W.H. Freeman and Co., New York, NY). The composition of the synthetic peptides or polypeptides may be confirmed by amino acid analysis or sequencing (e.g., procedure of degradation on Adminu; Creighton, supra). In addition, the amino acid sequence of a peptide or polypeptide, or any portion thereof may be altered during direct synthesis and/or combined by chemical methods with sequences from other proteins, or any part of them, to politicisation molecule.

In order to Express a biologically active peptides or polypeptides, nucleotide sequences, coding sequences or functional equivalents, may be inserted into a suitable expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted coding sequences. Can be used in ways well known to specialists in this field, to construct expression vectors containing sequences encoding a peptide or polypeptide, and appropriate controls transcription and translation. These methods includein vitromethods of recombinant DNA and synthetic techniques and genetic recombinationin vivo. Such methods are described in Sambrook, J.et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, as well as in Ausubel, F.M.et al.(1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY.

A number of expression vectors and systems host cells can be used for the retention and expression of sequences encoding peptides or polypeptides according to the invention. They include, but are not limited to the above, microorganisms such as bacteria transformed with recombinant phage, plasmid or kominami DNA expression vectors; yeast transformed yeast vectors expre the FIC; system of the insect cells infected with viral expression vectors (e.g. baculovirus); plant cell transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g. plasmids Ti or pBR322); any system of animal cells. For bacteria useful plasmids include pET, pRSET, pTrcHis2 and pBAD plasmid from Invitrogen, pET and pCDF plasmids from Novagen, as well as Director™ plasmid from Sigma-Aldrich. For methaneproducing useful plasmids include, but are not limited to the above, pME2001, pMV15 and pMP1. The invention is not limited only used expression vectors or cells of the host.

The "control elements" or "regulatory sequences" are those non-translated region of vectors, enhancers, promoters, 5'- and 3'-noncoding region, which interact with proteins in host cells for transcription and translation. Such elements may vary in their effectiveness and specificity. Depending on the vector system and used host cells can be used any number of suitable elements for transcription and translation, including constitutive and inducible promoters. For example, when cloning in bacterial systems can be used to the s-induced promoters, such as the hybrid lacZ promoter family BLUESCRIPT (Stratagene, LaJolla, CA) or plasmids pSPORTI (Life Technologies) and the like. Baculovirus polyhedrin promoter may be used in insect cells. Promoters and enhancers, formed from the genome of plant cells (e.g., genes, proteins, heat shock, RUBISCO and spare protein) or of plant viruses (for example, viral promoters or leader sequences), can be cloned in this vector.

In bacterial systems a number of expression vectors may be selected depending on the application, expected for a peptide or polypeptide. For example, when you have a large number of peptide or polypeptide, can be used in vectors designed for high level expression of a chimeric protein, which can easily be cleaned. Such vectors include, but are not limited to the above, the multipurpose cloning vectors and expression inE. colisuch as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide, can be Legerova in the vector in the same reading frame with sequences aminoanisole Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein produced; pIN vectors (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like.

pGEX vectors (Promega, Madison, WI) can also be used DL is the expression of peptides or polypeptides in the form of a chimeric protein with glutathione-S-transferase (GST). Typically, these chimeric proteins are soluble and can easily be purified from lysed cells by adsorption on pellets glutathione-agarose followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin or factor XA cleavage sites of the protease and interest to the cloned peptide or polypeptide can be released from the GST fragment discretion. In the yeastSaccharomyces cerevisiaecan be used a number of vectors containing constitutive or inducible promoters such as alpha factor, alcoholecstasy and PGH. For an overview, see Ausubelet al.(above), as well as Grantet al.(1987) Methods Enzymol. 153:516-544.

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding peptides or polypeptides according to the invention. Such signals include the initiating ATG codon and adjacent sequences. In cases where sequences encoding a peptide or polypeptide, his initiating codon and sequences in the 3'-5' direction, inserted into the appropriate expression vector, no additional transcriptional or translational control signals. However, in cases when inserted only serial encoding inost or its fragment, must be provided by exogenous translational control signals, including the initiating codon ATG. In addition, the initiating codon must be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiating codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers that are intended for specific cellular system, for example, described in the literature (Scharf, D.et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, the strain host cells can be selected by its ability to modulate the expression of the inserted sequences or subjected to processing downregulation of the peptide or polypeptide in the desired manner. Such modifications sequences include, but are not limited to the above, acetylation, carboxylation, glycosylation, phosphorylation, lipidization and acylation. Post-translational processing which cleaves a "pre-Pro" form of the peptide or polypeptide can also be used to ensure correct insertion, folding and/or function. Different cell owners who have specific cellular device and characteristic mechanisms for post-translational activities, may be the pic is of aulani American type culture collection (ATCC; Bethesda, MD) and can be chosen to ensure the correct modification and processing of the sequence. Specific cell hosts include, but are not limited to the above, cells methaneproducing, such as cellsMethanobrevibacterin particularM. ruminantiumor cellsM. smithii. Cell hosts include, for example,Rhodotorula, Aureobasidium, Saccharomyces, Sporobolomyces, Pseudomonas, ErwiniaandFlavobacterium; or such other organisms asEscherichia, Lactobacillus, Bacillus, Streptomycesand like them. Specific cell hosts includeEscherichia colithat is especially suited for use in the present invention,Saccharomyces cerevisiae,Bacillus thuringiensis, Bacillus subtilis, Streptomyces lividansand similar to them.

There are several methods of introducing nucleic acids into eukaryotic cells, culturedin vitro. These include chemical methods (Feigneret al., Proc. Natl. Acad. Sci., USA, 84:7413 7417 (1987); Bothwellet al., Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jones and Bartlett Publishers, Inc., Boston, Mass. (1990), Ausubelet al., Short Protocols in Molecular Biology, John Wiley and Sons, New York, NY (1992); and Farhood, Annal. NY Acad. Sci., 716:23-34 (1994)), the use of protoplasts (Bothwell,aboveor electrical impulses (Vatteroniet al., Mutn. Res., 291:163-169 (1993); Sabelnikov, Prog. Biophys. Mol. Biol., 62:119-152 (1994); Bothwellet al.,above; and Ausubelet al.,above), the use of attenuated viruses (Daviset al., J. Virol. 1996, 70(6), 3781-3787; Brinsteret al.J. Gen. Virol. 2002, 83(Pt 2), 369-381; Moss, Dev. Biol. Stan., 82:55 to 63 (1994); and Bothwellet al. above)and physical methods (Fynanet al.Int J Immunopharmacol. 1995 Feb; 17(2):79-83; Johnstonet al., Meth Cell Biol., 43(Pt A): 353-365 (1994); Bothwellet al.,above; and Ausubelet al.,above).

Successful delivery of nucleic acids in animal tissues can be achieved using cationic liposomes (Watanabeet al., Mol. Reprod. Dev., 38:268-274 (1994)), direct injection deproteinizing DNA or RNA in muscle tissue of animals (Robinsonet al., Vacc, 11:957-960 (1993); Hoffmanet al., Vacc. 12:1529-1533; (1994); Xianget al., Virol., 199:132-140 (1994); Websteret al., Vacc, 12:1495-1498 (1994); Daviset al., Vacc, 12:1503-1509 (1994); Daviset al., Hum. Molec Gen., 2:1847-1851 (1993); Dalemanset al.Ann NY Acad. Sci. 1995, 772, 255-256. Conry,et al.Cancer Res. 1995, 55(7), 1397-1400) and embryos (Naitoet al., Mol. Reprod. Dev., 39:153-161 (1994); and Burdonet al., Mol. Reprod. Dev., 33:436-442 (1992)), intramuscular injection of self-replicating RNA vaccines (Daviset al.J Virol 1996, 70(6), 3781-3787; Balasuriyaet al.Vaccine 2002, 20(11-12), 1609-1617) or intradermal injection of DNA technology, using a "gene gun" (Johnstonet al.,above).

In this area there are many protocols for detection and evaluation of expression of peptides or polypeptides according to the invention, using either polyclonal or monoclonal antibodies specific for this protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay analysis (RIA) and fluorescent sorting cells (FACS). Two-layer enzyme-linked immunosorbent assay based on mo is oclonal antibodies can be applied using monoclonal antibodies, reacting with two reiterferien the epitope peptide or polypeptide, but may also be applied to the analysis of competitive binding. These and other tests in addition to other places described in Hampton, R.et al.(1990; Serological Methods, a laboratory Manual, APS Press, St Paul, MN), and Maddox, D.E.et al.(1983; J. Exp. Med. 158:1211-1216).

Specialists in this field there are a large variety of labels and conjugation methods, and it can be used in various analyses of nucleic acids and amino acids. Means for receiving labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligo-labeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternative sequences encoding peptides or polypeptides, or any fragments thereof, or variants, can be cloned into a vector to obtain an mRNA probe. Such vectors are known in the field that are commercially available and can be used to synthesize RNA probesin vitroby adding the appropriate RNA polymerase such as T7, T3 or SP6, and labeling of nucleotides. These procedures may be conducted using a variety of commercially available kits from Amersham Pharmacia Biotech, Promega, and US Biochemical. Suitable reporter molecules or labels, suitable for ease of detection include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like.

