Clostridial toxin netb

FIELD: medicine.

SUBSTANCE: invention discloses a purified and/recombinant antigen polypeptide possessing toxin activity, recovered from Clostridium perfringens with specified amino acid sequence. The invention discloses the recovered or recombinant polynucleotide coding such polypeptide, an expression vector and a host cell expressing the polypeptide. The invention discloses a method for preparing the polypeptide, an antibody specifically bound with the polypeptide, immunogenic compositions and vaccines containing the given polypeptide or a polynucleotide thereby providing a specifically immune response to the polypeptide. There are disclosed a method for inducing the immune response, a method of determining the fact whether an individual has been exposed to a pathogen (versions), a method of screening, an agonist or an antagonist modulating activity of the polypeptide, a method of animal vaccination, e.g. hens for inducing active immunity, as well as passive immunity in hen off-springs which becomes less sensitive to clostridial diseases. What is disclosed is a transgenic plant containing the exogenous polynucleotide coding the polypeptide under the invention, applicable for animal feeding.

EFFECT: polypeptide is used as an ingredient of a forage or a beverage for preventing a disease caused by bacteria expressing the polypeptide under the invention.

39 cl, 8 dwg, 6 tbl, 11 ex

 

The technical field to which the invention relates.

The invention relates to a new toxin. The present invention relates to immunogenic compositions containing the toxin and the methods of vaccination of animals such as chickens, so that they become less sensitive to clostridial diseases.

Prior art

The genus Clostridium consists of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the gut of humans and other animals. Despite the identification of approximately 100 species of Clostridium, only a small amount was recognized as etiological agents of medical or veterinary importance. However, these species are associated with serious diseases, including botulism, tetanus, anaerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis and clostridial gastroenteritis.

Clostridium perfringens is the causative agent for numerous clostridial diseases found in economically valuable animals. Necrotic enteritis (NE) is one example clostridiales intestinal diseases caused by C. perfringens. Necrotic enteritis leads to the development of necrotic lesions in the intestinal wall, resulting in the results to morbidity and mortality in poultry. He is also a multi-factorial disease with a complex and partially unknown epidemiology and pathogenesis (Kaldhusdal, 1999). The bacterium C. perfringens is commonly found in the gastrointestinal tract of poultry (Tschirdewahn et al., 1991), however, the incidence of necrotic enteritis is sporadic (Cowen et al., 1987). However, food infected with C. perfringens was considered to be implicated in outbreaks of necrotic enteritis in chickens (Kaldhusdal, 1999). Studies have also shown that healthy chickens have a relatively low number of C. perfringens in their gastro-intestinal tracts, although the increase in the concentration of these bacteria can lead to necrotic enteritis (Craven et al., 1999).

It is believed that clinical necrotic enteritis occurs when C. perfringens proliferate to high numbers in the small intestine and produces extracellular toxins, which destroys the gut. It is believed that the main participating toxin is alpha-toxin, but its exact role in the disease process is not yet fully understood. Alpha-toxin is secreted zinc-metallothionen, which has the activity of phospholipase C, and the activity sphingomyelinase and is the major toxin involved in the pathogenesis of gas gangrene human (Awad, et al., 1995; Songer, 1997). All five types of toxin C. perfringens (A-E) carry and Express the structural the Yong alpha-toxin, plc.

To date no other toxin has not been identified as idiopathic virulence factor in necrotic enteritis.

The invention

The authors of the present invention have identified a new clostridial toxin. The authors of the present invention called the polypeptide NetB.

Thus, the present invention relates to essentially cleared or the recombinant polypeptide, where the polypeptide contains:

i) the amino acid sequence represented in SEQ ID NO:2 or SEQ ID NO:3,

ii) the amino acid sequence that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3, or

iii) a biologically active and/or antigenic fragment of i) or ii).

In one embodiment, the polypeptide has toxin activity.

In another embodiment, the polypeptide has reduced toxin activity in comparison with the polypeptide encoded by SEQ ID NO:2 and/or SEQ ID NO:3.

In a preferred embodiment, the polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or SEQ ID NO:3.

In one embodiment, the polypeptide can be purified from bacteria of the genus Clostridium. Preferably, the polypeptide may be purified from Clostridium perfringens.

In others the GOM embodiment of the present invention, this polypeptide is a toxoid.

In another embodiment, the polypeptide is fused protein containing at least one other polypeptide sequence.

At least one other polypeptide can be, for example, a polypeptide that enhances the stability of the polypeptide of the present invention, or a polypeptide that facilitates cleaning of this fused protein or polypeptide, which enhances immunological properties of the polypeptide of the present invention.

In another embodiment, the polypeptide of the present invention is a synthetic polypeptide.

The present invention additionally relates to isolated and/or recombinant polynucleotide containing:

i) the nucleotide sequence presented in SEQ ID NO:1,

ii) a nucleotide sequence encoding a polypeptide according to any one of items 1-8,

iii) a nucleotide sequence that is at least 40% identical to SEQ ID NO:1, and/or

iv) a sequence that hybridizes with any of i)-iii) under stringent conditions, or its reverse complement.

In one embodiment, the selected or recombinant polynucleotide contains a nucleotide sequence that is at least 40% identical to the nucleotide 226-1194 SEQ ID NO:1.

Present from reenie additionally relates to a vector, containing polynucleotide of the present invention.

Preferably, polynucleotide in the vector is functionally linked to a promoter.

In one embodiment, the vector is a viral vector or a plasmid vector.

The present invention additionally relates to the cell host containing the polypeptide of the present invention, polynucleotide of the present invention and/or a vector according to the present invention.

Cell host according to the present invention can be any cell capable of producing at least one polypeptide of the present invention, and include animal cells, plant, bacterial, fungal (including yeast) cells, cells of parasites and arthropods.

Preferably, a host cell is a bacterium.

In one embodiment, the bacterium is E. coli. In a more preferred embodiment, the bacterium is E. coli, selected from CCEC22, CCEC31 and CCEC59.

The present invention additionally relates to a method for producing a polypeptide of the present invention, involving the cultivation of a host cell of the present invention, or the vector of the present invention, encoding the specified polypeptide, under conditions that allow expression of polynucleotide encoding this polyp is Ted.

Preferably, this method further provides for the allocation of the specified polypeptide.

The present invention additionally relates to essentially purified antibody or fragment that specifically binds to the polypeptide of the present invention.

The present invention additionally relates to compositions containing the polypeptide of the present invention, polynucleotide of the present invention, the vector according to the present invention, the cell host according to the present invention and/or antibody of the present invention.

In one embodiment, the composition is an immunogenic composition.

In one embodiment, the immunogenic composition further comprises adjuvant and/or pharmaceutically acceptable carrier.

The present invention additionally relates to a vaccine containing the antigen, where the antigen contains the polypeptide of the present invention.

In one embodiment, the vaccine contains an adjuvant and/or pharmaceutically acceptable carrier.

In one embodiment, the vaccine further comprises one or more additional antigens.

The present invention additionally relates to a DNA vaccine containing polynucleotide encoding the polypeptide under this image is in the shadow, moreover, after administration to a subject, the polypeptide is expressed, and there is an immune response to the polypeptide.

The present invention additionally relates to attenuated bacteria, which produces the polypeptide containing the amino acid sequence that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3, where this bacterium produces a reduced amount of the polypeptide in comparison with the wild-type bacterium and/or has reduced toxin activity in comparison with the polypeptide in bacteria of the wild type.

In one embodiment, this attenuated bacterium does not expresses the polypeptide.

In another embodiment, this attenuated bacterium additionally modified for the expression of heterologous polypeptide. The heterologous polypeptide can be, for example, a biologically active polypeptide or antigen. Examples of biologically active polypeptides include cytokines, growth factors and enzymes. This antigen can be, for example, the agent of bacterial, fungal, parasitic or viral diseases.

Preferably, the attenuated bacterium belongs to the genus Clostridium. In the most preferred embodiment, the bacterium is Clostridium perfringens.

The present invention additionally is relates to a method for attenuative virulence of the bacteria, which expresses a polypeptide containing the amino acid sequence at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3, providing matirovanie polynucleotide sequence to decrease the expression and/or toxin activity of the given polypeptide, resulting in attenuated bacterium has reduced toxin activity in comparison with aattanayagan bacteria.

The present invention additionally relates to a method for increasing the immune response in the subject, introducing this subject polypeptide of the present invention, polynucleotide of the present invention, the vector according to the present invention, compositions of the present invention, the vaccine of the present invention, the host cell of the present invention and/or bacteria of the present invention.

In one embodiment, the cell of the host or the bacteria are alive.

In another embodiment, the data polypeptide, polynucleotide, composition, vector, cell host or the bacteria are deliveredin ovo.

The present invention additionally relates to a method for determining, did the subject of the impact of the pathogen that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID O:2 and/or SEQ ID NO:3, defining the presence or absence of a given polypeptide in the sample obtained from the subject, where the presence of this polypeptide is an indicator of exposure to this pathogen.

The present invention additionally relates to a method for determining, did the subject of the impact of the pathogen that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3, defining the presence or absence of antibodies in the sample that specifically bind to a polypeptide of the present invention, where the presence of antibodies is an indicator of exposure to this pathogen.

The present invention additionally relates to a method for determining, did the subject of the impact of the pathogen, which expresses polynucleotide containing a nucleotide sequence that is at least 40% identical to SEQ ID NO:1, providing for the determination of the presence or absence of this polynucleotide in the sample obtained from the subject, where the presence of this polynucleotide polypeptide is an indicator of exposure to this pathogen.

Can be used any suitable method of determining the presence or absence of polynucleotide. In the example, the presence or absence of polynucleotide can be detected by hybridization, for example, a blot on the Southern or amplification of this polynucleotide, for example, by using PCR.

In one embodiment, the pathogen belongs to the genus Clostridium. In a preferred embodiment, the pathogen is Clostridium perfringens.

In one embodiment, methods of the present invention the subject is a bird.

Preferably, the subject is poultry. For example, the subject is a chicken, Turkey, pheasant, quail, duck, ostrich or other poultry usually bred in commercial quantities.

In the most preferred embodiments of implementation of the subject is a chicken.

The present invention additionally relates to a method of screening for an agonist or antagonist that modulates the activity of the polypeptide of the present invention, involving the contacting of the polypeptide of the present invention with connection-kandidata and determining increases or decreases the specified connection toxin activity of the polypeptide of the present invention. In one embodiment, this compound is an antagonist. Preferably, this compound is an antibody.

The present invention additionally relates to the person sample testing for toxin activity providing:

(a) the division of the sample in which suspect the presence of the polypeptide according to any one of items 1 to 8, for at least the first and second parts of the sample,

(b) contacting the first part of the sample with an antagonist of the polypeptide according to any one of items 1 to 8, and

(c) determining whether the first and second parts of the sample toxin activity,

where the absence of toxin activity in the first part of the sample and the presence of toxin activity in the second part of the sample is indicative of the presence of the polypeptide that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3.

In one embodiment, stage (C) provides independent incubation of the first and second parts of the sample with the cells of the animal under conditions and for a period of time sufficient for the manifestation of the cytopathic effect, and the presence or absence of cytopathic effect in these cells.

In the preferred embodiment, this antagonist is an antibody.

The present invention additionally relates to food (feed) and drink containing antagonist of the polypeptide of the present invention.

Preferably, the antagonist is an antibody of the present invention.

The present invention additionally relates to the use of food and/or beverage according to the present image is meniu to reduce infection and/or colonization of animal bacteria, which expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3.

The present invention additionally relates to a transgenic organism, not a person, containing exogenous polynucleotide encoding the polypeptide of the present invention. Preferably, the data of the transgenic non-human organism is a plant.

The present invention also relates to food and/or drink containing the polypeptide of the present invention.

Preferably, the polypeptide induces an immune response against a bacterial pathogen, when transgenic organism (non-human) administered orally to the subject. Bacterial pathogen may be a pathogen of the genus Clostridium, such as Clostridium perfringens. Preferably, the subject is a bird, such as chicken, Turkey or duck.

As will be clear to the person skilled in the art, the transgenic non-human organism and/or the food or drink of the present invention can be used for introduction of the polypeptide of the present invention to a subject so that the subject is induced immune response against this polypeptide.

Thus, in one embodiment, the present invention relative to the camping to a method of inducing immune response against the polypeptide of the present invention, providing oral administration to a subject transgenic non-human organism according to the present invention and/or food and/or beverage of the present invention.

The present invention additionally relates to a method for providing passive immunity in the offspring of birds of females, providing for the introduction of the polypeptide of the present invention, polynucleotide of the present invention, the vector according to the present invention, a host cell of the present invention, compositions of the present invention, the vaccine of the present invention, attenuated bacteria of the present invention, transgenic non-human organism according to the present invention and/or food and/or beverage of the present invention bird-a female before laying eggs of this bird is a female, containing the offspring, resulting in the offspring provides passive immunity against bacteria that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3.

The present invention additionally relates to the use of the polypeptide of the present invention, polynucleotide of the present invention, the vector according to the present invention, compositions of the present invention, the vaccine really hard is retenu, the host cell of the present invention, the bacteria of the present invention, transgenic non-human animal of the present invention and/or food and/or beverage of the present invention in the manufacture of drugs to induce immune response in the subject.

The present invention additionally relates to the use of the polypeptide of the present invention, polynucleotide of the present invention, the vector according to the present invention, compositions of the present invention, the vaccine of the present invention, the host cell of the present invention, the bacteria of the present invention, transgenic non-human animal of the present invention and/or food and/or beverage of the present invention as a drug to induce immune response in the subject.

As will be apparent, the preferred features and characteristics of one aspect of the present invention is applicable to many other aspects of the present invention.

Throughout the description, the word "include" or variations such as "contains" or "containing", will be understood as the inclusion of the specified element, integer or step or group of elements, integers or steps, but not the exclusion of any other element, integer or step or group of e is of the elements, integers or steps.

Further, the present invention is described using the following not limiting the invention examples and with reference to the accompanying figures.

A brief description of the accompanying figures

Figure 1.ClustalW-alignment NetB and beta-toxin of C. perfringens; "*" means that the residues or nucleotides in that column are identical in all sequences in the alignment; ":" means that the observed conservative substitutions; "." means that there are semi-conservative substitutions.

