Cell-penetrating peptides and polypeptides for microorganism cells

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.

EFFECT: invention enables providing higher methane-producing cell permeability.

18 cl, 35 dwg, 3 ex

 

RELATED APPLICATIONS

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

The scope to which the invention relates

The present invention relates to compositions and methods for delivery of inhibitory molecules into microbial cells, in particular methaneproducing cells. In particular, the present invention relates to signal peptides and polypeptides containing these peptides, as well as polynucleotides that encode these peptides or polypeptides. The present invention also relates to expression vectors and cells-owners to obtain these peptides or polypeptides. The present invention additionally relates to methods of identifying, directed delivery, penetration and inhibition of microbial cells, in particular, methaneproducing cells using the described peptides or polypeptides, polynucleotides, expression vectors and host cells.

Prerequisites to the creation of inventions

In New Zealand agriculture is responsible for most greenhouse gas emissions. Therefore, the decrease in agricultural is on greenhouse gas emissions is important to meet New Zealand's Kyoto Protocol. In accordance with this Protocol are required to reduce greenhouse gases to 1990 levels by the end of the first period of commitment (2008-2012). By the end of this period, the group's agricultural sector and the New Zealand government approved the research consortium on greenhouse gases (Pastoral Greenhouse Gas Research Consortium (PGGRC)) to establish ways to reduce greenhouse gas emissions from agriculture in New Zealand.

An important part of the activities of the PGGRC was a study in reducing methane emissions from new Zealand pastures. Reducing methane emissions from ruminants is of commercial interest for two reasons. First, the failure to comply with obligations under the Kyoto Protocol will force the government to acquire quotas on carbon emissions. Currently the cost of this is estimated at $350 million. Secondly, the formation of methane leads to the loss of 8-12% of the total energy produced in the rumen. This energy could be used instead to improve the productivity of some animal.

Methane is produced in the rumen by microorganisms called methaneproducing, which are part of the taxonomy typeEuryarchaeotain the Kingdom ofArchaea. Most methanogens grow in CO2and H2as the only sources of energy, but some may use for the growth of acetate or methyl the s connection. In the rumen, there are several different kinds of methaneproducing archaea, but the species of the genusMethanobrevibacterin particular,M. ruminantiumandM. smithiiconsidered to be the predominant methaneproducing from ruminant New Zealand.M. ruminantiumis currently the subject of a project to sequence the genome funded PGGRC. This project represents the first genome sequencing of methaneproducing scar, and its purpose is to improve understanding of the biologyMethanobrevibacterto detect targets for the inhibition of methane formation.

To reduce the production of methane in the rumen requires inhibition of methaneproducing or inactivation their way of formation of methane. By way of inhibiting the formation of methane is the delivery of specific inhibitory molecules in cells methaneproducing. This can be achieved, for example, a compound inhibiting molecules from penetrating into the cell peptides. In microbial cells signal peptides mediate the translocation of extracellular proteins from the internal environment of the cells in the external, and are suitable for migration inhibitory molecules. Therefore, it would be useful to identify signal peptides, which have the ability to penetrate into cells methaneproducing and deliver inhibitors.

The signal peptide or signal sequence, usually included the in protein precursor, secreted from prokaryotic and eukaryotic cells. Signal peptides are part penetrating into the cell elongation on the N-end predecessor. The primary amino acid sequence of the signal peptide is not conservative, except for the cleavage site for signal peptidases (von Heijne, 1985). In addition, signal peptides have structural similarities. Signal peptides typically include from one to five positively charged N-terminal amino acid residues (n-region) followed by 10 to 15 hydrophobic amino acid residues (h-region). The residue of glycine or Proline is usually located within the hydrophobic domain, and the residue (remainder) of threonine and/or serine to form polar domain (c-region) near the site of cleavage (Inouye and Halegoua, 1980; Vlasuk et al., 1983, von Heijne, 1985).

Was proposed loop model of translocation signal peptide (Inouye et al., 1977; Inouye and Halagoua, 1980) whereby positively charged N-terminal signal peptide interacts with the negatively charged inner surface of the cell membrane. The hydrophobic domain is then immersed in the hydrophobic lipid bilayer membrane by formation of a loop. This loop eventually includes the cleavage site, which is subjected to signal peptidases to remove the signal is of eptide. One of the obstacles for inhibiting or limiting the formation of methane is the ability to deliver inhibiting components in cells methaneproducing. Thus, there is a need to identify signal peptides that are able to join the cell membranes and transfer of molecules through the lipid bilayer, in suitable media for inhibitors of the cell.

Summary of the invention

The invention relates to the selection signal peptide or polypeptide containing this peptide, which contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172. In a separate aspect, the peptide or polypeptide contains at least one amino acid sequence KKLIIILLLLILLLSI sequence SEQ ID NO:117, or at least one amino acid sequence KKIIIILLLLILLLISI sequence SEQ ID NO:119. In another aspect, the peptide or polypeptide includes a fragment of, for example, containing at least one amino acid sequence containing amino acids 3-14, 3-16, or 2-16 sequence SEQ ID NO:117, or at least one amino acid sequence containing amino acids 3-15, 3-17, or 2-17 sequence SEQ ID NO:119. In an additional aspect, the peptide or polypeptide contains the fragment, containing at least one conservative crustal sequence of the sequence SEQ ID NO:1-172, described in this application. In another additional aspect, the peptide or polypeptide encoded by at least a fragment of polynucleotide selected from the group consisting of SEQ ID NO:173-341 or SEQ ID NO:342-533.

The present invention also relates to the selected polynucleotide containing the coding sequence of at least one signal peptide or polypeptide containing this peptide. In one aspect, this polynucleotide contains the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172. In a specific aspect, polynucleotide contains the coding sequence of at least one amino acid sequence KKLIIILLLLILLLSI sequence SEQ ID NO:117, or the coding sequence of at least one amino acid sequence KKIIIILLLLILLLISI sequence SEQ ID NO:119. In another aspect, polynucleotide contains a fragment of the coding sequence, for example, the coding sequence of at least one amino acid sequence containing amino acids 3-14, 3-16, or 2-16 sequence SEQ ID NO:117, or the coding sequence of at least one amino is islotes sequence, containing amino acids 3-15, 3-17, or 2-17 sequence SEQ ID NO:119. In an additional aspect, polynucleotide contains a fragment of the coding sequence, for example, a nucleotide sequence that encodes at least one conservative crustal sequence described in the present application sequence SEQ ID NO:1-172.

In an additional aspect, the present invention relates to selected polynucleotide containing a nucleic acid sequence selected from the group consisting of SEQ ID NO:173-341 or SEQ ID NO:342-533. In a specific aspect, this polynucleotide contains the nucleic acid sequence SEQ ID NO:531, 532 or 533. In another aspect, polynucleotide is a fragment or oligonucleotide, for example, contains a sequence of nucleic acids, the length of nucleotides 7-42, 7-48 or 4-48 sequence SEQ ID NO:531, 532 or 533. In addition, the invention encompasses a dedicated polynucleotide or its fragment that's hybrid with any of the nucleic acid sequences SEQ ID NO:173-341 or SEQ ID NO:342-533. The present invention additionally encompasses a dedicated polynucleotide containing complementary, complementary back, reverse the sequence, or fragments thereof, of any of the nucleic acid sequences encoding the signal peptide is ID or polypeptide, containing this peptide.

The present invention relates to an expression vector containing polynucleotide, which contains the coding sequence of at least one signal peptide or polypeptide containing this peptide. In one aspect, the expression vector contains the coding sequence of at least one amino acid sequence selected from the group consisting of sequences SEQ ID NO:1-172. In a specific aspect, the expression vector contains the coding sequence of at least one amino acid sequence KKLIIILLLLILLLSI sequence SEQ ID NO:117, or the coding sequence of at least one amino acid sequence KKIIIILLLLILLLISI sequence SEQ ID NO:119. In another aspect, the expression vector contains the coding sequence of at least one amino acid sequence, stretching from amino acids 3-14, 3-16 or 2-16 sequence SEQ ID NO:117, or the coding sequence of at least one amino acid sequence containing amino acids 3-15, 3-17, or 2-17 sequence SEQ ID NO:119. In another aspect, the present invention relates to a cell-master, for example, microbial cell host containing at least one expression vector.

The present invention in cast the STI refers to the antibody, the aim of the peptide, polypeptide, or polynucleotide described in this application. In certain aspects, this antibody is directed by at least one sequence of the signal peptide selected from the group consisting of SEQ ID NO:1-172, or its modified sequence. In alternative aspects, the antibody is directed to at least a fragment of the sequence of the signal peptide, for example, conservative crustal sequence of a sequence selected from the group consisting of SEQ ID NO:1-172. In an additional aspect, the antibody binds to a polypeptide containing the sequence of the signal peptide of any of the sequences SEQ ID NO: 1-172. In alternative aspects, this antibody is directed to at least a fragment of polynucleotide selected from the group consisting of SEQ ID NO:173-341 or SEQ ID NO:342-533, or its complement, or a modified sequence. In another aspect, the antibody contains one or more fusions or conjugates of at least one cell inhibitor, for example, the compounds against the formation of methane (for example, bromoethanesulfonate acid), antibodies and fragments of antibodies, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, are described in detail in this application.

p> The present invention also relates to a modified signal peptides and polypeptides containing these peptides, and antibodies directed to these peptides or polypeptides, including biologically active changes, fragments, variants and derivatives described in this application. Also described polynucleotide encoding these modified peptides or polypeptides, as well as modifications, fragments, variants and derivatives of the described polynucleotides, expression vectors containing these nucleic acid sequences, and cell-hosts containing these vectors. In particular aspects, the compositions and methods according to the invention are those modified polynucleotide, polypeptides or antibodies, or the corresponding expression vectors or cells of the hosts. In specific aspects, the peptides or polypeptides are obtained in the form of mergers or conjugates at least one cell inhibitor, for example, the compounds against the formation of methane (for example, bromoethanesulfonate acid), antibodies and fragments of antibodies, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, are described in detail in this application.

The invention additionally relates to compositions containing the selected signal peptide (for example, at least one of SE ID NO:1-172, or a modified sequence or the polypeptide containing this peptide, or antibody directed to the peptide or polypeptide. Also described composition containing the selected polynucleotide (for example, at least one of SEQ ID NO:173-341 or SEQ ID NO:342-533, or their complementary or a modified sequence). Additionally described composition, which contains an expression vector, or a host cell containing the expression vector, in accordance with the present invention. The composition may contain any biologically active changes, fragments, variants and derivatives described in this application. The composition may additionally contain at least one cell inhibitor, and can be obtained, for example, in the form of pharmaceutical compositions or food supplements, in particular, components of feed for ruminant animals.

In a specific aspect, the invention relates to compositions according to the invention as part of a kit for determining and/or measuring, or directed delivery, penetration and/or inhibiting microbial cells, particularly cells of methaneproducing, in accordance with the described methods. These kits contain: a) at least one composition described in this application; and (b) optionally, instructions for use, for example, targeted delivery of Il is the penetration into cells or inhibition of cell growth, or replication for methaneproducing or other microorganisms. In particular aspects, the peptide or polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or its modified sequence.

The present invention relates to a method of receiving a signal peptide or polypeptide containing this peptide, the method includes: a) culturing the expression vector or host cell containing the expression vector, which contains the coding sequence of at least one signal peptide or polypeptide containing this peptide under conditions suitable for expression of the peptide or polypeptide; and b) removing the peptide or polypeptide from the culture. Also describes how to get the described compositions. In particular aspects, the peptide or polypeptide contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or its modified sequence.

The present invention also relates to a method for receiving a signal peptide or polypeptide containing this peptide, which contains a fusion or conjugate with at least one cell inhibitor, for example, the compounds against the formation of methane (for example, bromoethanesulfonate Ki is lateu), the antibodies and fragments of antibodies, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, is described in detail in this application. This method comprises: a) culturing the expression vector or host cell containing the expression vector, which contains the coding sequence of at least one peptide or polypeptide under conditions suitable for expression of the peptide or polypeptide; (b) education merger or conjugate (for example, by expression of the fused sequence or chemical conjugate with the cellular inhibitor); and c) removing the fusion or conjugate. In particular aspects, the signal peptide or the polypeptide contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or its modified sequence.

The present invention relates to a method of penetration into the microbial cell, in particular, the cell methaneproducing, providing: a) optionally, obtaining or extracting at least one signal peptide or polypeptide containing this peptide; and b) contacting the cells with a signal peptide or a polypeptide. In a specific aspect, the peptide or polypeptide contains at least one amino acid sequence selected from the GRU is dust, consisting of SEQ ID NO: 1-172, or its modified sequence. In additional aspects, the peptide or polypeptide contains a fusion or conjugate with at least one cell inhibitor, for example, one or more compounds against the formation of methane (for example, bromoethanesulfonate acid), antibodies and fragments of antibodies, political fragments, peptide-nucleic acids, antimicrobial peptides or other antibiotics, are described in detail in this application.