The expression vectors or cells of hosts transformed with the expression vectors, may be cultured under conditions suitable for the expression and excretion of the peptide or polypeptide from the culture. Culture can include components for the expression ofin vitroorin vivo. Components of the expressionin vitroinclude those for lysate of rabbit reticulocytes, lysatesE. coliand extracts of wheat germ, for example, systems Expressway™ or RiPs from Invitrogen, system Genelator™ from iNtRON Biotechnology, systems EcoPro™ or STP3™ from Novagen, systems TNT® Quick Coupled from Promega and the EasyXpress system from QIAGEN. The peptide or polypeptide derived from the culture, can secretariats or contained within the cell depending on the sequence and/or used in the vector. In specific aspects of expression vectors, encoding the peptide or polypeptide can be constructed containing signal sequences which direct secretion of the peptide or polypeptide through a prokaryotic or eukaryotic cell membrane.

Other designs can include amino acid domain, which facilitates purification of the peptide or polypeptide. Such domains include, but are not limited to the above, the metal-Hel is yousie domains such as histidine-tryptophan (e.g., 6X-HIS (SEQ ID NO:1396)) modules that allow purification on immobilized metals, protein domains And that allow purification on immobilized immunoglobulin, and the domain used in inserzione/affinity purification system FLAG® (Immunex Corp., Seattle, WA). Suitable epitope markers include 3XFLAG®, HA, VSV-G, V5, HSV, GST, GFP, MBP, GAL4 and β-galactosidase. Suitable plasmids include those that contain biodynamy marker (e.g., plasmids PinPoint™ from Promega), calmoduline protein (e.g., plasmids pCAL from Stratagene), strategicinitiatives peptide (e.g., plasmids InterPlay™ from Stratagene), c-myc or FLAG® marker (e.g., plasmids thus from Sigma-Aldrich) or his-tag token (e.g., plasmids QIAExpress from QIAGEN).

To facilitate cleaning, the expression vectors can include biodegradable linker sequence, such as specific for factor XA or enterokinase (Invitrogen, San Diego, CA). For example, the vector may include one or more linkers between the purification domain and the peptide or polypeptide. One such expression vector provides the opportunity for expression of a chimeric protein comprising a peptide or polypeptide according to the invention and a nucleic acid encoding 6 histidine residues (SEQ ID NO:1396)preceding thioredoxin or enterokinase the site of cleavage. Residues of histidine facilitate cleaning and the IMAC (affinity chromatography on immobilized metal ions, as described in Porath, J.et al.(1992) Prot. Exp. Purif. 3:263-281), while enterokinase the cleavage site provides a means for the purification of a peptide or polypeptide from the chimeric protein. Discussion of vectors containing chimeric proteins found in Kroll, D.J.et al.(1993; DNA Cell Biol. 12:441-453).

Antibodies and vaccines

Antibodies according to the invention can be obtained using methods well-known in this field. Specifically, purified peptides, polypeptides or polynucleotide can be used to generate antibodies in accordance with known methods. Such antibodies may include, but are not limited to the above, polyclonal, monoclonal, chimeric, and single-chain antibodies, Fab fragments and fragments obtained by expressing the Fab library. Neutralizing antibodies (i.e. those that inhibit the function) is particularly preferred for use with vaccines.

To produce antibodies can be immunized with various media, including goats, rabbits, rats, mice, humans, and others, by injection of the peptide, polypeptide, polynucleotide or any fragment, which possess immunogenic properties. Depending on the species of the carrier can be used in a variety of adjuvants to enhance the immune response. Such adjuvants include, but are not limited to the above, hell is uwant's adjuvant, such mineral gels such as aluminum hydroxide, and such surfactants as lysolecithin, plutonomy polyols, polyanion, peptides, oil emulsions, hemocyanin fiorelli and dinitrophenol. Among adjuvants used in humans, particularly preferred BCG (bacilli Calmette-Guerin) andCorynebacterium parvum.

Preferably, the peptides, polypeptides or fragments used to induce antibodies, had the amino acid sequence comprising at least five amino acids and more preferably at least 10 amino acids. In addition, it is desirable that they were identical to part of the amino acid sequence of the natural protein, and they can contain a complete amino acid sequence of the small molecules of natural origin. Short stretches of amino acids may be fused with fragments of another protein, such as hemocyanin fiorelli and the antibody raised against the chimeric molecule.

Monoclonal antibodies can be produced using any method which provides for the production of antibodies stable cell lines in culture. These include, not limited to the above, hybridoma method, the hybridoma method based on human B-cells and hybridoma method based virus Epstein-Barr (Kohler, G.et al.(1975) Nature 256:495-497; Kozbor, D.et al.(198) J. Immunol. Methods 81:31-42; Cote, R.J.et al.(1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S.P.et al.(1984) Mol. Cell Biol. 62:109-120).

In addition, can be used the methods developed for the production of "chimeric" antibodies, for example, a combination of antibody genes of the mouse genes and antibodies person to obtain a molecule with appropriate antigen specificity and biological activity (Morrison, S.L.et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M.S.et al.(1984) Nature 312:604-608; Takeda, S.et al.(1985) Nature 314:452-454). An alternative can be adapted to the methods described for obtaining single-chain antibodies using methods known in this field, to obtain specific single-chain antibodies. Antibodies with similar specificnosti, but different antiidiotypic composition can be obtained by shuffling circuits from random combinatorial immunoglobulin libraries (Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Experts in the field relevant to the present invention, will appreciate, the terms "diately" and "Triatel". It is a molecule containing the variable domain of the heavy chain (VH)connected to the variable domain of the light chain (VL) short peptide linker that is too short to allow pairing between the two domains on the same chain. This promotes the formation of pairs with complementary domains of one or the more chains and promotes the formation of dimeric or trimeric molecules with two or more functional binding sites of the antigen. The resulting molecule antibodies can be a monospecific or multispecific (for example, bivalent if diatel). Such antibody molecules can be created from two or more antibodies using the methodology, standard for the field to which the invention relates; for example, described in Todorovskaet al.(Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods. 2001 Feb 1; 248(1-2):47-66).

Antibodies can also be obtained inducyruyain vivoproducts in a population of lymphocytes or scrinia immunoglobulin libraries or panels of highly specific binding reagents as described in the literature (Orlandi, R.et al.(1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G.et al.(1991) Nature 349:293-299).

Can be obtained antibody fragments which contain specific binding sites. For example, such fragments include, but are not limited to the above, F(ab')2fragments which can be obtained pepsinogen by proteolysis of the molecule antibodies, and Fab fragments, which can be created by the restoration of the disulfide bridges of F(ab')2the fragments. An alternative can be created Fab expressing the library for quick and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D.et al.(1989) Science 254:1275-1281).

Various ELISA tests can be used for screening the ha, to identify antibodies having binding specificity. Numerous protocols for competitive binding or radioimmunoassay analysis using either polyclonal or monoclonal antibodies with established specificnosti well known in this field. Such enzyme-linked immunosorbent assays typically include the control of the formation of complexes between the peptide, polypeptide or polynucleotide and-specific antibodies. Two-layer enzyme-linked immunosorbent assay based on monoclonal antibodies can be applied using monoclonal antibodies reactive with two reiterferien the epitope peptide or polypeptide, but may also be applied to the analysis of competitive binding (Maddox,above).

Described in the present description, the antibodies have the ability to target and/or to suppress cells and is also useful as carrier molecules for the delivery of additional inhibitory molecules into microbial cells. Chemistry connections to join the amino acids is well developed, and a number of different types of molecules can be bound by antibodies. The most versatile methods of Association based on the available amino- (alpha-amino - or Lys), sulfhydryl (Cys) or carboxyl groups (Asp, Glu or alpha-carboxyl). Can be used ways in which soedineniya, to connect antibody with cell inhibitor through the carboxy - or aminoterminal balance. In some cases, the sequence includes several residues that can react with the chosen chemistry. This can be used to obtain multimers containing more than one cell inhibitor. An alternative antibody can be shortened or selected so that the reactive residues were localized either at the amino or carboxyl end of the sequence.

For example, a reporter molecule, such as fluorescein, can be specifically attached to the lysine residue (Onoet al., 1997) using N-α-Fmoc-Nε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ilidene-3-methylbutyl)-L-lysine during synthesis of the polypeptide. After synthesis can be attached 5 - and 6-Succinimidyl esters of carboxyfluorescein after removal of 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene by treatment with hydrazine. Thus, it can be achieved accession of the inhibitor molecule to the antibody by including a lysine residue in the sequence of the polypeptide, and then reaction with a specially converted cell inhibitor.

Can also be used EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) or carbodiimide method of joining. Carbodiimide can and tigerbeat carboxyl group of the side chain of aspartic and glutamic acids, as carboxyterminal group, to turn them into reactive sites for attachment of primary amines. The activated antibody is mixed with the cellular inhibitor of receiving the final conjugate. If the cellular inhibitor of the activated first, the EDC is a way to attach cellular inhibitor through the N-terminal alpha amine and, possibly, through the amine side chain of Lys, if it is present in the sequence.

m-Maleimidomethyl-N-hydroxysuccinimide ester (MBS) is heterobifunctional reagent that can be used for the connection of antibodies with cell inhibitor through cysteine. Accession shall be effected by thiol groups of cysteine residues. If the selected sequence does not contain Cys, is a common connection Cys residues at the N - or C-Termini, in order to obtain a high degree managed stitching antibodies with cell inhibitor. For the purposes of synthesis may be useful to cysteine was on the N end of antibodies. MBS is particularly suitable for use in the present invention.