Figure 2.Schematic diagram of the region of chromosome NE18-ΔnetB. MutantsnetBdesigned allelic changes using suicide plasmids containing insertion inactivated genenetBapproximately 2 TPN homologous DNA on each side of this gene, and introduced into EHE-NE18.

Figure 3.Analysis of cytotoxicity culture supernatant of C. perfringens EHE-NE18 in LMH cells. a. Cultural environment TPG (undiluted); b. the culture supernatant of C. perfringens EHE-NE18 (dilution 1:16); c. the culture supernatant of C. perfringens JIR325 (strain 13 - strain necroticism enteritis) (dilution 1:2); d. the culture supernatant of C. perfringens NE18-M1 (mutantplc) (dilution 1:16).

Figure 4.Analysis of cytotoxicitynetB-negative derivatives of the culture supernatant EHE-NE18 in LMH cells. a. Culture with pernament ENE-NE18 (dilution 1:16); b. culture supernatant NE18-deleteriouslynetB1 (dilution 1:2); c. culture supernatant NE18-deleteriouslynetBλ+pJIR.1457 (Shuttle plasmid) (dilution 1:2); d. culture supernatant NE18-deleteriouslynetBX+pALK20 (complementation plasmidnetB) (dilution 1:16); e. cultural environment TPG (undiluted); f. Purified on a column of recombinant NetB (dilution 1:8).

Figure 5.Analysis of lactate dehydrogenase cytotoxicity of LMH cells treated NetB. LDH released into the supernatant was measured as an indicator of cytolysis with a set of Cyto-Tox (Promega) and represented as the percentage of cytotoxicity. Each dilution was analyzed in three repetitions for each dilution was calculated SEM.

6.PCR screening of strains of NE and non-NE C. perfringens in the presence ofnetB. a. strains NE; b. strains of non-NE.

Fig.7.Survey of Western blot for the presence of protein in different strains of C. perfringens (NE and non-NE). Western blot analysis of screening NE strains and strains of non-NE on the expression of NetB. Strains of C. perfringens were grown in medium TPG, until they reached OD600 nm of 0.6, and cultural supernatant were separated by electrophoresis on LTO-PAG. Separated proteins were transferred to a PDVF membrane and probed with anticorodal rNetB from rabbits. Brackets show the strains of NE and non-NE C. perfringens.

Fig.Western blot analysis of sera from chickens vaccinated with NetB. is orozca 1: molecular weight marker (pre-painted Invitrogen SeeBlue ®Plus2 standard); Lanes 2-14: serum from vaccinated birds #1-#13.

The key to the list of sequences

SEQ ID NO:1 is the nucleotide sequence encoding the NetB toxin of Clostridium perfringens.

SEQ ID NO:2 is the Mature amino acid sequence of NetB toxin of Clostridium perfringens.

SEQ ID NO:3 is the Amino acid sequence of NetB toxin of Clostridium perfringens, containing the sequence of the signal peptide.

SEQ ID NO:4 is the Amino acid sequence of beta-toxin of Clostridium perfringens.

SEQ ID No:5-10 - Oligonucleotide primers.

Detailed description of the invention

General methods and definitions

Unless specifically stated otherwise, all technical and scientific terms used in this description, should be understood as having the meaning commonly understood by a person skilled in the art (for example, in Microbiology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise stated, the methods of production of recombinant protein, cell culture, microbiological and immunological methods used in this invention are standard procedures, well known to specialists in this field. Such methods are described and explained in the literature as J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarbourLaboratory Press (1989), T.A.Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.Glover and B.D.Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updated publications to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E.Coligan et al., (editors) Current Protocols in Immunology, John Wiley&Sons (including all updated publications to date), which are incorporated in this description by reference.

As used herein, the term "subject" refers to an animal, for example, bird or mammal. In a preferred embodiment, the subject is a bird, such as chicken. In another embodiment, the subject is man. Other preferred embodiments of include animals-Pets, such as cats and dogs, as well as related to livestock animals such as horses, cattle, sheep and goats.

As used herein, the term "poultry" refers to any kind, subspecies or race of organism of the taxonomic class Aves (birds), such as, but without limitation, chicken, Turkey, duck, goose, quail, pheasants, parrots, small birds (finches, finches), small birds of prey, crows and becciliya birds, including ostrich (African), EMU and cassowary. This term includes different from the local Gallus gallus, or chickens (for example, white Livorno, brown Livorno, striped plymouthrock, Sussex, new Hampshire, Rhode island, australorp, Cornish, Minorca, amrox, California Gray, Italian Partidge-colored), as well as race turkeys, pheasants, quail, ducks, ostriches and other poultry usually bred in commercial quantities.

As used in this description, "toxin activity" refers to the ability of the polypeptide or peptide (e.g., toxin NetB) to kill cells of animals or to cause cytopathic effect in animal cells. In some cases it may be desirable that the polypeptide had reduced toxin activity in comparison with the NetB toxin. Reducing toxin activity is preferably at least 50, 60, 70, 80, 90, 95 or 99%. Reducing toxin activity can be measured, for example, by reduction of the cytopathic effect.

As used in this description, the terms "cytopathic effect" or "CPE" describe changes in the structure of cells in the cellular activity of the toxin (i.e. pathological effect). Normal cytopathic effects include the destruction of cells, round cells, the formation of entities (i.e. merged giant cells), formation of vacuoles and the formation of Taurus inclusion. CPE comes from the effects of the toxin on cells, which have a negative impact on the ability given what's cells to perform their functions, necessary to ensure that cells remained viable. In cell cultures in vitro CPE is evident when cells as part of a confluent monolayer, find the area of lack of confluently after contact with the sample, which contains the toxin. Cytopathic effects are easily recognizable and distinguished experts in this field.

As used herein, the term "toxoid" refers to any at least partially inaktivirovannaja toxin, but is not intended to limit in any way the specific methods of inactivation of the toxin to obtain toxoid. Such inactivating technologies include: (i) chemical methods, which modify the intact toxin, for example, treatment with formaldehyde or glutaraldehyde; (ii) physical methods such as heating; (iii) enzymatic methods that modify the toxin, such as proteiny, wherein the toxin is cleaved into fragments; (iv) recombinant methods, such as genetic engineering gene toxin removal or alteration of the enzymatic sites of the toxin, but save one or more antigenic epitopes.

The term "wild type"as used in this description against bacteria, refers to the natural bacteria that produce the polypeptide containing aminoisophthalate, at least 40% identical, more preferably at least 90% identical to SEQ ID NO:2 or SEQ ID NO:3, which has toxin activity.

As used in this description, the terms "treating", "treat" or "treatment" includes the introduction of a therapeutically effective amount of the polypeptide, polynucleotide, vectors, host cells, compositions, vaccines and/or attenuated bacterium according to the present invention, sufficient to reduce or remove at least one symptom of the disease caused by infection by the bacterium expressing a polypeptide possessing toxin activity.

The term "warning" ("prevention") refers to the protection of the subject, which may be in contact with bacteria from the development of at least one symptom caused by infection and/or colonization with this bacterium, or reduce the severity of a symptom of an infection and/or colonization of a subject exposed to this bacteria.

Polypeptides/peptides

The terms "polypeptide" and "protein" are usually used interchangeably and refer to a single polypeptide chain, which may or may not be modified by adding deamination groups. It should be understood that such polypeptide chain can be associated with other polypeptides or proteins or other supposedly kulami, such as cofactors. As used in this description, the terms "proteins" and "polypeptides" include variants, mutants, biologically active fragments, modifications, analogues and/or derivatives described in this description of the polypeptides. The term "essentially purified polypeptide" or "isolated polypeptide" the authors mean a polypeptide, which was usually separated from the lipids, nucleic acids, other peptides and other contaminating molecules with which it is associated in its native state. Preferably, essentially purified polypeptide at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is associated natural.

The term "recombinant" in the context of a polypeptide refers to the polypeptide when it is produced by the cell or in the cell-free expression system, in an altered amount or at a modified speed compared to its native state. In one embodiment, the cell may be a cell that nature does not produce this polypeptide. However, this cell can be a cell that contains a non-endogenous gene, which causes an altered, preferably increased, the amount of polypeptide, which must be producerof is N. Recombinant polypeptide of the present invention includes polypeptides that have been separated from other components transgenic (recombinant) cells, or cell-free expression system, in which it is produced and polypeptides produced by such cells or cell-free systems, which are then purified from at least some other components.

The percent (%) identity to the polypeptide determined using GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a penalty (fine) create gap=5, penalty (fine) extension gap=0,3. The requested sequence has a length of at least 15 amino acids, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the requested sequence has a length of at least 50 amino acids, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the requested sequence has a length of at least 100 amino acids, and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the requested sequence has a length of at least 250 amino acids, and the GAP analysis aligns the two sequences over a region of at least 250 is minislot. More preferably, two sequences are aligned across their entire length.

With regard to the designated polypeptide, it will be understood that the figures % identity, higher than the above, will include the preferred options for implementation. Thus, when it is acceptable, in light of the minimum digits % identity, preferably the polypeptide contains an amino acid sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1 per cent, more prefer is Ino at least 99.2%, more preferably at least 99.3 percent, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%and more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant specified SEQ ID NO.

Mutants of the amino acid sequences of the polypeptides of the present invention can be obtained by introducing appropriate nucleotide changes into a nucleic acid of the present invention or the synthesis in vitro of the desired polypeptide. Such mutants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence. The combination of deletions, insertions and substitutions can be made for the final design, provided that the final peptide product has the desired characteristics.

Mutant (altered) polypeptides can be obtained using any suitable method known in this field. For example, polynucleotide of the present invention may be subjected to mutagenesis in vitro. Such methods of mutagenesis in vitro include subclavian of polynucleotide into a suitable vector, transforming this vector into a strain of “mutator”, such as E. coli XL-I red (Stratagene) and reproduction is the group of transformed bacteria within the appropriate number of generations. In another example, polynucleotide of the present invention are methods of DNA shuffling, widely described Harayama (1998). Such methods of DNA shuffling may include encoding the toxin genes related genes of the present invention, such as genes from bacteria other than Closrtidium perfringens. Products derived from mutated/altered DNA can readily be subjected to screening with the use described in this description of how to determine whether or not they have toxin activity.

In the design of mutants with amino acid sequences, the location of the site of mutation and the nature of the mutation will depend on the properties (properties), which shall matirovanie. The sites of mutation can be modified individually or in series, for example, (1) replacement of the first preferred conservative amino acids and then a more radical choice depending on the obtained results, (2) deleterevision balance target, or (3) insertional other residues, found adjacent to the site.

Deletions of amino acid sequences are typically in the range of about 1-15 residues, more preferably about 1-10, and usually about 1-5 adjacent residues.

The replacement mutants have at least one remote amino acid residue in the molecule, polypeptid the a and the remainder, insertional in his place. Of greatest interest sites for replacement mutagenesis include sites identified as the active site (active site). Other interest sites are those in which particular residues obtained from different strains or species are identical. These provisions can be important for biological activity. Sites, particularly sites within the sequence of at least three other identical conservative sites, substitute, preferably a relatively conservative manner. Such conservative substitutions are shown in table 1 under the heading “examples of substitutions”.

Table 1
Examples of substitutions
Initial balanceExamples of the substituents
Ala (A)val; leu; ile; gly
Arg(R)lys
Asn(N)gln; his
Asp (D)glu
Cys (C)ser
Gin (Q)asn; his
Glu (E)asp
Gly (G)pro ala
His (H)asn; gln
Ile (I)leu; val; ala
Leu (L)ile; val; met; ala; phe
Lys (K)arg
Met (M)leu; phe
Phe (F)leu; val; ala
Pro (P)gly
Ser (S)thr
Thr (T)ser
Trp (W)tyr
Tyr (Y)trp; phe
Val (V)ile; leu; met; phe, ala

In the scope of the present invention also includes biologically active fragments of the polypeptides of the present invention. As used herein, "biologically active fragment" is part of the polypeptide of the present invention, which retains a specific activity of full-length polypeptide. Biologically active fragments can in order to be of any size, while they retain some activity. Preferably, the biologically active fragment retains at least 10% of the activity of the full-size protein. Specialists in this area known methods identification of biologically active fragment of the polypeptide. For example, a fragment of the polypeptide of the present invention can be tested in the appropriate analysis to determine whether this fragment toxin activity, for example, by determining whether the fragment to induce a cytopathic effect in the cell. In one embodiment, a biologically active fragment has a length of at least 100 amino acids, more preferably has a length of at least 110 amino acids, more preferably has a length of at least 120 amino acids, more preferably has a length of at least 130 amino acids, more preferably has a length of at least 150 amino acids, more preferably has a length of at least 175 amino acids, more preferably has a length of at least 175 amino acids, or more preferably has a length of at least 200 or more amino acids.

The terms "antigen" and "antigen" are well known in this field and are part of the macromolecule, which is specifically recognized by a component of the immune system, for example, the antibody is whether the antigen receptor of T cells. The term "antigen" refers to a peptide, polypeptide or other macromolecule, in respect of which the owner may be induced immune response. Thus, the present invention includes an antigenic fragment of the polypeptide of the present invention. Preferably, the antigenic fragment is capable of increasing an immune response against a bacterial pathogen Clostridium, including, but without limitation, Clostridium perfringens. In one embodiment, the antigen is an epitope of the polypeptide of the present invention. In one embodiment, the antigenic fragment has a length of 6 amino acids, more preferably has a length of 7 amino acids, more preferably has a length of 8 amino acids, more preferably has a length of 9 amino acids, more preferably has a length of at least 10 amino acids. Alternatively, this antigenic fragment has a length of at least 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids. In one embodiment, the antigen when administered to a subject is able to induce an immune response against the polypeptide containing the amino acid sequence represented in SEQ ID NO:2 and/or SEQ ID NO:3.

In addition, if desired, the polypeptides of the present invention can be introduced non-natural amino acids or chemical analogs of amino acids. Such is minamikata include, but not limited to, the D-isomers of the common amino acids 2,4-diaminopentane acid, α-aminobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-aminohexanoic acid, 2-aminoethanol acid, 3-aminopropionic acid, ornithine, norleucine, Norvaline, hydroxyproline, sarcosine, citrulline, homocitrate, cysteine acid, tert-butylglycol, tert-butylene, phenylglycine, cyclohexylamine, β-alanine, germinability designed amino acids such as β-methylaminomethyl, S-α-methylaminomethyl, N-α-methylaminomethyl, and similar amino acids in General.