The present invention also relates to a method of inhibiting microbial cells (e.g., inhibiting growth or replication), in particular, cells methaneproducing, providing: a) optionally, obtaining or allocating at least a signal peptide or polypeptide containing this peptide, which further comprises at least one cellular inhibitor; and b) contacting the cells with a signal peptide or a polypeptide. In a specific aspect, the peptide or polypeptide contains at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-172, or its modified sequence. In an additional aspect, the cellular inhibitor selected from compounds against the formation of methane (for example, bromoethanesulfonate acids), antibodies and fragments of antibodies which, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, is described in detail in this application.

The present invention also relates to a method of inhibiting microbial cells (e.g., inhibiting growth or replication), in particular, cells methaneproducing, providing: a) optionally, obtaining or extracting at least one signal peptide or polypeptide containing this peptide, which further comprises at least one cellular inhibitor; and b) contacting the cells with a signal peptide or a polypeptide. In a specific aspect, the peptide or polypeptide contains at least one amino acid sequence from the group consisting of SEQ ID NO:1-172, or modified sequences. In an additional aspect, the cellular inhibitor selected from compounds against the formation of methane (for example, bromoethanesulfonate acids), antibodies and fragments of antibodies, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, is described in detail in this application.

The present invention also relates to a method for determining and/or measuring the levels of the signal peptide or the polypeptide or polynucleotide providing: 1) contacting the sample obtained the t of the individual, with antibody directed by the signal peptide (for example, at least one of SEQ ID NO:1-172, or modified sequences) or the corresponding polypeptide or polynucleotide; and 2) determining the presence or levels or antibody-based test of the complex formed with the signal peptide or the polypeptide or polynucleotide in the sample. Such methods can also be used to determine and/or measure the levels of microbial cells, in particular cells of methaneproducing.

The present invention also relates to a method for determining and/or measuring the levels of the signal sequence polynucleotide (for example, the coding sequence of the signal peptide, or the corresponding coding sequence of the polypeptide), providing: 1) contacting the sample obtained from the individual, with complementary polynucleotides (for example, a sequence complementary to any of sequences of SEQ ID NO:173-341, or modified sequences); and 2) determining the presence or levels of hybridization complex formed with the signal sequence of polynucleotide in this sample. Such methods can also be used to determine and/or measure the levels of microbial cells, in particular cells of methaneproducing.

In sabyasachi, in the methods according to the invention are usedin vivoorin vitrocomponents of the expression. In other aspects, methods are peptides or polypeptides produced by recombinant, synthetic or semi-synthetic methods, or peptides or polypeptides produced by endogenous methods.

Other aspects and embodiments of the present invention are described below.

Brief description of drawings

The present invention is described with reference to specific variations in its implementation and with reference to the drawings.

FIG. 1A-1C: Comparison of the genomes of methanobacteria (FIG. 1A); the statistics of the genomeM. ruminantium(FIG. 1B); the genes presumably involved in the formation of methane in a species of methanobacteria (FIG. 1C).

FIG. 2: Alignment of the signal peptideMethanobrevibacter ruminantium. The crust conserved region of each peptide is shown in bold.

FIG. 3A: Protein sequence logo of 102 sequences created using LogoBar. FIG. 3B: Protein sequence logo of 102 sequences created using LogoBar showing the most conservative amino acid residues. FIG. 3C: the Crust of the consensus sequence of the signaling protein forM. ruminantium. FIG. 3D: Amino acid sequence ofM. ruminantiumpenetrating into the cell peptide with addition of N-con is avago lysine-fluorescein.

FIG. 4: Permeability in cellsM. ruminantiumpeptide labeled fluorescent.

FIG. 5: Venn Diagram showing the preliminary determination signal peptide SignalP 3.0-HMM, using three different models forM. ruminantiumthe signal sequence M1093 ORF.

FIG. 6: GenesM. ruminantiumthe signal peptides.

FIG. 7: GenesM. ruminantiumand the appropriate signal peptides.

FIG. 8: Coding sequences of the signal peptides from FIG. 7.

FIG. 9: Coding sequences of the signal peptides from FIG. 7, with codons optimized for expression inE. coli.

Detailed description of the invention

Definitions

"Altered" nucleic acid sequences encoding signal peptides in the context of the present invention include sequences with deletions, insertions, or substitutions of different nucleotides, resulting in polynucleotide that encodes the same or functionally equivalent peptides. The encoded peptide can also be "altered" and contain deletions, insertions or substitutions of amino acid residues, which give a silent change and result in a functionally equivalent peptide. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, rest is remote, hydrophobicity, hydrophilicity, and/or amphipatic nature of the residues, while maintaining the biological activity (e.g., Association with the cell or penetration into the cell), or immunogenic activity of this peptide. For example, negatively charged amino acids may include aspartic and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar terminal groups having similar hydrophobicity values may include leucine, isoleucine and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.

"Amino acid sequence" in the context of the present invention, refers to an Oligopeptide, peptide, polypeptide, or protein sequence, or their fragments, and to the natural, recombinant, or synthetic or semi-synthetic molecules. Sequence according to the invention (for example, SEQ ID NO:1-172) contain at least 5, 6, 7, 8, 9, 10, 11, 12, 15, 17, 19, or 22 amino acids, preferably at least 5-10, 5-15, 10-15, 12-15, 15-17, 17-19, or 17-22 amino acids, and preferably retain biological activity (for example the Association of the cage or penetration into the cell) or immunological activity (for example, at least one binding site from an antibody) of the original series is doomed. In cases where "amino acid sequence", cited in the present description, refers to the amino acid sequence of the molecule of the natural peptide or polypeptide, it is considered that the amino acid sequence and similar terms do not limit the amino acid sequence to the complete, natural amino acid sequence associated with a full-sized molecule.

"Amplification" in the context of the present invention relates to the production of additional copies of a nucleic acid sequence and is generally provided using the techniques of polymerase chain reaction (PCR), well known from the prior art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY).

The term "antibody" should be understood in its broadest sense, and it is intended to include intact monoclonal antibodies and polyclonal antibodies. This term also encompasses fragments and derivatives of antibodies, provided that they exhibit the desired biological activity. Antibodies cover of the immunoglobulin molecule and an immunologically active portion of the molecules of immunoglobulins (Ig), i.e., molecules that contain an antigen binding site which specifically binds (immunological interacts with the antigen. Antibodies include, but not limited to, polyclonal, monoclonalny, chimeric, single-chain, Fc, Fab, Fab', and Fab2fragments and expression of the Fab library.

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 in the molecule. These molecules also include subclasses such as IgG1, IgG2, and others. Light chain can be a Kappa chain or a lambda chain. Reference in the present description antibody includes a reference to all the classes, subclasses and types. Also includes chimeric antibodies, e.g. monoclonal antibodies or fragments thereof that are specific to more than one source, for example, one or more sequences of mouse, human or some animal. Also includes antibodies of camelids or nanotesla. It will be understood that each reference to an "antibody" or any similar term, in the present description includes intact antibodies, and any fragments thereof, measurement, derivatives or variants.

The terms "biologically active" or "functional"in the context of the present description, refers to a peptide or polypeptide that retains one or more structural, immunogenic or biochemical functions (e.g., Association with cell or penetration into the cell) natural sequence. As one example, a functional sequence includes Myung is our least one of crustal conservative areas, described in this application.

The terms "cellular inhibitor" or "inhibitor", in the context of this Scripture refers to funds that reduce or inhibit the growth or replication of microbial cells, particularly cells of 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 may also reduce or block the activity of enzymes involved in the cascade of methane formation. The inhibitor can target cell recognition components of the immune system. Inhibition of cells also includes the destruction of cells and cell death, for example, in the lysis, apoptosis, necrosis, etc. Suitable inhibitors include, but are not limited to, compounds against the formation of methane (for example, bromoethanesulfonate acid), antibodies and antibody fragments, lytic enzymes, peptide-nucleic acids, antimicrobial peptides, and other antibiotics, is described in detail in this application.

The terms "complementary" or "complementarity", in the context of the present invention, refers to the natural binding of polynucleotides in permissive salt and temperature conditions by mating the basis of the rd. For the sequence A-G-T, a complementary sequence is T-C-A, back of the complementary sequence represents A-C-T, and the reverse sequence is a T-G-A. Complementarity between two single-stranded molecules may be partial, in which only some of the nucleic acids bind, or it may be complete when there is an absolute complementarity between 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 in amplification reactions, which depend upon binding between the chains of nucleic acids and in the creation and application of RNA molecules.

The term "derivative" in the context of the present invention relates to chemical modification of a nucleic acid that encodes a signal peptide, or nucleic acid complementary to it. Such modifications include, for example, replacement of hydrogen by alkyl, acyl, or amino group. In preferred aspects, the derived nucleic acid encodes a peptide that retains the biological or immunological function of the natural molecule. The derived peptide is a peptide which is modified by glikos the modeling, paglierani or any similar method that retains one or more biological functions (e.g., Association with cell or penetration into the cell) or immunogenic function sequence derived which he is.

The term "homology" in the context of the present invention refers to the degree of complementarity. Homology may be partial (i.e. identity is less than 100%) or fully (i.e. 100% identity). A partially complementary sequence that at least partially inhibits an identical hybridization sequence of the target nucleic acid, referred to using the functional term "essentially homologous". The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (southern or Northern blot hybridization in solution and the like) under conditions of low stringency. Essentially homologous sequence or hybridization probe will compete for binding or inhibiting the binding of a completely homologous sequence to the target sequence under conditions of low stringency. Not to say that conditions of low rigidity such that Pets nonspecific binding; conditions of low stringency is ask, to the binding of two sequences to each other was specific (i.e. selective) interaction.

The term "hybridization" in the context of the present invention refers to any process by which a chain of nucleic acid binds with a complementary chain by mating grounds.

"Insert" or "add" in the context of the present invention refers to a change in amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, compared with the natural molecule.

"Methaneproducing" in the context of the present invention relates to microorganisms that produce methane gas, which include theMethanobrevibacter,Methanothermobacter,Methanomicrobium,MethanobacteriumandMethanosarcina. Typical methaneproducing include, but not limited to,Methanobrevibacter ruminantium,Methanobrevibacter smithii,Methanobrevibacter acididurans,Methanobrevibacter thaueri,Methanobacterium bryantii,Methanobacterium formicicum,Methanothermobacter marburgensis,Methanothermobacter wolfeii,Methanosphaera stadtmanae,Methanomicrobium mobile,Methanosarcina barkeri,Methanosarcina mazei,Methanococcoides burtoniiandMethanolobus taylorii. All kinds and types of methaneproducing covered by this term.

"Microbial cells in the context of the present invention relate to natural or genetic and modified microbial cells, 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" refers to the altered sequences and fragments, variants and derivatives of the sequences described in the present invention.

"Nucleic acid sequence" or "nucleotide sequence" in the context of the present invention refers to the sequence of polynucleotide, the oligonucleotide or their fragments, and to DNA or RNA of natural, recombinant, or synthetic or semi-synthetic origin which may be single or double helix, and may represent the sense or antisense chain, and coding or non-coding region. The sequence of the present invention most preferably include sequences encoding the polypeptide (for example, SEQ ID NO:173-341 or 342-533, or their complementary or modified sequences)that contain at least 15, 18, 21, 24, 27, 30, 33, 36, 39, 45, 51, 57 or 66 nucleotides, preferably at least 15 to 30, 15 to 45 30 to 45, from 36 to 45, 45 to 51, 51 to 57, or from 51 up to 66 nucleotides, or at least 100 nucleotides, or at least 1000 is ucleotide. It will be understood that each reference to a "nucleic acid sequence" or "nucleotide sequence" in the present invention will include a full-sized native sequence (e.g., SEQ ID NO:173-341 or 342-533), as well as any complementary sequences, fragments, modifications, derivatives, or variants.

The term "oligonucleotide" refers to a nucleic acid sequence containing at least 6, 8, 10, 12, 15, 18, 21, 25, 27, 30 or 36 nucleotides, or at least from 12 to 36 nucleotides, or at least 15 to 30 nucleotides (e.g., at least a fragment of the sequence SEQ ID NO:173-341 or 342-533, or their complementary sequence), which can be used in PCR amplification, sequencing or hybridization assays. In the context of the present invention the oligonucleotide is essentially equivalent to the terms "amplier", "primers", "oligomers", "oligi" and "probes"as usually defined in this field.

The term "polynucleotide"used in the singular or plural, generally refers to any nucleic acid sequence, for example, any polyribonucleotide or polymethacrylamide, which can be unmodified RNA or DNA or modified RNA or DNA. This t is pmin includes, without limitation, single-stranded and double-stranded DNA, DNA containing single and double helix region, single and double helix RNA and RNA containing single and double helix region, hybrid molecules containing DNA and RNA that may be single or, more commonly, double helix, or contain single and double helix region. Also included are RNA or DNA containing transferline region, or both RNA and DNA. In particular, enabled mRNA, cDNA and genomic DNA, and any fragments thereof. The term includes DNA and RNA, which contain one or more modified bases, such as bases, labeled with tritium, or unusual bases such as inosine. Polynucleotide of the present invention can include coding or non-coding sequence, or a sense or antisense sequence, or RNC, such as siRNAs. It will be understood that each reference to "polynucleotide" or a similar term in the context of the present invention, will include a full-size sequence, and any of their complementary sequences, fragments, measurement, derivatives or variants.