Glutaraldehyde can be used as a bifunctional reagent connection that links two connections via their amino groups. Glutaraldehyde provides extremely flexible spacer between the antibody and cellular inhibitor suitable for expos the AI. Glutaraldehyde is a very reactive compound and reacts with Cys, Tyr, and to a limited extent with His. Glutaraldehyde method of joining is particularly useful when the polypeptide contains only one free amino group at its aminocore. If the antibody contains more than one free amino group, can form a large multimeric complex.

In one aspect, the antibodies of the invention can be chimeric (e.g., cloned in a common reading frame) or related (for example, by chemical absorption) with the cell inhibitors, such as antimicrobials. These include antimicrobial peptides, for example a protein that increases the bactericidal/permeability, cationic antimicrobial proteins, lysozyme, lactoferrin and cathelicidin (e.g. neutrophils; see, for example, Hancock and Chappie, 1999, Antimicrob. Agents Chemother.43:1317-1323; Ganz and Lehrer, 1997, Curr. Opin. Hematol. 4:53-58; Hancocket al., 1995, Adv. Microb. Physiol. 37:135-175). Antimicrobial peptides also include defensin (for example, epithelial cells or neutrophils) and bactericidal proteins of platelets (see, for example, Hancock and Chappie, 1999, Antimicrob. Agents Chemother. 43:1317-1323). Additional antimicrobial peptides include, but are not limited to the above, gramicidin S, bacitracin, polymyxin B, theplain, bactenecin (for example, bactenecin large horns which CSOs cattle), ranalexin, cecropin a, indolicidin (for example, indolicidin cattle) and lowlands (e.g., bacterial lowlands).

Also included as antimicrobial agents the ionophores, which contribute to the transport of ions (such as sodium) through the lipid barrier, such as the cell membrane. Two ionophoric compounds particularly suitable for this invention are rumensin (RUMENSIN™) (Eli Lilly) and lasalocid (Lasalocid) (Hoffman LaRoche). Other ionophores include, but are not limited to the above, salinomycin, avoparcin, arizin and actaplanin. Other antimicrobials include monensin (Monensin™) and azithromycin, metronidazole, streptomycin, kanamycin and penicillin, as well as, usually, β-lactams, aminoglycosides, macrolides, chloramphenicol, novobiocin, rifampicin and fluoroquinolones (see, for example, Hornet al., 2003, Applied Environ. Environ. 69:74-83; Eckburget al., 2003, Infection Immunity 71:591-596; Gijzenet al., 1991, Applied Environ. Environ. 57:1630-1634; Boneloet al., 1984, FEMS Environ. Lett. 21:341-345; Huseret al., 1982, Arch. Environ. 132:1-9; Hilpertet al., 1981, Zentbl. Bakteriol. Mikrobiol. Hyg. 1 Abt Orig. C 2:21-31).

Particularly useful inhibitors are compounds that block or inhibit methanogenesis him, including bromoethanesulfonate acid, such as 2-bromoethanesulfonate acid (BES), or its salt, e.g. sodium salt. Molybdate (Mo) sodium is an inhibitor of sulphate recovery and can be used with bromoethanesulfonate acid. Other compounds that are effective against methanogenesis, include, but are not limited to the above, nitrate, formate, vermeil, chloroform, chloral hydrate, sodium sulfite, ethylene and unsaturated hydrocarbons, acetylene, fatty acids such as linoleic acid and CIS-oleic acid, saturated fatty acids, such as Baganova and stearic acid, as well as lunasin (for example, 2,4-pteridinyl). Additional compounds include 3-bromopropane-sulfonate (BPS), propionic acid and ethyl-2-butynoate.

Further included as antimicrobial agents lytic enzymes, including phage lysozyme, endolysin, lysozyme, lysine, lysine phage, morality, muramidase and virulizin. Useful enzymes demonstrate ability to hydrolysis of specific links in the cell wall of bacteria. Specific lytic enzymes include, but are not limited to the above, glucosaminidase that hydrolyzing glycosidic bonds between amino sugars (e.g., N-acetylmuramic acid and N-acetylglucosamine) peptidoglycan, amidase that cleave N-acetylmuramyl-L-alanine amide bond between pianoboy chain and cross-linking peptide, and endopeptidase that hydrolyzing interpeptide communication (e.g., cysteine of endopeptidase), and endosomatic, which destroy pseudomurein of methaneproducing familyMethanobacteriaceae.

More is positive as antimicrobial agents included PNA. PNA represents a peptide-nucleic acid hybrids, in which the phosphate skeleton was replaced with achiral and neutral frame of N-(2-amino-ethyl)-glycine units (see, for example, Eurekah Bioscience Collection. PNA and Oligonucleotide Inhibitors of Human Telomerase. G. Gavory and S. Balasubramanian, Landes Bioscience, 2003). Bases A, G, T, C attached to the amide nitrogen of the frame through methylanthranilate communication (P.E. Nielsenet al., Science 1991. 254:1497-1500; M. Egholmet al.Nature 1993. 365:566-568). PNA bind complementary sequences with high specificity and higher affinity compared to the same DNA or RNA (M. Egholmet al.,above). PNA/DNA or PNA/RNA hybrids also showed higher thermal stability than the corresponding DNA/DNA or DNA/RNA duplexes (M. Egholmet al.,above). PNA also have high chemical and biological stability due to unnatural amide of the frame, which is not recognized by nucleases and proteases (V. Demidovet al., Biochem Pharmacol 1994, 48:1310-1313). Usually PNA have a length of at least 5 bases and include terminal lysine. PNA can be paglierani for additional lengthening life expectancy (Nielsen, P.E.et al.(1993) Anticancer Drug Des. 8:53-63).

In one particular aspect of the antibodies according to the invention can be chimeric or associated with other antibodies or their fragments. Added antibodies or fragments of the Academy of Sciences is ITIL can be directed against microbial cells, or specifically against cells methaneproducing or against one or more components of the cell. For example, the target may serve as cell surface proteins, for example the outer cell receptors. In some aspects, the antibodies or fragments of antibodies can be constructed containing sequences expressed exactly by the subjects, for example the sequence of human or ruminant. Also includes chimeric antibodies, for example monoclonal antibodies or fragments thereof specific against more than one source, for example against one or more sequences of mouse, human or ruminant. Added "camel" antibodies or nanotesla.

Antibodies according to the invention find their practical use for targeting microbial cells, in particular cells of methaneproducing. In some aspects, these antibodies can be used to associate or bind to the cell wall or membrane and/or to inhibit the growth or replication of cells. For this reason, these antibodies can be used for short-term or long-term attachment to the cell or to serve as intermediaries in the destruction or absorption cells and/or lysis. To implement targeting microbial cell can be subjected to the action of antibodies, vydelennogo is from the host organism, or produced by expression vectors and/or cells by owners or by using synthetic or semi-synthetic chemistry, as described in detail in the present description. Alternatively, antibodies can be produced by the host organism in response to the introduction of peptides, polypeptides or polynucleotides disclosed in the present description. It is clear that the antibodies according to the invention, as well as the corresponding polynucleotide, expression vectors, cell-owners, peptides and polypeptides, can be used for targeting a variety of microbes, such asMethanobrevibacter ruminantiumwho are the main methaneproducing the ruminant, andMethanobrevibacter smithiiwho are the main methaneproducing people. In particular aspects, the antibody or the corresponding polynucleotide, expression vectors, cell-owners, peptides or polypeptides are delivered to subjects in the form of the composition, are described in detail in the present description, for example, by using vnutrinosovyh devices slow release.

In various aspects of the means according to the invention (for example, one or more peptides, polypeptides, polynucleotides and antibodies) can be incorporated into compositions such as pharmaceutical compositions, particularly vaccine compositions. This composition contains, for example: (a) vydeleny the th peptide or its modification, a fragment, variant or derivative; (b) the selected polypeptide or its modification, fragment, variant or derivative; (c) selected polynucleotide or its modification, fragment, variant or derivative; (d) an expression vector comprising this polynucleotide; (e) cell host comprising the expression vector; or (f) the antibody or its modification, fragment, variant or derivative. The composition of the invention can be specially packaged as part of a set for targeting and/or inactivation of microbial cells, especially cells methaneproducing, in accordance with the disclosed methods. The kits contain at least one composition as described in the present description, and instructions for use for targeting cells or to inhibit cell growth or replication of methaneproducing or other microbes.

Vaccines can be used a number of approaches to increase the immunogenicity of antigens, for example, using antigenic particles; antigenic polymers and polymerization; emulsifiers; microcapsulation antigens killed bacteria and bacterial products; chemical adjuvants and cytokines; and means targeting antigens to antigen-presenting cells (for review, see Paul, Fundamental Immunology, 1999, Lippincott-Raven Publishers, New York, NY, p. 1392-1405).