In the scope of the present invention also includes polypeptides of the present invention, which are differentially modified during synthesis or after synthesis, e.g., bioterrorism, benzylalkonium, by glycosylation, acetylation, phosphorylation, amidation, derivatization known defensive/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Such modifications may serve to increase the stability and/or bioactivity of the polypeptide of the present invention.

The polypeptides of the present invention can be obtained in various ways, including the production and extraction of natural polypeptides, obtaining and the attraction of recombinant polypeptides and chemical synthesis of polypeptides. In one embodiment, the selected polypeptide of the present invention is produced by culturing cells able to Express the polypeptide, under conditions effective to produce a given polypeptide, and the extraction of the given polypeptide. The preferred cell for cultivation is a host cell of the present invention. Effective culturing conditions include, but are not limited to, effective environment, bioreactor, temperature, pH and oxygen conditions that make possible the production of the polypeptide. An effective medium refers to any medium in which a cell is cultured to obtain the polypeptide of the present invention. Such an environment typically contains the aquatic environment, having assimilated sources of carbon, nitrogen and phosphate and a suitable salt, mineral compounds, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter tablets and Petri dishes. The cultivation can be carried out at a temperature, pH and oxygen content appropriate for recombinant cells. Such culturing conditions are within the competence of the specialist in this field.

Antibodies

The term "anti-Christ. ate", as used in the description of the present invention includes polyclonal antibodies, monoclonal antibodies, bespecifically antibodies, dimeric antibodies (diabodies), trimeric antibodies (triabodies), heteroconjugate antibodies, chimeric antibodies, including intact antibodies and fragments thereof such as Fab, F(ab')2and Fv, which are capable of binding the epitope determinants, and other antibody-like molecules.

Antibody fragments retain some ability to selectively bind with their antigen or receptor and are defined in this case as follows:

(1) Fab, the fragment which contains a monovalent antigennegative fragment of the antibody molecules, can be obtained by splitting a full antibody with the enzyme papain to obtain intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of the antibody molecules, can be obtained by processing a full antibody with pepsin, followed by reduction and obtaining intact light chain and part of the heavy chain; two Fab fragment' get on one molecule of the antibody;

(3) F(ab')2fragment of an antibody, which can be obtained by processing a full antibody with the enzyme pepsin without subsequent recovery; F(ab')2is a dimer of two Fab fragments', held together by two disulfide bonds;

(4) Fv, defined Leamy as a genetically engineered fragment, containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) single-chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of light chain, the variable region of the heavy chain associated with a suitable polypeptide linker as a genetically fused single-chain molecule.

Methods of obtaining such fragments are known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), included in this description by reference).

(6) the Antibody single-domain, usually variable domain of the heavy chain, devoid of light chains.

Polyclonal antibodies

The antibody of the present invention may be a polyclonal antibody. Methods of obtaining polyclonal antibodies are known to the person skilled in the art. Polyclonal antibodies can be induced in the mammal or bird, for example, one or more injections of cells expressing the polypeptide, and, if desirable, the adjuvant. Usually, cells and/or adjuvant is injected to the mammal or bird of multiple subcutaneous or intraperitoneal injections. Examples of adjuvants that can be used include beta-blockers and adjuvant MPL-TDM (monopost the RIL-Lipid A, synthetic dikarenakan trehalose). The immunization Protocol may be selected by a person skilled in the art without undue experimentation.

Monoclonal antibodies

Antibodies obtained by the method according to the present invention, can be, alternatively, monoclonal antibodies. Monoclonal antibodies can be obtained hybridoma methods, such as methods described by Kohler and Milstein, (1975). In one hybridoma method, a mouse, hamster, or other appropriate animal host is usually subjected to immunization with cells expressing consider the polypeptide of the first type obtained from the transgenic mammal to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind with the polypeptide of the first type.

Usually use either peripheral blood lymphocytes ("PBL"), if desired cells, human-derived, or spleen cells or cells of lymph nodes, if desired sources mammals, non-human. Then lymphocytes merge with immortalizing cell line using a suitable merge agent such as polyethylene glycol, with the formation of hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). An immortalized cell line usually is about are transformed mammalian cells, in particular, myeloma cells derived from rodent, cow or human. Usually use a line of myeloma cells rat or mouse. Hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival nesmith, immortalized cells. For example, if the parent cells lack the enzyme gipoksantin-guanine-phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include gipoksantin, aminopterin and thymidine (“medium HAT”), substances that prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are lines that effectively merge, maintain a stable expression of antibodies of high level producing the selected antibody cells are sensitive to this environment, as the environment NAT. More preferred termed cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and American Type Culture Collection, Manassas, Va. Myeloma cell lines of human and heteromyinae cell line mouse-people have also been described for obtaining monoclonal human antibodies (Kozbor, 1985; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New ork, 1987, pp. 51-63).

Then the cultural environment in which the cultured hybridoma cells can be analyzed for the presence of monoclonal antibodies directed against a polypeptide of the first kind. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or analysis of binding in vitro, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such methods and assays known in the field. The binding affinity of the monoclonal antibody can be, for example, determined by analysis of Scatchard Munson and Pollard, (1980).

After identifying the desired hybridoma cells, the clones may be subcloned by methods of limiting dilutions and grown by standard methods. Suitable culture media for this purpose include, for example, modified by way of Dulbecco Wednesday Needle and medium RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

Monoclonal antibodies secreted by these subclones may be isolated or purified from the culture medium or ascitic fluid common methods of purification of immunoglobulins, such as, for example, chromatography on protein a-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis Il is affinity chromatography.

Monoclonal antibodies can also be obtained by means of recombinant DNA, such as the methods described in U.S. patent 4816567. DNA encoding the monoclonal antibodies of the present invention, can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are able to bind specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells according to the present invention serve as a preferred source of such DNA. After separation, the DNA may be placed in expressing vectors, which are then transferout in cell host, such as COS cells monkeys, cells of the Chinese hamster ovary (Cho or myeloma cells that otherwise do not produce the protein of the immunoglobulin, to obtain the synthesis of monoclonal antibodies in the recombinant cell host. DNA can also be modified, for example, by replacing the coding sequence constant domains of the heavy and light chain human instead of the homologous murine sequences (U.S. patent 4816567) or covalent joining the coding sequence of the immunoglobulin whole or part of the coding sequence of the polypeptide, non-immunoglobulin. Such a polypeptide, not awlays is the action scene immunoglobulin, can replace the constant domains of the antibodies of the present invention or can replace the variable domains of antigennegative site antibodies of the present invention to create a chimeric bivalent antibody.

Such antibodies can be monovalent antibodies. Methods of obtaining monovalent antibodies are well known in this field. For example, one method involves recombinant expression of light chain immunoglobulin and a modified heavy chain. This heavy chain usually shortened at any point in the Fc-region to prevent fusion of the heavy chain. Alternatively, the relevant cysteine residues substituted with other amino acid residue or deleteroute to prevent stitching.

In vitro methods are also suitable for the production of a monovalent antibody. Cleavage of the antibodies for their fragments, in particular, Fab fragments, can be performed using routine methods known in this field.

These antibodies can also be obtained in the form of affine Mature antibodies using known methods of selection (selection) and/or mutagenesis, as is well known in this field. Preferred affinity Mature antibodies have affinity, which is five times, more preferably 10 times, even more preferably 20 or 30 times higher than the original antibody from which vtorogo was obtained Mature antibody.

Bespecifically antibodies

Bespecifically antibodies are monoclonal antibodies that have the binding specificity against at least two different antigens. For example, one of specificdate binding may be in respect of the polypeptide of the present invention, a different binding specificity can be against any other antigen, and preferably in the ratio of protein or receptor or receptor subunit cell surface.

Methods of obtaining bespecifically antibodies known in the field. Traditionally, the recombinant getting bespecifically antibodies is based on the co-expression of two pairs of heavy chain/light chain immunoglobulin, where the two heavy chains have different specificities (Milstein and Cuello, 1983). Due to the random rearrangement of the heavy and light chains of immunoglobulin, hybridoma (quadroma) produce a potential mixture of ten different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule is usually performed stages affinity chromatography. Such methods are described in WO 93/08829 and in Traunecker (1991).

Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically F(ab')2-antibodies). Methods of generating b the specific antibodies of fragments of the antibodies described in the literature. For example, bespecifically antibodies may be produced using chemical binding. Also describes the various methods of obtaining and allocating bespecifically fragments of antibodies directly from a culture of recombinant cells.

Hollinger et al. (1993) proposed an alternative mechanism for obtaining bespecifically fragments of antibodies. The fragments contain the variable domain of the heavy chain (VH)connected to the variable domain light chain (VL) a linker that is too short to allow pairing between the two domains on the same chain. Thus, the domains of the VHand VLone fragment are forced to pair with the complementary domains of the VLand VHanother fragment by the formation of two antigen-binding sites. Another strategy to obtain bespecifically fragments of antibodies using single-chain Fv-dimers (scFv) have also been reported Gruber et al. (1994).

It also considers antibodies with more than two valencies. For example, can be obtained thespecification antibodies (Tutt et al. (1991)).

Heteroconjugate antibodies

Heteroconjugate antibodies are also included in the scope of the present invention. Heteroconjugate antibodies consist of two covalently United antibodies. Such antibodies have been proposed, for example, to Nazeli the project for immune system cells to unwanted cells (U.S. patent 4676980) and for the treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is assumed that such antibodies can be obtained in vitro using known methods of chemical synthesis of proteins, including methods involving crosslinking agents. For example, immunotoxins can be constructed using the reaction of disulfide exchange or formation of thioester linkages. Examples of suitable reagents for this purpose include aminothiols and methyl-4-mercaptopyrimidine and reagents described, for example, in U.S. patent 4676980.

Polynucleotide

Under "selected polynucleotide" the authors mean polynucleotide, which, as a rule, was selected from a polynucleotide sequence with which it is associated or linked in its native state. Preferably, the selected polynucleotide at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is associated natural. In addition, the term "polynucleotide" is used herein interchangeably with the terms "nucleic acid molecule", "gene" and "mRNA".

The term "recombinant" in the context of polynucleotide refers to polynucleotide, when it is present in the cell or the cell-free expression system, in a modified amount in comparison with its native state. what one embodiment, the cell can be a cell, which is natural does not contain polynucleotide. However, this cell can be a cell that contains a non-endogenous polynucleotide, leading to an altered, preferably increased, the number receiving the encoded polypeptide. Recombinant polynucleotide of the present invention includes polynucleotide that have not been separated from other components transgenic (recombinant) cells or cell-free expression system, in which it is present, and polynucleotide produced in such cells or cell-free systems, which are then purified from at least some other components.

"Polynucleotide" refers to an oligonucleotide, polynucleotide or any of their piece. It can be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein or other substances to perform a specific particular in this description of the activity.

percent identity of polynucleotide determined using GAP analysis (Needleman and Wunsch, 1970) (GCG program) with a penalty (fine) create gap=5, penalty (fine) extension gap=0,3. The requested sequence has a length of at least 45 nucleotides, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably the, the requested sequence has a length of at least 150 nucleotides, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. Even more preferably, the requested sequence has a length of at least 300 nucleotides, and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. More preferably, two sequences are aligned across their entire length.

With regard to the designated polynucleotides, it will be understood that the figures % identity higher than the above, will include the preferred options for implementation. Thus, when it is acceptable, in light of the minimum digits % identity, preferably polynucleotide contains a polynucleotide sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 80%, more preferably m is Nisha least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1 per cent, more preferably at least 99.2%, more preferably at least 99.3 percent, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%and more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant specified SEQ ID NO.

Polynucleotide of the present invention may have, in comparison with the natural molecules, one or more mutations are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either natural (i.e. isolated from natural source), or synthetic (for example, obtained by performing site-directed mutagenesis or DNA shuffling to the above nekleenov the th acid). Thus, it is obvious that polynucleotide of the present invention can be either natural or recombinant.

Polynucleotide of the present invention include polynucleotides that hybridize under strict conditions with polynucleotides containing a nucleotide sequence that is at least 40% identical, more preferably at least 90% identical, SEQ ID NO:1. The term "stringent hybridization conditions", etc. refers in this context to the parameters that are known in this area, including temperature variation hybridization depending on the length of the oligonucleotide. The parameters of the hybridization of nucleic acids can be found in the links that unite such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra). For example, stringent hybridization conditions, in this context, may refer to hybridization at 65°C in buffer for hybridization by (3.5×SSC, 0.02% Ficoll, of 0.02% polyvinylpyrrolidone, 0.02% of bovine serum albumin, 2.5 mm NaH2PO4(pH 7), 0.5% of LTOs, 2 mm EDTA).

Vectors and cells-owners

One variant of implementation of the present invention includes a recombinant vector, which contains at least one selected molecule polynucleotide of the present invention, insertional into any vector capable of delivering a molecule polynucleotide in cell-hosei the and. Such a vector contains heterologous polynucleotide sequence, i.e. the polynucleotide sequence, which are not detected natural related polynucleotide molecules of the present invention and which preferably derived from a species different from the species from which you obtained this polynucleotide molecule (from which you obtained polynucleotide molecules). This vector can be either RNA or DNA, either prokaryotic or eukaryotic, and is usually the transposon (such as transposon described in US 5792294), virus or plasmid.

The term "functionally linked”, as used herein, refers to a functional relationship between two or more segments of nucleic acid (e.g. DNA). Usually, it refers to the functional relationship of the regulatory element, the transcription with the transcribed sequence. For example, a promoter functionally linked to the coding sequence such as defined in the present description polynucleotide, if it stimulates or modulates the transcription of the coding sequence in a suitable cage. Typically, promoter regulatory elements of transcription, which is functionally linked to the transcribed sequence, are physically adjacent to this Tran is kabirwala sequence, i.e. they are CIS-acting. However, some regulatory elements of transcription, such as enhancers, should not be located adjacent or in close proximity to coding sequences, the transcription of which they reinforce.

As used in this description, expressing the vector is a DNA or RNA vector that is capable of transforming a cell of the host and execute the expression specified polynucleotide molecules. Preferably, expressing the vector able to replicate in the cell host. Expressing vectors can be either prokaryotic or eukaryotic, and are usually viruses or plasmids. Expressing the vectors of the present invention include any vectors that function (i.e. control gene expression) in recombinant cells of the present invention, including bacterial, fungal cells, cells of endoparasites, arthropod, animal and plants.

