Prophylaxis, treatment and diagnostics of infection caused by p.gingivalis bacteria

FIELD: biotechnologies.

SUBSTANCE: invention describes a composition for induction of immune response against P. gingivalis, which contains an effective amount of one of the above chimeric or hybrid proteins, a prophylaxis method of a state or a disease related to P. Gingivalis, and a method for reduction of incidence or severity of the state or disease related to P. gingivalis with their application. Besides, the invention describes use of the above chimeric or hybrid proteins for determination of antibodies to P. Gingivalis in a biological specimen.

EFFECT: invention allows effective induction of immune response against the specified etiologic agent.

16 cl, 7 dwg, 4 tbl, 22 ex

 

The technical field

The present invention relates to peptides and chimeric or hybrid proteins and the use of these proteins for the preparation of cellular and humoral response for the prevention and treatment of conditions and diseases caused by the bacterium P. Gingivalis.

The level of technology

Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth, leading to resorption of the alveolar process and teeth loss. This disease is an acute problem in health care in all countries and is estimated to affect up to 15% of the adult population, with 5-6% of cases it takes a severe form.

The development and progression of chronic periodontitis is associated with specific gram-negative bacteria in subgingival plaque. The presence of bacteria Porphyromonas gingivalis in subgingival the plaque is closely associated with the disease.

It was reported that the continued presence of the bacteria P. gingivalis in the subgingival plaque of patients with periodontitis after treatment (removal of subgingival deposits and polishing of tooth roots) is closely associated with progressive destruction of the alveolar process. Moreover, it was shown that increasing the number of cells of the bacterium P. gingivalis in subgingival the plaque correlates with disease severity, which is determined by the loss of attachment of the tooth, CH is Bina periodontitis pockets and bleeding upon probing.

It is shown that the infection of the oral cavity P. gingivalis causes destruction of periodontal bone in mice, rats and primates other than man. Also there is increasing evidence of the relationship between periodontal disease and infection with P. gingivalis and cardiovascular diseases and some types of cancer.

It was reported that a number of virulence factors contributes to the pathogenicity of P. gingivalis, including LPS, fimbria, hemagglutinin, hemolysin and extracellular hydrolytic enzymes (especially rq-X and Lys-X - specific proteases), known as "trypsin-like enzymes of P. gingivalis".

This health problem is so great that there is a need for the development of the immune serum, in particular specific antibodies, which provide a strong protective response to infection with P. Gingivalis, and means for its implementation.

One of the problems is that it is unclear how to obtain a strong protective response to infection with P. gingivalis despite the fact that there are many virulence factors from which to choose.

Remain not fully understood and comparative immunogenicity of epitopes of virulence factors, as well as comparative immunogenicity of epitopes specific factor, in particular when it is not clear there still remain unknown epitopes.

One specific problem is that many f the Torah virulence consist of multiple domains, and they are difficult to Express in such a way that the factors had the same conformation, which have factors of P. gingivalis. In addition, when the expression of these domains as separate units, i.e., separately from other domains of virulence factors, they tend to acquire a conformation different from that which are the factors of P. gingivalis.

It is also not known which of the many different ways to modify the immunogenicity of virulence factor most likely to provide a protective immune response.

In the course of the research underlying the present invention, the inventors have discovered peptides whose amino acid sequences identical or homologous amino acid sequence, which forms the site of trypsin-like enzyme from P. gingivalis, where this plot determines the site of the specified enzyme for cleavage of the peptide bond located on the With-end with respect to residues Lys or rq in peptide containing Lys or rq, and built like peptide in chimeric or hybrid protein, which when used as a vaccine provides the best protection of periodontal tissue from destruction than the purified proteinase complex and adesina, educated natural trypsin-like enzyme P. Gingivalis, or killed whole cells.

Brief description of the invention

who according to one aspect of the present invention is proposed hemery or hybrid protein to induce an immune response to infection R. gingivalis, while this protein contains the first peptide, connected directly or through a linker to the second peptide, thus:

(A) said first peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:1;

or

(ii) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:2;

(B) the specified second peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. gingivalis; or

(ii) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. Gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain qA bacteria P. gingivalis.

According to another aspect of the present invention provides chimeric or hybrid protein to induce immune response to infection with P. gingivalis, and this protein contains the first peptide, the connected bar is dstone or through a linker with the polypeptide, when you do this:

(A) the specified peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous sequence represented by SEQ ID No:1;

or

(ii) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:2;

and

(B) the specified polypeptide contains:

(i) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. Gingivalis; or

(ii) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain rq-X-proteinase bacteria P. Gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain q bacteria P. gingivalis.

According to another aspect, the present invention provides a peptide to stimulate immune response to infection with P. gingivalis, and the peptide contains the sequence:

(i) which is identical or homologous to one of the sequences presented in SEQ ID No: 64-66; and

(ii) which is identical or homologous after which outermost, presented in SEQ ID No: 67 or 68.

According to one aspect, the peptide containing the sequence that is identical or homologous to one of the sequences presented in SEQ ID nos: 64-68, may be provided in the form of chimeric or hybrid protein in which the peptide is connected directly or through a linker to the second peptide, the second peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous sequences adhesive; domain Lys-X - proteinase bacteria P. gingivalis; or

(ii) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. Gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Nada bacteria P. gingivalis.

According to another aspect of the present invention provides a composition, such as antigenic composition, in particular a vaccine composition comprising a chimeric or hybrid protein or peptide, described above, possibly in combination with an auxiliary substance.

According to this aspect of the present invention also provides a method of preventing or is Nigeria frequency or severity of conditions or diseases, caused by infection with P. gingivalis, the entity that includes the introduction of a specified subject chimeric or hybrid protein as described above or a composition as described above.

According to this aspect of the present invention also provides the use of chimeric or hybrid protein as described above or a composition as described above in the composition of the medicinal product or for the preparation of a medicinal product for preventing or reducing the frequency or severity of conditions or diseases caused by infection with P. gingivalis, the subject.

According to another aspect of the present invention provides an antibody, particularly a monoclonal antibody raised against a chimeric or hybrid protein or peptide, described in detail above.

According to this aspect of the present invention also provides a method of preventing or reducing the severity of conditions or diseases caused by infection with P. gingivalis, the entity that includes the introduction of a specified subject antibodies as described above.

According to this aspect of the present invention also provides the use of antibodies as described above in a drug and money or for the preparation of a medicinal product for preventing or reducing the frequency or severity of diseases or conditions caused by infection R. Gingivalis, the subject.

According to another aspect of the present invention also provides a nucleic acid molecule containing a nucleotide sequence encoding a chimeric or hybrid protein as described above, may functionally connected to at least one regulatory element.

According to this aspect of the present invention also provides a vector containing a nucleic acid molecule, as well as prokaryotes or eukaryotes containing the specified nucleic acid molecule.

According to this aspect of the present invention also provides a method of preventing or reducing the severity of conditions or diseases caused by infection with P. Gingivalis, the subject, which includes an introduction to the subject the nucleic acid molecule as described above, a vector as described above or cells, prokaryotes or eukaryotes, as described above.

According to this aspect of the present invention also provides the use of nucleic acid molecules as described above, a vector as described above or cells prokaryotes or eukaryotes as described above in the composition of the medicinal product or for the preparation of a medicinal product for preventing or reducing the severity of diseases or conditions caused by infection with P. Gingivalis, sub the KTA.

According to another aspect of the present invention provides a method of diagnosing or monitoring a condition or disease caused by infection with P. Gingivalis, the entity that includes the use of chimeric or hybrid protein as described above for the detection of antibodies to P. gingivalis in a sample of biological material obtained from the specified entity.

According to this aspect of the present invention also provides the use of chimeric or hybrid protein as described above for the detection of antibodies to P. gingivalis in a sample of biological material obtained from the specified entity.

According to another aspect of the present invention provides a method of diagnosing or monitoring a condition or disease caused by infection with P. Gingivalis, the entity that includes the use of antibodies as described above to determine the presence of P. gingivalis in the sample of biological material obtained from the specified entity.

According to this aspect of the present invention also provides the use of antibodies as described above to determine the presence of P. gingivalis in the sample of biological material obtained from the specified entity.

According to another aspect of the present invention provides use of a peptide containing part of the sequence and the full sequence, which is identical or homologous to the sequence Lys-X or AGD-X-proteinase bacteria P. gingivalis, or nucleic acid that encodes the specified peptide, to obtain a chimeric or hybrid protein to stimulate immune response to infection with P. gingivalis. According to this aspect of the indicated peptide may contain the sequence represented in one of SEQ ID No: 17, 18, 25, or 26.

Brief description of drawings

The Figure 1 shows the results of staining of Kumasi gel after SDS-PAGE with recombinant proteins CRA. Band 1=KAS2-KLA1, band 2=KLA1, band 3=KsA1, band 4=KAS1-KsA1. The molecular weight markers are indicated in kDa.

The Figure 2 shows the detection antibody peptide KAS2 and killed by formaldehyde cells of P. gingivalis W50. (A) Peptide KAS2 investigated in the reaction with serum produced by formalin killed cells of P. gingivalis W50 (FK-W50), recombinant proteins KAS1-KsA1, KAS2-KLA1, and synthetic conjugate KAS2-DT PBS and ELISA method. (V) formalin killed cells of P. gingivalis W50 was investigated in the reaction with serum produced by formalin killed cells of P. gingivalis W50 (FK-W5D) recombinant proteins KAS1-KsA1, KAS2-KLA1, KLA1 PBS using ELISA method. Antibody responses were expressed in the resulting titer ELISA at OD415 for vicecomodoro background levels, each value of the title represents the mean ± standard deviation of three values.