To bring the antigens in disperse the condition, can be used for the deposition of alum. When using aluminum hydroxide or aluminum phosphate target antigen is introduced into an insoluble, gel-like precipitate or bound with preformed gel by electrostatic interactions. Antigens can be subjected to mild thermal aggregation. Antigens, demonstrating Samegrelo, can also be used. Liposomes, virosome and claim-vaccine complexes (ISCOM) is also useful for the formation of particles.

To promote polymerization, as an additive to adjuvants can be used non-ionic block copolymers, such as polymers of polyoxypropylene and polyoxyethylene, which can be associated antigen. They were selected as components of combination adjuvant compositions of the two companies: Syntex (SAF-1, Syntex Adjuvant Formulation-1) and Ribi Chemical Co. Carbohydrate polymers of mannose (e.g., mannan) or β1-3 glucose (e.g., glucan) can be used similarly (Okawa Y., Howard C.R., Steward M.W. Production of anti-peptide antibody in mice following immunization of mice with peptides conjugated to mannan. J Immunol Methods 1992; 142:127-131; Ohta M., Kido, N., Hasegawa,T.,et al.Contribution of the mannan side chains to the adjuvant action of lipopolysaccharides. Immunology 1987; 60:503-507).

Various means can be used for emulsification, including emulsion, water-in-oil", such as adjuvants's adjuvant (e.g., incomplete is duvant's adjuvant), or other mixture comprising microscopic inclusions of water, stabilized by surfactant, such as mandalorean in the dispersion phase of mineral oil or other oils such as squalane. An alternative approach is to use emulsions oil-in-water", such as MF5963 (Chiron), or other mixtures containing oil inclusions of squalene and the mixture of emulsifiers TWEEN80 and SPAN85 and chemical immunomodulators, such as derivatives muramyl-dipeptide, for example muramyl-Tripeptide-phosphatidylethanolamine (MTP-PE) (Valensi J-PM, Carlson J.R., Van Nest GA. Systemic cytokine profiles in Balb/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants. J Immunol 1994; 153:4029-4039). A small amount of Polysorbate 80 and sorbitrate can also be used in these mixtures. As another example, can be used SAF-165 (Syntex) or other mixture of the oil-in-water, including Pluronic L121, squalene and TWEEN80.

Microcapsules, in particular biodegradable microcapsules, can be used for vaccine manufacturing controlled release (Chang TMS. Biodegradable, semi-permeable microcapsules containing enzymes hormones, vaccines and other biologicals. J Bioeng 1976; 1:25-32; Langer R. Polymers for the sustained release of macromolecules: their use in a single-step method of immunization. Methods Enzymol 1981; 73:57-75). Cyanoacrylate represent some form of biodegradable polymers. So, for example, as an adjuvant for oral immunization can be the used poly(butyl 2-cyanoacrylate) (O Hagan D.T., Palin K.J., Davis S.S. Poly (butyl-2-cyanoacrylate) particles as adjuvants for oral immunization. Vaccine 1989; 7:213-216). Microcapsules useful for mucosal vaccination. Particularly suitable particles of very small size (nanoparticles). Gastric digestion can resist covered intersolubility shell polymer, and optionally coating substances that enhance the absorption in the intestine.

Various bacteria in addition to the deadM. tuberculosiscan be used as adjuvants. Where the drug killed the bacteria itself is vysokopatogennym, adjuvant properties apply to the jointly-administered antigen. Useful organisms includeBordetella pertussis, Corynebacterium parvumandNippostrongylus brasiliensis. Peptide and lipid components of bacteria can also be used. Illustrative components include acetylmuramyl-L-alanyl-D-isoglutamine or muramyl-dipeptide (MDP) (F. Ellouz, Adam A., Ciorbaru R., Lederer E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycans. Biochem Biophys Res Commun 1974; 59:1317-1325), MDP (murabutide) (Chedid L. Parant M.A., Audibert F.M.,et al.Biological activity of a new synthetic muramyl dipeptide devoid of pyrogenicity. Infect lmmun 1982; 35:417-424), threonyl-MDP (Allison A.C., Byars N.E. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and cell-mediated immunity. J Immunol Methods 1986; 95:157-168) and MTP-PE. Lipid adjuvants may include lipopolysaccharide endotoxins of gram-negative bacteria such asEscherichia, SalmonellaandPseudomoas . In some approaches, the structure of lipid a can be chemically modified to reduce toxicity but retain immunogenicity, for example, as for monophosphorylated lipid A (MPL) (Johnson A.G., Tomai, M., L. Solem, L. Beck, E. Ribi Characterization of non-toxic monophosphoryl lipid. Rev Infect Dis 1987; 9:S512).

Various chemicals can be used as adjuvants, including polynucleotide, such as poly-I:C and poly A:U, vitamin D3, sulfate, dextran, inulin, dimethyldioctadecyl ammonium (DDA), avidin, carbohydrate polymers, similar to mannan, and dimycolate trehalose (Morein B., Lovgren-Bengtsson K, Cox J. Modern adjuvants: functional aspects. In: Kaufmann SHE, ed. Concepts in vaccine development. Berlin: Walter de Gruyter, 1996:243-263). Also included is polyphosphazene (originally used as tools to facilitate slow release) and proteinLeishmania, LeIF. Cytokines can also be used as adjuvants, for example, IL-2, IL-4, IL-6, IL-10, GM-CSF and IFN-γ.

For targeting antigen presenting cells can be used C3d domains, Fc domains and CTB domains (Dempsey P.W., Allison MED, Akkaraju, S., Goodnow C.C., D.T. Fearon C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 1996; 271:348-350; Sun J-B, Holmgren j, Czerkinsky C. Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc Natl Acad Sci USA 1994; 91:10795-10799; Sun J-B, Rask, C., Olsson, T., Holmgren j, Czerkinsky C. Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit. Proc Natl Acad SciUSA 1996; 93:7196-7201).

Can also be used of special adjuvants for delivery via the mucous membranes, such as CT, LT, and fragment C of tetanus toxin (C.J. Elson, Ealding, W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol 1984; 132:2736-2743; Holmgren J., Lycke N., Czerkinsky C. Cholera toxin and cholera B subunit as oral - mucosal adjuvant and antigen vector systems. Vaccine 1993; 11:1179-1184; J.D. Clements, Hartzog N.M., Lyon F.L. Adjuvant activity ofEscherichia coliheat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens. Vaccine 1988; 6:269-277; Gomez-Duarte O.G., Galen J., Chatfield S.N., Rappuoli R., L. Eidels, Levine M.M. Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of reagent grade toxin in Salmonella typhi CVD 908 vaccine strain. Vaccine 1995; 13:1596-1602).

Therapy and diagnostics

Peptides, polypeptides, polynucleotides and antibodies of the present invention are useful for health. In specific aspects of vaccines aimed at methaneproducing, can be used for recovery of the energy content of a subject, which is usually lost in the form of methane. The invention is for this reason that associated with the pharmaceutical composition (particularly a vaccine composition in combination with pharmaceutically acceptable medicinal basis for use with any of the ways discussed above. Such pharmaceutical compositions can include a peptide, polypeptide or antibody in combination with cell inhibitor. Alternative pharmaceutical compositions can include polynucleotide, in ctor expression or cells of hosts, as described in detail herein. Song data can be entered separately or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical basis, including, not limited to the above, in saline solution, buffered saline solution, glucose and water. These compositions can be administered to the subject separately or in combination with other agents, drugs (e.g., antimicrobial drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and auxiliary ingredients that facilitate processing of the active compounds into preparations which can be used pharmaceutically. More detailed information about the methods of preparation of the formulation and introduction can be found in the latest edition Remington''s Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). The pharmaceutical compositions used in this invention can be defined any number of ways, including, but not limited to the above, oral, intravenous, intramuscular, intraarterial intramedullary, intrathecal, intraventricular, transdermal, on the skin, intraperitoneal, intranasal, enteral, local, sublingual, or rectal means.

Pharmaceutical compositions for oral administration can be created using pharmaceutically acceptable dosage bases known in this field, in dosages suitable for oral administration. Such medicinal basis enable the pharmaceutical compositions to be created in the form of tablets, pills, coated tablets, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the subject. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding the appropriate additional ingredients, if it is desirable to obtain core tablets or pills. Suitable excipients are carbohydrate or protein fillers, as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potatoes and other plants; cellulose, such as methylcellulose, hydroxyethylcellulose or carboxymethylcellulose sodium; resin, including gum Arabic and tragakant; and proteins such as gelatin and collagen. If desired, can be added disintegrity is s or solubilizing means, such as cross-crosslinked polyvinylpyrrolidone, agar, alginic acid or its salt, such as sodium alginate.

Pharmaceutical preparations which can be used orally include collapsible hard capsules made of gelatin, and also soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Collapsible hard capsules can contain the active ingredients mixed with a filler or binders such as lactose and starch, softening ingredients such as talc, magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in appropriate liquids, such as fatty oils, liquid or liquid polyethylene glycol in the presence or absence of stabilizers. Index of the core can be used in combination with suitable coatings, such as concentrated sugar solutions, which may also contain gum Arabic, talc, polyvinylpyrrolidone, gel of carbopol, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes and pigments can be added to the coating of tablets or dragee to identify the product or as a distinctive sign of the number is as active substance, i.e. dosage.