"Peptide nucleic acid" or "NCP" in the context of the present invention relates to antisense molecule, or proteogenomic means that sod is RIT Foundation, associated through peptide skeleton.

The term "ruminant" in the context of the present invention refers to animals that have the scar as a special type of digestive organ. Ruminants include, but not limited to, cattle, sheep, goats, oxen, moose, caribou and deer.

"Signal peptides" in the context of the present invention relates to the selected peptides according to the invention, obtained from any species, preferably microorganisms, from any source whether natural, synthetic, semi-synthetic or recombinant. In particular, the signal peptide may be derived from cells of methaneproducing, such as cellsMethanobrevibacterin particular, cellsM. ruminantiumorM. smithii. For recombinant receiving the signal peptide according to the invention can be derived from microbial or eukaryotic cells, for example,Escherichia,Streptomyces,Bacillus,Salmonella, yeast, insect cells, such asDrosophila, animal cells such as COS cells and CHO, or plant cells. It will be understood that each reference to "a peptide" in the context of the present invention will include a full-size sequence (e.g., SEQ ID NO:1-172)and any modifications, fragments, derivatives or variants.

The terms and conditions of rigidity or stiffness" in the context of this izopet the deposits belong to the conditions of hybridization, defined nucleic acid, salt and temperature. These conditions are well known in the art and can be modified in order to identify or 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 equivalent conditions involving either the low or high rigidity, depend on such factors as the length and nature of sequence (DNA, RNA, nucleotide composition), the nature of the target (DNA, RNA, nucleotide composition), environmental conditions (in solution or immobilized on a solid substrate), the concentration of salts or other components (for example, formamide, dextran sulfate and/or polyethylene glycol), and temperature reactions (within the interval 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 stiffness that is different from the above conditions, but equivalent.

The term "individual" includes humans and animals, non-people. Animals that are not relevant to humans include, but not limited to, birds and mammals, such as ruminants, and in particular, mice, rabbits,cats, dogs, pigs, sheep, goats, cows and horses.

The terms "essentially purified" or "isolated" in the context of the present invention relates to sequences of nucleic or amino acids that are isolated from their cell, recombinant or synthetic environment, and at least 60% free, preferably 75% free, and most preferably at least 90% free, or at least 99% free from other components with which they are associated in the cell, recombinant or synthetic environment.

"Transformation" in the context of the present invention describes a 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 of insertion sequences alien nucleic acid in a prokaryotic or eukaryotic cell host. The method is chosen based on the type of the transformed host cell, and may include, but not limited to, viral infection, electroporation, heat shock, lipofection and the bombarding particles. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication l the Bo in the form of Autonomous plasmids can replicate, either as part of the chromosome of the host. They also include cells that transtorno Express the inserted DNA or RNA for limited periods of time.

"Variant" of a peptide or polypeptide in the context of the present invention refers to an amino acid sequence that is altered by one or more amino acids. Variant of polynucleotide modified by one or more nucleotides. Variant may lead to a "conservative" changes, in which a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may lead to a "nonconservative" changes, e.g., replacement of glycine with tryptophan. Similar minor modifications can also include amino acid deletions or insertions, or both. A guide to determining which amino acid residues can be substituted, inserted, or deleted without loss of biological or immunogenic activity, can be found using computer programs well known in this field, for example, the software LASERGENE (DNASTAR).

The present invention also encompasses variants that are at least keep one biological activity (e.g., Association with cell or penetration into the cell) or the functional activity of the peptide or polypeptide. The preferred option is option, the sequence of which at least 80%, and more preferably at least 90%identical to the described sequence. The most preferred option is option, the sequence of which at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%or at least 99.9% identical to the sequence described in the present invention. The percentage identity is determined by aligning the two compared sequences, as described below, determine the number of identical residues in the aligned part, divide this number by the total number of residues in the sequence according to the invention (required), and multiplying this result by 100. Suitable equalization program is AlignX (Vector NTI).

Description of the invention

Methane is formed in the front part of the digestive tract of ruminants by methaneproducing, which act as the final reducing carbon in the system of the scar. Multi-stage process of formation of methane is well lit, mainly from the study of methaneproducing not ruminants, but adaptations that allow methaneproducing to grow and persist in the rumen, it is not clear.Methanobrevibacter ruminantiumis WPI is a unique methaneproducing from new Zealand ruminants. As described in this application, the size of the pre-genomic sequencesM. ruminantiumis approximately 3.0 Mb, and the GC content is 33,68%. As the important information, it was found that the genome ofM. ruminantiumcontains sequences of the signal peptides for use in targeted delivery and penetration into cells. The present invention therefore relates to signal peptides, including those that contain SEQ ID NO:1-172, as well as polypeptides containing these peptides, and their alterations, fragments, variants and derivatives.

The present invention relates to the use of these peptides or polypeptides for directed delivery and penetration into microbial cells, particularly cells of methaneproducing. The present invention additionally relates to the use of peptides or polypeptides to inhibit the growth or replication of such cells. The peptides and polypeptides of the present invention can be expressed and used in various assays to determine their biological activity. These peptides and polypeptides can be used for large-scale synthesis and allocation protocols, for example, for commercial receipt. Such peptides and polypeptides can be used for the induction of antibodies, to highlight the corresponding sequence is of elesta antibodies and for the quantitative determination of the levels of amino acid sequences.

The polypeptides of the present invention can also be used as compositions, such as pharmaceutical compositions, and as additives to food, for example, components of the feed for ruminant animals. The peptides and polypeptides of the present invention also benefit health. For example, in the aspects related to health, inhibitors methaneproducing can be used to return energy to the individual, which is usually lost in the form of methane. In specific aspects, the device slow release in the rumen can be used in conjunction with the peptides, polypeptides and compositions (such as pharmaceutical compositions and additives for animal feed) of the present invention.

The peptides and polypeptides of the present invention contain at least one sequence selected from the group consisting of: (a) peptides or polypeptides that contain at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants, or derivatives; (b) a peptide or polypeptide containing the functional domain (e.g., conservative crustal area described in this application) at least one amino acid sequence is lnasty, selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants, or derivatives; and (c) peptides or polypeptides that contain at least specifically specified number of contiguous residues of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants or derivatives thereof. All these sequences together in the present description are referred to as peptides and polypeptides of the present invention. In one embodiment, the invention relates to the selected peptide or polypeptide containing the amino acid sequence of at least one of SEQ ID NO:1-172.

The present invention also relates to polynucleotides that encode at least one signal peptide including SEQ ID NO:1-172, as well as polypeptides containing such peptides and their changes, fragments, variants or derivatives.

The present invention encompasses the use of these polynucleotides to obtain expression vectors and host cells for directed delivery and penetration into microbial cells, particularly cells of methaneproducing. The present invention additionally encompasses the use of these polynucleotides for inhibiting the growth or replication of such cells. Selected polynucleotide for this is the invention are also applicable in genome mapping, in physical mapping and cloning of genes more or less related bacteria. Probes obtained using polynucleotides of the present invention, can be used to detect the presence and study the expression profiles of genes in any organism having sufficiently homologous DNA and RNA sequences in their cells, using methods that are well known in this area, such as a slot-blot or analysis on the microarray. The primers obtained from polynucleotides of the present invention, can be used for sequencing and PCR amplification.

Polynucleotide of the present invention can also be used as compositions, such as pharmaceutical compositions, and as feed additives, for example, components of the feed for ruminant animals. Polynucleotide the present invention also benefit health. For this application polynucleotide can be represented as expression vectors or host cells containing the expression vectors. In specific aspects, can be used device for slow release in the rumen together with polynucleotides, vectors, cells masters and compositions (such as pharmaceutical compositions and feed additives) according to the present invention.

olignucleotides of the present invention contain at least one sequence, selected from the group consisting of: (a) a sequence containing the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants, or derivatives; (b) complementary sequences, reverse sequences and complementary back coding sequences of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants, or derivatives; (c) open reading frames contained in the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants, or derivatives; (d) functional domains (e.g., crustal conserved regions described in this application) the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, or alterations, fragments, variants, or derivatives; and (e) sequences containing at least specifically specified number of contiguous residues of the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172, is whether it changes, fragments, variants or derivatives thereof. Also presents oligonucleotide probes and primers. All of these polynucleotide and oligonucleotide probes and primers together in the present description are called polynucleotide of the present invention. In one embodiment, the present invention encompasses a dedicated polynucleotide containing the coding sequence of at least one amino acid sequence selected from the group consisting of SEQ ID NO:1-172.

Specialists in this field will be clear that as a result of the degeneracy of the genetic code, can be obtained many nucleotide sequences that encode the peptides of the present invention, to some extent, bearing minimal homology in relation to the nucleotide sequences of any known and natural gene. Therefore, in the present invention are considered without exception, all possible variants of the nucleotide sequence, which could be obtained by selecting combinations based on possible elections codons. These combinations receive in accordance with the standard triplet genetic code as applied to the natural amino acid sequence, and all such variations are considered to be particularly described.

Nucleotide sequences that encode with Gnarnia peptides or polypeptides, or modified sequence, preferably capable of hybridisierung with nucleotide sequence of a natural sequence in the respectively selected conditions of stringency. However, it may be preferable to obtain the nucleotide sequence encoding the peptide or its derivatives possessing essentially different frequency of use of the codon. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are used by this host. For example, codons can be optimized for expression inE. colifor example, in accordance with SEQ ID NO:342-533. Other reasons for changes largely nucleotide sequence that encodes the peptide and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a longer half-life, than transcripts produced from natural sequence.

The present invention also comprises obtaining DNA sequences, or their fragments which encode peptides or polypeptides, or modified sequences, iscrucial is but by synthetic chemistry. After receiving the synthetic sequence can be embedded in any of the many available expression vectors and cell systems using reagents that are well known in the field. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding a peptide or polypeptide, or any of their modifications, variants, derivatives or fragments. The invention also encompasses polynucleotide sequences that are capable of hybridisierung with the claimed nucleotide sequences, and, in particular, is shown in SEQ ID NO:173-341 or 342-533, or their complements, under various conditions of stringency, as described in Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol. 152:507-511).

Methods of DNA sequencing are well known and generally available in this area, and can be used to implement any of the embodiments of the present invention. The methods can be used such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (U.S. Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase Amersham Pharmacia Biotech (Piscataway, NJ), or combinations of polymerases and corrective economies, such as prisoners in the ELONGASE Amplification System marketed by Life Technologies (Gaithersburg, MD). Preferably, this is about the ESA automated, devices 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 20TM(Roche Diagnostics).

Nucleic acid sequence encoding these peptides, can be extended using partial nucleotide sequence and employing various methods known in the field to detect upstream sequences such as promoters and regulatory elements. For example, in one method that can be applied PCR site-restriction", uses universal primers to search for 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 a known area. Amplificatoare sequence was then subjected to a second round of PCR with the same linker primer and other specific primer located inside the first. The products of each round of PCR transcribed with the appropriate RNA polymerase, followed by use of reverse transcriptase.

System capillary electrophoresis, which are commercially available, can be used to analyze the size or confirm the nucleotide sequence of secv the treatment or PCR products. In particular, capillary sequencing can be used flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide)which are activated by the laser, and the definition of the emitted wavelengths by using the camera on charge-coupled devices. Output/light intensity of radiation 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 electronic data display may be controlled by a computer. Capillary electrophoresis is particularly preferred for sequencing small pieces of DNA that may be present in limited amounts in a particular sample.

In another embodiment of the present invention, polynucleotide or fragments thereof that encode peptides or polypeptides may be used in recombinant DNA molecules to direct the expression of the peptides, polypeptides or modified sequences in the appropriate cell hosts. Due to inherent genetic code degeneracy, can be obtained by other DNA sequences which encode essentially the same or functionally equival NTUU amino acid sequence, and these sequences can be used for cloning and expression of signal peptides or polypeptides. The nucleotide sequence of the present invention can be engineered using methods generally known in this field to change the sequences encoding amino acids for a variety of reasons, including, but not limited to, changes which modify the cloning, processing and/or expression of the gene product. DNA shuffling by random fragmentation and PCR rebuild of gene fragments and synthetic oligonucleotides may be used to design the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, changes in the glycosylation profiles, change the preference of codons, the introduction of mutations, and so on.

In another embodiment of the present invention, natural, modified, or recombinant nucleic acid sequence encoding the peptides or polypeptides can be legirovanyh with a heterologous sequence to encode a fused protein. For example, it may be useful to encode a chimeric sequence that can be recognized by a commercially available antibody. Protein also which may be constructed, thus, in order 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 split and clean the heterologous part.