The Figure 3 shows razor the tion of the bones of the molars of the upper jaw in a horizontal plane, caused by infection with P. Gingivalis in mice immunized separately recombinant proteins and recombinant chimeric proteins, formalin killed cells of P. gingivalis and auxiliary substance (PBS, IFA), or uninfected oral (unimmunized) mice. In the figure 3 KAS2-KLA1 depicted in the form of AS2-LA1, KLA1 depicted as LA1, KAS1-KsA1 depicted as AS1-sA1, KsA1 depicted as sA1. The bone resorption was expressed in square millimeters (mm2from the cement-enamel junction (CEJ) to the alveolar crest of bone (ABC) buccal side of each molar of the upper jaw on the left and right sides of the jaw. The data had a normal distribution according to the criterion of homogeneity of variance of Leuven, and are presented as mean (n=12) in mm2; they were analyzed using one-way analysis of variance and T3-criterion of Dunnet. (* indicates group, which was significantly (P<0.001) less bone loss compared with the control (infected) group; t represents a group in which there were significantly (P<0,001) large bone resorption compared with a group of AS2-LA1).

The Figure 4 shows the responses of subclasses of antibodies in the serum of immunized mice in a model of periodontitis. Serum of mice: A (to oral immunization) and (after oral immunization) immunized with recombinant proteins KsA1, KL1, KAS1-KsA1 and KAS2-KLA1 and formalin killed cells of P. gingivalis strain W50, used for the production of ELISA with formalin killed P. gingivalis strain W50 as immobilized antigen. The responses of IgG antibodies (black bars), lgG1 (gray bars), lgG2a (the white stripes), lgG2b (bars with horizontal striation), lgG3 (strips with diagonal striation) expressed in the form of ELISA titer (log 2) minus the background level, each value of the title represents the mean ± standard deviation of three values.

The Figure 5 shows the results of PEPSCAN analysis of peptide-specific reactivity of the antibodies to overlapping peptides representing the sequence 433-468 peptide KAS2. (A) KAS2 overlapping peptides (overlapping peptides) in the reaction with sera KAS1-KsA1 (the white stripes), KAS2 - KLA1 (black bars). (C) KAS2 overlapping peptides (overlapping peptides) in the reaction with the serum to the conjugate KAS2-DT. Each strip shows antibody reactivity (optical density [OD] at 415 nm).

In Figure 6 it is shown that the hybrid AS2-LA1 causes an immune response in outbred mice, which are recognized by whole cells of P. Gingivalis and complex RgpA-Kgp.CD1 outbred mice were immunized hybrid AS2-LA1 (50 mg/mouse) and the obtained serum was used for staging the ELISA with AS2-LA1 (A), formalin killed cells of P. gingivalis strain W50 (B) complexa RgpA-Kgp (S) as the immobilized antigens. This Figure KAS2-KLA1 depicted in the form of AS2-LA1. Determined the titer of each isotype of immunoglobulin and the data obtained were expressed as obtained titer ELISA ('000) net double the background level, where each value of the title represents the mean ± standard deviation of three values.

The Figure 7 depicts the CRA proteases. KAS2 [Asn433-Lys468], (A) KAS4 [Asp388-Val395] (), KAS5 [Asn510-Asp516] (C) and KAS6 [lle570-Tyr580] (D).

Detailed description of the invention

It should be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual signs mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

The authors of the present invention have found that the sites of trypsin-like enzymes bacteria P. gingivalis, which are arranged on both sides or otherwise determine the catalytic or active site of cleavage of the peptide bond, have a high immunogenicity and indeed sufficient to ensure that the humoral response to infection with P. gingivalis. In particular, it was found that the chimeric or hybrid protein containing one or more of these sites, provides protection from destruction of the alveolar ridge of the jaw is greater than the immune sivaram is a, raised against whole cells, and other immunogen. This discovery is particularly unexpected because until now believed that the catalytic domain of trypsin-like enzymes bacteria P. gingivalis is relatively weakly immunogenic.

According to one aspect of the present invention provides chimeric or hybrid protein to stimulate response to infection with P. gingivalis, and this protein contains the first peptide, connected directly or through a linker to the second peptide, thus:

(A) said first peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:1;

or

(ii) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:2;

and

(B) the specified second peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. gingivalis; or

(ii) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. gingivalis; or

p> (iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Nada bacteria P. gingivalis. In the present description, the term "peptide" refers to a sequence containing up to 40 amino acid residues, preferably 5-40 amino acid residues.

According to one implementation variant, instead of the term "second peptide" is used the term "polypeptide". The term "polypeptide" refers to amino acid sequence comprising a length of at least about 40 amino acid residues.

Thus, according to another aspect provides chimeric or hybrid protein to stimulate immune response to infection with P. gingivalis, while this protein contains a peptide that is connected directly or through a linker with the polypeptide, thus:

(A) the specified peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:1;

or

(ii) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:2;

and

(B) the specified polypeptide contains:

(i) part of a sequence or a full sequentially is th which is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. Gingivalis; or

(ii) part of a sequence or a fullsequence that is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Nada bacteria P. gingivalis.

According to another aspect of the present invention provides a peptide to stimulate immune response to infection with P. Gingivalis selected from the group consisting of:

(i) sequence that is identical or homologous sequence represented in one of SEQ ID No: 64-66; and

(ii) a sequence that is identical or homologous to the sequence presented in SEQ ID No: 67 or 68.

According to one aspect of the invention, in which the peptide contains the sequence of SEQ ID No: 64-68, this peptide may be provided in the form of chimeric or hybrid protein in which the peptide is connected directly or through a linker to the second peptide. According to this variant implementation of the second peptide of a chimeric or hybrid protein contains:

(i) part of a sequence, or p is low sequence, which is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. gingivalis; or

(ii) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequence adhesives domain q bacteria P. gingivalis.

According to the above implementation, the term "polypeptide" is used instead of the term "second peptide". Thus, according to another aspect provides chimeric or hybrid protein to stimulate immune response to infection with P. gingivalis, while this protein contains a peptide that is connected directly or through a linker with the polypeptide, thus:

(A) the specified peptide contains:

(i) sequence that is identical or homologous sequence represented in one of SEQ ID No: 64-66; or

(ii) a sequence that is identical or homologous to the sequence presented in SEQ ID No: 67 or 68; and

(C) the specified polypeptide contains:

(i) part of a sequence or a full sequence that is identical or homologic the second sequence adhesieve domain Lys-X-proteinase bacteria R. gingivalis; or

(ii) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Nada bacteria P. gingivalis.

In the present description, the term "homolog" of a peptide or polypeptide means a peptide or polypeptide containing the amino acid sequence that is homologous or has similarity with the amino acid sequence specified first peptide or polypeptide, preferably at least 90% sequence preferably at least 95% sequence more preferably at least 98% sequence when compared with the method of the BLAST algorithm, and the algorithm parameters are selected so as to obtain maximum compliance with the analyzed sequence along the entire length of sequence comparison. The term "sequence similarity" refers to an exact match between the amino acids of the compared sequences. Such homologues can be obtained from natural variants or isolates Lys-X - proteinase or AGD-X-proteinase P. gingivalis. In another case, homolog may be a variant of the peptide or polypeptide Lys-X-proteinase or AGD-X-proteinase R. gingivalis, having a "conservative substitution", in which one or more amino acid residue replaced without altering the overall conformation of the function of the peptide or polypeptide, including, but not limited to, replacement of amino acids to an amino acid with similar properties. Amino acids with similar properties are known in the art. For example, a polar/hydrophilic amino acids that can be used interchangeably, include asparagine, glutamine, serine, cysteine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; non-polar/hydrophobic amino acids that can be used interchangeably, include glycine, alanine, valine, leucine, isoleucine, Proline, tyrosine, phenylalanine, tryptophan, and methionine; the acidic amino acids that can be used interchangeably, include aspartic and glutamic acids, and essential amino acids which can be used interchangeably, include histidine, lysine and arginine. Preferably such variants with conservative substitutions are less than 20, more preferably less than 15, more preferably less than 10 and most preferably less than 5 substitutions of amino acids.

Plot trypsin-like enzyme of the bacteria P. Gingivalis, in particular Lys-X-proteinase (CRA) or AGD-X-proteinase (RgpA), which determines the site for cleavage of the peptide bond, you can determine the agreement is but the present description, in particular, the relative Figures 7 and Example 9, which is an example of a method for predicting the three-dimensional conformation of the catalytic site in the form in which it is present in P. Gingivalis for Lys-X-proteinase. Example 10 provides a method for modeling three-dimensional conformation rq-X-proteinase.

According to some versions of the invention, chimeric or hybrid protein, or the first and second parts of the peptide, can be formed from peptidomimetic. Peptidomimetic is a molecule that mimics one or more property of the peptide, such as conformation, and which consists of amino acid residues, some of which may not exist in nature.

Identifying immunogenic sites of the catalytic site, the inventors have determined the sequence of the different peptide immunogens, against which can be acquired immune response. In particular, it identified the following 6 sections, which are arranged on both sides or otherwise, determine the catalytic site: KAS1/RAS1, KAS2/RAS2, KAS3/RAS3, KAS4/RAS4, KAS5/RAS5 and KAS6 (see Table 1). Taking into account the information received, the inventors analyzed a database of protein sequences to identify peptides that have homology with amino acid sequences that form the sites located n the both sides of the catalytic site, and so are immunogenic epitopes found in the P. gingivalis. The sequences of these peptides are determined by structural formulas.

Table 1. Sequences that are located on both sides of the active site of the RIC and RgpA.