Pharmaceutical compositions suitable for parenteral administration, can be created on the basis of aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, ringer's solution, or physiological buffer solution. Aqueous suspension for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be obtained in the form of an oil suspension for injection. Suitable lipophilic solvents or inert bases include fatty oils such as sesame oil, or synthetic esters of fatty acids, such as etiloleat or triglycerides, or liposomes. Non-lipid poly-aminopolymers can also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or tools that increase the solubility of compounds in order to obtain highly concentrated solutions. For local injection or a nasal formulation used wetting agents suitable for penetration through the concrete barrier. Such wetting agents are widely known in this field.

The pharmaceutical compositions of the present invention can is to be made by way known in this field, for example, by means of conventional mixing, dissolving, crushing, production of drops, grinding into powder, emulsifying, filling the capsules, encapsulation in liposomes or freeze-drying. The pharmaceutical composition can be prepared as a salt and can be formed with many acids, including, not limited to the above, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts are usually more soluble in water or other protonated solvents than the corresponding form of their free bases. In other cases, the preferred preparation may be a lyophilized powder which may contain any choice or all of the following: 1-50 mm histidine, of 0.1%-2% sucrose and 2%-7% mannitol, pH range from 4.5 to 5.5, that is combined with buffer prior to use. After the manufacture of pharmaceutical compositions can be placed in a suitable standard containers and label the label for the treatment of these diseases. For the introduction of the compositions according to the invention, this marking must include the amount, frequency and method of administration.

Pharmaceutical compositions suitable for use according to the invention include compositions where the active ingredients are contained in amounts effective for achieving the set of goals. For any connection, therapeutically effective dose can be estimated initially either in tests on cells, such as microbial cells or, in particular, on the cells of methaneproducing, or in animal models, usually mice, rabbits, dogs and pigs, or species of ruminants, such as sheep, cow, deer and goats. The animal model can also be used to determine the appropriate concentration and route of administration. Such information can be used to determine the effective dose and route of administration. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending on route of administration. Methodological principles for determining the particular dosages and methods of delivery are given in the literature and generally available to practitioners in this field. Specialists in this field will use a different recipe for polynucleotides and polypeptides. Similarly, the delivery of peptides or polypeptides, polynucleotides or antibodies will be specific to particular cells, diseases, places, etc.

The exact dosage will be determined by a physician taking into account factors related to the subject, which need treatment. The method of application and doses are adjusted to provide sufficient levels of the active funds is because to maintain the desired effect. Factors that may be taken into account include the severity of the disease condition, General health of the subject, age, weight, sex, diet, time and frequency of administration, drug(s) combination(s), sensitivity reactions, and tolerance/response to therapy. Pharmaceutical compositions prolonged action, you can enter every 3-4 days, every week or every two weeks depending on half-life and renal clearance of specific composition. Compositions can be administered together with one or more additional antimicrobial agent, including compounds against methanogenesis (e.g., bromoethanesulfonate), antibodies and fragments of antibodies, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, as described in detail herein. Co-administration can be simultaneous or sequential or interleaved with the re-introduction.

Especially useful for the compositions according to the invention (e.g., pharmaceutical compositions) structures or mechanisms for slow release. For example, vnutrichasovye devices include, but are not limited to the above, Time Capsule™ Bolus range from Agri-Feeds Ltd., New Zealand, originally developed within the company AgResearch Ltd., New Zealand, as p is hidden in WO 95/19763 and NZ 278977, and CAPTEC from Nufarm Health & Sciences, division of Nufarm Ltd., Auckland, New Zealand, as disclosed in AU 35908178, PCT/AU81/100082 and Labyet al., 1984, Can. J. Anim. ScL 64 (Suppl), 337-8, and all incorporated herein by reference. As a specific example, the device may include a spring and a piston that squeezes the composition, overcoming the resistance of the holes in the end of the cylinder.

As an additional variant implementation of the invention relates to a composition for water additives, for example, a composition for injection into the oral cavity of an animal or a food additive, for example a component of ruminant feed, for use with any of the ways discussed above. In a particular aspect of this dietary Supplement contains at least one edible plant material, as well as peptide or polypeptide according to the invention. Alternative food additive contains at least one edible plant material and the polypeptide or peptide or polynucleotide encoding the peptide or polypeptide disclosed in the present description, for example, the expression vector or the cell host comprising the expression vector. In particular, the composition also includes a cellular inhibitor as heriditary or associated with the resulting sequence. Preferred plant material includes any of the hay, t is ava, grain or flour, such as legume hay, grass hay, corn silage, grass silage, legume silage, corn, oats, barley, grains distilleries, brewery grains, flour soybean flour and cotton seeds. In particular, grass silage, useful as an integral part of the food of ruminants. The plant material can be genetically modified to contain one or more components according to the invention, for example one or more polypeptide or peptide, polynucleotide or vector.

In another embodiment, antibodies which specifically bind to the peptides, polypeptides or polynucleotide according to the invention can be used to determine the presence of microbes, especially methaneproducing, or in tests for control of titles such microbes. These antibodies are useful for diagnostic purposes, can be obtained in the same manner as described above. Diagnostic tests include methods that use the data of the antibody and a label for detection of the peptide or polypeptide in the body fluids of a person or extracts of cells or tissues. These antibodies can be used with or without modification, and can be labeled by joining, either covalently or ecovalence reporter molecule. Can be used with a wide range of reporter molecules, Izv the STN in this area, some of which are described above.

A variety of protocols for measuring levels of the peptide, polypeptide or polynucleotide known in this field (e.g., ELISA, RIA, and FACS) and serve as the basis for diagnosing the presence or titles of microbes, especially methaneproducing. Normal or standard titles are established by combining body fluids or cell extracts taken from normal subjects, for example in healthy humans or ruminants, with the antibody under conditions suitable for the formation of the complex. The number of standard formations of complexes can be calculated in various ways, but preferably by photometric means. The number of peptide, polypeptide or polynucleotide expressed by the subject, the control and treated samples (e.g. samples from vaccinated subjects) compared with the standard values. Deviation values for the subjects from standard values establishes the parameters for determining the presence or titles of this microbe.

In another embodiment, the invention these polynucleotide can be used for diagnostic purposes using specific hybridization and/or amplification methods. Polynucleotide, which can be used include oligonucleotides, complementary RNA molecules and DNA, and PNA. Data is olignucleotides can be used for detection and quantitative analysis of gene expression in samples in which expression may correlate with the presence or titer of a microbe. You can use a diagnostic test to differentiate between absence, presence and change of microbial titles, as well as to control titles for therapeutic intervention.

In one aspect, hybridization with PCR probes can be used to identify nucleic acid sequences, particularly genomic sequences encoding the peptides or polypeptides according to the invention. The specificity of the probe, whether created from a highly specific region, for example, 10 unique nucleotides in the 5' regulatory region, or from a less specific region, for example in the 3' coding region, and the stringency conditions of hybridization or amplification (maximal, high, medium, or low) will determine whether the probe to identify only the sequence of natural origin, alleles, or related sequences. Probes may also be used for the detection of related sequences and should preferably contain at least 50% of the nucleotides from any of the coding sequences. Hybridization probes on a "linked" to the invention can be DNA or RNA and can occur from the nucleotide sequence of SEQ ID NO:703-1395 or their complements, or modified sequences, or genome the x sequences, including promoter and enhancer elements of natural origin.

Methods of obtaining specific hybridization probes for DNA include the cloning of nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known in the field that are commercially available and can be used to synthesize RNA probesin vitroby adding the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled with a different reporter groups, such as radionuclides, such as32P or35S, or enzymatic labels such as alkaline phosphatase attached to the probe via avidin/bitenova system connections, and the like. These polynucleotide can be used in the southern blot or Northern blot analysis, dot-blot analysis, or other membrane technologies, in PCR technology, either Express sample imprintirovannymi substrate (dipstick), when screening randomized peptide libraries immobilized on a solid pins (pin), enzyme-linked immunosorbent assays (ELISA) or microarrays utilizing fluids or tissue biopsies of the subject to detect the presence or title of a microbe. Such qualitative and quantitative methods well known in the field.

In a specific aspect in the ranks of the analyses can be useful sequence data nucleic acids, labeled by standard methods, and added to the sample fluid or tissue of a subject under conditions suitable for hybridization and/or amplification. After an appropriate incubation period, the sample is washed and the signal quantitatively evaluate and compare with the standard value. If the total amount of signal in the sample is significantly different from comparable control sample, the presence of altered concentrations of the nucleotide sequences in the sample indicates the presence or title of a microbe. Such analyses can also be used to evaluate the effectiveness of a particular mode of vaccination in animal studies, in clinical trials or monitoring treatment of a subject.

To ensure the basics of diagnosing the presence or titles microbe has a normal or standard profile for expression. This can be accomplished by combining body fluids or cell extracts taken from normal subjects, with polynucleotide or fragment under conditions suitable for hybridization and/or amplification. Standard titles can be quantified by comparing the values obtained from normal subjects with values obtained in the experiment, which uses a known amount practically cleared of polynucleotide. Standard the e values, obtained for normal samples may be compared with values obtained for samples from subjects exposed to microbial growth. Deviation between standard and subject is used to establish the presence or titles of this microorganism.