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, M. 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 peptide or polypeptide may be produced using chemical methods to synthesize the amino acid sequence or its fragment. For example, peptide synthesis 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) Proeins Structures and Molecular Principles, WH Freeman and Co., New York, NY). The composition of the synthetic peptides or polypeptides can be confirmed by using amino acid analysis or sequencing (e.g., using a splitting technique for Adminu; Creighton, supra). Additionally, the amino acid sequence of the peptide or polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any parts thereof, to obtain a modified molecule.

For the expression of biologically active peptides, nucleotide sequences encoding the peptide or functional equivalents, may be incorporated into the appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the built-in coding sequence. Methods which are well known to specialists in this field can be used to construct expression vectors containing sequences encoding this peptide and appropriate transcriptional and translational control elements. These methods includein vitrorecombinant DNA technology, synthetic methods, and genetic recombinationin vivo. Such technologies are described in 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.

A number of expression vectors and systems of the hosts can be used for content and expression of sequences encoding the peptides according to the invention. They include, but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or expression vectors kosmidou DNA; yeast transformed with yeast expression vectors; cellular system of insects 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., Ti or pBR322 plasmids); or animal cells. For bacteria, suitable plasmids include pET, pRSET, pTrcHis2, and pBAD plasmid from Invitrogen, pET and pCDF plasmids from Novagen, and DirectorTMplasmids from Sigma-Aldrich. For methaneproducing suitable plasmids include, but not limited to, pME2001, pMV15, and pMP1. In particular,Escherichia colican be used with the expression vector pET. The present invention is not limited to used expression vector or the host-cell.

The "control elements" or "regulatory sequences" are untranslated region of vectors such as enhancers, promoters and 5' and 3' netr slirvey areas which interact with host cell proteins for transcription and translation. Such elements can vary in intensity and specificity. Depending on the vector system and host, can be used any number of suitable transcription and translation elements, including constitutive and inducible promoters. For example, when cloning in bacterial systems, can be used inducible promoters such as the hybrid lacZ promoter BLUESCRIPT phagemid (Stratagene, LaJolla, CA) or pSPORT1 plasmid (Life Technologies) and the like. Polyhedrally the promoter of the baculovirus can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO genes spare protein) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into this vector.

In bacterial systems, a number of expression vectors may be selected depending on the application of this peptide. For example, in those cases when you need a large amount of a peptide can be used for vectors which direct high level expression of the fused proteins, which are easy to clean. Such vectors include, but not limited to, multifunctionalE. colicloning and Express the respective vectors, such as BLUESCRIPT (Stratagene), in which the sequence encoding the peptide may be Legerova into the vector in frame with sequences for aminoanisole Met and the subsequent 7 residues of β-galactosidase, so as to form a hybrid protein; 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 for expression of foreign peptides in the form of a fused protein with glutathione S-transferase (GST). Basically, such fused proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins obtained in such systems can be created containing the sites of cleavage of heparin, thrombin or factor Xa protease, so that the cloned polypeptide of interest can be released from the GST portion on request. In yeast,Saccharomyces cerevisiaecan be used a number of vectors containing constitutive or inducible promoters such as alpha factor, alcoholecstasy, and PGH. For an overview, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding the peptides according to the invention. Such signals include the start of the first ATG codon and adjacent sequences. In cases where sequences encoding a peptide, a start codon, and upstream sequences are integrated into the appropriate expression vector, additional transcriptional or translational control signals may not be needed. However, in cases where built only the coding sequence, or a fragment should be given exogenous translational control signals, including the start codon ATG. Moreover, the start codon must be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and start codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are using a particular cellular system, such as that described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, the strain host cell can be selected by its ability to modulate the expression of the built-in sequences or processional downregulation of the peptide or polypeptide in the desired way. Such modifications sequences include, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, limitirovanie and acylation. Post-translational PR is cessing, which digests "shall" form of the peptide or polypeptide can also be used to facilitate a correct embedding, formation of the folded structure and/or function. Different cell owners who have specific cellular mechanism and characteristic mechanisms for post-translational activities, available from the American type culture collection (ATCC; Bethesda, MD) and can be chosen to ensure the correct modification and processing of the sequence. Special cell hosts include, but are not limited to, cells methaneproducing, such as cellsMethanobrevibacterin particular, the cells ofM. ruminantiumorM. smithii. Cells are the owners of interest include, for example,Rhodotorula,Aureobasidium,Saccharomyces,Sporobolomyces,Pseudomonas,ErwiniaandFlavobacterium; or other organisms, such asEscherichia,Lactobacillus,Bacillus,Streptomycesand like that. Special cell hosts includeEscherichia colithat is particularly suited for use in the present invention,Saccharomyces cerevisiae,Bacillus thuringiensis,Bacillus subtilis,Streptomyces lividansand similar.

There are some methods of introducing nucleic acids into eukaryotic cells, culturedin vitro. These methods include chemical methods (Felgner et al., Proc. Natl. Acad. Sci., USA, 84:7413 7417 (1987); Bothwell et al., Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jones and Bartlett Publishers, Inc.,Boston, Mass. (1990), Ausubel et 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, supra) or electrical impulses (Vatteroni et al., Mutn. Res., 291:163 169 (1993); Sabelnikov, Prog. Biophys. Mol. Biol., 62: 119 152 (1994); Bothwell et al., supra; and Ausubel et al., supra), the use of attenuated viruses (Davis et al., J. Virol. 1996, 70(6), 3781 3787; Brinster et al. J. Gen. Virol. 2002, 83(Pt 2), 369 381; Moss, Dev. Biol. Stan., 82:55 63 (1994); and Bothwell et al., supra)and physical methods (Fynan et al., supra; Johnston et al., Meth. Cell Biol., 43(Pt A):353 365 (1994); Bothwell et al., supra; and Ausubel et al., supra).

Successful delivery of nucleic acids in animal tissues can be achieved using cationic liposomes (Watanabe et al., Mol. Reprod. Dev., 38:268 274 (1994)), direct injection of naked DNA or RNA in muscle tissue of an animal (Robinson et al., Vacc., 11:957 960 (1993); Hoffman et al., Vacc. 12:1529 1533; (1994); Xiang et al., Virol., 199:132 140 (1994); Webster et al., Vacc., 12:1495 1498 (1994); Davis et al., Vacc., 12:1503 1509 (1994); Davis et al., Hum. Molec. Gen., 2:1847 1851 (1993); Dalemans et al. Ann NY Acad. Sci. 1995, 772, 255 to 256. Conry, et al. Cancer Res. 1995, 55(7), 1397-1400), and embryos (Naito et al., Mol. Reprod. Dev., 39:153 161 (1994); and Burdon et al., Mol. Reprod. Dev., 33:436 442 (1992)), intramuscular injection of self-replicating RNA vaccines (Davis et al., J Virol 1996, 70(6), 3781 3787; Balasuriya et al. Vaccine 2002, 20(11 12), 1609 1617) or intradermal injection of DNA technology, using a "gene gun" (Johnston et al., supra).

A number of protocols for detecting and measuring the expression of peptides or polypeptides according to the invention, using either polyclonal or monoclone is lnyh antibodies specific to this protein, known from the prior art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated sorting of cells (FACS). An immunological assay based on monoclonal antibodies from two sites can be used with monoclonal antibodies reactive against two reintervenciisa epitopes peptide or polypeptide, but can also be used in the analysis of competitive binding. These and other assays are described, among other things, 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).

A number of labels and methods of conjugation are known to specialists in this field and can be used in various analyses of nucleic acids and amino acids. Methods of obtaining labeled hybridization or PCR probes for detecting sequences related to polynucleotides include Oligocene, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequence encoding the peptide of any of the polypeptides containing this peptide, or any modified sequence may be cloned into a vector to obtain an mRNA probe. Such vectors are known in the field, are commercially available may be used to synthesize RNA probes in vitroby adding the appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. These techniques can be performed using a number of commercially available kits Amersham Pharmacia Biotech, Promega, and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, such as substrates, cofactors, inhibitors, magnetic particles and the like.

The expression vectors or cells of hosts transformed with the expression vectors, can be cultured under conditions suitable for the expression and recovery of peptide or polypeptide from the culture. This culture may contain components forin vitroorin vivoexpression. Componentsin vitroexpression include components for lysate of rabbit reticulocytes, lysatesE. coliand extracts of wheat germ, for example, systems Expressway™ or RiPs from Invitrogen, system GenelatorTMfrom iNtRON Biotechnology, EcoPro™ or system STP3™ from Novagen, systems TNT® Quick Coupled from Promega, and the EasyXpress system from QIAGEN. Peptides or polypeptides derived from culture may be secreted or contained intracellularly depending on the sequence and/or vector. As will be clear to experts in the field, expression vectors, the coding for the Ute these peptides or polypeptides preferably manufactured so that to contain a signal sequence to direct secretion of this peptide through a prokaryotic or eukaryotic cell membrane.

Other designs can include amino acid domain which will facilitate purification of the peptide or polypeptide. Such domains include, but not limited to, metal chelating peptides such as modules histidine-tryptophan (for example, 6X-HIS (SEQ ID NO: 514))that allow purification on immobilized metals, protein domains And that allow purification on immobilized immunoglobulin, and the domain used in the system extension/affinity purification of FLAG® (Immunex Corp., Seattle, WA). Suitable epitope tags include 3XFLAG®, HA, VSV-G, V5, HSV, GST, GFP, MBP, GAL4, and β-galactosidase. Suitable plasmids include plasmids containing bitenova label (for example, PinPoint™ plasmid from Promega), calmodulin binding protein (e.g., pCAL plasmids from Stratagene), streptavidin binding protein (e.g., InterPlay™ plasmid from Stratagene), tag, c-myc or FLAG® (e.g., plasmids thus from Sigma-Aldrich), or his-tag tag (for example, QIAExpress plasmids from QIAGEN).

For easy cleaning can be used biodegradable linker sequence, for example, specific factor Xa or enterokinase (Invitrogen, San Diego, CA). For example, this vector can include one or the more of the linkers between the purification domain and the peptide or polypeptide. In one aspect, the expression vector can provide the expression of the fused protein containing the peptide or polypeptide of the present invention, and the nucleic acid encoding 6 histidine residues (SEQ ID NO: 514), standing in front of thioredoxin or site of cleavage by enterokinase. Residues of histidine facilitate the IMAC purification (affinity chromatography with immobilized metal ion described in Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), while the site of cleavage by enterokinase provides a means of purification of the peptide or polypeptide of the fused protein. Review of vectors, which contain fused proteins, presented in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

Antibodies of the present invention can be obtained using methods, mainly known from the prior art, for example, for use in cleaning or diagnostic methods. In particular, purified peptides, polypeptides or polynucleotide can be used to generate antibodies in accordance with well-known protocols. Such antibodies may include, but not limited to, polyclonal, monoclonal, chimeric, and single-chain antibodies, Fab fragments and fragments obtained using a Fab expression library. Neutralizing antibodies (i.e., antibodies that inhibit the function), are especially preferred for use in this the future of invention.

To generate antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection of the peptide, polypeptide, polynucleotide or any fragment, which has immunogenic properties. Depending on the type of host that can be used in a variety of adjuvants to enhance the immune system. Such adjuvants include, but not limited to, beta-blockers, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomy polyols, polyanion, peptides, oil emulsions, hemocyanin lymph snails and dinitrophenol. Among adjuvants used in humans, particularly preferred are BCG (Bacillus of Calmet-guérin (BCG) andCorynebacterium parvum.

Preferably, the peptides, polypeptides or fragments used to induce antibodies, had the amino acid sequence containing at least five amino acids and more preferably at least 10 amino acids. It is also preferred that they were identical part of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small natural molecules. Short stretches of amino acids may be fused with those of another protein, for example, hemocyanine lymph snail and ant the body, obtained against the chimeric molecule.

Monoclonal antibodies can be obtained. using any technique which provides for the receipt of antibody molecules using stable cell lines in culture. These techniques include, but not limited to, hybridoma technology, hybridoma technology In human cells and the EBV-hybridoma technology (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) 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). Antibodies can also be obtained by inductionin vivoproducts of lymphocyte population or by screening libraries of immunoglobulins 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).

Additionally, there may be used methods for the purification of "chimeric antibodies", for example, the Association of antibody genes 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). Alternatively, techniques described for obtaining single-chain antibodies may be adapted, using methods known in this field, to obtain specific single-chain antibodies. Antibodies with related specificity, but the other idiotypes composition, can be obtained by celevogo of shuffling from random combinatorial libraries of immunoglobulins (Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Specialists in this field to which the present invention will be understood the terms "diately" and "Triatel". They are molecules that contain 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 be able pairing between the two domains on the same chain. This helps to pair with complementary domains of one or more other circuits, and stimulates the formation of dimeric or trimeric molecules with two or more functional antihistamine sites. The resulting molecule antibodies can be monospecificity or multispecificity (for example, bispecific if diatel). Such molecules antibodies can be obtained from two or more antibodies, using a methodology that is standard in the field to which the present invention; for example, as described in Todorovska et al. (Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods. 2001 Feb 1;248(1-2):47-66).