In particular, expressing the vectors of the present invention contain regulatory sequences, such as regulatory sequences, transcription regulatory sequence broadcast, the sites of initiation of replication, and other regulatory sequences that are compatible with the recombinant cell and that reguliruyut the expression of the polynucleotide molecules of the present invention. In particular, recombinant molecules of the present invention include a regulatory sequence for transcription. Regulatory sequence for transcription are sequences that regulate the initiation, elongation and termination of transcription. Especially important regulatory sequences for the transcription are sequences that regulate transcription initiation, such as promoter, enhancer, operator and repressure sequence. Suitable regulatory sequence for transcription include any regulatory sequence for transcription, which can function in at least one of the recombinant cells of the present invention. Various such regulatory sequences of known transcription specialists in this field.

Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., the nucleic acid sequences of the signal segment) for secretion of the expressed polypeptide of the present invention from a cell which produces the polypeptide, and/or (b) to contain the merged sequences that lead to the expression of the nucleic acid molecules of the present invention in the form of a fused protein. Examples of suitable si the national segments include any signal segment, able to control the secretion of the polypeptide of the present invention. Recombinant molecules can also include intermediate and/or not transmitted sequence surrounding the nucleic acid sequences and/or in the sequence of the nucleic acid molecules of nucleic acids of the present invention.

Another variant of implementation of the present invention includes a cell host containing one or more recombinant molecules of the present invention. The transformation of the polynucleotide molecules in the cell can be performed by any method by which polynucleotide molecule can be insertion in this cell. Methods of transformation include, but are not limited to, transfection, electroporation, microinjection, lipofection, the adsorption and fusion of protoplasts. The recombinant cell can be unicellular or can grow into the tissue, organ or multicellular organism. Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e. recombinant) cells in such a way that preserves their ability to be expressed.

Suitable for transformation of a cell hosts include any tile is in, which can be transformed polynucleotide of the present invention. Cell host according to the present invention either can be endogenously (i.e. natural) and are able to produce the polypeptides of the present invention or can be capable of producing such polypeptides after transformation of at least one polynucleotide molecule of the present invention. Cell host according to the present invention can be any cell capable of producing at least one protein of the present invention, and include animal cells, plant, bacterial, fungal (including yeast) cells, cells of parasites and arthropods. Preferably, a host cell is a bacterial cell. In one preferred embodiment, the host-cell is an E. coli strain with an antigen serotype H, H10. Examples of suitable E. coli strains include CCEC22, CCEC31 and CCEC59, as described in WO 2007/025333.

Recombinant DNA technology can be used to improve the expression of the transformed polynucleotide molecules by manipulating, for example, kopiosto this polynucleotide molecule in the cell host, the efficiency with which are transcribed polynucleotide molecules, the efficiency with which the received broadcast transcripts, and efficiency postre kationnyh modifications. Recombinant methods used to increase expression of polynucleotide molecules of the present invention, include, but are not limited to, functionally related polynucleotide molecules with plasmids high copy number, integration polynucleotide molecule into one or more chromosomes of host cells, adding sequences to the stability of the vectors to plasmids, substitutions or modifications of the signals in the regulation of transcription (e.g., promoters, operators, enhancers), substitutions or modifications of signals regulation of translation (for example, binding sites of the ribosome, sequences Shine-Dalgarno), modified polynucleotide molecules of the present invention, in order to match the frequency of use of codons of the host cell and deletions sequences that destabilize transcripts.

Detection of polynucleotides

Can be used in any suitable way, which makes it possible to detect polynucleotide of the present invention, including methods, which allow to quantify the level of expression of this polynucleotide in the tissue and/or cell. For example, the presence or levels of the transcribed gene can be determined by Northern-blot and/or amplification of this polynucleotide, such as PCR. Maybe DM is Lano comparison by reference to a standard control. For example, levels of transcribed gene can be determined by Northern-blot and/or RT-PCR. With the advent of quantitative (real-time) PCR, quantitative analysis of gene expression can be achieved by using appropriate primers for the gene of interest. The nucleic acid may be labeled and hybridized on the matrix genes, and in this case, the concentration of the gene will be directly proportional to the intensity of the radioactive or fluorescent signal generated in the matrix.

"Polymerase chain reaction" ("PCR") is a reaction in which are formed up replicate of polynucleotide target using a pair of primers or primer sets"consisting of "upper" and "lower" primer and catalyst for polymerization such as DNA polymerase, and usually thermostable polymerase enzyme. PCR methods known in this field and are described, for example, in "PCR" (Ed. M.J. McPherson and S.G Moller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can be performed on cDNA obtained by reverse transcription of mRNA isolated from a biological sample. However, the easier way is usually PCR performed on genomic DNA.

Primer is usually the oligonucleotide, usually from about 20 nucleotides, with a minimum amount of about 15 nucleotides, which is capable of hybridiza is the substance of the sequence-specific manner with a sequence of target and extended during PCR. Amplicons or PCR products or PCR fragments or amplification products are products of elongation, which contain primer and new synthesized copies of the sequences of the target. Multiplex PCR system contain multiple sets of primers that result in the simultaneous production of more than one amplicon. Primers can closely match the target sequence or they may contain internal mistakenly mating grounds, which may lead to the induction of a restriction enzyme or catalytic sites of the recognition/cleavage of nucleic acids in a specific sequence target. The primers may also contain additional sequences and/or modified or labeled nucleotides to facilitate capture or detection of the amplicons. Repeated cycles of denaturation by heating the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers by the polymerase leads to exponential amplification of a target sequence. The terms target sequence of a target or matrix refer to sequences of nucleic acids that are amplified.

The person skilled in the art will understand that there are numerous alternative methods of amplification of polynucleotide what about the present invention. Examples of other methods of amplification include polymerase chain reaction with reverse transcription (RT-PCR), ligase chain reaction ("LCR"), and also includes methods isothermally amplification, such as amplification preemptive chain (SDA) - mediated loop isothermally amplification of DNA (LAMP).

Alternatively, polynucleotide of the present invention can be detected in the sample by using appropriate methods of amplification, for example, hybridization of a southern blot with appropriately labeled probes. "Probe" is single-stranded molecule of DNA or RNA sequence that can base pairing with the second molecule of DNA or RNA that contains a complementary sequence (the target). The stability of the resulting hybrid molecules depends on the mating grounds, which takes place, and is influenced by such parameters as the degree of complementarity between the probe molecule and the target, and the degree of stringency of hybridization conditions. Probes specific for the described in this description polynucleotides or parts thereof, may vary along their length as any integer of at least 8 nucleotides, about 500 nucleotides, including any value in between, depending on the purpose for which you use this the DME, and conditions when using this probe. For example, the probe may have a length of at least 8, 10, 15, 20 or 25 nucleotides, or may have a length of at least 30, 40, 50, or 60 nucleotides, or may have a length of more than 100, 200, 500, or 1000 nucleotides. Probes that are specific in relation described in this description polynucleotides, usually by at least 40%, 50%, 55% or 60%, or at least 65%, 75%, 80%, 85%, 90% or 95%or even 96%, 97%, 98% or 99% identical to the sequences of nucleic acids that are described in this description, using the program align (Myers and Miller, 1989).

The term "hybridization", as used herein, refers to the Association of two molecules to each other through the formation of hydrogen bonds. Factors that affect this binding, include the type and amount of solvent; reaction temperature; hybridization; mixing agent for blocking nonspecific attaching molecules of the liquid phase with solid media (denhardt's reagent or BLOTTO); the concentration of molecules; the use of compounds to increase the rate of Association of molecules (doctranslate or polyethylene glycol) and the severity of the washing conditions after hybridization (see Sambrook et al., Molecular Cloning; A Laboratory Manual, Second Edition (1989)). In accordance with these principles, the inhibition of hybridization of complementary molecules with molecule the target can be tested by analysis of hybridization; essentially homologous molecule having a higher degree of homology will compete for binding and to inhibit the binding of a completely homologous molecules with molecule-target under various conditions of stringency, as described in Wahl et al., (1987).

As used in this description in relation to hybridization, "stringent conditions" are conditions that (1) employ low ionic strength and high temperature for washing, for example, 0,015M NaCl/0,0015M sodium citrate/0.1% of NaDodSO4at 50°C; (2) employ during hybridization, denaturing agent, such as formamide, for example, 50% (vol./about.) formamide with 0.1% bovine serum albumin, 0.1% Picollo, 0.1% polyvinylpyrrolidone, 50 mm sodium phosphate buffer at pH 6.5 with 750 mm NaCl, 75 mm sodium citrate at 42°C; or (3) employ 50% of formamide, 5×SSC (0,75M NaCl, 0,075M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5× denhardt's solution, processed by the ultrasound DNA salmon sperm (50 g/ml), 0.1% of LTOs and 10% extrasolar at 42°C in 0.2×SSC and 0.1% - ordinator.

Attenuated bacteria

Methods attenuative virulence of bacterial pathogens known in this field. Usually, in the bacterial genome introduce mutations to prevent or reduce the expression of toxins or other virulence genes for deletion or inactivation of this gene. In some cases, they Ave the lead to a complete knockout of the functions of this gene. This can be accomplished either by elimination of the synthesis of any of the polypeptide generally of this gene, or by formation of mutations, which leads to the synthesis of non-functional polypeptide. For the abolition of the synthesis of the polypeptide can be deleterows either the entire gene or the 5'-end. Deletion or insertion in the coding sequence of a gene can be used to create a gene that synthesizes only non-functional polypeptide (e.g., a polypeptide that contains only the N-terminal sequence of the protein of wild type). In the case of a toxin gene, this mutation may make the product of this gene is non-toxic.

"Mutation" includes any change in DNA sequence, i.e. the genome of the organism in comparison with the parent strain. Such changes can occur when exposure to this organism mutagenic stimulus, such as a mutagenic chemical, energy, radiation, recombinant methods, crossing or any other method used to modify DNA. The mutation may include a change in any of the described nucleotide sequences, or may include a change in the nucleotide sequence that encodes any of the polypeptides described.

Mutation can attenuate virulence", if the result of this mutation, the level of virulence of this mutant the pension is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, in comparison with the parent strain. The decrease in virulence can be measured by the reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% expression and/or toxin activity of the polypeptide, for example, a polypeptide containing the amino acid sequence, to essentially identical sequences of SEQ ID NO:2 or SEQ ID NO:3, a fragment or a variant, mutant strain compared to the parental strain.

The person skilled in the art will understand that attenuated bacterial pathogen of the present invention may be suitable for delivering one or more biologically active polypeptides to a subject. Examples of biologically active polypeptides suitable for the delivery of attenuated bacteria of the present invention include polypeptides, which can exhibit endocrine activity, acting locally or on the metabolism of the whole organism.

In one embodiment, the biologically active polypeptide can be a heterologous polypeptide. The term "heterologous polypeptide" is well understood in this field and refers to a polypeptide that is not endogenous to the cell. The nucleic acid molecule encoding an polypeptide can be from any organism capable of producing p is establishi polypeptide of interest, or may be completely synthetic genome. The nucleic acid molecule encoding the polypeptide, can be added to a cell, for example, infection, transfection, microinjection, electroporation, delivery “micronarrative” or similar

As an example, the biologically active polypeptide can be a polypeptide which is able to adjust immunocompetency system. Alternatively, the biologically active polypeptide can be a polypeptide which is able to affect the viability, growth and differentiation of a variety of normal or neoplastic cells in the body. Alternatively, the biologically active polypeptide can be a polypeptide which is able to influence immune regulation or induction of inflammatory acute phase reactions to injury and infection. Alternatively, the biologically active polypeptide can be a polypeptide which is able to increase or induce resistance to infection of cells and tissues, mediated by the action of chemokines to their receptors on target cells, or proliferation of epithelial cells or stimulation of wound healing.

Specific examples of such polypeptides include insulin, growth hormone, prolactin, calcitonin, luteinizing hormone, parathyroid hormone, somatostatin, thyroid stimulating (thyrostimulin) hormone, vasoactive is Chechnya polypeptide, cytokine structural group 1, such as IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-23, IL-24, IL-25, IL-26, IL-32, cMGF, LT, GM-CSF, M-CSF, SCF, IFN-γ, IFN-λ, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFNα/β, a cytokine structural group 2, such as TNF-family cytokines, such as TNFα, TNFβ, CD40, CD27 or FAS-ligand, IL-1 family of cytokines, a family of fibroblast growth factors, platelet-derived growth factors, transforming growth factor β and nerve growth factors, cytokine structural group 3, for example, the family of epidermal growth factor cytokines, chemokines, insulin-like cytokines, cytokine structural group 4, such as heregulin or neuregulin, for example, EGF.

Alternatively, the biologically active polypeptide can be a receptor or an antagonist for biologically active polypeptides defined above.

In another embodiment, the biologically active polypeptide is an antibody, preferably a recombinant antibody.

Alternatively, the biologically active polypeptide can be an antimicrobial peptide or a synthetic option. Antimicrobial peptides include cecropin, magainin and defensive. Cecropin were the first well-characterized family of structurally related antimicrobial peptides have been found in a broad spread of insects (Boman, 2003). In vertebrates, the class of magainin antimicrobial peptides were isolated from skin glands and the gastrointestinal tract Xenopus laevis, and believed that they form the basis for the protective system of the mucous surfaces of amphibians against infection (Soravia et al., 1988).

Defensin are antimicrobial peptides found in phagocytic cells isolated from several mammalian species, including humans, and can be characterized by eight invariant residues in the sequence (Gabay et al., 1989). The mechanism of antimicrobial activity of these peptides, as defensin, through selective destruction of membranes, leading to the characteristic broad spectrum antibiotic activity (Boman, 1995). Antimicrobial spectrum of defensins includes gram-positive and gram-negative bacteria, mycobacteria, many fungi and some with the shell of the virus.

Antimicrobial peptides of bacterial origin known as microzine, colicins and bacteriocins (Jack et al., 1995; Ingham et al., 2003). It is known that the sequence, structure and mechanism of activity of bacteriocins diverse. The most abundant and extensively studied bacteriocins include bacteriocins of class I (lantibiotic) and class II bacteriocins (small stable against heat, does not contain lanthionine peptides) (Ennahar et al., 2000). Bacteriocins of class II constitute an important subgroup because of their activity and potential use. Bacteriocins Klah is sa IIa include piscicole 126, leucocin and enterocin R, among others. Bacteriocins of class IIa have a common N-terminal motif: YGNGVXaaCXaa(K/N)XaaXaaCXaaV(N/D)(W/K/R)Xaa(G/A/S)(A/N), where residues with higher variability presents Xaa (Bhugaloo-Vial et al., 1996). In the example, demonstrating the antimicrobial properties of bacteriocins, it was shown that, when injected to mice, piscicole 126 exhibits antimicrobial activity in vivo and significantly reduces the load Listeria in the liver and spleen (Ingham et al., 2003).