PlotThe CRA Lys - X (numbering according to SEQ ID No.62)q Lys - X consensusRgpA Arg-X (numbering according to SEQ ID No.61)RgRA Arg-X consensus
PAS1K/ PAS1RPAS 1 (432-453)LNTGVSFANYT AHGSETAWAD R (SEQ ID NO: 30)PAS1R (426-446)FNGGISLANYTG HGSETAWGT (SEQ ID NO: 34)
KAS1/ RAS1KAS1 (432-454)LNTGV[G/S]FAN YTAHGSET[S/A] WADP[S/L] (SEQ ID NO: 27)RAS1 (426-448)FNGGISL[V/A]NY TGHGSETAWGT SH (SEQ ID NO: 31)
KAS2/ RAS2KAS2 (433-468)NTGV[G/S]FANY TAHGSET[S/A] WADP[S/L][L/V]T [A/T][T/S]Q[V/L]K ALTNK[D/N]K (SEQ ID NO: 28)RAS2 (427-462)NGGISL[V/A]NYT GHGSETAWGTS HFGTTHVKQLTN SNQ (SEQ ID NO: 32).
KAS3/RAS3KAS3 (436-455)V[/S]FANYTAH GSET[S/A]WAD P[S/L][L/V] (SEQ ID NO: 29) RAS3 (430-449)ISL[V/A]NYTQH© SETAWGTSHF (SEQ ID NO: 33)
KAS4/RAS4KAS4 (388-395)D[S/Y][Y/S]WN[P /S][K/Q][I/V] (SEQ ID NO: 64)RAS4 (379-386)EGGPSADN (SEQ ID NO: 67^
KAS5/RAS5KAS5 (510-516)NSYWGED (SEQ ID NO: 65)RAS5 (508 to 514)[N/D]Q[S/Y]WA[S/ P]P (SEQ ID NO: 68)
KAS6KAS6 (570-580)IGN[V/I]THIGAH Y (SEQ ID NO: 66)

The inventors have found that chimeric proteins containing these peptides, can be used in different ways. For example, according to the present description, some of them induce a humoral response, which provides high protection in the treatment or prevention of bone destruction that is observed in periodontitis. These peptides can also be used to conduct diagnostic tests, in which they can detect or observe the characteristics of the patient serum that enables you to determine with the patient and, if he is infected, do I need treatment and if the treatment was done - did it effect the poor.

It should be understood that the plot trypsin-like enzyme from P. gingivalis, which determines the site of the enzyme responsible for cleavage of the peptide bond located at the end from the residues Lys or rq, does not contain the full sequence Lys-X-proteinase or rq-X-proteinase.

In the present description, the terms "heterologous protein" or "chimeric or hybrid protein" refers to a protein that consists of functional units, domains, sequences or portions of the amino acids obtained from various sources, or obtained from one source and collected in such a way that their organization is different from the molecules of cotapos unit, domain, sequence or plot were obtained. A common feature of chimeric or hybrid proteins according to the present invention is that they contain at least one peptide containing amino acid sequence which is identical or has homology with the sequence of the trypsin-like enzyme from P. gingivalis, which determines the catalytic site responsible for the cleavage of peptide bonds.

According to a preferred implementation variant, if the first peptide includes a peptide of the plot KDR [432-468], it preferably represents (i) a peptide contains a sequence selected from: VSFANYT and VGFANYT, valueproposition, sequence selected from GVSFANYT, GVGFANYT, VSFANYTA and VGFANYTA; or (i i) the peptide contains a sequence selected from:1 ETAWAD, ETSWAD, TAWADP and TSWADP, preferably a sequence selected from: SETAWAO, SETSWAD, ETAWADP, ETSWADP, TAWADPL and TSWADPL, more preferably a sequence selected from: GSETAWAD, GSETSWAD, SETAWADP, SETSWADP, ETAWADPL, ETSWAD PL, TAWADP LL and TSWADPLL. More preferably, the peptide is chosen among peptides KAS1 [432-454]; KAS2[433-468] and KAS3[436-455], shown in Table 1. In another case, the peptide can be a peptide PAS1K[432-453], also known as PAS1(K48), described in international patent application PCT/AU 98/00311 (WO 98/049192). Pointers sequences corresponding to the peptides listed in Table 3.

Similarly, according to another preferred implementation variant, in which the first peptide includes a peptide of the plot RgpA [426-462], this peptide is preferably chosen among peptides RAS1[426-448], RAS2[427-462] and RAS3[430-449], shown in Table 1. In another case, the first peptide may be a peptide PAS1 R[426-446], also known as PAS1(R45), described in international patent application PCT/AU 98/00311 (WO 98/049192).

In chimeric or hybrid protein according to the present description of the second peptide can be a peptide adhesieve domain trypsin-like enzyme of the bacteria P. gingivalis, such as Lys-X - proteinase (CRA) or AGD-X-crack is back (RgpA) or Nada (see Table 2). In some cases, these domains are known as hemagglutinin. In the Lys-X-proteinase preferred domains are KA1, CA, KAZ, CA, CA shown in Table 2. Arg-X-proteinase preferred domains are RA1, RA2, R3 and RA4 are shown in Table 2. In Nada preferred domains are q1, q1* and q1**.

Table 2. Adhesive domains proteases CRA and RgpA.

A1sA1LA1A2A3A4A5
The CRA Lys-X-proteinase SEQ ID No. 62KA1 (738-1099) SEQ ID NO: 35KsA1 (759-989) SEQ ID NO:36KLA1 (751-1056) SEQ ID NO:37KA2 (1157-1275) SEQ ID NO:40CA (1292-1424) SEQ ID NO:41KA4 (1427-1546) SEQ ID NO:42KA5 1 (1548-1732) SEQ ID NO:43
RgpA Arg-X-proteinase SEQ ID No. 61RA1 (720-1081) SEQ ID NO:38RsA1 (831-971) SEQ ID NO:39-RA2 (1139-1257) SEQ ID NO: 44RA3 (1274-1404) SEQ ID NO: 45RA4 (1432-176) SEQ ID NO: 46 -
HagA SEQ ID NO. 63HagA1 (26-351) (SEQID NO: 80), HagA1* (366-625) (SEQID NO:81), HagA1** (820-1077) (SEQID NO:82) or HagA1** (1272-1529) (SEQID NO:82)

In addition to improved humoral response to the peptide according to the present invention, such as KAS1, KAS2, KAS3, KAS4, KAS5 and KAS6 or RAS1, RAS2 and RAS3, RAS4 and RAS5, when enabled in conjunction with the peptide in the composition of the chimeric or hybrid protein, adhesively domain also contains immunogenic epitopes, which creates various features to run a protective immune response. The discovery that immunogenic epitopes adhesieve domain are stored in a form appropriate to the trypsin-like enzyme of the bacteria P. gingivalis, while ensuring as part of a chimeric or hybrid protein is unexpected.

It should be understood that in these embodiments of the invention, chimeric or hybrid protein may contain any one or more peptide selected among KAS1/RAS1, KAS2/RAS2, KAS3/RAS3, KAS4/RAS4, KAS5/RAS5 and KAS6/RAS6, jointly with any one or more adhesives domain trypsin-like enzyme of the bacteria P. gingivalis, in particular, with any one or more adhesives domain Lys-X-proteinase (KA1, CA CA, CA and CA) or AGD-X-proteinase (RA1, RA2, RA3 and RA4) or domains Nada, Nada, Nada* and Nada**.

Also keep in mind that adhesively domain does not necessarily have to be completely the domain of trypsin-like enzyme of the bacteria P. Gingivalis. For example, adhesively domain can represent a fragment of such a domain, in particular, preferred fragments are fragments of KsA1 and KLA1 domain A1 Lys-X-proteinase (see Table 2). If the domain is a fragment adhesieve domain, it usually contains one or more specific adhesieve domain epitope. Pointers sequences corresponding to adesina related peptides, are shown in Table 3.

According to one implementation variant, the second peptide or polypeptide contains the sequence represented in one or more of SEQ ID No: 69-79 or one or more of SEQ ID No 83-85.

Chimeric or hybrid protein according to the present invention may contain additional peptides selected from the CRA area [432-468]Lys-X - proteinase, and/or one or more additional peptide selected from the RgpA[426-462] rq-X-proteinase.

According to preferred variants of implementation of the present invention chimeric or hybrid protein contains one or more of the KAS1, KAS2, KAS3, KAS4, KAS5 and KAS6, or one or more of RAS1, RAS2, RAS3, RAS4 and RAS5 together with KsA1 or KLA1.

Thus, when according to some variants of realization of chimeric or hybrid protein may contain at least one additional peptide, while this additional peptide contains:

(i) part of a sequence or a full sequence that is identical or homologous sequence represented by SEQ ID No:1; or

(ii) part of a sequence or a full sequence that is identical or homologous to the sequence presented in SEQ ID No:2; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. gingivalis, or

(iv) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. gingivalis; or

(v) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain q bacteria P. gingivalis.

Other examples of domains units, sequences or sections that can be included in the composition of the chimeric or hybrid protein according to the present description, include domains for binding to receptors or ligands, such as binding site Fc or Fc receptors, domains for longer half-lives, such as albumin, or domains to facilitate expression or separation is of chimeric or hybrid protein.

In chimeric or hybrid proteins according to the present invention the C-terminal residue of the first peptide may be covalently linked to N-terminal residue of the polypeptide adhesieve domain or N-terminal residue of the first peptide may be covalently linked to the C-terminal residue adhesieve domain polypeptide. According to this variant of realization of the first peptide and the polypeptide adhesieve domain is denoted by the term "directly connected" or "adjacent".