Can also be derived antibody fragments which contain specific binding sites. For example, such fragments include but not limited to, F(ab')2fragments which can be obtained by pepsin cleavage of the molecule antibodies, and Fab fragments, which can be obtained by restoring the disulfide bridges of fragments F(ab')2. Alternatively, expression libraries of Fab can be constructed quickly and easily carry out the identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays can be used for screening to identify antibodies having binding specificity. Numerous protocols for competitive binding or quantitative radioimmunoassays using either polyclonal or monoclonal antibodies with established specificnosti, well known in this field. Such immunoassays typically involve the measurement of complex formation between the peptide, polypeptide or polynucleotide and specific to him antibody. An immunological assay based on monoclonal antibodies from two sites using monoclonal antibodies reactive against two reintervenciisa epitopes is preferred, but can also be used in the analysis of competitive binding (Maddox, supra).

Signal peptides described in this invention have the pic is the institutional capacity to enter cells and, therefore, suitable as molecules carriers for delivery of inhibitory molecules in microbial cells. The chemical reaction for amino acids and compounds that are well designed and the number of different types of molecules can be connected to the signal peptide. The most common connection methods are based on the available amino (alpha-amino or Lys), sulfhydryl (Cys), or carboxyl groups (Asp, Glu, or alpha carboxyl). Connection methods can be used for connecting peptide with cell inhibitor through the carboxy - or aminobenzene balance. In some cases, the sequence includes numerous residues that can interact with a selected chemistry. This can be used to obtain multimers containing more than one cell inhibitor. Alternatively, the peptide or polypeptide can be shortened or selected so that the reactive residues were located at either the amino or carboxyl end of the sequence.

For example, a reporter molecule, such as fluorescein, may be specifically included in the lysine residue (Ono et al., 1997) usingN-α-Fmoc--1-(4,4-dimethyl-2,6 dioxocyclohex-1-ilidene-3-methylbutyl)-L-lysine during peptide synthesis. After synthesis, esters of 5 - and 6-carboxyfluorescein is cinemedia can be connected, then 4,4-dimethyl-2,6 dioxocyclohex-1-ilidene removed by treatment with hydrazine. Therefore, the connection is inhibitory molecules with signal peptide or polypeptide can be done by including lysine residue in penetrating the sequence, then the interaction with a suitable derivative of cellular inhibitor.

Can also be used EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) or carbodiimide method of connection. Carbodiimide can activate the carboxyl group of the side chain of aspartic and glutamic acid, as well as carboxykinase group to make them reactive sites for coupling with primary amines. Activated peptides mixed with cellular inhibitor for the final conjugate. If the cellular inhibitor of the activated first, the method of the EDC cell inhibitor will be connected via N-terminal alpha amine and, possibly, through the amine in the side chain of Lys, if present in the sequence.

Ester m-maleimidomethyl-N-hydroxysuccinimide (MBS) is heterobifunctional reagent, which can be used for connection of peptides with cell inhibitors through cysteine. The connection is with the thiol group of cysteine residues. If the selected sequence is not who will win Cys, normal is the location of the Cys residue at the N - or C-end to obtain vysokokontrastnoe connection peptide with cell inhibitor. For the purposes of synthesis, cysteine may be more appropriate location for the N-end of the peptide. MBS is particularly suitable for use in the present invention.

The glutaraldehyde can be used as a bifunctional linking reagent, which connects the two connection via their amino groups. The glutaraldehyde provides a spacer high flexibility between the peptide and cellular inhibitor suitable for presentation. The glutaraldehyde is a very reactive compound and will interact with Cys, Tyr, and His to a limited extent. The way glutaraldehyde crosslinking is particularly suitable in cases where the peptide contains only one free amino group on its aminocore. In cases where the peptide contains more than one free amino group, can be formed large multimeric complexes.

In one aspect, the peptides or polypeptides of the present invention can be merged (for example, by cloning inside the frame) or custom made (for example, by chemical compounds) with cell inhibitors, such as antimicrobials. Among these tools included antimicrobial peptides, for example, bactericidal/reinforcing pronice is the outer coat protein, cationic antimicrobial proteins, lysozyme, lactoferrin and cathelicidin (e.g. neutrophils; see, for example, Hancock and Chapple, 1999, Antimicrob. Agents Chemother. 43:1317-1323; Ganz and Lehrer, 1997, Curr. Opin. Hematol. 4:53-58; Hancock et al., 1995, Adv. Microb. Physiol. 37:135-175). Antimicrobial peptides further include defensin (for example, epithelial cells or neutrophils) and platelets Microbiocide proteins (see, for example, Hancock and Chapple, 1999, Antimicrob. Agents Chemother. 43:1317-1323). Additional antimicrobial peptides include, but not limited to, gramicidin S, bacitracin, polymyxin B, theplain, bactenecin (for example, bactenecin cattle), ranalexin, cecropin a, indolicidin (for example, indolicidin cattle)and lowlands (e.g., bacterial lowlands).

As antimicrobial agents include ionophores, which facilitate the transport of the ion (for example, sodium), through the lipid barrier, such as the cell membrane. Two ionogennymi compounds, particularly suitable for the present invention are RUMENSINTM(Eli Lilly) and Lasalocid (Hoffman LaRoche). Other ionophores include, but not limited to, salinomycin, avoparcin, arizin and actaplanin. Other antimicrobial agents include Monensin™ and azithromycin, metronidazole, streptomycin, kanamycin and penicillin, as well as, in General, ß-lactams, aminoglycosides, macrolides, chloramphenicol, novobiocin, rifampin and fluoro enology (see, for example, Horn et al., 2003, Applied Environ. Environ. 69:74-83; Eckburg et al., 2003, Infection Immunity 71:591-596; Gijzen et al., 1991, Applied Environ. Environ. 57:1630-1634; Bonelo et al., 1984, FEMS Environ. Lett. 21:341-345; Huser et al., 1982, Arch. Environ. 132:1-9; Hilpert et al., 1981, Zentbl. Bakteriol. Mikrobiol. Hyg. 1 Abt Orig. C2:21-31).

A particularly effective inhibitors are compounds that inhibit or prevent the formation of methane, including bromoethanesulfonate acid, for example, 2-bromoethanesulfonate acid (BES) or their salts, e.g. the sodium salt. The sodium molybdate (Mo) is an inhibitor restore sulfate, and can be used with bromoethanesulfonate acid. Other compounds against metanoobrazovanie include, but not limited to, nitrate, formate, methylphenid, chloroform, chloral hydrate, sodium sulfate, 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-proprofessional (BPS), propionic acid and ethyl 2-butynoate.

Additionally included as antimicrobial agents are lytic enzymes including lysozyme, endolysin, lysozyme, lysine, phage lysin, morality, muramidase, and virality. Suitable enzymes demonstrate the ability to hydrolysis specific connection is in the cell wall of bacteria. Specific lytic enzymes include, but are not limited to, glucosaminidase that hydrolyzing glycosidic bonds between amino sugars (e.g., N-acetylneuraminic acid and N-acetylglucosamine) peptidoglycan, amidase, which decompose N-acetylmuramyl-L-alaninato between pianoboy chain and cross-linking the peptide and endopeptidase that hydrolyzing interpeptide bond (for example, cysteine of endopeptidase) and antisapated that affect pseudomurein of methaneproducing familyMethanobacteriacaea.

Additionally, the NCP included as antimicrobial agents. NCP represent a hybrid peptide-nucleic acid, in which the phosphate skeleton was replaced with achiral and neutral skeleton formed from 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 join aminoazo skeleton through methylanthranilate communication (P.E. Nielsen et al., Science 1991. 254: 1497-1500; M. Egholm et al., Nature 1993. 365: 566-568). NCP bind complementary sequences with high specificity and higher affinity compared to the same DNA or RNA (M. Egholm et al., supra). Hybrids PNK/DNA or PNK/RNA also exhibit higher thermal stability compared with the corresponding control is laxami DNA/DNA or DNA/RNA (M. Egholm et al., supra). NCP also have high chemical and biological stability due to unnatural amide skeleton, which is not recognized by nucleases or proteases (V. Demidov et al., Biochem Pharmacol 1994. 48: 1310-1313). Usually NCP represent at least 5 bases in length, and include terminal lysine. NCP can be paglierani for additional increase in life expectancy (Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63).

In one particular aspect, the peptides or polypeptides according to the invention can be fused (e.g., cloning inside the frame) or custom made (for example, by chemical compounds) with cell inhibitors, such as antibodies or fragments thereof. Antibodies or fragments of antibodies can be directed to microbial cells or specific cell methaneproducing, or one or more components of the cell. For example, cell surface proteins, such as receptors, can be a target. Includes the immunoglobulin molecule and an immunologically active portion of the molecules of immunoglobulins (Ig), i.e., molecules that contain antigennegative site which specifically binds (immunological interacts with the antigen.

Peptides or polypeptides according to the invention are of practical use in targeted delivery in the microbial cell, particularly a cell of methaneproducing. In some the deeper aspects, the peptides and polypeptides can be used to bind to the cell wall or membrane and/or permeability in the cell. As such, peptides or polypeptides can be used for transient or long-term connection to the cell, or for penetration of the cell wall or membrane and/or accumulation in the extracellular environment. It is clear that the peptides, polypeptides, and the corresponding polynucleotide, expression vectors, cell-hosts and antibodies according to the invention can be used for directions to various microorganisms, for example,Methanobrevibacter ruminantiumthat is the usual methaneproducing in ruminants, andMethanobrevibacter smithiithat is a common methaneproducing people. To implement directional, microbial cell can be brought into contact with the signal peptide or polypeptide containing this peptide selected from one or more natural sources or obtained by using expression vectors and/or host cells, or synthetic or semi-synthetic chemistry, as described in detail in this application. In specific aspects, the peptide or polypeptide is delivered to individuals in the form of compositions described in this application, for example, through the use of the device for slow release in GVA the different animals.

In some embodiments, implementation, polypeptide fused or sewn with cell inhibitor, for example, the connection against the formation of methane (for example, bromoethanesulfonate acid), an antibody or antibody fragment, a lytic enzyme, a peptide-nucleic acid, an antimicrobial peptide, or another antibiotic. This peptide-inhibitor or polypeptide-inhibitor is delivered to the individual in the form of a composition for inhibiting the growth or replication of microbial cells, in particular cells of methaneproducing. This composition contains, for example: (a) a selected signal peptide or polypeptide containing this peptide, or its modification, fragment, variant or derivative; (b) the selected polynucleotide, or modify, fragment, variant or derivative; (c) an expression vector containing this polynucleotide; or (d) cell host containing the expression vector. The composition of the invention can be Packed in a special way as part of sets for directed delivery, penetration and/or inhibiting microbial cells, particularly cells of methaneproducing, in accordance with the described methods. These kits contain at least one composition presented in the present description and instructions for use for permeability in cells or inhibiting cell growth or replication methaneproducing is in or other microorganisms.

As an additional variant implementation of the invention relates to pharmaceutical compositions in combination with pharmaceutically acceptable carrier, for use with any of the methods described above. Such pharmaceutical compositions may contain a signal peptide or polypeptide containing this peptide, in combination with cell inhibitor. Alternatively, the pharmaceutical compositions may contain the expression vector or the cell-master, described in detail in this application. The composition can be introduced separately or in combination at least one other agent, such as stabilizing compound, which may be entered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose or water. The composition can be supplied by the individual alone or in combination with other agents, drugs (e.g., antimicrobial agents), or hormones.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers contain excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Additional sublattice is the competitiveness of production technology and the introduction can be found in the latest edition Remington''s Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). The pharmaceutical compositions used in the present invention, can be introduced by any means, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, local, sublingual, or rectal means.

Pharmaceutical compositions for oral administration can be obtained using pharmaceutically acceptable carriers well known in the prior art in dosages suitable for oral administration. Such carriers enable you to obtain a pharmaceutical composition in the form of tablets, pills, coated tablets, capsules, liquids, gels, syrups, suspensions and the like for oral administration. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, followed by the addition of suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; CE is lulose, for example, methylcellulose, hypromellose or carboxymethylcellulose sodium; gum, including Arabian and tragacanth; and proteins such as gelatin and collagen. If desired, can be added disintegrant or soljubilizatory, such as poperechnyy polyvinylpyrrolidone, agar, alginic acid or its salt, such as sodium alginate.

Pharmaceutical preparations which can be used orally include capsules, easy to swallow, made of gelatin, and also soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Convenient for swallowing capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid or liquid polyethylene glycol with or without stabilizers. The dragee cores can be used in combination with suitable coatings, such as concentrated solutions of sugars, which may also contain the Arabian gum, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, the solution is blue, and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the coating of tablets or dragee to identify the product or to characterize the quantity of active substance, i.e. the doses.