Alternatively, the biologically active polypeptide can be an enzyme. This enzyme can be any enzyme having the desired activity. For example, it may be desirable delivery of the enzyme, which plays a role in improving the ability of digestion or removal antinutritive compounds. For example, to increase the digestibility of food can be delivered degrading polysaccharides and fibroticheski enzymes, such as xylanase (Liu et al., 2005), glucanase (Cho et al., 2000), cellulase (Liu et al., 2005), amylase, Lavansaari and inflacarata. Proteases, peptidases and lipases can also be delivered to increase the nutritional value of eaten food. Phytase (Vohra and Satyanarayana, 2003; Nahashon et al., 1994) and acid phosphatase (Palacios et al., 2005) can be delivered to reduce antinutritive effects of phytate, which is found in the seeds of plants.

In another embodiment, the implementation of the Oia attenuated bacterial pathogen of the present invention can Express the antigen. If this antigen is, for example, the agent of bacterial, fungal, parasitic or viral diseases, attenuated bacterial strain can be used for vaccination of a subject against disease caused by such agents. For example, attenuated bacterial strain can be used to deliver antigen of an avian pathogen. Such microorganisms include, but are not limited to, Corynebacteria species, Mycoplasma, Listeria, Borrelia, Chlamydia, Clostridia, Coxiella, Eysipelothrix, Flavobacteria, Staphylococcus, Escherichia, Salmonella, Campylobacter and Streptococcus. Examples of fungal and parasitic avian pathogens, known to infect poultry, are the kinds of Amoebotaenia, Aproctella, Ascaridia, Aspergillus, Candida, Capillaria, Cryptosporidium, Cyathostroma, Dispharynx, Eimeria, Fimbriaria, Gongylonemia, Heterakis, Histomonas, Oxyspirura, Plasmodium, Raillietina, Strongyloides, Subulura, Syngamus, Tetrameres and Trichostrongylus. Viruses known that they infect poultry, include adenoviruses (e.g., virus, hemorrhagic enteritis), astroviruses, coronaviruses (e.g., infectious bronchitis virus), paramyxoviruses (e.g., Newcastle disease virus), picornaviruses (e.g., avian encephalomyelitis), poxvirus, retrovirus (e.g., avian leukosis/avian sarcoma), reoviruses and rotaviruses. Specific examples include avian influenza, a virus disease Mare who and chicken anemia virus. Preferred gene products for use as antigens are polypeptides and peptides, including glycoproteins and lipoproteins. Antigenaemia genes of prokaryotic and eukaryotic organisms can be cloned and expressed in attenuated bacteria using standard methods.

Composition and introduction

"Immunogenic composition" is a composition that contains materials that induce a desired immune response, and includes a "vaccine". The term "vaccine" includes any composition that induces at least partially immune response against the pathogen target or which effectively protects against the pathogen; for example, after the introduction or injection of the animal (for example, the representative of the bird, such as chicken, or representative of pork, such as a pig), induces at least partially protective immune response against a pathogen (e.g., C. perfringens). Subunit of a pathogen, for example, the antigen or immunogen or epitope selected from the pathogen, and the composition of the subunits contains essentially one or more antigens, immunogens or epitopes or essentially consists of one or several antigens, immunogens or epitopes selected from this pathogen. Under the induction of at least partially protectively the immune response mean the vaccine reduces infection and/or colonization of bacteria expressing the polypeptide of the present invention, or reduces at least one symptom caused by infection with bacteria expressing the polypeptide of the present invention.

The immunogenic composition may select, activate, or reproduced in cells of the immune system, including b - and T-memory cells, for example, to create the possibility of elimination of infectious agents, such as bacterial pathogens expressing the polypeptide containing the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO:3, or antigenic fragments.

In some embodiments, the implementation of the immunogenic composition comprises a suitable carrier such as an adjuvant, which is an agent that acts non-specific way, to increase the immune response to a particular antigen or group of antigens, making it possible to reduce the amount of antigen in any specific dose or frequency of doses required to generate the desired immune response. The desired immune response may include, for example, full or partial protection from the spread of bacterial pathogen (presence in the feces of an infected animal such as a mammal or bird) or colonization (presence in the intestine infection is consistent animal, for example, a mammal or bird) bacterial pathogen. For example, the desired immune response may include any value from 10%-100%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, protection from the spread of bacterial pathogen or bacterial colonization of the pathogen in the vaccinated animal compared to unvaccinated animals.

Adjuvants can be used to improve the immune response and/or increase the stability of vaccines. Adjuvants are usually described as non-specific stimulators of the immune system, but can also be used for targeting specific branch of the immune system. One or more compounds that possess such activity, can be added to the vaccine. Thus, a specific vaccine of the present invention optionally contain adjuvant. Examples of chemical compounds that can be used as adjuvants include, but are not limited to, aluminum compounds (e.g., aluminum hydroxide), and metabolisable not metabolisable oil, mineral oil, including derivatives oleate manned in a solution of mineral oils (e.g., MONTANIDE ISA 70 from Seppic SA, France), and light mineral oil such as DRAKEOL 6VR, block copolymers, ISCOM (immune stimulating complexes), vitamins and minerals (including, but not exhaust initialdata them vitamin E, vitamin A, selenium and vitamin B12) and CARBOPOL®.

Other suitable adjuvants, which are sometimes referred to as Immunostimulants include, but are not limited to: cytokines, growth factors, chemokines, supernatant from cell cultures of lymphocytes, monocytes, cells from lymphoid organs, drugs, cells and/or extracts from plants, bacteria or parasites (drugs Staphylococcus aureus or lipopolysaccharide preparations) or mitogens.

Typically, the adjuvant is administered at the same time as the antigen of the present invention. However, adjuvants may also, or alternatively, be introduced within a two-week period before vaccination and/or within a certain period of time after vaccination, i.e. until the antigen, for example, a polypeptide containing the amino acid sequence represented in SEQ ID NO:2 or SEQ ID NO:3, or antigenic fragment, stored in the tissues.

Immunogenic compositions of the present invention may include those described in this description of the molecule polypeptides and nucleic acids, or immunogenic fragments, and can be entered using any form of injection, known in the field or described in this specification. In some embodiments, implementation of the present invention the immunogenic composition or vaccine may include live bacterial pathogen killed bacterially pathogen or their components. Live bacterial pathogens that may be introduced in the form of an oral vaccine can be attenyerevan so that the reduced virulence of this bacterial pathogen, but not its induction of immune responses. Live vaccine may be able to colonize the intestine inoculated animal, such as birds.

In some embodiments, the implementation described in this description of the molecule polypeptides and nucleic acids, or antigenic fragments or mutated bacteria (e.g., attenuated bacteria), described herein, can be administered to poultry, such as chicken, ducks, turkeys, etc. for the induction of an immune response, for example, induction of antibodies in poultry. Eggs or products derived from such poultry, which show an immune response against the described molecules, polypeptides, and nucleic acids, or immunogenic fragments can be administered to an animal, such as humans, cattle, goats, sheep, etc. to induce the immune response to molecules described polypeptides and nucleic acids of the animal. Methods of inducing antibodies in poultry and the introduction of such antibodies are described, for example, in U.S. patent 5750113 and U.S. patent 6730822.

Immunogenic compositions and vaccines of the present invention may be more the positive supplemented by the addition of other recombinant or purified antigens, which can lead to the production of antibodies, various specificdate when administered to a subject animal. Not all of these antibodies should be protective against the disease. In a particular embodiment of this type, such antigens are also of C. perfringens. Thus, the vaccine of the present invention may contain various other active or inactivated pathogenic factors, together with the polypeptide of the present invention. Thus, in accordance with the present invention, the polypeptide of the present invention may be combined with other clostridial or clostridial cells, anatoxine and extracts.

Additional antigens may contain viral antigen and/or bacterial antigen, and/or parasitic antigen. For example, the antigen can be obtained from a microorganism, including, but not limited to, Corynebacteria species, Mycoplasma, Listeria, Borrelia, Chlamydia, Clostridia, Coxiella, Eysipelothrix, Flavobacteria, Staphylococcus, Escherichia, Salmonella, Campylobacter and Streptococcus. Examples of fungal and parasitic avian pathogens, known to infect poultry, include species Amoebotaenia, Aproctella, Ascaridia, Aspergillus, Candida, Capillaria, Cryptosporidium, Cyathostroma, Dispharynx, Eimeria, Fimbriaria, Gongylonemia, Heterakis, Histomonas, Oxyspirura, Plasmodium, Raillietina, Strongyloides, Subulura, Syngamus, Tetrameres and Trichostrongylus. Viruses known that they infect poultry, clucalc adenoviruses (e.g., the haemorrhagic enteritis), astroviruses, coronaviruses (e.g., infectious bronchitis virus), paramyxoviruses (e.g., Newcastle disease virus), picornaviruses (e.g., avian encephalomyelitis), poxvirus, retrovirus (e.g., avian leukosis/avian sarcoma), reoviruses and rotaviruses.

Polyvalent vaccine of the present invention may also contain one or more of the following antigens: beta-toxin of C. perfringens beta-2-toxin of C. perfringens enterotoxin of C. perfringens Epsilon-toxin of C. perfringens iota-toxin of C. perfringens, Kappa-toxin of C. perfringens, lambda-toxin of C. perfringens, theta-toxin of C. perfringens, hemorrhagic toxin of C. sordellii lethal toxin of C. sordellii And C. difficile toxin, C. difficile toxin, alpha toxin of C. septicum alpha-toxin of C. novyi and beta-toxin C. novyi.

Immunogenic compositions and vaccines of the present invention can be administered in the form of liquid, emulsion, dried powder and/or “fog” by any parenteral way, intravenously, intraperitoneally, intradermally, scarification, subcutaneously, intramuscularly or inoculants through the mucous membrane, for example, oral, intranasal, aerosol, using eye drops, the introduction ofin ovoor be implanted in the form of lyophilized powder.

The introduction of the described molecules, polypeptides, and nucleic acids or their IMM is noennig fragments, mutated bacteria (e.g., attenuated bacteria) and/or described immunogenic compositions may conveniently be achieved by injection into the egg birds (e.g., poultry) and usually by injection into the air bag. Despite the fact that the air bag is the preferred method of introduction ofin ovoother sites, such as yolk SAC or chorioallantoic fluid may also be inoculated by injection. Speed liluplanet may drop slightly when the air bag is not a target for the introduction, though not necessarily in commercially unacceptable levels. The injection mechanism is not crucial for the practical application of the present invention, although it is preferable that the needle did not cause excessive damage to the eggs or tissue and organs of the developing embryo or unembryonated membranes surrounding the embryo.

As a rule, is appropriate syringe for subcutaneous injection with needle gauge 22. The method according to the present invention is particularly well adapted for use with an automated injection system, such as the injection system described in US 4903635, US 5056464, US 5136979 and U.S. patent 20060075973.

The present invention also relates to a method of providing passive immunity to the offspring, a female animal (for example, pregnant females), preduster is that the introduction of the vaccine of the present invention the female animal (for example, the mother before the birth of her child. "Passive immunity" is called transfer of immunity from mother to child, and it can be done inter alia through the ingestion of colostrum, as it occurs in mammals, or by absorption of antibodies in the blood from the yolk, as it happens with birds.

In one embodiment, the female is a bird, and the vaccine is administered to the female birds before laying eggs, which contain the seed. The way her offspring provides passive immunity. In one such embodiment, the poultry is poultry. Preferably poultry is chicken, Turkey or duck.

Immunogenic compositions and vaccines of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carrier includes acceptable in the veterinary carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory Agency of the Federal government or the state government or listed in the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia for use in animals and, more specifically, to the people. The term "carrier" refers to a diluent, excipient or filler, which introduced this therapeutic tool. Such pharmaceutical carriers can be tellinii liquids, such as water and oils, including oils derived from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc.

Usually, the ingredients of the preparations according to the present invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.

The provided compositions containing the polypeptide of the present invention, polynucleotide of the present invention, the vector according to the present invention and/or the cell host according to the present invention. As will be clear to the person skilled in the art, these compositions may contain suitable carriers or excipients.

DNA vaccine

DNA vaccination involves direct introduction of in vivo DNA that encodes the antigen, b cells and/or tissue of the subject for expression of this antigen in the cells of the tissue of the subject. Such vaccines referred to in this description of "DNA vaccine" or "vaccine based on nucleic acids". Examples of DNA vaccines described in US 5939400, US 6110898, WO 95/20660 and WO 93/19183. The ability of directly injected DNA that encodes the antigen to induce protective immune the reaction has been demonstrated in numerous experimental systems (see, for example, Conry et al., 1994; Cardoso et al., 1996; Montgomery et al., 1993; Yang et al., 1997).

Factor, which is known that it affects the immune response induced by DNA immunization is the method of delivery of DNA, for example parenteral methods, can give low rates of gene transfer and to produce significant variability in gene expression (Montgomery et al., 1993). High speed inoculation of plasmids, using a gene gun, accelerated immune response of mice (Fynan et al., 1993), presumably due to the greater efficiency of transfection of DNA and more effective antigen presentation by dendritic cells. Vectors containing vaccine on the basis of the nucleic acids of the present invention, can also enter the desired host in other ways known in this field, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextranomer method, precipitation with calcium phosphate, lipofectin (merge liposomes), or a DNA vector as a carrier.

Vaccines derived from transgenic plants

The term "plant" refers to whole plants, plant organs (e.g. leaves, stems, roots, etc.) seeds, plant cells, etc. Plants considered for use in the practice of the present invention, include both monocotyledonous and dicotyledonous rasteniyami dicotyledonous plants include corn, tomato, potato, beans, soybeans, etc. Usually, the transgenic plant is routinely used as a source of feed products for livestock, particularly chickens.

Transgenic plants that are defined in the context of the present invention include plants (as well as parts and cells of these plants and their progeny which have been genetically modified using recombinant DNA to produce or increase production of at least one polypeptide according to the present invention in the desired plant or organ of a plant.