According to other variants of realization of chimeric or hybrid protein contains a linker sequence to connect the first peptide with a polypeptide adhesieve domain. The linker as amino acid, and amino acid, can connect peptide and the polypeptide. Preferably, the linker is not immunogenic. Suitable linkers can contain up to 15 amino acids in length, but preferably contain less than five amino acids in length. The linker may serve to bring the first peptide and polypeptide itinalaga domain in closer spatial arrangement than the typical trypsin-like enzyme of the bacteria P. gingivalis. In another case, he can pass the first peptide and the polypeptide, adhesivos domain.

Chimeric or hybrid proteins according to the present invention can be obtained by using systems Express the recombinant molecules (for example, methods recombinant DNA) or by chemical synthesis (e.g., solid phase peptide synthesis). These methods are well known in the art.

Heterologous or chimeric protein is particularly promising, because it improves the humoral response compared to the response caused by applying the first or second peptide component chimeric or hybrid protein.

The inventors have found that chimeric proteins containing such peptides, have many useful properties. For example, according to the present description, some of them induce a humoral response, which is vysokoproduktivnyi for the treatment or prevention of bone destruction that occurs in chronic periodontitis. These peptides can also be used to conduct diagnostic tests, in which they identify or observe the characteristics of the serum of the patient, whereby is determined with the patient, and if he is infected, do I need treatment or if treatment has already been carried out, whether it is effective.

According to one implementation variant chimeric or hybrid protein induces a protective immune response, which at least reduces to a minimum level or limits the destruction of the connective tissue caused by infection with P. gingivalis. who according to one implementation variant of the protective immune response to at least reduce to a minimum level or limits the destruction of bones, caused by infection with P. gingivalis. In the present description discusses a model system for the measurement of bone destruction caused by infection with P. gingivalis. Normally protective immune response is primarily humoral response. According to some variants of the implementation of a protective immune response also includes cell response.

The present invention also provides a composition comprising a chimeric or hybrid protein described above. Typically, the composition is antigenic or immunogenic. In particular, the present invention provides a composition suitable for stimulating a protective or therapeutic immune response to infection with P. gingivalis, containing chimeric or hybrid protein, optionally together with an adjuvant. This composition may also contain another component to modulate or enhance the immune response. According to one implementation variant, the composition is represented in the form of a vaccine.

There are various excipients that can be used in conjunction with vaccines. Excipients allow through modulation of the immune response to achieve a more robust immunity when using smaller amounts or doses of antigen as compared with the use of only vaccine. Examples of adjuvants include incomplete the excipient's adjuvant (IFA), excipient 65 (containing peanut oil, monooleate manned and aluminum monostearate), oil emulsions, excipient, Ribi, a surfactant, a polyol, polyamine, avidin, Quil A, saponin, MPL, QS-21, mineral gels such as aluminum salts and calcium, nanoparticles such as hydroxyapatite, calcium phosphate, aluminum salts, Sugar oligomers and polymers, such as mannan, chitosan. Other examples include emulsion oil-in-water", such as SAF-1, SAF-0, MA, Seppic ISA720, and other aerosol auxiliary substances, such as ISCOM™ and ISCOM matrix™. Extensive, but not exhaustive list of examples of auxiliary substances is presented in Sokh and Coulter 1992 [In: Wong WK (ed.) Animals parasite control utilising technology. Bocca Raton; CRC press, 1992; 49-112]. In addition to the excipients in the composition of the vaccine may include pharmaceutically acceptable carriers, excipients, buffers or diluents, as needed. One or more dose of the vaccine containing excipient, you can enter in the preventive or therapeutic purposes developed for the treatment of periodontitis.

According to a preferred implementation variant chimeric or hybrid protein combined with mucosal auxiliary substance and administered orally, transbukkalno or nasal. Examples of mucosal adjuvants include nanoparticles, cholera toxin and those who maleberry toxin E. Li, non-toxic B-subunit of these toxins, mutant forms of these toxins, which have low toxicity. Other methods that can be applied for the delivery of antigenic proteins oral/transbukkalno/nasal, include the embedding of the protein or in the absorption of the protein on the particles of the biodegradable polymer (e.g., acrylates or polyether) method microencapsulate to improve uptake of the microspheres from the gastrointestinal tract or other mucous membranes and to protect proteins from degradation. Liposome, ISCOM™ hydrogels represent examples of possible methods that can be improved by including guides molecules such as LTBJ STV, or pectins for the delivery of antigenic proteins to the immune system, mucous membranes. In addition to antigen, proteins and mucosal excipients or delivery system, the vaccine composition may include conventional pharmaceutically acceptable carriers, fillers, shell, dispersion media, antibacterial or antifungal agents, and buffers or diluents, if necessary.

According to this aspect of the present invention also provides a method of preventing or reducing the incidence or severity of condition or disease caused by infection with P. Gingivalis, the entity that includes the introduction of the decree is nomu subject chimeric or hybrid protein, as described above, or a composition as described above. The subject may be a human or other animal, and preferably is a human.

Usually the condition or disease caused by infection with P. gingivalis, is a chronic periodontitis, but also it can be a destruction of the bones, in particular, the destruction of the bone of the alveolar process or the coronary arteries.

There are many ways the vaccine composition to a human or animal, including but not limited to these, percutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal sublingual, transbukkalno and oral route of administration. These techniques are particularly useful for vaccination.

According to another aspect of the present invention provides an antibody, preferably a monoclonal antibody raised against a chimeric or hybrid protein according to the detailed description above.

These antibodies can be obtained by standard methods and used for passive immunization of a subject. Accordingly, according to this aspect of the present invention also provides a method of preventing or reducing the severity of the condition or disease caused by P. gingivalis, in a patient, which includes the introduction of pale is the antibody as described above.

According to another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric or hybrid protein according to the detailed description above, optionally functionally connected to at least one regulatory element. According to one implementation variant nucleic acid is provided in a dedicated or substantially purified form.

The nucleic acid molecule can be, for example, built into the appropriate expression vector for production of the chimeric protein in the form of a recombinant protein by introduction of a vector in a prokaryotic or eukaryotic cell host. For successful recombinant expression vector, it is necessary that the expression vector contained regulatory elements of transcription and translation that are compatible with and recognized by specific cell of the host used for expression. For expression recombinant protein you can use different types of host cells, which include, but are not limited to these, bacteria, transformed with vectors based on bacteriophage, plasmid and kosmidou; yeast containing yeast vector, mushrooms, containing the vectors on the basis of mushrooms; line of insect cells infected with virus (e.g., baculovirus), and lines of mammalian cells is affected, transfetsirovannyh expression vectors based on the plasmid or virus, or infected with recombinant virus (e.g. vaccinia virus, adenovirus, adeno-associated virus, retrovirus, and so on).

Methods known in the field of molecular biology, the expression vector can be administered in a number of promoters and enhancers to increase expression of recombinant protein, provided that the increased expression of amino acid sequences compatible (for example, non-toxic) with a specific used the host-cell.

The choice of promoter depends on the expression system. Promoters vary in strength, i.e. the ability to enhance transcription. Usually it is desirable to use a strong promoter to achieve a high level of transcription of the coding nucleotide sequence of the expression of the recombinant protein. For example, known in the art promoters of bacteria, phages or plasmids, which enjoyed a high level of transcription in the system of host cells, including E. Li, include a promoter, lac operon, trp operon, the promoter goes, the rRNA promoter, promoters PRand PL, lacUV5, ompF, bl, lpp, etc., and can be used to provide transcription built nucleotide sequence that encodes the amino acid sequence.

Other regulatory elements effective transcription or translation include enhancers and regulatory signals. Sequence-enhancers are DNA elements, which, apparently, increase the efficiency of transcription is relatively regardless of the location and orientation relative to the nearest coding nucleotide sequence. Thus, depending on the system expression vector host cell enhancer may be placed in the forward or reverse direction relative to the embedded coding sequences to increase the efficiency of transcription. For regulating the expression of the coding sequence, you can use other regulatory sequences, for example, signals the initiation of transcription or translation.

According to another implementation variant, the vector may be a viral or bacterial vaccine vector and can be used for recombinant viral vaccines, recombinant bacterial vaccine, recombinant attenuated bacterial vaccine or an inactivated recombinant viral vaccine. Vaccinia virus is the most famous example of infectious virus in the art, which has been modified by the methods of engineering so that they are capable of expression is activated vaccine antigens obtained from other organisms. Recombinant live virus of cowpox, which weakened or otherwise processed so that it does not cause disease itself, is used to immunize the host. Subsequent replication of the recombinant virus in the host organism provides constant stimulation of the immune system, antigens, vaccines, through providing long-lasting immunity.

Other live vaccine vectors include adenovirus cytomegalovirus and, preferably, poxviruses, such as vaccinia virus [Paoletti and Panicali, US Patent No. 4603112] and attenuated strains of Salmonella [Stocker et al., U.S. patent 5210035; 4837151; and 4735801; and Curtiss et al., 1988, Vaccine 6:155-160]. Live vaccines are particularly preferred, because they are constantly stimulate the immune system, which can provide very long-lasting immunity. If the immune response is protective against subsequent infection by P. gingivalis, itself a live vaccine can be used in a prophylactic vaccine against infection with P. gingivalis. In particular, the live vaccine can be created on the basis of commensal bacteria in the oral cavity. This bacterium can be transformed with the vector carrying the recombinant chimeric protein, and then it can be used to check the oral cavity, in particular the mucous membrane of the oral cavity. After colonization by the bacteria to the mucosa of the glasses oral expression of the recombinant protein will stimulate the production of neutralizing antibodies lymphoid tissue of the mucous. To further illustrate the present embodiments the nucleotide sequence encoding the chimeric proteins using molecular biology techniques known in the art, can be inserted into the genomic DNA vaccine virus to the website, which promotes the expression of epitopes, but has not had a negative effect on the growth or replication of the vector-based vaccine virus. The obtained recombinant virus can be used as the immunogen in the vaccine composition. Similar methods can be used to create a dosage form of inactivated recombinant viral vaccine, except when the recombinant virus is inactivated by chemical methods known in the art, before using as immunogen and without significant effect on the immunogenicity of the expressed immunogen. Inactivated recombinant vaccine can be prepared with the inclusion of appropriate excipients to enhance the immune response to vaccine antigens.