Pharmaceutical compositions suitable for parenteral administration, can be obtained in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as carboxymethylcellulose sodium, sorbitol, or dextran. Additionally, suspensions of the active compounds can be obtained in the form of appropriate oily injection suspensions. Suitable lipophilic solvents or carriers 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 enhance the solubility of the compounds to be able to get the solutions of high concentration. For local or nasal administration, the composition used penetrants appropriate to the barrier, liable the mu penetration. Such penetrants are mostly known from the prior art.

The pharmaceutical compositions of the present invention can be obtained by a method known in this field, for example by conventional mixing, dissolving, granulation, making pills, otmuchivanie, emulsifying, encapsulating, enable, or lyophilization processes. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than the corresponding form of the free bases. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mm histidine, of 0.1%-2% sucrose, and 2-7% mannitol, at a pH in the range from 4.5 to 5.5, combined with buffer prior to use. After receiving pharmaceutical compositions, they may be placed in an appropriate container and labeled for treatment of the specified condition. For applying the composition according to the invention, such a label would contain the number, frequency and method of administration.

Pharmaceutical compositions suitable for use in the present invention include to whom notizie, in which the active ingredients are contained in an effective amount to achieve the goal. For any compound, therapeutically effective dose can be determined initially either in cell research, for example, microbial cells, or, in particular, in cells methaneproducing, or in animal models, usually mice, rabbits, dogs, or pigs, or from ruminant animals such as sheep, cattle, deer and goats. The animal model can also be used to determine the appropriate concentration range and route of administration. This information can then be used to determine appropriate dosages and routes of administration to the individual. The normal number of doses can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending on route of administration. Guidance on specific doses and delivery methods presented in the literature and generally available to practitioners in this field. Specialists in this field will use other structures for polynucleotides than for peptides or polypeptides. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Drugs based on peptides and polypeptides are well known, and methods for such to the positions are common in the prior art. Cited as an example of peptide and polypeptide drugs and obtaining them are described, for example, for denileukin of tipitaka, octreotide, vapreotide, lanreotide, peptides series RC-3940, decapeptyl, lupron, Zoladex, cetrorelix (see, for example, Lu et al., 2006, AAPS J 8:E466-472), genociding, statenew (see, for example, Dubin et al., 2005, Acta Biochemica Polonica, 52:633-638)and indolizidine, defensins, lantibiotics, microcilia B17, gestation and maganini (see, for example, Yeaman and Yount, 2003, Pharmacol Rev 55:27-55). General guidelines for peptide and polypeptide drugs can also be found at Degim et al., 2007, Curr Pharm Des 13:99-117 and Shai et al., 2006, Curr Prot Pept Sci, 7:479-486. Approved recently drugs based on peptides include Hematide™ (synthetic tool for peptide-based, stimulating erythropoiesis, Affymax, Inc.), exenatide (synthetic Asendin-4, Amylin/Eli Lilly), natrecor (nesiritide, naturethese peptide, Scios), plenaxis (abarelix, Praecis Pharmaceuticals)and SecreFlo (secretin, Repligen).

The exact dose will be determined by a practising professional, taking into account factors related to the individual, for which treatment is required. The dose and the introduction of set to provide sufficient levels of the active agent or to maintain the desired effect. Factors that may be taken into account include the severity of the pathological condition, about what her state of health of the individual, age, weight and sex of the individual, diet, time and frequency of administration, combination of(I) drugs, reaction sensitivity and toleratethe/response to treatment. Pharmaceutical compositions of long-term action, you can enter every 3 to 4 days, every week or once every two weeks depending on half-life and rate of excretion of the specific composition.

Especially suitable for compositions according to the invention (e.g., pharmaceutical compositions) structures or mechanisms for slow release. For example, vnutrichasovye devices include, but not limited to, Time Capsule™ Bolus from Agri-Feeds Ltd., New Zealand, originally developed in AgResearch Ltd., New Zealand, as described in WO 95/19763 and NZ 278977, and CAPTEC company Nufarm Health & Sciences, Department of Nufarm Ltd., Auckland, New Zealand, as described in AU 35908178, PCT/AU81/100082, and Laby et al., 1984,Can. J. Anim. Sci.64 (Suppl.), 337-8, each of which is incorporated into this description by reference. As a specific example, the device may include a spring and a piston that pushes the song to the hole on the end of a vessel.

As an additional variant implementation, the present invention relates to compositions for water additives, for example, irrigating composition or feed additives, for example additives to feed for ruminant animals, for use with any of the ways discussed above. In Conques is to maintain aspects, feed additive contains at least one edible vegetable substance and the peptide or polypeptide of the present invention. Alternatively, feed additive contains at least one edible vegetable substance and the polypeptide or peptide, or polynucleotide encoding the peptide or polypeptide described in this application, for example, in the form of an expression vector or host cell containing the expression vector. In particular, the composition further includes a cellular inhibitor, merged or combined with the resulting sequence. Preferred plant material includes any of hay, grass, grain or fodder cereals, for example, legumes, hay, grass hay, corn silage, silage made from cereals, legume silage, corn grain, oats, barley, grains, brewer's grain, soy flour and flour from the seeds of the cotton plant. In particular, the silage from cereals is applicable as a feed composition for ruminant animals. The plant material can be genetically modified so that it contains one or more components according to the invention, for example, one or more polypeptides or peptides, polynucleotides or vectors.

In another embodiment, antibodies which specifically bind to peptides, polypeptides or polynuclear the DAMI according to the present invention, can be used to determine the presence of microorganisms, in particular, methaneproducing, or in assays to monitor levels of these microorganisms. Antibodies suitable for diagnostic purposes, can be obtained in the same manner as described above. Diagnostic studies include methods that use an antibody and a label to detect peptide or polypeptide in the body fluids of a person or extracts of cells or tissues. Antibodies can be used with or without modification modification and can be labeled by joining them, either covalently or ecovalence, with a reporter molecule. Can be used a number of reporter molecules, which are known from the prior art, some of which are described above.

A number of protocols for measuring levels of the peptide, polypeptide or polynucleotide known in this field (e.g., ELISA, RIA, FACS, and blotting), and provide the basis for determining the presence or levels of the microorganism, in particular, methaneproducing. Normal or standard levels established by combining body fluids or cell extracts taken from normal individuals, for example, healthy people or ruminant animal, with the antibody under conditions suitable for the formation of the complex. The value of standard compleksoobrazutee the Oia can be quantified in various ways, but preferably by photometric means. The number of peptide, polypeptide or polynucleotide expressed by the individual, and in the treated samples (e.g. samples from individuals treated), compared with standard values. The deviation between the standard values and the values obtained from the individual, sets the parameters for determining the presence or levels of the organism.

In a specific embodiment of the present invention, polynucleotide can be used for diagnostic purposes using specific hybridization and/or amplification techniques. Polynucleotide, which can be used include oligonucleotides, complementary RNA and DNA molecules and the NCP. Polynucleotide can be used for detection and quantitative analysis of gene expression in samples in which expression may correlate with the presence or levels of the microorganism. Diagnostic analysis can be used to distinguish between absence, presence, and changing levels of microorganisms and for monitoring levels during therapeutic intervention.

In one aspect, hybridization with PCR probes can be used to identify sequences of nucleic acids, in particular genome is of posledovatelnostei, which encode the peptides or polypeptides of the present invention. The specificity of the probe, if it is from a highly specific region, eg, 10 unique nucleotides in the 5'regulatory region, or a less specific region, e.g., in the 3'coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate or low) will determine identifies whether the probe is only natural sequences, alleles, or related sequences. The probes can 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 of the subject invention can be a DNA or RNA and can occur from the nucleotide sequence of SEQ ID NO:173-341 or 342-533, or compliments, or modified sequences, or from genomic sequence including promoter, enhancer elements, and introns of the natural sequence.

Methods of obtaining specific hybridization probes for DNA include the cloning of nucleic acid sequences in the vectors to obtain mRNA probes. Such vectors are known in the prior art, commercially available and can be used to synthesize RNA probesin vitroby the adding the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be in the state next reporter groups, for example, radionuclides such as32P or35S, or enzymatic labels such as alkaline phosphatase, connected to the probe via system connection avidin/Biotin, and the like. Polynucleotide can be used in southern or Northern analysis, dot-blot, or other membrane techniques; PCR methods; or in the test strips, rods, ELISA assays or microarrays using fluids or tissues from biopsies of the individual to detect the presence or levels of the microorganism. Such qualitative and quantitative methods known in this field.

In a specific aspect, the nucleic acid sequence can be used in various analyses, labeled by standard methods and added to a fluid or tissue sample from the individual under conditions suitable for hybridization and/or amplification. After an appropriate incubation period, the sample is washed, and the signal is measured and compared with a standard value. If the signal value in the test sample is significantly altered in comparison with the signal value of the control sample, the presence of altered levels of nucleotide sequences in the sample indicates the presence or levels of the microorganism. Such analyses can also be used DL shall evaluate the effectiveness of a specific treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual.

To provide a framework for diagnosing the presence or levels of the microorganism, establish a normal or standard profile for expression. This can be accomplished by combining body fluids or cell extracts taken from normal individuals, with polynucleotides or its fragment, under conditions suitable for hybridization and/or amplification. Standard levels can be quantified by comparing the levels obtained from healthy individuals with levels of the experiment, which uses a known amount of essentially purified polynucleotide. Standard values obtained from normal samples may be compared with values obtained from samples from individuals treated against microbial growth. The deviation between the standard values and the values of the individual is used to measure the presence or levels of the organism.

After identification of the microorganism and the beginning of the implementation of the Protocol processing, hybridization and/or amplification tests can be repeated regularly to determine the start of reduction of the level of expression of the individual compared to the level observed in normal individuals. The results obtained from subsequent analyses, can the be used to demonstrate the efficacy of treatment over a period of time in the range from several days to months.

Specific diagnostic uses for oligonucleotides derived from the sequences of nucleic acids may include the use of PCR. Such oligomers can be synthesized chemically, obtained by enzymatic or receivedin vitro. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'.fwdarw.3') and another with antisense orientation (3'.fwdarw.5')used in the optimized conditions for identification of a specific gene or condition. The same two oligomer, a set of oligomers for nested-PCR, or even a degenerate pool of oligomers can be used in less stringent conditions for detection and/or quantitative analysis of closely related DNA or RNA sequences.

Methods that can also be used for quantitative analysis of gene include radioactively labeled or biotinylated nucleotides, co-amplification of a control nucleic acid, and standard curves onto which interpolatory the results of the experiments (Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The speed of the quantitative analysis of numerous samples can be increased by conducting this research in the ELISA format where the oligomer of interest is in a different dilutions and spec is reformationstag or colorimetric response gives rapid quantitation.

In additional embodiments, implementation, oligonucleotides or longer fragments derived from any of polynucleotides described in this application can be used as targets in a microarray. The microchip can be used for monitoring the level of expression of a vast number of genes simultaneously (for capturing an image of the transcript), and to identify genetic variants, mutations and polymorphisms. This information can be used to determine gene function, to understand the genetic basis of disease, diagnosis of disease and for the development and monitoring of the activities of therapeutic agents. In one embodiment, the microchip is received and used in accordance with methods known in this field, for example, described in PCT application WO 95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and 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 microchip, using methods chemicals and apparatus, ink-jet deposition, for example, described in PCT application WO95/251116 (Baldeschweiler et al.). In another aspect of the matrix "grid" is similar to a dot or slot the blot (HYBRIDOT apparatus, Life Technologies) can be used for organizing and connecting fragments of cDNA or oligonucleotide the s with the surface of the substrate, using a vacuum system, thermal, UV, mechanical method or means of chemical binding. In another aspect, the matrix can be obtained manually or by using available devices, materials, and devices (including multichannel microdonation or robotic tools; Brinkmann, Westbury, N.Y.) and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other multiple of the value from 2 to 1000000, which is suitable for the efficient use of commercially available application of technical means.

For analysis of the sample with the microarray, polynucleotide extracted from a biological sample. Biological samples can be obtained from any body fluids (blood, urine, saliva, sputum, gastric juice, etc), cultured cells, biopsies, or other tissue preparations. To obtain probes polynucleotide extracted from the sample, is used to produce sequences of nucleic acids that are complementary to nucleic acids on a microchip. If the microchip consists of cDNA, antisense RNA are appropriate probes. Therefore, in one aspect, the mRNA is used to produce cDNA, which, in turn and in the presence of fluorescent nucleotides, is used to obtain fragments or EN is Ismailovich RNA probes. These fluorescently labeled probes are incubated with a microchip, so that the sequence of the probe was hybridizations with cDNA oligonucleotide microchip. In another aspect, the nucleic acid sequences used as probes may contain polynucleotide, fragments and complementary or antisense sequences, obtained using restriction enzymes, PCR methods and kits Oligocene (Amersham Pharmacia Biotech), well known in the field of hybridization technology.