There are several ways of introducing foreign genetic material into a plant cell and receiving plants that stably retain and Express the introduced gene. Such methods include the acceleration of genetic material, deposited in the form of a coating on the particles directly into cells (see, for example, US 4945050 and US 5141131). Plants may be transformed using Agrobacterium technology (see, for example, US 5177010, US 5104310, US 5004863, US 5159135). The electroporation technology is also used for plant transformation (see, for example, WO 87/06614, US 5472869, 5384253, WO 92/09696 and WO 93/21335). In addition to numerous technologies for the transformation of plants can also vary the type of tissue that result in contact with alien genome. This is Cagnes may include, but not limited to, embryogenic tissue, callus tissue type I and II, hypocotyl, meristem, etc. Almost all tissues of plants can be transformed during the development and/or differentiation by means of suitable described methods.

A number of vectors suitable for stable transfection of plant cells have been described, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989, and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Usually, expressing vectors of plants include, for example, one or more cloned plant genes under the transcriptional control of 5'- or 3'-regulatory sequences and a dominant breeding marker. Such expressing vectors of plants can also contain a promoter regulatory region (e.g., a regulatory region regulating the inducible or constitutive, regulated environment or stage of development or kletka - or tissue-specific expression), the site of transcription initiation, the binding site of the ribosome, the signal processing RNA, the site of transcription termination and/or polyadenylation signal.

Examples of promoters in plants include, but are not limited to, the promoter of the small subunit of ribulose-1,6-bisphosphatase, the promoter of the beta-better promoter phaseolina, the ADH promoter, the promoters of elkow heat shock and tissue-specific promoters.

Promoters may also contain specific enhancer elements in the sequence, which can improve the efficiency of transcription. Typical enhancers include, but are not limited to, Adh-intron 1 and Adh-intron 6.

Constitutive promoters control the continuous gene expression in all cell types and at all time points (for example, promoters of actin, ubiquitin, CaMV 35S). Tissue-specific promoters are responsible for gene expression in specific cell types or tissues, such as leaves or seeds (for example, promoters of Zein, oleosin, napin, ASR, globulin and the like), and such promoters can also be used. Promoters can also be active during a certain stage of plant development, and is also active in the tissues or organs of plants. Examples of such promoters include, but are not limited to, specific for pollen-specific embryo-specific silk (aggregate columns in the cob) corn, specific to cotton fiber-specific root specific to the endosperm of the seeds promoters, etc.

Under certain circumstances, it may be desirable to use inducible promoter. The inducible promoter is responsible for expression of genes in response to a specific signal, such as physical stimuli (genes Belk is in heat shock); light (RUBP-carboxylase), hormone (Em); metabolites and stress. Can be used other desired elements of transcription and translation, which function in plants.

In addition to the promoters of plant promoters from different sources can be used effectively in plant cells for expression of foreign genes. For example, can be used promoters of bacterial origin, such as the promoter octopunctata, the promoter napaliensis, the promoter Monophysites; promoters of viral origin, such as the promoter of the virus of the mosaic disease of cauliflower (35S and 19S), etc.

The number obtained from the fit for eating plants vaccines developed currently for pathogens of both animals and man (Hood and Jilka, 1999). The immune response was also received oral immunization by transgenic plants producing virus-like particles (the VLP), or chimeric viruses of plants, detect antigenic epitopes (Modelska et al., 1998; Kapustra et al., 1999). There was supposed to consist of particles form the VLP or chimeric viruses can lead to a higher stability of the antigen in the stomach, with an effective increase in the amount of antigen available for absorption in the intestine (Modelska et al., 1998).

Food

In one embodiment, the composition according to the present invention is a food with the (food) or food (food) product. For the purposes of the present invention, "food" or "food" includes any food or any drug for human consumption or animals (such as cattle, horses, goats and sheep) (including enteral and/or parenteral consumption), which when ingested (and) are used for food or build tissue or power supply; and/or (b) retain, restore or maintain nutritional status or metabolic function.

Food products include food substances, such as good food macronutrients, vitamins and/or minerals in amounts desired for a particular application. The amount of ingredients will vary depending on, is the song for the application of normal individuals or individuals having individualized needs, for example, individuals suffering from metabolic disorders, or other

Examples of substances with nutritional value include, but are not limited to, macronutrients, such as good food fats, carbohydrates and proteins. Examples of such suitable for food fats include, but are not limited to, coconut oil, borage oil, fungal oil, black currant oil, soybean oil, and mono - and diglycerides. Examples of such carbohydrates include (but are not limited to): glucose suitable for food lactose and hydrolyzed starch. In addition, examples of proteins that can be used in food compositions of the present invention, include, but are not limited to, soybean protein, electrodialyzer whey, electrodialyzer separated milk, whey or protein hydrolysates.

With regard to vitamins and minerals, the following can be added to food compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine and vitamins a, E, D, C, and complex Century Can also be added to other similar vitamins and minerals.

The components used in the food compositions of the present invention, can be procisceni or cleared. Under polyoxidonium or purified mean the material, which was obtained by the treatment of natural material or by de novo synthesis.

In one embodiment, the polypeptide of the present invention is used in cooking (food). For example, the food containing the polypeptide of the present invention, can be used to vaccinate animals to provide at least partial protection against infection and/or colonization by bacterial pathogen expressing the polypeptide with toxin activity. Preferably, the bacterial pathogen expresses polypeptid is, containing the amino acid sequence that is at least 40% identical to SEQ ID NO:2 and/or SEQ ID NO:3. In one embodiment, the bacterial pathogen belongs to the genus Clostridium, for example, the bacterial pathogen is Clostridium perfringens.

In another embodiment, the food contains a transgenic plant of the present invention and/or some of the listed plants, and/or extract specified plants.

Agonists and antagonists - Analyses and molecules

The polypeptides of the present invention can be used in the method of screening for compounds which activate (agonists) or inhibit (antagonists) toxin activity in question polypeptide.

Examples of potential antagonists include antibodies, oligonucleotides and their derivatives. Potential antagonists include a small molecule that binds to the polypeptide of the present invention, making it inaccessible to substrate polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules. Small molecules can mimic the structure of the substrate polypeptide of the present invention.

The present invention also includes analyses of high-throughput screening (HTS) to identify compounds that interact with the polypeptide or inhibit who have biological activity (i.e. affect enzymatic activity) of the polypeptide possessing toxin activity. HTS assays allow for efficient screening of large numbers of compounds. HTS assays are designed to identify "hits" or "compounds of examples with the desired property, modifications which can be designed to improve the desired properties. Chemical modification of the "hit" or "connection example" is often based on an identifiable relationship structure activity between "hit" and toxin polypeptide.

Antagonists of the polypeptide of the present invention can be used to protect animals from disease by adding them to food or drink animals. This treatment can reduce the active load originating from the environment of organisms with which the animal is in contact. Thus, the present invention relates to food and/or drink containing antagonist of the polypeptide of the present invention. The present invention also relates to the use of food and/or beverage containing such antagonists to reduce infection and/or colonization of the animal by a bacterium that expresses the polypeptide of the present invention.

EXAMPLES

Example 1. The NetB toxin of Clostridium perfringens

Sequencing of the gene encoding NetB

10 µg genomeid, isolated from a strain of Clostridium perfringens EHE-NE18, used to obtain indicators of reading sequences of Contigo and points of quality sequences. Amino acid sequence transcribed from a nucleotide sequence. Prediction of signal peptide were performed using the program SignalP v 3.0 (Bendtsen et al., 2004). Sequences homologous to the decoded amino acid sequences were subjected to search using geervani program BLAST (Altschul et al., 1997).

The nucleotide sequence of the gene encoding NetB, presented as SEQ ID NO:1 and amino acid sequence NetB, including the signal sequence presented as SEQ ID NO:3. The sequence of the signal peptide is cleaved from the Mature secreted protein (SEQ ID NO:2). A BLAST search identified beta-toxin of C. Perfringens as having less than 39%identity with NetB (figure 1).

Purification of recombinant NetB and generation of rabbit anti-rNetB-antisera

GenenetBPCR-amplified and cloned into pENTR/SD/D-TOPO and was subcloned into the frame read in expressing vector pDest41BA. The protein was purified on an affinity column of Nickel, with subsequent gel filtration (S200). Fraction peaks were pooled, split TEV and re-applied on the column with Nickel to remove undigested be the Cove and TEV. Recombinant protein (~1.3 mg) was sent to Chemicon to obtain antibodies (Chemicon-Millipore, CA, USA). Anti-rNetB-anticigarette used in Western blot analysis of strains of C. perfringens and research neutralization.

Clearing of native NetB

C. perfringens EHE-NE18 were grown in broth TPG to OD600nm of 0.6. Culture supernatant (3 l) was obtained by centrifugation at 18000 g for 15 minutes at 4°C. the Obtained supernatant was concentrated 5-fold using ultrafiltration (Amicon 8400) through a 10 kDa membrane (DIAFLO®YM 10-76 mm, Amicon), followed by precipitation of 40% (weight/volume) (NH4)2SO4at 4°C over night and were separated by centrifugation at 18000 g for 2 hours at 4°C. the Precipitate containing the toxin was concentrated 20 times (a total of 100 times) and deliberately against 10 mm Tris-HCl pH 7.2, for 48 hours at 4°C. Proteins were chromatographically using anion-exchange resin Q Sepharose FF (GE) with Tris-buffer (pH 8.5) and passing through a column of the solution was collected.

Example 2. The generation of a mutant strain of C. perfringens, devoid of toxin

Manipulation of DNA was carried out according to standard methods. The oligonucleotides used in the construction of suicide plasmids were AKP60 (SEQ ID NO:7), AKP61 (SEQ ID NO:8), AKP58 (SEQ ID NO:9) and AKP59 (SEQ ID NO:10). All amplificatoare products were cloned into the system cloning vector pGEM®-TEasy (Promega) and then was subcloned into if necessary. Labeled, partially depletirovannoi suicide plasmid, pALK.16, designed by cloning fragments of the gene regionnetBon either side of the cassettecatPin pALK1 and received a deletion 541 BP genenetB. FirstMfeI-SpeI-fragment amplificatory using AKP60 and AKP61, directionally cloned in the siteEcoRI-SpeIpALK1, with subsequent cloningBamHI-NheIslice 1937 gel, amplified using AKP58 and AKP59 in the siteBamHI-NheIthe resulting plasmids. Finally,ermBand oriT, amplificatoare of pJIR1457, cloned with zatuplenie all in the siteSmaI. The final suicide plasmid pALK16 was introduced into C. perfringens strain EHE-NE18, as described previously (Scott and Rood, (1989)). After growth at 37°C in TSC, supplemented by thiamphenicol, colony cross inflicted on TSC, supplemented with erythromycin, to confirm what took place a double crossover. Colonies that grew on the appropriate antibiotics were selected for additional analysis. Received chromosomal DNA and used PCR and blot analysis for Southern to confirm that the obtained mutants came from the events of double crossover in the field of genenetB. Designed completation plasmid, pALK20, by cloning the full-size genenetBin the Shuttle vector pJIR1457 C. perfringens and introduced in both specimens. The complementation was confirmed using selection with Erie what fumicino and tested in the analysis of cytotoxicity. Schematic diagram of the region of chromosome NE18-ΔnetBshown in figure 2.

Example 3. Analysis activity NetB

Analysis of cytotoxicity were carried out in the culture supernatant of C. perfringens EHE-NE18. The LMH cells were cultured to 70% of confluently 26-hole tablets coated with 0.2% gelatin, and were grown in EMEM medium at 37°C. the Culture supernatant was added to the environment with 2-fold dilution across the Cup to 1:32, and incubated for 16 hours at 37°C. LMH Cells were incubated in the presence of undiluted culture medium TPG (figa); the culture supernatant of C. perfringens EHE-NE18, a dilution of 1:16 (fig.3b); culture supernatant necroticism strain 13 C. perfringens enteritis JIR325, dilution 1:2 (figs); or the culture supernatant of C. perfringens NE18-M1 (mutantplcnot expressing alpha-toxin), a dilution of 1:16 (fig.3d). Cytopathic effects (CPE) were observed under a light microscope at magnification 100×.

Normal cells (figa) look healthy, but adding the culture supernatant of the strain producing NetB, causes the cells to be rounded and die (fig.3b). Supernatant from strain, which expresses not NetB, had no effect on these cells (figs). The deletion of the gene alpha-toxin does not affect the ability of culture supernatants to kill cells (fig.3d).

Example 4. The complementation of mutants of C. perfringens, lichen the x toxin NetB

NE18-deleteriouslynetB1strain (netB-negative strain) complementarily complementations the plasmid pALK20netB. Then complementary strain of C. perfringens were tested for toxin activity. For analysis of cytotoxicity, LMH cells were cultured to 70% of confluently in 24-hole tablets coated with 0.2% gelatin, and grew: from culture supernatant EHE-NE18, dilution 1:16 dilution (figa); culture supernatant NE18-deletirovanienetB1dilution 1:2 (fig.4b); culture supernatant NE18-deletirovanienetB1+pJIR.1457 (Shuttle plasmid), dilution 1:2 (figs); culture supernatant NE18-deletirovanienetB1+pALK20 (netB-complementation plasmid), a dilution of 1:16 (fig.4d); undiluted culture medium TPG (file); purified on a column of recombinant NetB, a dilution of 1:8 (fig.4d).

The deletion of the genenetBdestroyed killing activity in the analysis of cell culture. The complementation of this mutant gene cloned on a plasmid restored the killing activity. Recombinant NetB protein kills cultured cells.

Example 5. Quantitative analysis of killing cells toxin protein

To determine the ability NetB to kill cells, analysis of cytotoxicity using lactate dehydrogenase were performed on the LMH cells treated NetB. Cells LMH, cultive is ovali up to 70% confluently in 96-well tablets, coated with 0.2% gelatin, and were grown in EMEM medium at 37°C. Paleoceanic NetB from NE18-M1 was added to the medium with 2-fold dilution across the plate before dilution 1:128, and incubated for 4 hours at 37°C. LDH released into the supernatant was measured as an indicator of cytolysis using a set of Cyto-Tox (Promega) and were expressed as percent cytotoxicity. Each dilution was represented in three repetitions and SEM was calculated for each dilution (figure 5).