The present invention also provides use of a nucleic acid molecule containing a nucleotide sequence encoding a chimeric or hybrid protein according to the invention directly in the form of a dosage form of the vaccine. The nucleotide sequence is eljnosti, encoding chimeric proteins that are functionally connected to one or more regulatory element, can be entered directly for vaccination of the patient ("targeted gene transfer") against pathogenic strains of P. gingivalis. Directed gene transfer imparted to the patient, leading to the expression of the genetic material in cells inoculated with the patient, such as endothelial cells, and tissues of major organs was shown using methods known in the art, such as intravenous injection expressing plasmids: a complex of cationic liposomes [Zhu et al., 1993, Science 261:209-211]. Other effective methods of delivery of the vector DNA into target cells is known in the art. In one example, purified recombinant plasmid DNA containing viral genes used for injection parenteral, mucosal, or using the gene gun) in a vaccine to stimulate a protective immune response [Fynan et al., 1993, Proc Natl Acad Sci USA 90:11478-11482]. In another example, cells derived from the patient can be transliterate or you can spend a standard electroporation techniques known in the art, which leads to the penetration of DNA recombinant vector into the target cells. Cells containing the recombinant DNA vector can be extracted using methods known in the art, e.g. the measures by incorporating marker selection in the vector, and the selected cells can be returned to the patient for expression of recombinant protein.

According to this aspect of the present invention also provides a method of preventing or reducing the frequency or severity of condition or disease caused by infection with P. gingivalis, the subject, which includes an introduction to the subject the nucleic acid molecule, as described above, a vector as described above, or the cells of prokaryotes or eukaryotes, as described above.

According to other variants of the proposed pharmaceutical composition comprising a chimeric or hybrid protein or antibody as described above. This composition may further contain a diluent, filler, carrier or chemotherapeutic agent for treatment of a condition or disease caused by infection with P. gingivalis, and may be adapted for oral administration. The composition according to the present illness can be included in the composition of lozenges or chewing gums and other products, for example, by blending in to warm the basis of chewing gum or coating the outer surface of the base chewing gum, examples of which are jelutong, rubber latex, vinylite resin and so on, preferably with conventional plasticizers or softeners,sugar or other sweeteners, such as glucose, sorbitol, etc.,

Oral composition according to the present invention, which contains the above components can be prepared and applied in various forms suitable for oral administration, such as funds for the care of teeth, including tooth powders and tooth elixirs, mouthwashes for oral, lozenges, chewing gum, toothpaste, cream to massage the gums, dairy products and other food products. Oral composition according to the present invention may also include known ingredients depending on the type and shape of a particular oral composition.

According to some preferred forms of the invention the oral composition, essentially, can be a liquid, such as a rinse for the mouth. In such medicines carrier is typically a water-alcohol mixture, preferably containing moisturizing component as described below. Typically, the ratio by weight of water to alcohol is in the range from about 1:1 to approximately 20:1. The total amount of the mixture of water and alcohol mixture drug of this type is within approximately from approximately 70 to 99.9% by weight of the dosage form. Alcohol is typically ethanol or isopropanol, suppose the equipment ethanol.

the pH of such liquid and other dosage forms according to the present invention will typically range from about 5 to about 9 and generally from about 5.0 to about 7.0. The pH value can be adjusted by adding acid (e.g. citric or benzoic acid) or base (e.g. sodium hydroxide) or buffer (e.g., citrate, benzoate, carbonate, or sodium bicarbonate, dinitrigenoxide, nutregisterdevice and so on).

In the case of other desirable forms according to the present invention, a pharmaceutical composition may consist essentially of a solid or paste, for example, tooth powder, toffee or toothpaste (dental cream or gel toothpaste. The media in such solid or pasty oral dosage forms typically contain acceptable for teeth polishing material.

In toothpaste carrier liquid may contain water and a moisturizing component number in the range from about 10% to about 80% by weight of the dosage form. Examples of appropriate moisturizing components/carriers are glycerin, propylene glycol, sorbitol and polypropylenglycol. Also preferred are liquid mixture of water, glycerine and sorbitol. In transparent gels, in which an important indicator of the CoE is the rate of refraction, preferably used in approximately 2.5-30% by weight of water, about 70% by weight of glycerol and about 20-80% by weight of sorbitol.

Toothpastes, creams and gels typically contain a natural or synthetic thickener or gelling agent in a quantity of from about 0.1 to about 10, preferably from about 0.5 to about 5% by weight. Acceptable thickener is synthetic hectorite, a synthetic colloidal clay, which is a complex magnesium silicate and alkali metal, known as Laponite (for example, CF, SP 2002, D) produced by Laorte, Industrie Limited. Laponite D contains by weight approximately 58,00% by weight approximately 58,00% by weight SIO, SIS2, 25.40% by weight of MgO, 3.05% by weight of Na2Oh, 0.98% by weight of Li2Oh, a little water and metals in trace quantities. Its true specific gravity is 2.53, and apparent bulk density 1.0 g/ml at 8% moisture content.

Other useful thickeners include Irish moss, iota-carrageenan, traganou gum, starch, polivinilpirrolidon, hydroxyethylmethylcellulose, hydroxyethylmethylcellulose, hypromellose, hydroxyethyl cellulose (e.g. available under the name Natrosol), sodium carboxymethyl cellulose, and colloidal silica such as finely ground Silo is on (for example, Syloid 244)). You can also include soljubilizatory, such as moisturizing polyols, such as propylene glycol, dipropyleneglycol and hexyleneglycol, cellosolve, such as methylcellosolve and ethyl cellosolve, vegetable oils and waxes containing at least 12 carbon atoms in a straight chain such as olive oil, castor oil and petrolatum and esters, such as amylacetate, ethyl acetate and benzyl benzoate.

You must specify that, as usual, oral drugs should be sold or otherwise distributed in packages with corresponding labels. Thus, a vessel with means for rinsing the mouth should have a label describing the product composition, method of use for rinsing or washing of the mouth and tooth paste, cream or gel must be contained in a crushable tubes are usually made of aluminium, lead coated or plastic or kept in crushed relentlessly pumped or pressurized impeller measuring the contents and having a label describing the composition of toothpaste, gel or dental cream.

Organic surfactants used in the compositions of the present invention to achieve increased prophylactic action, to promote the full distribution in the oral cavity active agent, as well as to t the th, to make these compositions more cosmetically acceptable. Organic surface-active agents, by their nature, are preferably anionic, nonionic or ampholytic and preferably do not interact with the active agent. As surface-active substances it is recommended to apply detergent, giving the cleansing composition and penoobraznaya properties. Suitable examples of anionic surfactants include water-soluble (salt monosulfate of monoglycerides of higher fatty acids, such as sodium salt monochlorophenol monoglyceride fatty acids, hydrogenated coconut oil, higher alkyl sulphates, such as nutriceuticals, alkylarylsulfonates, such as nitrotoluenesulfonic, the highest alkylsulfonates, esters of higher fatty acids with 1,2-dihydroxydiphenylsulfone and saturated higher aliphatic acylamino lower aliphatic aminocarbonyl acids, such as containing 12-16 carbon atoms in jimokalos, the alkyl or acyl radical, etc., Examples of these last amides are N-lauroylsarcosine, as well as sodium, potassium and ethanolamine salt N-lauroyl-, N-myristoyl or N-palmitoylcarnitine, which must not contain soap or similar derivatives of higher fatty acids. Examples K is favorable for the application of water-soluble nonionic surfactants are condensation products of ethylene oxide with various reacts with her connections, having long hydrophobic (for example, aliphatic chain of from about 12-20 carbon atoms), and such condensation products ("autocamera") contain hydrophilic polyoxyethylene fragments, including condensation products of polyethylene oxide with fatty acids, fatty alcohols, fatty inorganic salts, polyhydric alcohols (e.g. sorbitol monostearate) and polypropyleneoxide (for example, planovye substances).

Surfactant is usually present and the amount of 0.1-5% by weight. It should be noted that a surfactant to promote dissolution of the active agent according to the present invention and, thereby, reduce the required number solubilizing humidifier.

In oral preparations of the present invention can be introduced and various other substances, such as illuminators, preservatives, silicones, chlorophyll compounds and/or ammoniagenesis compound, such as urea, diammonium phosphate and mixtures thereof. Such auxiliary substances if their presence is injected in the preparations in quantities that do not have undesirable effects on the desired properties and performance. It is necessary to avoid the presence of significant quantities of salts of zinc, magnesium and other metals, usually soluble, capable of forming complexes with active components this is the future of invention.

Can also be used any acceptable flavoring and sweeteners. Examples of acceptable flavoring components include the flavoring oils, e.g. oil of spearmint, peppermint, integranova oil, sassorosso oil, clove oil, sage oil, eucalyptus oil, maiorano oil, cinnamon oil, lemon oil and orange oil, and methylsalicylate. Acceptable sweeteners include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, peralatan of aspartylphenylalanine (AMF), saccharin, etc., it is Desirable to flavouring and sweetening substances separately or together are present in an amount of from 0.1% to 5% or more for the drug.