In another embodiment of the present invention, the peptides or polypeptides according to the invention or their functional or immunogene fragments or oligopeptides can be used for screening libraries of compounds in any of a number of methods of screening drugs. The fragment used in such screening may be free in solution, fixed to a solid substrate, transferred to the cell surface or located intracellularly. You can measure the formation of binding complexes between the peptide or polypeptide and the test substance.

One method of screening drugs which may be used provides for high-throughput screening of compounds having binding affinity of respect peptide or polypeptide, having presented the subsequent interest as described in published PCT application WO 84/03564. In this way a large number of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Test the connection lead into interaction with the peptide or polypeptide, or its fragments, and washed. Linked peptide or polypeptide is then detected by methods well known in the field. The purified peptide or polypeptide can also be applied directly to the plates for use in the above methods of screening drugs. Alternative, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid substrate.

In another way, it is possible to use a competitive analysis for the screening of drugs in which neutralizing antibodies capable of contacting a peptide or polypeptide specifically compete with a test compound for binding to this peptide or polypeptide. Thus, antibodies can be used to detect the presence of the test compound that shares with one or more antibody antigenspecific sites.

EXAMPLES

The examples described in this application are intended to illustrate embodiments of the crust is asego invention. Other embodiments of, methods and types of analyses are within the competence of specialists in the field of molecular diagnostics and need no detailed description. Other embodiments of the volume level of equipment are considered part of this invention.

EXAMPLE 1: Materials and methods

Estimation of genome size

The strain ofMethanobrevibacter ruminantiumM1T(DSM1093) were grown in the environment BY+ (minimum environment, Joblin et al., 1990), which consists 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), dH2O (360 ml), NaHCO3(5), resazurin (0.2 ml), L-cysteine-HCl (0.5 in), yeast extract (2), and a solution of trace elements Balch (10 ml) (added trace elements; Balch et al., 1979), which consists of (g/l) nitrilotriacetate acid (1,5), MgSO4.7H2O (3), MnSO4.H2O (0,5), NaCl (1), FeSO4.7H2O (0,1), CoCl2.6H2O (0,1), CaCl2(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 NISO4.6H2O (0,03), Na2SeO3(0,02), and Na2Wo4.2H2O (0,02). Genomic DNA was extracted using the method of grinding the freeze. The cells were collected by centrifugation, and cell debris in the escali in pre-cooled mortar, frozen with liquid nitrogen, and gently rubbed into a fine powder using a pre-chilled sterile mortar and pestle. Homogenates of cells were loaded in agarose plugs, and subsequent manipulations were carried out in these tubes to reduce the physical fragmentation of genomic DNA. Cleavage was performed by restriction endonucleases, and DNA fragments were separated using gel electrophoresis pulsed gradient fields (PFGE).

Cloning and DNA sequencing

DNA genomeM. ruminantiumwas sequenced Agencourt Biosciences Corporation (Massachusetts, USA) using a cloning approach stochastic genomic fragments (Fleischmann et al., 1995) and Macrogen Corporation (Rockville, MD, USA) using persecutione. Library DNAM. ruminantiumdesigned inEscherichia coliby random physical disruption of genomic DNA and separation of fragments by gel-electrophoresis. Large fragments in the range of 40 Kb were extracted from the gel and used to obtain large insertion fominoy library. The DNA fragments in the range from 2 to 4 Kb were extracted and used to obtain a small insertion plasmid library. Clones obtained from both large and small insertion libraries grew, their pomidou or plasmid DNA was extracted and sequenced, using the technology of high-performance sequencing. Matched with the existing clones sequenced to obtain, theoretically, 8 fold coverage of the genomeM. ruminantium. Persecutione conducted on randomly split fragments of genomic DNA to obtain the final theoretical 10 fold coverage.

Assembly and sequence analysis

DNA sequences were aligned to find the interleaved sequences and assembled in contiguous (contig) sequence, using the Paracel Genome Assembler (Paracel Inc., CA, USA) and Staden package (Stadenet al.,1998) in combination with a sequence from both standard and inverted PCR. Kontiki analyzed using the finder open reading frame (ORF) GLIMMER (GeneLocatorInterpolatedMarkovModelER,Salzberg et al., 1998) and each ORF was analyzed using BLAST (BasicLocalAlignmentSresearchTool (Altschul et al., 1997) regarding updated nucleotide and protein databases of the National center for biotechnology information (NCBI).

Contigo a multiple of 8 preliminary sequence connected randomly by artificial connection sequence to obtain "pseudomolecular", and was submitted in the Institute for genomic research (TIGR, DC, USA) for altaanastacia. Kontiki collected in the 10 fold persecutione, re-analyzed using GLIMMER, and ORF has autoantibody using GAMOLA (Global Annotation f Multiplexed On-site Blasted DNA sequences; Altermann and Klaenhammer, 2003). Automated annotation was subsequently checked manually. ORF classified by function, using the database of clusters ontologica of proteins (COG) (threshold 1e-02)(Tatusov et al., 2001).

Protein motifs were determined using HMMER (hypertext transfer protocol://hmmer.wustl.edu), using library PFAM HMM and TIGRFAM, and General and local alignment (hypertext transfer protocol://pfam.wustl.edu) and standard and fragmented model TIGRFAM HMM (hypertext transfer protocol://www.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 package KODON (AppliedMaths, Austin, TX, USA) and REPUTER (Kurtz and Schleiermacher, 1999). Genomic Atlas of visualizations was using GENEWIZ (Jensen et al., 1999). The path reconstructions of predictedM. ruminantiumORF conducted in conjunction with KEGG(Kyoto Encyclopedia of Genes and Genomes, Kanehisa et al., 2004) database on-line using software custom design (PathwayVoyager; Altermann and Klaenhammer, 2005).

Identification of signal peptide

To date, there is no model of the signal peptide forArchaea. There is just a little experimentally verified secretory proteins available forArchaeafor the formation of specific models. For this reason, the sequence of open reading frames (ORFS) were analyzed on cash the many signal peptides, using SignalP Version 3.0 (Bendtsenet al.,2004), prepared against gram-positive, gram-negative and eukaryotic models. SignalP-HMM (hidden Markov model) is used to distinguish between the ORF of the signal peptide and the non-signaled peptide, whereas SignalP-NN (neural network) used for the preliminary determination of the sites of attachment, as described Emanuelssonet al.,2007.

SignalP predicts the presence and localization of sites of cleavage of the signal peptide in the amino acid sequences from different organisms. This method includes a preliminary determination of cleavage sites and a preliminary definition of the signal peptide/non-signaled peptide based on a combination of several artificial neural networks and hidden Markov models. The sequence of the signal peptide identified from gram datasets, levelled and calculate a consensus sequence using the AlignX program of Vector NTI (version 9.1.0, Invitrogen Corporation). Conservative hydrophobic core identified by the analysis of amino acid hydrophobicity.

The consensus data set identified from all three models SignalP, and the corresponding sequences of the signal peptides were aligned using ClustalW (Larkinet al.,2007), and processed using BioEdit (hypertxt transfer protocol://world wide web.mbio.ncsu.edu/BioEdit/bioedit.html). Protein sequence logo (FIG. 3A) was obtained using LogoBar (Perez-Bercoffet al.,2006) to represent information in multiple sequence alignment. In this study, the ORF containing the signal peptide and three or more transmembrane domains, was considered to be membrane proteins and were excluded from further analyses. Best Y-criterion from each of the three models were adopted for the proposed cleavage site (FIG. 6). Optimized frequency of usage of the codon (FIG. 9) was calculated using a custom perl script: opt_codons.pl (Altermann, E) based on the table of codonsEscherichia coliK12.

Peptide synthesis and fluorescent labeling

The core consensus peptide was synthesized commercially, using Invitrogen's custom peptide service (Invitrogen NZ Ltd). This peptide was synthesized using Fmoc chemistry on a small scale (10-12 mg) and purified using HPLC to a purity >95%. This peptide was labelled with fluorescent on the N-terminal lysine (K).

The study of cellular permeability

The occurrence of the labeled peptide in cellsM. ruminantiumaccompanied by fluorescent analysis. CultureM. ruminantiumwere grown in 10 ml of medium BY+ and collected by centrifugation at 10000×gfor 10 min at 4°C. Cells was transferred into a 1.5 ml polypropylene Eppendorf tubes and washed in 1 ml of TE buffer (10 mm Tris-HCl, 1 mm ethylenediaminetetra ssnoi acid, pH 8) and collected by centrifugation in microcentrifuge at 13,000×gfor 10 min at 4°C. Cells (approximately 1×108) resuspendable in a total volume of 200 ál of TE buffer was added to 20 μg of fluorescently labeled peptide. This mixture is incubated for 30 minutes at 37°C, and then centrifuged at 13,000×gfor 10 min at 4°C. the Fluid over the cell debris was left, and was a fraction of the supernatant. Cell debris was washed 3 times with 200 ál of TE buffer by repeated resuspendable cells in the buffer, and centrifuged at 13,000×gfor 10 min at 4°C.

The wash solution was collected and cell wash fraction. Cellular debris remaining after the third washing, resuspendable in 200 ál of TE buffer containing 1% sodium dodecyl sulfate. Cells were centrifuged at 13,000×gfor 10 min at 4°C to precipitate the cells, and the liquid above the cells was collected and was cell-associated fraction. The remaining cell debris was frozen in liquid N2and the cells were physically destroyed by grinding frozen debris with a glass rod. The resulting cell homogenate was centrifuged at 20000×gfor 30 min at 4°C. the Liquid above the debris was collected and presented intracellular fraction, while the remaining precipitated substance p is suspended in TE buffer and represented the fraction of the cell wall/membrane. The fluorescence in each of these fractions was measured, the hermetic closure of the sample of each fraction in a glass capillary, and measure the emitted fluorescence when 510-533 relatively nm fluorescently labeled peptide standards, using a fluorescent probe (Channel 1) Lightcycler (Roche).

EXAMPLE 2: results of experiments

Estimation of genome sizeM. ruminantiumby splitting enzymes genomic DNA, and determining the size of the fragments by PFGE showed one chromosome of approximately 2,5-2,9 Mb. Initial sequencing of large and small insertion clones (6-fold) and the Assembly sequence in contiki, showed that the region of the genome 40 Kb was highly represented (>20 times), especially within a small part of the library. Because of this large distortion sequence, additional sequencing was performed (2 times theoretical coverage of the genome) only for large insertion clones with obtaining at the outlet end 8-fold coverage of the sequencing on Tengeru. 8x phase sequence collected in 756 of Contigo, which was connected through 105 frames. Further persecutione conducted to dopolnitelnogo ~10-fold coverage and the inclusion of these sequences in the Assembly, resulting in the number of Contigo, snausages is up to 27. Further closing of the gaps, using technologies inverted PCR and long PCR fragments, reduced the number of Contigo to 14.

The combined length of 14-contig sequence indicates that the genome little more (2937347 pairs of nucleotides)than the size estimated PFGE (FIG. 1A) and significantly more than its nearest relative,M. smithii(1,9 Mb). %G+C 32,64% close to the specified range of 27.5% to 31.6%, described for strains ofM. ruminantium(Balch et al, 1979). The sequence analysis suggests 2239 ORF and the total number of hits in the protein family (TIGRFam and PFam) and clusters ontologica groups (Clusters of Orthologous Groups (COG)) shown in FIG. 1B. Presents all the genes presumably involved in the formation of methane from H2+CO2and formate (FIG. 1C). However, the draft sequence ofM. ruminantiumdevoid system metalloenzyme reductase II (mcrII ormrt). Other methaneproducing, clustermcrIIencodes an isoenzyme methyl CoM reductase I, an enzyme that is activated during growth at high partial pressures of H2(Reeve et al., 1997). H2fast is used in the rumen and does not accumulate to high levels, so, apparently,M. ruminantiumadapted to use low levels of H2only through themcrI.

169 signal peptides containing the ORF was identified in Geno is e M. ruminantium. (FIG. 7). Of these 102 signal peptide was identified using all three models SignalP, and amino acid sequences of these signal peptides were aligned (FIG. 2) and received the protein sequence logo (FIG. 3A). Was identified crustal sequence of 17 hydrophobic amino acids (KKIIIILLLLILLLISI; SEQ ID NO:119). SignalP-HMM calculates the probability of the content in this sequence of the signal peptide. The probability of this signal peptide is a value from 0 to 1, with the set value cutoff of 0.5 to differentiate between the signal peptide and the non-signaled peptide for this analysis. SignalP-NN Y-criterion gives a better appreciation of the place of SP cleavage (FIG. 6). Y-criterion is defined as the geometric mean C-criterion (raw cleavage site score) and aligned slope of the S-criterion (criterion signal peptide)obtained by using the SignalP-NN. Y-criterion is a value from 0 to 1 with higher indices pointing to the correct prediction of the cleavage site.