Example 6.netB-mutant strains to model disease of chickens

A group of 11 birds were infected with either the wild-type strain C. perfringens (NE18), ornetB-deleteregvalue mutants of this strain (NE18-NetB-M1 and NE18-NetB-M2) at the age of 20 days and 21 days. In the 24 days of age birds were subjected to autopsy for evaluation in points of necrotic damage in the intestine. Segments of ileum or jejunum, approximately 2-4 cm, were collected in 10% neutral buffered phosphate sodium formaldehyde. Samples of the small intestine is cut transversely at intervals of 4 mm and the segments were processed, to obtain embedded in paraffin blocks for routine histology, did sections of 4-5 μm and stained them with hematoxylin and eosin (RE). Histological slides were examined by light microscopy. The intestines were evaluated in points according to the number of necrotic damage: 0 - no damage is calling, 1 - concistency and loose bowels, 2 - focal necrosis or ulceration (16 or more lesions), 3 - focal necrosis or ulceration (6-15 foci), 4 - focal necrosis or ulceration (16 or more lesions), 5 necrotic areas with a length of 2-3 cm, 6 - diffuse necrosis, typical for field cases. The wild-type strain showed a significant level of disease, whereas none of independently selected mutants did not show any signs of disease. It was concluded that NetB is the main virulence factor required for disease pathogenesis.

0
Table 2
NetB-mutant strains have reduced virulence in models of disease of chickens
The number of birdsStrain to infect
NE18NE18-NetB-MlNE18-NetB-M2
1300
2200
300
4000
5000
6400
7200
8300
9000
10300
11000
The average score of defeat1,5500
The number of affected birds in group600
Weighted grade point average×number 9,300

Example 7. Research on the presence of toxin in strains of C. perfringens

PCR examination for the presence of toxin in strains of C. perfringens

The presence of a gene ofnetBin strains of NE and non-NE C. perfringens was investigated using PCR. For each of the tested strains of C. perfringens only colony suspended in 0.1 ml of distilled water, boiled for 10 minutes and then centrifuged at 10,000 g for 10 minutes. Supernatant collected and used as DNA templates in PCR. PCR was performed in a total volume of 25 μl reaction mixture containing: 1× PCR buffer (containing no Mg2+); 2.5 mm MgCl2; a mixture of 0.2 mm dNTP; 2.5 units DNA polymerase Go Taq (Promega); 50 PM primers AK.P78 and AK.P79; and 5 μl of the solution matrix. In PCR used the following conditions: denaturation at 94°C for 2 min; 35 cycles of denaturation at 94°C for 30 s; annealing at 55°C for 30 s and elongation at 72°C for 1 min; with a final stage of elongation at 72°C for 12 min PCR Products were analyzed by electrophoresis on 1.5% agarose gels, as shown in Fig.6: and. strains NE; b. strains of Non-NE.netB-fragment of 384 BP is visible in most strains of C. perfringens isolated from cases of necrotic enteritis of chickens.netBspecific PCR fragment was not observed in any other strain. This witness is stuet about the presence of a gene ofnetBit was a good indicator of virulence of C. perfringens in chickens, and such analysis can be used to detect potentially virulent strains.

Western blot study on the presence of toxin in strains of C. perfringens

Strains of C. perfringens were grown in pre-boiled broths TPG to OD600nm ~0,6. The culture supernatant was obtained by centrifugation at 18000 g for 10 minutes. Supernatant were separated by electrophoresis in LTO-page (NuPAGE®Novex 4-12% Bis-Tris gel, Invitrogen) in working buffer MES-SDS (NuPAGE®MES SDS Running Buffer, Invitrogen). Proteins were transferred to a PDVF membrane (Millipore) and probed rabbit polyclonal antibody against rNetB (Chemicon, USA). The blots showed using the kit for Western blotting ECL (Amersham Biosciences, NJ, USA) and results were recorded on radioautographic film, as shown in Fig.7. Brackets indicate the strains of NE and non-NE C. perfringens. The results of Western blotting confirmed the PCR results of the study: this gene and protein are present in most of the NE strains, but not in strains of non-NE. This method of detecting an antibody-based is another way to detect potentially virulent strains.

Example 8. Protective efficacy of recombinant subunit vaccines NetB

The efficiency of recombinant protein NtB (SEQ ID NO:2) at delivery in the form of a subunit vaccine was tested in a study using vaccination.

A study using vaccination 1193-4

Broiler chickens Ross 308 (Aviagen) were vaccinated with 50 μg of recombinant NetB as antigen per dose in 0.5 ml of aluminum hydroxide as adjuvant. Birds were vaccinated at day 7 and day 14 and were infected on days 20 and 21 1.5 ml oral dose of the strain Clostridium perfringens EHE-NE18. To increase the susceptibility of birds to necrotic enteritis they were fed a diet high in protein, containing fish meal, during the period of infection. Birds were subjected to euthanasia and subjected to necropsy at day 25 for the evaluation points of damage of the intestine due to necrotic enteritis.

Damage was evaluated according to the following scheme:

0 No damage

1 Concistency and loose bowels

2 Focal necrosis or ulceration (1-5 foci)

3 Focal necrosis or ulceration (6-15 foci)

4 Focal necrosis or ulceration (16 or more foci)

5 Necrotic areas with a length of 2-3 cm

6 Diffuse necrosis, typical for field cases

Results

Average scores damage chickens vaccinated with recombinant antigen NetB, and chickens adjuvant control are presented in table 3.

Table 3
The average score of the lesions of chickens vaccinated with recombinant ant the NetB gene
GroupThe number of birdsThe average grade lesionThe number of affected birds (normalized with respect to the group of 10)Average × number (normalized with respect to the group of 10)
Adjuvant control (infection NE18)271,74to 5.9312,37
Vaccination NetB (infection NE18)180,212,10,46

Statistical analysis of the point estimates of the damage using the criterion of Mann-Whitney shows that the distinction between vaccinated NetB group and the group adjuvant control is statistically significant at a confidence greater than 95%.

Example 9. Western blot analysis of sera from vaccinated birds

Serum from vaccinated chickens were analyzed by Western blotting to determine, produce whether chickens serum antibodies to the protein NetB. 4 μg of recombinant NetB-antigen was applied to a well-polyacrylamide gel and subjected to electrophoresis on LTO-PAG. Be the OK to transfer on PVDF membrane by Western-blotting. Serum from vaccinated birds were diluted 1:1000 in 5% of separated milk in TBS/0.5% tween-20 and incubated with the membrane at room temperature for 1 hour. The membrane was washed 3 times with TBS/0.5% tween-20 and then incubated with conjugated with HRP goat antibodies against chicken antibodies (KPL; Cat# 14-24-06; Lot# 050860)diluted 1:10000 in 5% of separated milk in TBS/0.5% tween-20 for 1 hour at room temperature. After incubation the membrane was washed 3 times with TBS/0.5% tween-20. HRP-labeled secondary antibodies were detected by the reagents Western blotting GE Healthcare ECL (Cat# RPN2106) in accordance with the manufacturers instructions. Most birds vaccinated with recombinant NetB, was produced serum antibodies to the protein NetB (Fig), which indicated that the vaccine was able to induce a significant response to the antigen NetB.

Example 10. Repetition methods of vaccination and infection

The study vaccination 1219-1

The results obtained in the study 1193-4, tested for reproducibility by repeating the procedure of vaccination and infection with independently obtained by the party recombinant NetB protein. The results of this recurrence are presented in table 4.

Table 4
Vaccinated NetB group in comparison with adjus Tom as control
GroupThe number of birdsThe average grade lesionThe number of affected birdsAverage × number (normalized with respect to the group of 10)
Adjuvant control (infection NE18)92,33718,1
Vaccination NetB (infection NE18)110,6431,74

Statistical analysis of the point estimates of the damage using the criterion of Mann-Whitney shows that the distinction between vaccinated NetB group and the group adjuvant control is statistically significant at a confidence greater than 95%.

The study vaccination 1250-1

Using the same Protocol of immunization and infection, which was used in previous studies, the live weight of the birds were measured at autopsy. The average mass of each of negative control, positive control, and NetB-vaccinated birds are presented in table 5.

Table 5
Live weight NetB-vaccinated birds compared to the positive control group
GroupThe number of birdsAverage weight (g)The standard deviation (g)
Negative control (without infection)23930105,5
The positive control (infected NE18)22798107,5
NetB-vaccinated (infection NE18)2291485,7

Statistical analysis using unpaired t-test, the mass of birds shows that the difference of masses between NetB-vaccinated group and the positive control group is statistically significant at a confidence greater than 99% (P=0,0004685). Vaccinated birds were protected from delays weight gain, which affects unprotected infected birds positive control.

Example 11. Protective efficacy of alternative vaccines based on NetB

The study vaccination 1250-1, experienced a number of alternative the development of vaccines. Alternative vaccines were: bacterin plus NetB; vector live E. coli expressing NetB; and live C. perfringens (netB-deleteriously). Alternative vaccines described below, and live weight vaccinated birds compared to the positive control group are presented in table 6.

Bacterin plus NetB

Received overnight culture of C. perfringens strain EHE-NE18 (400 ml TPG). The culture was centrifuged and the sediment fraction of the cells and the supernatant was retained. Sediment cells resuspendable in 20 ml of buffered phosphate solution was treated with ultrasound for disclosing the cells and then treated with 0.3% formaldehyde. The supernatant was concentrated by ultrafiltration to a volume of 20 ml and then treated with 0.3% formaldehyde. Equal amounts of treated sludge cells, supernatant and adjuvant solutions were combined and added recombinant NetB protein to a final concentration of 100 µg/ml 0.5 ml of the prepared vaccine was used subcutaneously on one bird per vaccination.

Vector live E. coli expressing NetB

The strain of E. coli CCEC31m (described in WO 2007/025333) transformed with the plasmid expressing thenetBfrom its native promoter. NetB constitutively expressed from this plasmid. Each bird was injected oral dose of 0.5 ml overnight culture (broth, Luria) on day 2.

Live C. perfringens (netB-deleteriously)

i> netB-deleteriously mutant of C. perfringens EHE-NE18 were grown in liquid thioglycolate broth and 0.5 ml oral was inoculable 2-day birds.

Table 6
The live weight of vaccinated birds compared to the positive control group
GroupThe number of birdsAverage weight (g)The standard deviation (g)
Bacterin plus NetB2189597
Vector live E. coli expressing NetB23877127,5
Live C. perfringens (netB-delehanty)23924101

Statistical analysis using unpaired t-test, the mass of birds shows that the difference of mass between the group vaccinated bacteria plus NetB, and a positive control group is statistically significant with a confidence greater than 99% (P=0,003879), the mass difference between the group vaccinated with a live vector E. coli, xpression NetB, and the positive control group is statistically significant with a confidence greater than 95% (P=0,0217605), and the difference in mass between the group vaccinated with a live strain of C. perfringens with deletionism genenetBand the positive control group is statistically significant with a confidence greater than 99% (P=0,0003855). The results indicate that all vaccines can protect birds from delay weight gain that occurs in unvaccinated infected birds.

Specialists in this field will understand that can be made in numerous variations and/or modifications in relation to the present invention, shown in the specific embodiments, implementation, without deviating from the essence and scope of the present invention, described in General terms. Thus, embodiments of should be considered in all respects as illustrative and not restrictive.

All discussed and/or cited publications are included in this description in their entirety.

This application claims priority under US 60/942858, the full contents of which are incorporated in this description by reference.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in this description serves only the purpose of providing the context of the present invention. The discussion should not be construed as an admission that any or all of these documents form part of the basis of prior art or common General knowledge in the field to which the present invention that existed before the priority date of each claim of this application.

Sources of information

1. Essentially purified or recombinant antigenic polypeptide containing:
i) the amino acid sequence represented in SEQ ID NO:2, the polypeptide isolated from Clostridium perfringens, and he has toxin activity,
ii) the amino acid sequence that is at least 40% identical to SEQ ID NO:2, the polypeptide has toxin activity, or
iii) an antigenic fragment of i) or ii).

2. The polypeptide according to claim 1, where the polypeptide contains an amino acid sequence that is at least 90% identical to the sequence presented in SEQ ID NO:2.

3. The polypeptide according to claim 1, where a is a toxoid.

4. The polypeptide according to claim 1, where the polypeptide is a protein that contains at least one other polypeptide sequence.

5. Dedicated or recombinant polynucleotide encoding antigenic polypeptide, where polynucleotide contains:
i) sequence NUS is Evtimov, presented in SEQ ID NO:1, and polynucleotide isolated from Clostridium perfringens, and it encodes a polypeptide that has toxin activity,
ii) a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 4,
iii) a nucleotide sequence that is at least 40% identical to SEQ ID NO:1 and encodes a polypeptide that has toxin activity, and/or
iv) a sequence that hybridizes with any of i)-iii) under stringent conditions, or its reverse complement.

6. The expression vector containing polynucleotide according to claim 5.

7. The expression vector according to claim 6, where polynucleotide functionally linked to a promoter.

8. The expression vector of claim 6 or 7, which is a viral vector or a plasmid vector.

9. A host cell expressing the polypeptide according to any one of claims 1 to 4, wherein a host cell contains polynucleotide according to claim 5 or the expression vector according to any one of p-8.

10. A host cell according to claim 9, which is a bacterium.

11. A host cell of claim 10, where the bacterium is E. coli.

12. The method of producing the polypeptide according to any one of claims 1 to 4, providing for the cultivation of a host cell according to claim 9 or 11, or vector according to any one of p-8 encoding specified polypeptide, under conditions that allow expression of polynucleotide encoding this polypeptide.

13. The method according to item 12, further predusmatriva the in store selection of the specified polypeptide.

14. Essentially purified antibody that specifically binds to a polypeptide according to any one of claims 1 to 4.

15. Immunogenic composition comprising the polypeptide according to any one of claims 1 to 4 to obtain an immune response in a subject, where the introduction of the subject of get immune response to the polypeptide.

16. Immunogenic composition according to item 15, further containing adjuvant and/or pharmaceutically acceptable carrier.

17. The vaccine containing the antigen to obtain an immune response in a subject, where the antigen contains a polypeptide according to any one of claims 1 to 4, and where the introduction of the subject of get immune response to the antigen.

18. The vaccine 17, additionally containing adjuvant and/or pharmaceutically acceptable carrier.

19. A DNA vaccine containing polynucleotide encoding the polypeptide according to any one of claims 1 to 4, to obtain an immune response in the subject, where after the introduction of the subject polypeptide is expressed, and get the immune response to the polypeptide.