Compositions for oral administration can be prepared by any means known in the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of flavoring and sweetening agents, coloring agents and preserving agents, to provide pharmaceutically successful and pleasant to the taste of medicines. Tablets contain the active ingredients in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. Such fillers can provide the identification of a, for example, inert diluents, such as calcium carbonate, sodium bicarbonate, lactose, calcium phosphate or sodium granulating agents and disintegrating agents, for example corn starch or alginate acid; binding agents such as gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. Tablets can be coated or to be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract or periodontal pocket, whereby there is a constant action in a long time. For example, you can use the materials that obstruct the collapse, such as the monostearate of glycerol or distearate glycerin.

Dosage forms for oral administration can also be represented in the form of gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or in the form of soft gelatin capsules in which the active ingredient is mixed with water or oil, such as peanut oil or olive oil.

Aqueous suspensions contain the active materials in a mixture with fillers, suitable for receiving water suspensions. Such fillers are suspendresume agents, for example, arboxymethylcellulose is the sodium, methylcellulose, hypromellose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum acacia; dispersing or moisturizing agents may be a natural phosphatides, for example lecithin, or condensation products of oxides alkylene and fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecafluorooctane, or condensation products of ethylene oxide with partial esters of fatty acids and hexitol such as polyoxyethylene sorbitol, monooleate, or condensation products of ethylene oxide with partial esters of fatty acids and anhydrides hexitol, for example polyethylene sorbitan monooleate.

Aqueous suspensions can contain one or more preservatives or antimicrobial agents, such as benzoate, such as ethyl or n-propyl p-hydroxybenzoate, another example is chlorhexidine gluconate, one or more dyes, one or more flavoring agents, one or more sweeteners, such as sucrose or saccharin.

Oil suspensions can be prepared by dissolving the active ingredients in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid pairs of the fin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweeteners can be the above-mentioned agents, to obtain a palatable oral medicines, you can add flavorings. These compositions may be preserved by adding an antioxidant such as ascorbic acid.

According to another aspect of the present invention provides a method of diagnosing or monitoring a condition or disease caused by infection with P. gingivalis, the entity that includes the use of chimeric or hybrid protein as described above for the detection of antibodies to P. gingivalis in a sample of biological material obtained from the subject.

According to another aspect of the present invention provides a method of diagnosing or monitoring a condition or disease caused by infection with P. gingivalis, the entity that includes the use of antibodies as described above to determine the presence of P. gingivalis in the sample of biological material obtained from the subject.

According to another aspect of the present invention provides a peptide to stimulate immune response to P. gingivallis containing the sequence represented in one of: SEQ ID No: 17, 18, 25 and 26. According to one implementation variant PE the Ted contains the sequence homologous to one of: SEQ ID No: 17, 18, 25 and 26. The peptide may contain long 5-40 amino acids.

According to another aspect of the present invention provides nucleic acid encoding the peptide containing the sequence represented in one of: SEQ ID No: 17, 18, 25 and 26.

According to another aspect of the present invention provides use of a peptide containing the sequence represented in one of: SEQ ID No: 17, 18, 25 and 26, or nucleic acid encoding the peptide containing the sequence represented in one of: SBQ ID No:.17, 18, 25 and 26, to obtain chimeric or hybrid protein to stimulate immune response to infection with P. gingivalis.

According to another aspect of the present invention provides use of a peptide containing the sequence represented in one of: SEQ ID No: 17, 18, 25 and 26, or nucleic acid encoding the peptide containing the sequence represented in one of: SEQ ID No: 17, 18, 25 and 26, to stimulate immune response to infection with P. gingivalis. According to one implementation variant peptide is administered simultaneously or sequentially with a peptide containing:

(i) part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Lys-X-proteinase bacteria P. gingivalis; or

(ii) part of the placenta is successive or complete sequence, which is identical or homologous sequences adhesieve domain AGD-X-proteinase bacteria P. gingivalis; or

(iii) a part of a sequence or a full sequence that is identical or homologous sequences adhesieve domain Nada bacteria P. gingivalis.

Table 3

SEQID NO:Amino acid sequenceFragment
1LNTGV[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]T[An][T/S]Q[V /L]KALTNK[D/N]KKgp[432-468]
2FNGGISL[V/A]NYTGHGSETAWGTSHFGTTHVKQLTNSNQRgpA[426-4621
3VSFANYT
4VGFANYT
5GVSFANYT
6GVGFANYT
7VSFANYTA
8VGFANYTA
9 ETAWAD
10ETSWAD
11TAWADP
12TSWADP
13SETAWAD
14SETSWAD
15ETAWADP
16ETSWADP
17TAWADPL
18TSWADPL
19G SETAWAD
20GSETSWAD
21SETAWADP
22SETSWADP
23ETAWADPL
24ETSWADPL
25TAWADPLL
26TSWADPLL
27LNTGV[G/S]FANYTAHGSET[S/A]WADP[S/L]KAS1
28NTGV[G/S]FANYTAHGSET[S/A]WADP[S/L][UV]T[A/T][T/S]Q[V/ L]KALTNK[D/N]KKAS2
29V[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]KAS3
30LNTGVSFANYTAHGSETAWADPPAS1K
31FNGGISL[V/A]NYTGHGSETAWGTSHRAS1
32NGGISL[V/A]NYTGHGSETAWGTSHFGTTHVKQLTNSNQRAS2
33ISL[V/A]NYTGHGSETAWGTSHFRAS3
34FNGGISLANYTGHGSETAWGTPAS1R
35ANEAKWLAADNVWGDNTGYQFLLDADHNTFGSVIPATGPLFT GTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEWIPGGVY DYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTM RRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFE EDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQN LTGSSGQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESFENG IPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLG GIGVLTPDNYLITPALDLPNGGKLTFWVCAQDANYASEHYAVY ASSTGNDASNFTNALLEETITA KA1
36FLLDADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADP VVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGG NQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPAS YTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVKsA1

SPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTP NPNPNPNPNPGTTLSESF
37WGDNTGYQFLLDADHNTFGSVIPATGPLFTGTASSNLYSANFE YLIPANADPWTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGK MWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDM EVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYC VEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTL KWDAPNGTPNPNPNPNPNPGTTLSESFENGIPaswktidadgdghgyvkpgnapgiagynsngcvysesfglggigvltpdnyli7 PALDLPNGGKLA1
38SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHTL WPNCSVPANLFAPFEYTVPENADPSCSPTNMIMDGTASVNIPA GTYDFAIAAPQANAKIWIAGQGPTKEDDYVFEAGKKYHFLMKK MGSGDGTELTISEGGGSDYTYTVYRDGTKIKEGLTATTFEEDG VATGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTG SAVGQKVTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESFENG IPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLG GIGVLTPDNYLITPALDLPNGGKLTFWVCAQDANYASEHYAVY ASSTGNDASNFTNALLEETITARA1
39DDYVFEAGKKYHFLMKKMGSGDGTELTISEGGGSDYTYTVYR DGTKIKEGLTATTFEEDGVATGNHEYCVEVKYTAGVSPKVCKD VTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNPNPNP NPNPNPGTTTLSESFRsA1
40ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDWL TAHGGSNWSSFSWNGMALNPDNYLISKDVTGATKVKYYYAV NDGFPCDHYAVMISKTGTNAGDFTWFEETPNGIN,. L]-KA2
41 RQSWYIERTVDLPAGTKYVAFRHYNCSDLNYIllddluftmggi SPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNILEYCVEV KYTAGVSPKKCVNVTVNSTQFNPVQNLTAEQAPNSMDAILKW NAPASKA3
42AEVLNEDFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGH NSAICVSSASY1NFEGPQNPDNYLVTPELSLPGGGTLTFWVCA QDANYASEHYAVYASSTGNDASNFANALLEEVLTAKA4
43TWTAPEAIRGTRAQGTWYQKTVQLPAGTKYVAFRHFGCTDF FWINLDDWITSGNAPSYTYTIYRNNTQIASGVTETTYRDPDLA TGFYTYGVKWYPNGESAIETATLNITSLADVTAQKPYTLTWG KTITVTCQGEAMIYDMNGRRLAAGRNTWYTAQGGHYAVMW VDGKSYVEKLAVKKA5
44ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDWL TAHGGTNWSSFSWNGMALNPDNYLISKDVTGATKVKYYYAV NDGFPGDHYAVMISKTGTNAGDFTWFEETPNGINRA2
45PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGG SPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVatgnheycvev KYJAGYSPKKCVNVTVNSTQFNPVKNLKAQPDGGDVVLKWEA PSARA3
46ANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPATGPLFT GTASSDLYSANFESLIPANADPVVTTQNIIVTGQGEVVIPGGVY DYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTM RRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTETTYR DAGMSAQSHEYCVEVKYTAGVSPKVCVDYIPDGVADVTAQKP YTLTWGKTITVTCQGEAMIYDMNGRRLAAGRNTWYTAQGG YYAVMWVDGKSYVEKLAIKRA4

SEQ ID NO:The nucleotide sequence
47GACCATGGCTCATCACCATCACCATCACAATACCGGAGT CAGCTTTGCAKAS2-ol is my
48GACTCGAGTTATTTGTCCTTATTAGTGAGTGCTTTC,KAS2-reverse
49GACCATGGCTTGGGGAGACAATACGGGTTACKLA1 - direct
50GACTCGAGACCTCCGTTAGGCAAATCCKLA1-reverse
51CCGTATTGTCTCCCCATTTGTCCTTATTAGTGAGTGCTTT CKAS2-KLA1-reverse
52CACTAATAAGGACAAATGGGGAGACAATACGGGTTACKAS2-KLA1-direct
53CATGGATCTGAGACCGCATGGGCTGATCCACTTTTCTTG TTGGATGCCGATKAS1-KsA1-pryamoy
54CCATGGCTTTGAATACCGGAGTCAGCTTTGCAAACTATAC AGCGCATGGATCTGAGACCGCAKAS1-KSA1-pryamoy
55CTCGAGGAATGATTCGGAAAGTGTTKAS1-KsA1-reverse
56CCATGGCTGATTATAGCTGGAATTCCCAGGTAGTCAGCT TTGCAAACTATACAMulti-pryamoy
57CTTTGCAAACTATACAGCGCATGGATCTGAGACCGCATG GGCTGATCCACTTMulti-pryamoy
58ATGGGCTGTCCACTTCTGAATTCTTATTGGGGCGAGAT CGGCAATATTACC Multi-pryamoy
59GATCGGCAATATTACCCATATTGGTGCTCATTACGCTTGG GGAGACAATACGMulti-pryamoy
60CTCGAGACCTCCGTTAGGCAAATCCAATGCCGGTGTTAT CAGATAGTTGTCAMulti-reverse