As soon as the microorganism identified and initiated Protocol vaccinations on a regular basis may be repeated analyses of hybridization and/or amplification, to determine, begins to decrease the level of expression of the subject in relation to the observed in the normal subject. The results obtained from the sequential tests can be used to show the effectiveness of vaccination in the period from several days to several months.

Special diagnostic application of oligonucleotides were designed based on sequences of nucleic acids may include the use of polymerase chain reaction. Such oligomers can be chemically synthesized, generated enzymatically, or produced by thein vitro. The oligomers preferably consist of two nucleotide sequences, one in sense orientation (5'→3')and the other in antisense orientation (3'→5'), used under optimum conditions to identify specific genes and diseases. The same two olig the measure, built-in series of oligomers, or even a degenerate pool of oligomers can be used in less stringent conditions for detection and/or quantification of closely related DNA sequences or RNA.

Methods that can also be used for quantitative analysis of gene include tagging of radioisotopes or biotinylating nucleotides, joint amplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby, P.C.et al.(1993) J. Immunol. Methods 159:235-244; Duplaa, C.et al.(1993) Anal. Biochem. 229-236). Speed quantitative analysis of serial samples can be increased by running a test in an ELISA format where the desired oligomer is presented in various dilutions and a spectrophotometric or colorimetric signal gives rapid quantitation.

In further embodiments, the implementation of oligonucleotides or longer fragments derived from any of these polynucleotides described in the present description, can be used as targets in a microarray. The microchip can be used to control the level of expression of large numbers of genes simultaneously (image generation matrix), as well as to identify genetic variants, mutations and polymorphisms. This information can be used too to determine gene function, to understand the genetic basis of disease, diagnose disease, and to develop and monitor the effects of therapeutic agents. In one embodiment, the microchip is manufactured and used in accordance with methods known in this field that have been described in PCT application WO 95/11995 (Cheeet al.), Lockhart, D.J.et al.(1996; Nat. Biotech. 14:1675-1680) in Schena, M.et al.(1996; Proc. Natl. Acad. Sci. 93:10614-10619).

In one aspect, the oligonucleotides can be synthesized on the surface of the microarray using the procedure of chemical attach and apparatus of applying inkjet printing, such as described in PCT application WO 95/251116 (Baldeschweileret al.). In another aspect can be used in the matrix grid, similar to the dot-blot or slot-blot (apparatus HYBRIDOT, Life Technologies), to distribute and link cDNA fragments or oligonucleotides to the surface of the substrate using a vacuum system, thermal, UV, mechanical and chemical procedures for accession. In another aspect, the matrix can be done manually or by using available devices, materials, and machines (including multichannel pipettors and robotic tools; Brinkmann, Westbury, N.Y.) and may include, for example,, 24, 48, 96, 384, 1024, 1536 or 6144 point of recesses or holes (e.g. the, in advance tablet) or more, or any other number of departments, from 2 to 1000000, which makes it suitable for effective use of commercially available devices.

In order to conduct sample analysis using microarrays of biological samples was extracted with polynucleotide. Data biological samples can be obtained from any bodily fluids (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. For the manufacture of these probes polynucleotide extracted from the sample is used to create sequences of nucleic acids that are complementary to nucleic acids on a microchip. If the microchip contains cDNA, antisense RNA are appropriate probes. Thus, in one aspect, the mRNA is used to obtain cDNA, which in turn and in the presence of fluorescent nucleotides is used to obtain fragments or antisense RNA probes. These fluorescently labeled probes are incubated with a microchip, so that the sequence of the probe was hybridisable cDNA with oligonucleotide microarray. In another aspect, the nucleic acid sequences used as probes may include polynucleotide, fragments and complementary or antisense sequence, the floor is built using restricted, PCR technologies and kits for oligonucleotide labeling (Amersham Pharmacia Biotech), well known in the field of technology hybridization.

In another embodiment of the invention, the peptides or polypeptides according to the invention or their functional or immunogenic fragments or oligopeptides can be used for screening libraries of compounds in any of a variety of methods of screening for drugs. The fragment used in such screening may be free in solution, affixed to a solid substrate, to be on the cell surface or inside the cells. You can evaluate the formation of binding complexes between the peptide or polypeptide and the test agent.

One of the methods of screening for drugs which may be used provides for high-throughput screening of compounds having suitable binding affinity to the peptide or polypeptide of interest, as described in published PCT application WO 84/03564. In this method, a large number of different low-molecular test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Test compounds give the opportunity to react with the peptide, or polypeptide or fragments and washed. The bound peroxidase Pat the y or the polypeptide is then detected by methods well known in this field. The purified peptide or polypeptide can also be applied directly to the plates for use in the above methods of screening drugs. Alternatively can be used nanatsusaya antibodies to capture the peptide and its immobilization on a solid substrate.

In another method you can use competitive analysis screening of drugs in which neutralizing antibodies capable of binding the peptide or polypeptide specifically compete with a test compound for binding to this peptide or polypeptide. In this way, antibodies can be used for detection of the presence of the test compound that has one or more binding sites of the antigen with the antibody.

EXAMPLES

The examples described in the present description, are intended for purposes of illustration of embodiments according to the invention. Other embodiments of, methods and types of analysis are within the competence of the ordinary experts in the field of molecular diagnostics, and describe them further is not necessary. Other embodiments of within this area are considered part of this invention.

EXAMPLE 1: Estimation of the genome size

Methanobrevibacterruminantium strain M1T(DSM1093), exp is Diwali on the environment BY+ (minimal medium, Joblinet al., 1990)composed of (g/l): NaCl (1), KH2PO4(0,5), (NH4)2SO4(0,25), CaCl2·2H2O (0,13), MgSO4·7H2O (0,2), K2HPO4(1), clarified scar fluid (300 ml), distilled H2O (360 ml), NaHCO3(5), resazurin (0.2 ml), L-cysteine-HCl (0.5), and yeast extract (2) and trace element solution Balkh (10 ml) (additional trace elements; Balchet al., 1979), which contains (g/l): nitrilotriacetic acid (1,5), MgSO4·7H2O (3), MnSO4·H2O (0,5), NaCl (1), FeSO4·7H2O (0,1), CoCl2·6H20 (0,1), CaCl2·2H2O (0,1), ZnSO4·7H2O (0,1), CuSO4·5H2O (0.01), and AlK(SO4)2·12H2O (0.01), and H3BO3(0,01), Na2MoO4·2H2O (0.01), and with NISO4·6H20 (0,03), Na2SeO3(0.02) and Na2WO4·2H2O (0,02). Genomic DNA was extracted by freezing of cell debris in liquid N2and grinding using a pre-chilled, sterilized mortar and pestle. Cell homogenates were loaded in agarose inserts, and subsequent manipulations were carried out in boxes to reduce the physical fragmentation of genomic DNA. Cleavage was performed by restrictase and the DNA fragments were separated by gel electrophoresis pulsed field (PFGE).

EXAMPLE 2: Cloning and DNA sequencing

GE is UMNO DNA M. ruminantiumsequenced according Agencourt Biosciences Corporation (Massachusetts, USA) using the approach shot firing" stochastic cloning genomic fragments (Fleischmannet al., 1995), and according to Macrogen Corporation (Rockville, MD, USA) using persecutione. Briefly, inEscherichia colilibraries have been established DNAM. ruminantiumthrough stochastic physical destruction genomic DNAand separation of fragments by gel electrophoresis. Large fragments in the range of 40 KBP was extracted from the gel and used to create fominoy library large inserts. The DNA fragments in the range from 2 to 4 KBP was extracted and used to create a plasmid library of small inserts.

Clones resulting from additional rearing libraries large and small inserts, and their pomidou or plasmid DNA was isolated and determined their sequences by applying the technology of high-performance sequencing. Was sequenced sufficient number of clones that theoretically 8 times to block geneM. ruminantium. Additional overlapping sequences received by persecutione stochastically chopped fragments of genomic DNA (Macrogen Corporation) to a final theoretical overlap of the genome is approximately 10 times.

EXAMPLE 3: Sequence Assembly and annotation

On the sledovatelnot DNA was lined to find overlapping sequences, and mounted in a continuous (contiki) sequence using Paracel Genome Assembler (Paracel Inc., CA, USA) and service programs of Staden (Stadenet al., 1998) in combination with the sequence of the PCR both direct and reverse. Kontiki analyzed using detection of open reading frames (ORF) GLIMMER (Gene Locator interpolated Markov Model ER, Delcheret al., 1999), and each ORF were analyzed with the BLAST program (Basic Local Alignment Search Tool (Altschulet al., 1997), including breaks, relatively non-redundant databases of nucleotide and protein structures of the national center for biotechnology information (NCBI).

Kontiki from a sequence of 8-fold the preliminary stage were combined in a random way by artificial compounds sequences, creating a "pseudomolecular", and was submitted to the research Institute of genome - The Institute for Genomic Research (TIGR, DC, USA) - for autoantibody. Kontiki mounted on the results of 10-fold persecutione, re-analyzed using GLIMMER and ORF has autoantibody using GAMOLA (Global Annotation of Multiplexed On-site Blasted DNA sequences; Altermann and Klaenhammer, 2003). Found automatically annotate subsequently checked manually. ORF classified by function, using the database of clusters ontologica of proteins (COG) (threshold 1e-2) (Tatusov et al., 2001).