20. Method of inducing an immune response in the subject, introducing this subject polypeptide according to any one of claims 1 to 4, polynucleotide according to claim 5, vector according to any one of p-8, the host cell according to any one of p-11, the composition according to item 15 or 16 or vaccine according to any one of PP-19, which when administered to a subject is induced immune response to the polypeptide according to any one of claims 1 to 4.

21. The method according to claim 20, the de a host cell is alive.

22. The method according to claim 20 or 21, where the specified polypeptide, polynucleotide, composition, vector, a host cell can be delivered in ovo.

23. The method of determination, did the subject of the impact of the pathogen that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2, defining the presence or absence of a given polypeptide in the sample obtained from the subject, where the presence of this polypeptide is an indicator of exposure to this pathogen.

24. The method of determination, did the subject of the impact of the pathogen that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2, defining the presence or absence of antibodies in the sample that bind specifically to a polypeptide according to any one of claims 1 to 4, where the presence of antibodies is an indicator of exposure to this pathogen.

25. The method of determination, did the subject of the impact of the pathogen, which expresses polynucleotide containing a nucleotide sequence that is at least 40% identical to SEQ ID NO:1, providing for the determination of the presence or absence of this polynucleotide in the sample obtained from the subject, where the presence of the data is on polynucleotide is an indicator of exposure to this pathogen.

26. The method according to any of PP-25, where the subject is a bird.

27. The method according to p, where the subject is poultry.

28. The method according to item 27, where the subject is a chicken.

29. Method of screening for agonist or antagonist that modulates the activity of the polypeptide according to claim 1, involving the contacting of the polypeptide according to any one of claims 1 to 4 with the connection candidate and determining increases or decreases the specified connection toxin activity of the polypeptide according to claim 1.

30. Method sample testing for toxin activity, providing:
(a) the division of the sample in which suspect the presence of the polypeptide according to any one of claims 1 to 4, at least the first and second parts of the sample,
(b) contacting the first part of the sample with the antibody under 14 and
(c) determining whether the first and second parts of the sample toxin activity,
where the absence of toxin activity in the first part of the sample and the presence of toxin activity in the second part of the sample is indicative of the presence of the polypeptide that is at least 40% identical to SEQ ID NO:2.

31. The method according to item 30, where stage (c) provides independent incubation of the first and second parts of the sample with the cells of the animal, under conditions and for a period of time sufficient for the manifestation of the cytopathic effect of the polypeptide, and determining prisutstvie what I or absence of cytopathic effect in these cells.

32. The use of feed containing antibody at 14 to reduce infection and/or colonization of the animal by a bacterium that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2.

33. The use of drink, containing antibody at 14, to reduce infection and/or colonization of the animal by a bacterium that expresses a polypeptide containing an amino acid sequence that is at least 40% identical to SEQ ID NO:2.

34. Transgenic plant containing the exogenous polynucleotide encoding the polypeptide according to any one of claims 1 to 4.

35. The feed containing the polypeptide according to any one of claims 1 to 4 for feeding the animal to prevent diseases caused by bacteria expressing the polypeptide according to claims 1-4.

36. Drink, containing polypeptide according to any one of claims 1 to 4 for the introduction of the animal to prevent diseases caused by bacteria expressing the polypeptide according to claims 1-4.

37. Method of inducing an immune response against the polypeptide according to any one of claims 1 to 4 for a subject, involving oral administration of this subject transgenic plant according to clause 34, feed on p and/or drink p.

38. The method of providing passive immunity in the offspring of female birds, introducing a polypeptide according to any one of claims 1 to 4, palynol is otide according to claim 5, vector according to any one of p-8, the host cell according to any one of p-11, the composition according to item 15 or 16, the vaccine according to any one of PP-19, transgenic plants according to § 34, feed on p and/or drink up the female bird before oviposition by female birds, containing the offspring, resulting in the offspring provides passive immunity against bacteria that expresses a polypeptide according to any one of claims 1 to 4.

39. The use of the polypeptide according to any one of claims 1 to 4, polynucleotide according to claim 5, vector according to any one of p-8, the host cell according to any one of p-11, the composition according to item 15 or 16, the vaccine according to any one of PP-19, transgenic plants according to § 34, feed on p and/or drink p in the production of medicines for inducing an immune response to the polypeptide according to any one of claims 1 to 4 in the subject.

40. The use of the polypeptide according to any one of claims 1 to 4, polynucleotide according to claim 5, vector according to any one of p-8, the host cell according to any one of p-11, the composition according to item 15 or 16, the vaccine according to any one of PP-19, transgenic plants according to § 34, feed on p and/or drink p as a drug to induce an immune response to the polypeptide according to any one of claims 1 to 4 in the subject.



 

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13 cl, 7 ex

FIELD: medicine.

SUBSTANCE: claimed are expression vectors for obtaining IL-21 in E.coli cells. IL-21 coding nucleotide sequence, included in composition of novel vectors, contains modifications aimed at optimisation of codons and secondary mRNA structure for translation in E.coli. As a result of transformation with claimed vector structures E.coli strains suitable for industrial scale application have been obtained. Methods of wide scale IL-21 production which use said strains have been elaborated, allowing to obtain more than 1g/l of recombinant cytokine.

EFFECT: novel compounds possess useful biological properties.

14 cl, 1 dwg, 12 tbl, 19 ex

FIELD: biotechnology, in particular provision storage.

SUBSTANCE: bacteriocin represents polypeptide isolated from lactobacillus sakei 2512 and is capable to suppress lysteria growth and reproducing. Bactericin has specific amino acid sequence represented in claims. Nuclear acid sequence encoding said polypeptide is disclosed. Also disclosed is a vector including nuclear acid sequence for cloning and/or expression of polypeptide, for example in transformed cells, selected from Lactococcus, Lactobacillus, etc. Method for production of recombinant polypeptide is developed. Claimed bactericin or strain 2512 are used as component of bactericide composition, being capable to suppress growth of gram positive pathogenic bacteria, in particular Listeria monocytogenes.

EFFECT: large scale application of bacteriocin against pathogenic or undesired flora in food industry.

13 cl, 2 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method under the invention provides growing of an inoculate on the basis of BL21(DE3) E. coli strain carrying polymerase T7 RNA gene under control of lacUV5 promoter to the middle of a logarithmic phase of growth on a nutrient medium with the use of glucose in the concentration of 10 g/l as a carbon source at temperature 38°C, agitation at 200 rpm for 3 h; a fermenter is sown with the inoculate at a sowing dose of 10%; the microorganisms are cultured at 38°C, the dissolved oxygen concentration of 80% at maximum mixing rate 1200 rpm, maximum aeration level 1 volume of air /min: 1.2 volumes of a culture fluid, pH 7.00; termination of fermentation after 12-13 hours of culture; deposition of the cells of the E. coli producing strain. At the beginning of culture, 50% (wt %) glucose is added in the middle of the logarithmic phase of growth, 80% (wt %) glycerol is added in portions and the logarithmic phase of culture growth is terminated by fractional additions of 25% (wt %) lactose. After culture, the biomass containing recombinant end proteins in the form of insoluble inclusion bodies is deposited.

EFFECT: invention enables higher yield of the biomass and the end protein of a volume unit of the culture medium.

8 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to recombinant plasmid pVA891-2[mutR], a method of producing mutR gene-mutant strains of Streptococcus pyogenes and recombinant strains of Streptococcus pyogenes. The plasmid pVA891-2[mutR] is constructed based on the vector pVA891-2 which is incapable of replicating in cells of gram-positive microorganisms and contains an erythromycin resistance marker gene, by introducing a Pstl-Sacl fragment of the mutR gene of the Streptococcus pyogenes SF370 strain with size of 520 bps, shown on fig.1. To obtain mutant avirulent strains of Streptococcus pyogenes (SF3 70[mutR], No.97[mutR], No.152[mutR), the obtained plasmid is introduced into corresponding strains of S. pyogenes, which results in violation of the structural region of the mutR gene in the genome of such strains. Selection of clones with an inactivated mutR gene enabled to obtain strains in which exhibition of virulent properties is absent or considerably reduced.

EFFECT: group of inventions can be used in preventive medicine when producing vaccines.

5 cl, 8 dwg

FIELD: medicine.

SUBSTANCE: what is presented is a mutant adenylate cyclase having an amino acid sequence presented as SEQ ID NO: 2 wherein a residue of L-lysine in a position matched with position 432 in SEQ ID NO: 2 is substituted by a residue of L-glutamine, and a version of mutant adenylate cyclase having an amino acid sequence presented as SEQ ID NO: 2 wherein a residue of L-lysine in a position matched with position 432 in SEQ ID NO: 2 is substituted by a residue of L-glutamine containing a deletion, an insertion, an addition and/or a substitution of one or more amino acid residues and having activity of adenylate cyclase according to a sequence presented in SEQ ID NO: 2. What is presented is DNA coding mutant adenylate cyclase. What is also presented is a bacterium of Enterobacteriaceae family containing DNA coding mutant adenylate cyclase that is a producer of proteinogenic L-amino acid. What is disclosed is a method for producing proteinogenic L-amino acid involving bacterium growth in the nutrient medium containing ethanol and/or glycerol as a primary carbon source, and recovery of said L-amino acid from the culture fluid.

EFFECT: invention allows producing high efficacy L-amino acid.

10 cl, 3 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: inventions relate to a Corynebacterium ammoniagenes strain which produces inosine and a method of obtaining inosine using such a strain. The described strain is deposited in KSSM under number 10905 and has a blocked inosine catabolism pathway as a result of inactivation of genes which code ribonucleoside-hydrolase 1 and 2. The method of obtaining inosine involves culturing such a strain followed by extraction.

EFFECT: group of inventions enables to obtain inosine with high output in high concentration.

2 cl, 2 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: obtained is protein complex, possessing GPCRα1L affinity to ligand, which includes GPCRα1A and polypeptide with sequence of amino acids SEQ ID NO:1. Binding of said G-protein-conjugated receptor with polypeptide alters ligand affinity of the receptor. Also claimed are methods of screening agonists or antagonists of G-protein-conjugated receptor with application of transformant, in which said altered G-protein-conjugated receptor is expressed.

EFFECT: carrying out analysis of many supposed G-protein-conjugated receptors with still unknown structure.

12 cl, 3 dwg, 2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology. The invention discloses a composition for coexpression in an eubacterial host cell orthogonal tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synthetase (O-RS), which preferably aminoacylates said O-tRNA with an unnatural amino acid. The disclosed composition consists of two nucleic acid constructs: the first construct contains promoter and terminator nucleotide sequences derived from an E.coli proline tRNA gene and which is derived from the archaea of the expressed sequence, which encodes one O-tRNA or is a polycistronic operon, and the second construct contains a modified E.coli glnS promoter and the expressed nucleotide sequence which encodes the corresponding O-RS. Described is a translation system which includes the disclosed vector constructs, and a method of obtaining the polypeptide of interest which contains an unnatural amino acid in a genetically defined position.

EFFECT: obtaining properties with new properties, which are defined by inclusion of unnatural amino acids into a predetermined position.

54 cl, 8 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to microbiological industry and concerns the bacterium Escherichia coli that is a succinic acid producer, and a method for preparing succinic acid with the use of such bacteria. The described bacteria is modified by changing a nucleotide sequence of a promoter and a ribosome-binding site controlling expression of operon aceEF-lpdA genes in a bacterial chromosome so that expression of the aceE, aceF and lpdA genes in said bacteria is amplified. A method for preparing succinic acid consists in culture of such bacterium in a nutrient medium and recovery of succinic acid from a culture fluid.

EFFECT: invention provides producing a higher amount of succinic acid by microbiological procedure.

4 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention discloses RumC1, RumC2 and RumC3 peptides with antimicrobial activity, particularly on Clostridium perfringens, having molecular weight ranging from 4000 to 4600 Da and isolated from a mutant strain of Ruminococcus gnavus, deposited under number CNCM I-3705. The invention describes polynucleotides which code said peptides, an expression cassette which includes a promoter, said polynucleotide and a terminator sequence, as well as a polynucleotide-based cloning and expression vector. Described is a host organism which expresses said peptides, a composition having antimicrobial activity on Clostridium perfringens and containing a peptide, a host organism or a CNCM I-3705 strain, animal feed which is a food base and a composition.

EFFECT: use of the invention enables to produce a medicinal agent for preventing and treating diseases, particularly necrotic enteritis in pigs or poultry, and human gastrointestinal diseases.

16 cl, 9 dwg, 7 tbl

FIELD: chemistry.

SUBSTANCE: disclosed is an E.coli strain which produces a recombinant protein p30 of the African swine fever virus. The strain is homogeneous, stable during passage and culturing in liquid and solid culture media and is resistant to chloramphenicol.

EFFECT: invention can be used to produce a recombinant protein p30 of African swine fever virus for diagnostic purposes.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: what is presented is a vector for independent expression of at least two nucleic acid molecules which codes the same or different polypeptides of closely related human papilloma virus (HPV) serotypes and naturally contains the sequences having an identity of max. 80% on sites of the length of min. 40 sequential nucleotides. The invention also refers to a host cell containing the presented vector construct for multiple expression of HPV polypeptides, as well as to pharmaceutical compositions for prevention and therapy of the conditions caused by papilloma virus infection containing the vector or the vector-transformed host cells.

EFFECT: if involved in the vector, said molecules are modified so that to reduce percentage of identity of the sequences and reducing size of homology sites to the number of sequential nucleotides max 8.

32 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: hybrid cultured cell strain of the animals Mus museums 13F8 is produced by immunisation of Balb/c mice. The mice are immunised by four introductions of the recombinant antigen preparation F1 100 mcg/mouse. The third post-immunisation day is followed by splenocyte hybridisation of the immunised mice (1x108 cells) and mice myeloma cells P3-X63 Ag/8-653 (1×107cells). Polyethylene glycol (Sigma, the USA) is used as a fusion agent. The hybridisation is followed by hybridoma selection, screening, cloning and cryopreservation. Hybridoma is deposited in the State Collection of Pathogens and Cell Cultures of GKPM-Obolensk, No. N-18.

EFFECT: hybrid cultured cell strain producing monoclonal antibodies is applicable for constructing the based plague agent test systems.

7 dwg, 4 tbl, 8 ex

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