SEQID NO:Amino acid sequenceFull length
61MKNLNKFVSIALCSSLLGGMAFAQQTELGRNPNVRLLESTQQ SVTKVQFRMDNLKFTEVQTPKGIGQVPTYTEGVNLSEKG.MPTLPILSRSLAVSDTREMKVEVVSSKF^EKKNV,LIAPSKGMIMRNb DPKKIPYVYGKTYSQNKFFPGEIATLDDPFILrdvrgpyvnfal PLQYNPVTKTLRIYTEITVAVSETSEQGKNiLnkkgtfagfedt YKRMFMNYEPGRYTPVEEKQNGRMIVIVAKKYEGDIKDFVDW KNQRGLRTEVKVAEDIASPVTANAIQQFVKQEYEKEGNDLTYV LLIGDHKDIPAKITPGIKSDQVYGQIVGNDHYNEVFIGRFSCES KEDLKTQIDRTIHYERNITTEDKWLGQALCIASAEGGPSADNG ESDIQHENVIANLLTQYGYTKIIKCYDPGVTPKNIIDAFNGGISL ANYTGHGSETAWGTSHFGTTHVKQLTNSNQLPFIFDVACVNG DFLFSMPCFAEALMRAQKDGKPTGTVAIIASTINQSWASPMRGQDEMNEILCEKHPNNIKRTFGGVTMNGMFAMVEKYKKDGE KMLDTWTVFGDPSLLVRTLVPTKMQVTAPAQINLTDASVNVS CDYNGAIATISANGKMFGSAVVENGTATINLTGLTNESTLTLTV VGYNKETVIKTINTNGEPNPYQPVSNLTATTQGQKVTLKWDA PSTKTNATTNTARSVDGIRELVLLSVSDAPELLRSGQAEIVLEA HDVWNDGSGYQILLDADHDQYGQVIPSDTHTLWPNCSVPAN LFAPFEYTVPENADPSCSPTNMIMDGTASVNIPAGTYDFAIAA PQANAKIWIAGQGPTKEDDYVFEAGKKYHFLMKKMGSGDGT ELTISEGGGSDYTYTVYRDGTKIKETILTATRTFEEDGVATGNHE YCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKL VTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESFENGIPASWK TIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLT PDNYLITPALDLPNGGKLTFWVCAQDANYASEHYAVYASSTG NDASNFTNALLEETITAKGVRSPEAMRGRIQGTWRQKTVDLP AGTKYVAFRHFQSTDMFYIDLDEVEIKANGKRADFTETFESST HGEAPAEWTTIDADGDGQGWLCLSSGQLDWLTAHGGTNW SSFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDH YAVMISKTGTNAGDFTWFEETPNGINKGGARFGLSTEADGA KPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMG GSPTPTDYTYTVYRDGTKIKEGLTETTFEEDVATGNHEYCV EVKYTAGVSPKKCVNVTVNSTQFNPVKNLKAQPDGGDVVLK WEAPSAKKTEGSREVKRIGDGLFVTIEPANDVRANEAKWLAA DNVWGDNTGYQFLLDADHNTFGSVIPATGPLFTGTASSDLYS ANFESLIPANADPWTTQNIIVTGQGEWIPGGVYDYCITNPEP ASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGD pTDMEVEDDSPASYTYTVYRDGTKIKEGLTETtyrdapmsaq SHEYCVEVKYTAGVSPKVCVDYIPGGVADVTAQKPYTLTVVG KTITVTCQGEAMIYDMNGRRLAAGRNTVVYTAQGGYYAVMVV VDGKSYVEKLAIK RgpA
62MRKLLLLIAASLLGVGLYAQSAKIKLDAPTTRTTCTNNSFKQFD ASFSFNEVELTKVETKGGTFASVSIPGAFPTGEVGSPEVPAVR KLIAVPVGATPWRVKSFTEQVYSLNQYGSEKLMPHQPSMSK SDDPEKVPFVYNAAAYARKGFVGQELTQVEMLGTMRGVRIAA LTINPVQYDVVANQLKVRNNIEIEVSFQGADEVATQRLYDASF SPYFETAYKQLFNRDVYTDHGDLYNTPVRMLVVAGAKFKEAL KPWLTWKAQKGFYLDVHYTDEAEVGTTNASIKAFIHKKYNDG LAASAAPVFLALVGDTDVISGEKGKKTKKVTDLYYSAVDGDYF PEMYTFRMSASSPEELTNIIDKVLMYEKATMPDKSYLEKVLLIA GADYSWNSQVGQPTIKYGMQYYYNQEHGYTDVYNYLKAPYTKgp

GCYSHLNTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDK YFLAIGNCCITAQFDYVQPCFGEVITRVKEKGAYAYIGSSPNSY WGEDYYWSVGANAVFGVQPTFEGTSMGSYDATFLEDSYNTV NSIMWAGNLAATHAGNIGNITHIGAHYYWEAYHVLGDGSVMP YRAMPKTNTYTLPASLPQNQASYSIQASAGSYVAISKDGYLYG TGVANASGVATVSMTKQITENGNYPWITRSNYLPVIKQIQVGI EPSPYQPVSNLTATTQGQKVTLKVVEAPEAKKAEGSPCVKRIG DGLFVTIEPANDVRANEAKWLAADNVWGDNTGYQFLLDADH NTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPWTTQNII VTGQGEWIPGGVYDYCITNPEPASGKMWIAGDGGNQPARY DDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVY RDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVC KDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNP NPNPNPGTTLSESFENGIPASWKTIDADGDGHGWKPGNAPG AGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGGKLTF WVCAQDANYASEHYAVYASSTGNDASNFTNALLEETITAKGV RSPKAIRGRIQGTWRQKTVDLPAGTKYVAFRHFQSTDMFYIDL DEVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGW LCLSSGQLDWLTAHGGSNWSSFSWNGMALNPDNYLISKDV TGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEET PNGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAF RHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEG LTETTFEEDGVATGNHEYCVEVKYTAGVSPKKCVNYTVNSTQFNFNPVQNLTAEQAPNSMDAILKWNALPASKRAEVLNEDFENGP ASWKTLDADGDGNNWTTTPPPGGSSFAGRINSAICVSSASYIN FEGPQNPDNYLVTPELSLPGGGTLTFWVCAQDANYASEHYA VYASSTGNDASNFANALLEEVLTAKTWTAPEAIRGTRAQGT WYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDWITSGNAPS YTYTIYRNNTQIASGVTETTYRDPDLATGFYTYGVKVVYPNGE SAIETATLNITSLADVTAQKPYTLTVVGKTITVTCQGEAMIYDM NGRRLAAGRNTWYTAQGGHYAVMWVDGKSYVEKLAVK
63MRKLNSLFSLAVLLSLLCWGQTAAAQGGPKTAPSVTHQAVQK GIRTSKAKDLRDPIAGMARIILEAHDVWEDGTGYQMLWDAD HNQYGASIPEESFWFANGTIPAGLYDPFEYKVPVNADASFSPT NFVLDGTASADIPAGTYDYVIINPNPGIIYIVGEGVSKGNDYVVE AGKTYHFTVQRQGPGDAASVWTGEGGNEFAPVQNLQWSV SGQTVTLTWQAPASDKRTYVLNESFDTQTLPNGWTMIDADG DGHNWLSTINVYNTATHTGDGAMFSKSWTASSGAKIDLSPDN YLVTPKFTVPENGKLSYWVSSQEPWTNEHYGVFLSTTGNEAA NFTIKLLEETLGSGKPAPMNLVKSEGVKAPAPYQERTIDLSAY AGQQWLAFRHFGCTGIFRLYLDDVAVSGEGSSNDYTYTVYR DNVVIAQNLTATTFNQENVAPGQYNYCVEYKYTAGF DVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNPNPG TTTLSESFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGH NSAICVSSASYINFEGPQNPDNYLVTPELSLPNGGTLTFVVVCA QDANYASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAP EAIRGTRVQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWINLD DVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGWL CLSSGQLGWLTAHGGTNWASFSWNGMALNPDNYLISKDVT GATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTWFEETP NGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFR HYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGL TETTFEEDGVATGNHEYCVEVKYTAGVSPKECVNVTVDRVQF NPVQNLTGSAVGQKVTLKWDAPNGTPNPNPGTTTLSESFEN GIPASWKTIDADGDGNNWTTTPPPGGTSFAGHNSAICVSSASHagA