Protein motifs were determined using HMMER (hypertext transfer protocol://hmmer.wustl.edu), using library PFAM HMM and TIGRFAM, with General and local alignment (hypertext transfer protocol://pfam.wustl.edu), as well as standard and fragmented model TIGRFAM HMMs (hypertext transfer protocol://worid wide web.tigr.org/TIGRFAMs respectively (threshold 1e-02). tRNAs were identified using TRNASCAN-SE (Lowe and Eddy, 1997), and nucleotide repeats were identified using the software packages KODON (Applied Maths, Austin, TX, USA) and REPUTER (Kurtz and Schleiermacher, 1999). Genomic Atlas of images was created by GENEWIZ (Jensenet al., 1999). Reconstruction of the metabolic pathways of the estimated genome open reading frames (ORFeome)M. ruminantiumheld in conjunction with the online database of the Kyoto encyclopedia of genes and genomes (KEGG, Kanehisaet al., 2004) using self-developed software (PathwayVoyager; Altermann and Klaenhammer, 2005).

EXAMPLE 4: sequencing and analysis

Estimation of genome sizeM. ruminantiumby splitting restrictase genomic DNA and separation of fragments by size by PFGE indicate one chromosome length of approximately 2.5 and 2.9 MPa Initial sequencing of clones of large and small inserts (6-fold pre-overlapping) and the installation sequence in contiki showed that the region of the genome with a length of 40 KBP presents extremely huts is exactly (> 20-fold), especially in the small library inserts. Maybe it happened because of the high copy number plasmid (although there has been no extrachromosomal DNA) or because of a lysogenic bacteriophage, replicated with the growth of culture, used for extraction of DNA. Because of errors in the determination of these large sequences were performed additional sequencing (2-fold theoretical overlap of the genome) only for clones of large insertions that resulted in 8-fold overlap based on sequencing by Sanger. Sequence 8 times the preliminary stage was mounted in 756 of Contigo connected through 105 structural bridges. Conducted further persecutione up to an additional 10-fold overlap, and the inclusion of these sequences in the Assembly led to a reduction in the number of Contigo to 27. Further closing of the gaps using methods of inverse PCR and long PCR fragments reduced the number of Contigo to 14.

Combined sequence length of 14 Contigo shows that this gene are a few longer (2920443 BP)than the size estimated by PFGE (Fig. 1A), and significantly larger than the genome of its nearest relativeM. smithii(1,9 MPa). The overall G+C content equal to 32.7%, close to what was reported to range from 27,5% to 31.6%reported for strains ofM. ruminantiumBalch et al., 1979). The sequence analysis predicts 2672 ORF, and the total number of hits in the protein family (TIGRFam and PFam) and clusters ontologica groups (COG) are shown in Fig. 1B. Contains all the genes predicted involved in methanogenesis from H2+ CO2and formate (Fig. 1C and Fig. 6A-6C). However, the preliminary sequenceM. ruminantiumdoes not contain system the methyl-coenzyme a reductase II (mcr IIormrt). In other methaneproducing clustermcrIIencodes an isoenzyme of the enzyme methyl CoM reductase I, which is activated during growth at high partial pressure of H2(Reeveet al., 1997). H2fast is used in the rumen and does not accumulate in high concentrations, so thatM. ruminantiumseems to be adapted to use low concentrations of H2only through a system ofmcrI.

Comparison of the preliminary genomeM. ruminantiumwith the closely relatedM. smithiiandMt. thermoautotrophicusidentified a number of areas of differences. Some of the genetic differences encode a very large surface proteins from a large family asparagine/threonine-rich protein, which may contain repeated sequences CPOMP and DUF11 (polymorphic outer membrane proteins of chlamydia and a domain of unknown function, respectively), likely mediating interactions with over the awns or other microorganisms in the environment of the rumen (see Fig. 7A-7C). Similar repeated sequences are also found in large surface proteins encoded by genomes andMs. stadtmanae,andM. smithii(Samuelet al., 2007).

Earlier it was reported thatM. ruminantiumcapable of encapsulated (Smith and Hungate, 1958), and the sequence analysis shows that this process is encoded more than 50 genes (glycosyl transferring enzyme (GT), other transferring enzyme, epimers and transport proteins involved in the synthesis and export of exopolysaccharides, confirming the fact of finishing its surface polysaccharides (see Fig. 8A-8C). TheM. ruminantiumthere are at least 30 glycosyl transferring enzyme (6 GT1, 21 GT2, GT4 2 and 1 GT66; see Fig. 8A-8C) compared to 28 inM. smithii(1 GT1; 22 GT2; 4 GT4 and 1 GT66) and 41 atM. stadtmanae(2 GT1; 26 GT2; 12 GT4 and 1 GT66) (Samuel et al., 2007; Frickeet al., 2006; Coutinho and Henrissat, 1999). It is a fairly large number of genes, designed to encode these organisms surface polysaccharides, and it is assumed that this is an important factor for survival in the environment of the gastrointestinal tract.

Analysis of nucleotide repeats showed the presence in the genome ofM. ruminantiumat least two areas of direct repeats containing buried by spacers (SPIDR). SPIDR represent nucleotide repeats (usually less than 40 BP), composed of identical segments separated by heterologous sequences, and first described in prokaryotes (Jansenet l. , 2002). SPIDR IM. ruminantiumhas a unique organization of genes consisting of two identical structural repeats flanking region of length 17 KBP containing the cluster associatedcas-genes. Similar structural repetitions found in the genomes of a number of methaneproducing.Methanocaldococcus jannaschiicontains 18 copies multicopying repetitive nucleotide elements (Bult et al., 1996), consisting of a long (391-425 BP) recurring segment, followed by up to 25 short (27-28 BP) recurring segments, which are divided into unique sequences from 31 to 51 BP GeneMs. stadtmanaecontains the length of 4.8 KBP, in which the element length of 30 BP is repeated 59 times (Frickeet al., 2006).Mt. thermoautotrophicuscontains two extended repetition (of 3.6 and 8.6 KBP), which contain a repeating sequence of length 372 BP, and then 47 and 124 copies of the same repeating sequence length of 30 BP, separated by unique sequences from 34 to 38 BP in length (Smithet al., 1997). The biological function of these SPIDR is unknown, although the current hypothesis suggests that this system is a functional analogue systems eukaryotes small interfering RNA and is a protective system against alien replicons, operating on the principle of antisense RNA (Jansenet al., 2002; adhesiveet al., 2005; Godde and Bickerton/206; Makarovaet al., 2006).

In the genome ofM. ruminantiumalso coded a large number of ORF, presumably encode proteins with transmembrane domains, which, therefore, must contain a region exposed on the cell surface (Fig. 9A, 9B and 9C).

EXAMPLE 5: preparation and testing of antibodies

The drugs in the cell walls ofM. Ruminantium

Cell wallM. ruminantiumwas obtained by freezing the cell debris in liquid N2and crushing using a pre-chilled, sterilized mortar and pestle. Little cells resuspendable in trypsin-phosphate buffer (40 mg trypsin/200 ml of 0.1 M phosphate buffer, pH 7,9) and incubated at 37°C for 2 hours. The preparation was then centrifuged at 48000g for 30 minutes at 4°C and the precipitate washed twice with sterile distilled H2O and freeze-dried.

Antibodies

Nine peptide sequences, which are projected localized on the outer side of the cells identified in the genome ofM. ruminantiumand selected as potential antigens. Five milligrams of each of these peptides were synthesized (Invitrogen) and their purity was checked by mass spectroscopy. The encoding peptides and their sequences are shown in Fig. 4. The complete sequences of nucleic acids and AMI is ocelot shown in Fig. 5. Two milligrams of each peptide was left unconjugated for ELISA, and 3 mg conjugatively with hemocyanin fiorelli (KLH) for immunization of animals.

The vaccination program is shown in Fig. 2 and proceeded as follows. For each immunization used one sheep (1-3 years), which previously took so much blood to get 2-5 ml preimmune serum on day 0. This was followed by primary intradermal (ID) injection of 200 μg of conjugated peptide in CFA (complete beta-blockers) in 10-15 places in day 0. Intradermal (ID) injection of 200 μg KLH-peptide in IFA (incomplete beta-blockers) in 10-15 seats were made at day 14 and 200 μg KLH-peptide in CFA 10-15 places in day 28. Five intradermal (ID) injection of 200 μg KLH-peptide in IFA in 10-15 seats were made on days 56, 70, 84, 98 and 112. Four control blood samples (2-5 ml) was performed on days 42, 56, 84 and 112. Production drawing blood, giving about 1000 ml of antisera, conducted at the end of the standard Protocol.

Antibody titer was determined using an enzyme-linked immunosorbent assay (ELISA) with the chimeric peptide-GGG (peptide-goat gamma globulin), immobilized on a solid phase (0.1 ág/100 ál/well) highly-binding 96-well plate. First, the serum was diluted 50 times and then further diluted in 2-fold serial dilutions. ELISA-titer was calculated as the estimated ratios are the NT dilution, resulting in a value of OD at 405 nm of the order of 0.2 obtained from nonlinear regression analysis of the curve of serial dilutions. Detection was carried out using the conjugate secondary antibody with HRP and ABTS substrate.