A consensus amino acid sequence (FIG. 3C) was synthesized and conjugatively with a fluorescent label, a fluorescein via an additional N-terminal lysine residue (FIG. 3D), obtaining the finite length of the peptide of 17 amino acids. Purified FITC-peptide was tested for permeability in cellsM. ruminantium(F Is, 4). In the analysis of cellular permeabilityM. ruminantium23,5% of the peptide remained in the supernatant unattached to the cells for 30 minutes at 37°C. for Another 3.4% of the peptide could be removed from the cells by 3 washes with buffer. Approximately 62.9% of the peptide was recovered from a 1% SDS extraction cells, indicating that a large part of the peptide was associated with the cells. Of the remaining peptide of 5.8% was detected in the intracellular fraction and 4.4% was associated with the fraction of the cell wall/membrane. Therefore, 5.8% of the original peptide (equivalent to 1.16 µg) was able to contact theM. ruminantiumand pass through the cell membrane to enter the cytoplasm of a cell that is approximately 2.3×106peptide molecules on the cell.

EXAMPLE 3: Overview

Methanobrevibacter ruminantiumselected for sequencing of the genome, due to the dominance in the rumen under different conditions of supply (based on data from cultivation and molecular definitions), the availability of crops, facilities for routine cultivation in laboratory conditions, and the relatively large amount of preliminary research and primary sources available on this organism. A significant number of sequences inM. ruminantiumwas assigned function, and thus, was able to obtain a detailed picture of the lifestyle of this organism in PN is n. The dependence ofM. ruminantiumfrom simple substrates (H2+CO2, formate) and its interaction with the environment of the rumen via surface proteins and exopolysaccharides are targets for inhibition. These sequences explain the metabolism of this organism and its interaction with other microorganisms, and indicate conservative systems and components among 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 description, included as a reference.

Where the link is provided above description is given to integers having known equivalents, these equivalents are included as separately specified.

Although the present invention has been described with reference to particularly preferred options for implementation, it should be clear that the present invention as claimed should not be unduly limited to such specific implementation options.

It is clear that additional modifications can be made in relation to the present invention, as described in this application without deviating from the essence and scope of the invention.

1. The selected polypeptide or peptide, which is able to penetrate into the methane-producing cell, which is characterized by amino acid sequence SEQ ID NO:117, 118 or 119.

2. The selected polypeptide or peptide, which is able to penetrate into the methane-producing cell, which is characterized by:
a) amino acid sequence that is at least 90% identical to SEQ ID NO:117;
b) the amino acid sequence that is at least 95% identical to SEQ ID NO:118; or
c) the amino acid sequence, the cat heaven by at least 90% identical to SEQ ID NO:119.

3. The selected polypeptide or peptide, which is able to penetrate into the methane-producing cell, which is characterized by:
a) at least 15 sequential amino acids of the sequence SEQ ID NO:117 or 118; or
b) at least 15 sequential amino acids of the sequence SEQ ID NO:119.

4. Selected polynucleotide that encodes a polypeptide or peptide which is able to penetrate into metalproducing cell, characterized by:
a) a nucleotide sequence which encodes the amino acid sequence selected from the group consisting of SEQ ID NO:117, 118 or 119; or
b) a nucleotide sequence complementary to the sequence of (a).

5. Selected polynucleotide that encodes a polypeptide or peptide which is able to penetrate into metalproducing cell, characterized by:
a) a nucleotide sequence which encodes the amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:117;
b) a nucleotide sequence which encodes the amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO:118;
c) a nucleotide sequence which encodes the amino acid sequence that is at least 90% identical to p. the coherence SEQ ID NO:119; or
d) a nucleotide sequence complementary to the sequence of any of (a)-(c).

6. Selected polynucleotide that encodes a polypeptide or peptide which is able to penetrate into metalproducing cell, characterized by:
a) a nucleotide sequence which encodes 15 consecutive amino acids of the sequence SEQ ID NO:117 or 118;
b) a nucleotide sequence which encodes at least 15 consecutive amino acids of the sequence SEQ ID NO:119; or
c) a nucleotide sequence complementary to the sequence of any of (a) or (b).

7. Selected polynucleotide that encodes a polypeptide or peptide which is able to penetrate into metalproducing cell, characterized by nucleotide sequence SEQ ID NO:511, 512 or 513 or a nucleotide sequence complementary to them.

8. Cloning vector that contains polynucleotide according to any one of claims 4 to 7, or polynucleotide that encodes a polypeptide or peptide according to any one of claims 1 to 3.

9. The expression vector, which contains polynucleotide according to any one of claims 4 to 7, or polynucleotide that encodes a polypeptide or peptide according to any one of claims 1 to 3.

10. A host cell transformed by the vector of claim 8 or 9, for production of the polypeptide or peptide according to any of the at one of claims 1 to 3, or polynucleotide according to any one of claims 4-7.

11. A host cell for replication of the vector of claim 8 or claim 9, transformed with the vector of claim 8 or 9.

12. A host cell of claim 10 or 11, which is selected from prokaryotic cells or cells methaneproducing.

13. A host cell according to item 12, which is selected from Escherichia coli and Methanobrevibacter ruminantium, including Methanobrevibacter ruminantium strain M1T(DSM1093).

14. Conjugated molecule or fused molecule that can penetrate metalproducing the cell and which contains a polypeptide or peptide according to any one of claims 1 to 3, and the fluorescent tag on the N-terminal amino acid residue.

15. Method of increasing the permeability of methaneproducing cells, which includes: bringing the cell into contact with a polypeptide or peptide according to any one of claims 1 to 3.

16. Method of increasing the permeability of methaneproducing cells, comprising: bringing the cell into contact with a conjugate molecule or fused molecule by 14.

17. The method according to clause 16, in which the cell is a Methanobrevibacter ruminantium.

18. The method according to 17, in which the cell is a Methanobrevibacter ruminantium strain M1T(DSM1093).



 

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1 dwg, 1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: method comprises preparing a culture medium, its sterilisation and cooling. In the cooled culture medium the combined inoculum is added, containing separately activated by β-galactosidase bifidobacteria of strain Bifidobacterium longum B 379 M, propionic acid bacteria of strain Propionibakterium Freudenreichii Shermanii subsp. AC-2503, and the activated bacterial inoculum Lactobacillus acidophilus of viscous race taken in a ratio of 9:0.7:0.3, respectively, in an amount of 5% by volume of the culture medium. It is cultured for 14-16 hours at given process parameters, and the cells are separated from the culture liquid to obtain a bacterial mass. The resulting bacterial mass is mixed with the protective medium at a ratio of 1:1. It is bottled, sealed and frozen.

EFFECT: invention enables to intensify the process of fermentation and to improve the structural and mechanical properties of the clot.

2 cl, 7 dwg, 5 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a recombinant strain of Escherichia coli bacteria - a producer of biologically active flagellin. The described strain is obtained by transformation of an E. coli BL21[DE3] cell culture with recombinant plasmid DNA pET151FliC, which is obtained based on a pET151FliC vector in which was embedded a fliC gene which codes biologically active flagellin, having a nucleotide sequence represented in Seq ID No 3. The strain is deposited in the Russian National Collection of Industrial Microorganisms (RCIM) of the Research Institute for Genetics and Selection of Industrial Microorganisms under No B-11369.

EFFECT: present solution has higher production capacity with respect to recombinant flagellin, which is an effective adjuvant.

1 dwg, 2 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: strain Lactobacillus acidophilus No. 9-PS has biochemical activity and high acidity. The strain is deposited in the Departmental collection of beneficial microorganisms for agricultural purposes of Russian Agricultural Academy (RCAM) under the registration number of RCAM01850. The strain may find application in prevention and correction of disorders of microbiocenosis of the gastrointestinal tract.

EFFECT: invention enables to increase growth of mucous cultures of lactic acid bacteria and accelerates the process of colonisation of intestinal with beneficial microflora.

2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a plant, having high resistance to an AHAS-inhibiting herbicide, which includes at least one Shiloh-8 IMI nucleic acid, parts thereof, a plant cell and seeds. Described is a nucleic acid which encodes a polypeptide which increases herbicide resistance of a plant. Disclosed are an expression cassette and a plant transformation vector which include said nucleic acid. Described are methods of controlling weeds growing near a plant having high resistance to an AHAS-inhibiting herbicide. Disclosed is a method of producing a plant having high resistance to an AHAS-inhibiting herbicide, as well as a method of increasing AHAS activity in a plant. Described is a method of selecting a cell transformed by a vector containing IMI nucleic acid. Also disclosed is a method of increasing resistance to an AHAS-inhibiting herbicide and a weed control method which includes treatment with an AHAS-inhibiting herbicide.

EFFECT: invention enables to obtain a plant which is resistant to an AHAS-inhibiting herbicide, which provides effective control of weeds growing near said plant.

57 cl, 3 dwg, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to methods for preparing next generation drug preparations and biologically active additives in bioreactors on the basis of transgenic producing mammals. The method for creating transgenic animals producing a protein with stable and high expression in milk, involves producing transgenic mammals with using a vector containing a reporter gene coding a target protein that is a goat beta-casein gene promoter, a bovine growth factor terminator and effective two-fold transcription terminators. The terminators surround an expression cartridge and possess an ability to break genome transcripts effectively in a mammalian genome by the effective protection of transgene expression in the mammalian genome against further repression. The effective two-fold terminators represent any mammalian genome site fulfilling the following conditions: 3'-sites of the two simultaneously expressing and opposite genes containing a site of the second-to-last exon, the last intervening sequence, the last exon and a polyadenylation signal, a space of two polyadenylation signals at different DNA strands is no more than 100 base pairs.

EFFECT: method can be used for creating the transgenic animals with high and stable target protein production in milk for medical and research purposes.

4 dwg, 4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to the field of biotechnology. Synthetic 5'UTR regions are applied to enhance the transgene expression, with the 5'UTR regions being located between a promoter and a sequence, presenting an interest in an expression vector. The claimed invention also claims vectors, which contain the 5'UTR regions, and a method of enhancing the transgene expression in their application.

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.

EFFECT: invention makes it possible to controllably vary a period of activity of clostridial neurotoxins.

12 cl, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a recombinant strain of Escherichia coli bacteria - a producer of biologically active flagellin. The described strain is obtained by transformation of an E. coli BL21[DE3] cell culture with recombinant plasmid DNA pET151FliC, which is obtained based on a pET151FliC vector in which was embedded a fliC gene which codes biologically active flagellin, having a nucleotide sequence represented in Seq ID No 3. The strain is deposited in the Russian National Collection of Industrial Microorganisms (RCIM) of the Research Institute for Genetics and Selection of Industrial Microorganisms under No B-11369.

EFFECT: present solution has higher production capacity with respect to recombinant flagellin, which is an effective adjuvant.

1 dwg, 2 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: carrier is proposed for targeted delivery of nucleic acids to cells expressing the receptor CXCR4, which consists of a sequence-ligand to the receptor CXCR4 with the amino acid sequence KPVSLSYRSPSRFFESH, the linker part of two molecules of ε-aminohexanoic acid linking the sequence-ligand to the sequence for compaction of nucleic acids, the sequence providing compaction of nucleic acids and the complex output from endosomes CHRRRRRRHC.

EFFECT: invention can be used for targeted delivery of genetic structures into cells with the receptor CXCR4 on the surfaces, such as malignant tumour and stem cells, in order to correct genetic defects, influence on processes of implementation of the genetic information and prevention of diseases.

3 cl, 7 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology and genetic engineering and can be used in veterinary for creation of vaccines regulating the sexual function in animals. A gene of a hybrid protein GHbc is obtained by the PCR method and is inserted into a polylinker region of a plasmid vector pUC9.

EFFECT: claimed is recombinant DNA, coding the hybrid vaccine protein GHbc, consisting of a nucleocapsid protein of human hepatitis B virus, fused with gonadoliberin.

2 cl, 4 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to polypeptides, which are the inhibitors of methanogen cell and biological markers for detection of φmru phage, as well as polynucleotides, which code the said polypeptides. Disclosed are expression vectors and cloning vectors, which contain the said polynucleotides and host cells, containing the said vectors. Described are conjugated or fused molecules, which are the inhibitors of the methanogen cell and biological markers for detection of φmru phage, as well as antibodies, binding with the said polypeptides. Also disclosed is φmru phage, isolated with application of the described polypeptides. The invention also relates to pharmaceutical compositions and methods of inhibiting the methanogen cell with application of the described polypeptides, conjugated or fused molecules.

EFFECT: application of the invention makes it possible to lyse the methanogen cells inhibiting a paunch of ruminants, and/or deliver inhibitors of the methanogen cells.

24 cl, 8 dwg, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention is a set of synthetic oligonucleotides for species identification of roseroot (Rhodiola quadrifida (Pall.) Fisch. et Mey). The set comprises species-specific areas to create a forward, reverse primers and a destructible probe, namely forward primer - CGGCAACGGATATCTCGGCT-3', the reverse primer - 5'-GGCCTCGCAACCACCACTTGTC-3', the destructible probe - (fluorescent label)-5'-CCGTGAACCATCGAGTTTTT-3'-(quencher).

EFFECT: invention enables to identify reliably, fast and with high sensitivity the species of medicinal plant in the course of screening of plant raw material.

1 dwg, 1 ex

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