The formation of sheep antibodies in response to vaccination is shown in Fig. 3. All sheep serum for 6 weeks had titers that were at least 32 times higher than before immunization (1:1600). The most antigenic preparation wasmtrD-peptide, whose title was in 1024 times higher than the titer prior to immunization. The least immunogenic preparations weremtrE-peptide, ORF508 and ORF819 peptides surface protein. The drugs in the cell walls ofM. ruminantiumcaused a positive immune response (256 times higher than preimmune titles), but they were not sustained for more than 15 weeks, despite several booster immunizations.

The binding of the antibody with cellsM. ruminantium

For control of antibody binding to cellsM. ruminantiumthe ELISA test was carried out as follows. Tablets for ELISA MaxiSorp (Nunc) were coated with whole cellsM. ruminantium(40 μl of cells in 2 ml natrocarbonatite buffer) and fractionscytosolic proteinsM. ruminantium. Samples of serum were diluted 1/20 (25 ál 475 ál of diluent) in PBS/tween-20 containing 1% casein (weight/volume), and incubated at room temperature for 1 hour. The tablet was washed 6 times with PBS/tween-20. When calculating the Uch is Tivoli negative control serum, from the sheep, which was not colostrum as a lamb.

For detection used conjugate IgG-HRP donkey against sheep/goats (Batch 061005 Star 88P from Serotec) and 50 μl of 1/5000 dilution (2 μl in 10 ml solvent) was added to each well. Then added (50 μl/well) substrate 3,3',5,5'-tetramethylbenzidine (TMB), and the reaction was incubated at room temperature in the dark for 15 minutes Then add the stop solution (0.05 M H2SO4, 50 μl/well) and the testimony of the tablet is removed at 450 nm.

Analysis of the inhibition of growth ofM. Ruminantium

Samples of the immune sera were thawed in anaerobic laminar flow Cabinet, and 0.1 ml each of 10 samples were placed in a test tube of 1.5 ml for microcentrifuge. The mixture (1 ml) were incubated at room temperature with the lid open in anaerobic laminar flow Cabinet overnight to remove dissolved oxygen. Preimmune serum served as a negative control. The combined sample serum (0.3 ml) was added in three repetitions to 5 ml of the growing in test tubes of Hungate cultureM. ruminantiumin anaerobic laminar flow Cabinet. In tubes of Hungate was pumped gas (80% H2and 20% CO2) and the culture incubated at 39°C on a shaker (100 rpm). Growth methaneproducing controlled by measuring OD at 600 nm on a spectrophotometer and gas-chromatographic determination of water consumption is kind and methane production.

The ELISA analyses showed that antibodies generated against each of the antigens associated with the cellM. ruminantiumimmobilized on tablets for micrometrology. It was shown that antibodies bind with cellsM. ruminantiumin vitroalthough drugs of the same antibody that was added to the culturesM. ruminantiumnot inhibit the growth of methaneproducing or do not reduce the number of formed methane. However, a drug consisting of the combined samples of antisera for each of 10 different antigens, apparently, enhances the aggregation of cells when added to cultureM. ruminantium.

EXAMPLE 6: a Short description

Methanobrevibacterruminantiumwas selected for genome sequencing because of its predominance in the rumen under different food conditions (including cultivation and molecular data detection), the availability of crops, facilities for normal growth under laboratory conditions, as well as the relatively large number of previous studies and reference books for this organism. For a significant number of genesM. ruminantiumwere defined functions, and thereby establish detailed picture of the life of this organism in the rumen. The dependence ofM. ruminantiumfrom simple substrates (H2+ CO2, formate) and its interaction with the environment of the rumen via surface proteins and eksopolis the IDA are important targets for suppression. Similarly SPIDR promising for specific targetingM. ruminantiumand for future genetic manipulation, facilitating the identification of gene function. Data sequences explain the metabolism of this organism and how it interacts with other microbes and indicates conservatism systems and components between methaneproducing, which can be inactivated to prevent or reduce the formation of methane in the rumen.

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All publications and patents mentioned in the above patent application, is hereby incorporated into this description by reference. Where in the above description, reference was made to a unit, with their known equivalents, these equivalents are included in the present description as individually described. Although the invention has been described with reference to specific preferred options for implementation, it should be understood that the invention as claimed should not be unduly limited to such specific choices of implementation. Note that the invention can be subjected to further modification, as described in the present description, without departing from the spirit and scope of the invention.

1. The selected polypeptide that is a biological target for inhibition of cell-methaneproducing:
a) characterized by the sequence SEQ ID NO:641;
b) which is a functional variant of SEQ ID NO:641 characterizes the I sequence, which at least 90% identical to the full size of the sequence SEQ ID NO:641; or
c) which is the functional fragments of SEQ ID NO:641.

2. Selected polynucleotide, which is the biological target for inhibition of cell-methaneproducing and is characterized by:
a) a sequence that encodes a full-sized sequence of SEQ ID NO:641;
b) SEQ ID NO:1312;
c) a sequence that encodes a polypeptide that is at least 90% identical to the full size of the sequence SEQ ID NO:641; or
d) a sequence that is complementary to any of sequences (a)-(c).

3. The expression vector:
a) which contains polynucleotide encoding the polypeptide according to claim 1; or
b) containing polynucleotide according to claim 2.

4. Cloning vector:
a) which contains polynucleotide encoding the polypeptide according to claim 1; or
b) containing polynucleotide according to claim 2.

5. A host cell to obtain a polypeptide according to claim 1 or polynucleotide according to claim 2:
a) which is genetically modified so that it contains the polypeptide according to claim 1;
b) which is genetically modified so that it contains polynucleotide according to claim 2; or
c) which contains the vector according to claim 3.

6. A host cell for replication, containing the vector according to claim 3.

7. A host cell according to claim 6, which is a prokaryotic cell or methaneproducing.

8. A host cell according to claim 7, which is a cellEscherichia coliorMethanobrevibacterruminantiumincluding the strain ofMethanobrevibacterruminantiumM1T(DSM1093).

9. The antibody or the functional fragment of the antibody for inhibition of cell-methaneproducing, which binds to the polypeptide according to claim 1, where the antibody or fragment obtained with this polypeptide.

10. The antibody or antibody fragment according to claim 9, which is monoclonal.

11. Molecule is conjugated or fused molecule for the inhibition of cell-methaneproducing, which contains:
a) the polypeptide according to claim 1; or
b) the antibody or the antibody fragment according to claim 9 or 10; and
which additionally contains a compound against methanogenesis, signal sequence, a lytic enzyme, a peptide-nucleic acid, an antimicrobial peptide or an antibiotic.

12. Pharmaceutical composition for inhibiting cell-methaneproducing containing an effective quantity:
a) the antibody or antibody fragment according to claim 9 or 10; or
b) molecule conjugate or fused molecule according to item 11.

13. The method of identifying cells of methaneproducing, including: bringing the cells into contact with the antibody or antibody fragment according to claim 9 or claim 10.

14. Method of inhibiting cell-methaneproducing, including: bringing the cells into contact with the antibody or antibody fragment according to claim 9 or claim 1.

15. The method of identifying cells of methaneproducing, including: bringing the cells into contact with a molecule is conjugated or fused molecule according to item 11.

16. Method of inhibiting cell-methaneproducing, including: bringing the cells into contact with a molecule is conjugated or fused molecule according to item 11.

17. The method according to any of p-16, in which the cell is methaneproducing some animal or isolated from it.

18. The method according to any of p-16, in which the cell is aMethanobrevibacterruminantium.

19. The method according to p, in which the cell is aMethanobrevibacterruminantiumstrain M1T(DSM1093).



 

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FIELD: medicine.

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1 tbl, 1 ex

FIELD: medicine.

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FIELD: medicine.

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5 ex, 6 tbl

FIELD: medicine.

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3 ex

FIELD: medicine, pharmaceutics.

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35 cl, 15 tbl, 5 ex, 4 dwg

FIELD: medicine, pharmaceutics.

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17 cl, 2 tbl, 2 ex

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FIELD: medicine.

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57 cl, 3 dwg, 5 tbl, 3 ex

FIELD: medicine.

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FIELD: medicine, pharmaceutics.

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EFFECT: claimed invention provides the synthetic 5'UTR regions, which contain the first polynucleotide fragment in the form of the second intron of gene of calcium ATphase of the sarcoplasmic/endoplasmic reticulum and the second polynucleotide fragment, represented by a part of the 5' untranslated region (5'UTR) of casein gene.

25 cl, 17 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biochemistry, in particular to clostridial neurotoxins with a modified persistence. Claimed is a polypeptide, containing HC-domain, the first and, at least, one additional LC-domain with amino acid sequences, at least, 90% identical to the respective sequences of a neurotoxic component of botulotoxin of a serotype A, B, C1, D, E, F or G. Also claimed are nucleic acid, an expression vector and a host cell, intended for the expression of the said polypeptide. Also claimed are a method of obtaining and application of the said polypeptide, including as a component of a pharmaceutical composition, for treatment of a condition, associated with hyperactive cholinergic innervations of a muscle or a exocrine gland, and for cosmetic procedures, associated with wrinkles.

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12 cl, 1 tbl, 4 ex

FIELD: chemistry.

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1 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

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EFFECT: invention may be used in medicine for treating and preventing a staphylococcal infection.

23 cl, 8 tbl, 7 dwg, 8 ex

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