YINFEGPQNPDNYLVTPELSLPNGGTLTFWVCAQDANYASEH YAVYASSTGNDASNFANALLEEVLTAKTVVTAPEAIRGTRVQG TWYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDVEIKANGKR ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDW LTAHGGTNWASFSWNGMALNPDNYLISKDVTGATKVKYYYA VNDGFPGDHYAVMISKTGTNAGDRTVVFEETPNGINKSGRR GLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNIL LDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVA TGNHEYCVEVKYTAGVSPKECVNVTVDPVQFNPVQNLTGSA VGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKTIDA DGDGNNWTTTPPPGGTSFAGHNSAICVSSASYINFEGPQNPD NYLVTPELSLPNGGTLTFWVCAQDANYASEHYAVYASSTGND ASNFANALLEEVLTAKTWTAPEAIRGTRVQGTWYQKTVQLPA GTKYVAFRHFGCTDFFWINLDDVEIKANGKRADFTETFESSTH GEAPAEWTTIDADGDGQGWLCLSSGQLGWLTAHGGTNWA SFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHY AVMISKTGTNAGDFTWFEETPNGINKGGARFGLSTEANGAK PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGG SPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEV KYTAGVSPKECVNVTINPTQFNPVQNLTAEQAPNSMDAILKW NAPASKRAEVLNEDFENGIPASWKTIDADGDGNNWTTTPPPG GSSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPGGGT LTFWVCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTA KTVVAPEAIRGTRVQGTWYQKTVPLPAGTKYVAFRHFGCTD FFWINLDDWITSGNAPSYTYTIYRNNTQIASGVTETTYRDPDL ATGFYTYGVKVWPNGESAIETATLNITSLADVTAQKPYTLTVV GKTITVTCQGEAMIYDMNGRRLAAGRNTVVYTAQGGHYAVM VWDGKSYVEKLAVK

SEQ ID NO:Amino acid sequenceFragment
64D[S/Y][Y/S]WN[P/S][K/Q][I/V]KAS4
65NSYWGEDIKAS5
66IGN[V/I]THIGAHYKAS6
67EGGPSADNRAS4
68[N/D]Q[S/Y]WA[S/P]PRAS5
69PVSNLTATTQGQKVTLKWDAPSTABM1-RapAcat
70PVSNLTATTQGQKVTLKWEAPSAABM1-Kgpcat
71PVQNLTGSSVGQKVTLKWDAPSTABM1-KgpA1
72PVQNLTGSAVGQKVTLKWDAPNGABM1-RgpA1 & RgpAA3
73PVKNLKAQPDGGDWLKWEAPSAABM1-HagAA1*/**
74PVQNLTAEQAPNSMDAILKWNAPABM1-KgpA3 HagAA3
75PVQNLTQWSVSGQTVTLTWQAPASABM2-HagAA1
76YTYTVYRDGTKIKEGLTETTFEEDGVAABM2-ABM2-RgpAA4/td>
77YTYTVYRDNVVIAQNLTATTFNQENVAABM2-HagA1*
78YTYTVYRDGTKIKEGLTA/ETTFEEDGVAABM2 any Other adhesin
79PNGTP(NP)1-6GTT(T)LSESFAVM - all adhesin
80GGPKTAPSVTHQAVQKGIRTSKAKDLRDPIPAGMARIILEAHDV WEDGTGYQMLWDADHNQYGASIPEESFWFANGTIPAGLYDP FEYKVPVNADASFSPTNFVLDGTASADIPAGTYDYVIINRNRGII YIVGEGVSKGNDYVVEAGKTYHFTVQRQGPGDAASVVVTGEG GNEFAPVQNLQWSVSGQTVTLTWQAPASDKRTYVLNBSFPTH QTLPNGWTMIDADGDGHNWLSTINVYNTATHTGDGAMFSKS WTASSGAKIDLSPDNYLVTPKFTVPENGKLSYWVSSQEPWTN EHYGVFLSTTGNEAANFTIKLLEETLGSGNada [26-351]
81APAPYQERTIDLSAYAGQQVYLAFRHFGCTGIFRLYLDDVAVS GEGSSNDYTYTVYRDNVVIAQNLTATTFNQENVAPGQYNYCV EVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLK WDAPNGTPNPNPGTTTLSESFENGIPASWKTIDADGDGNNWT TTPPPGGSSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELS LPNGGTLTFWVCAQDANYASEHYAVYASSTGNDASNFANALL EEVLTANada* [366-625]
82PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGG SPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEV KYTAGVSPKECVNVTVDPVQFNPVQNLTGSAVGQKVTLKWDANada** [820-1077] or

PNGTPNPNPGTTTLSESFENGIPASWKTIDADGDGNNWTTTP PPGGTSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPN GGTLTFWVCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTANada* [1272-1529]
83 PYQPVSNLTATTQGQAVM[436-450]
84EGLTATTFEEDGVAAABM2 [672-686]
85GTPNPNPNPNPNPNPGTAVM [455-471]

The following examples are presented to illustrate, but not limit, this invention.

Example 1

Materials and methods.

The bacterial strains and culturing conditions. Lyophilized culture of Porphyromonas gingivalis W50 was cultured under anaerobic conditions at 37°C on blood agar with the addition of 5 μg/ml hemin, 0.5 μg/ml cysteine (LC agar, <10 passages). After 3-4 days the colonies were inoculable environment for infusion with extracts of tissues of the brain and heart with the addition of 5 μg/ml hemin, 0.5 μg/ml cysteine (1). Periodic culture was cultured under anaerobic conditions at a workstation MKZ (Don Whitley Scientific Ltd., Adelaide, Australia). Cells were harvested during exponential phase growth method centrifugation (7500 l, 30 min, 4°C) and washed twice with PG buffer (50 mm Tris-HCl, 150 mm NaCl, 5 mm CaCl2and 5 mm cysteine-HCl, pH 8.0) in the anaerobic workstation. The periodic growth of cultures was monitored at a wavelength of 650 nm on a spectrophotometer (model E, Perkin-Elmer). Purity of cultures was checked by a routine method of staining Gram microscopically and using the discrepancies between the different biochemical tests according to the Slots (2).

Creating designs RET containing sequence adesina and sequence adesina added to M-end sequences of the CRA proteases. The remains of the CRA, representing peptides and chimeric peptides active site (AS) and domain adesina CDR (A1), expressed at high levels in E. Li in the form of recombinant proteins (g) with hexa-His tags using expression vectors pet (Novagen). Recombinant proteins represented rKAS2 and rKLA1, and r-chimeric proteins represented rKAS2-KLA1, rKAS1-KsA1 and rKAS4-KAS3-KAS5-KAS6-KLA1 (also denoted by the term S-KLAI). Amino acid sequences representing different domains A1 and AS presented in Tables 1 and 2. Different domains KAS and KA1 kgp gene amplified on the basis of pNS1 (3.5 kb BamHI lys fragment in pUC18) or genomic DNA of P. gingivalis, respectively, using primers listed in Table 4, Taq DNA polymerase (Invitrogen) and thermal cycler PC-960 (Corbett Research Technologies). To obtain PCR products encoding KAS2 and KLA1, respectively, used a pair of primers: S2-direct and S2-back and KLA1-direct and KLA1-reverse under the following reaction conditions: 94°C 3 min, then 28 cycles at 94°C, 45 s (denaturation); 62°C, 40 s (annealing) and 72°C, 20 C (elongation), then the last cycle at 72°C, 5 minutes

Chimeric PCR product KAS2-KLA1 was obtained by PCR SOing as follows: received PCR product is using primers KAS2-direct and KAS2-KLA1-Chimera-back and KAS2-KLA1-Chimera-direct and KLA1-back under the above conditions. Then, annealing was performed PCR products and the final PCR using primers: KAS2-direct and KLA1-back 94°C, 2 min, then 28 cycles at 94°C, 30 s; 50°C, 30 s and 72°C, 40 C, then a final cycle at 72°C, 5 minutes

To obtain a PCR product KAS1-KsA1 spent two consecutive PCR using primers KAS1-KsA1-back with each of primers KAS1-KsA1-direct 1 and 2 at a time (reaction conditions: 94°C 2 min, then 35 cycles at 94°C, 15 s; 63°C, 30 s and 72°C, 2 min to obtain a PCR product, KAS1-KsA1. Primers KAS1-KsA1-direct and KAS1-KsA1-pryamoy and KAS1-KsA1-pryamoy at the 3'end contains a sequence overlapping with b'-end of the previous PCR product.

To obtain the fragment of the multi-KAS-KLA1 PCR was performed 4 consecutive PCR using primer multi-backward with each of the primers: multi-direct 1, 2, 3, and 4 turns (reaction conditions 95°C, 2 min, then 35 cycles at 95°C, 20 s; 68°C, 1.5 min) to obtain a PCR product from the multi-KAS-KLAI. Each primer multi-straight at the 3'end contains a sequence overlapping with the 5'-end of the previous PCR product.

All PCR products encoding KAS2, KLA1, KAS2-KLA1, KAS1-KsA1 and multi-KAS-KLAI, was purified on columns (Qiagen) was subjected to ligation of the cloning vector pGem-T Easy (Promega) and transformed to E. Li JM109 according to the manufacturer's instructions. Purified recombinant constructs pGemT-Easy, was treated with Ncol and Xhol, konyrov is whether processed Ncol/Xhdl pET28b (Novagen) and transformed cell host, in which there occurs the expression, E. coli, JM109 [DH5a]. Recombinant constructs RET was purified and transformed cell host, which is the expression, E. Li, BL21 (DE3) [HMS174(DE3)] (Novagen) and were selected on LB medium containing 50 μg/ml kanamycin according to the manufacturer's instructions. The integrity of each insert was confirmed by sequencing method.

Designed oligonucleotide primers (table 4) with built-in restriction sites, stop codons and hexa-His tags if necessary. Designed the primers used for rKAS2, rKLA1 and 1-KAS2-KLA1, with limited inclusion of g-proteins foreign coding sequences to no more than three amino acid and hexa-His tag. rKAS1 and rKLA1 designed so that they contain hexa-His tag at N-end-end, respectively, so that they can be directly compared with rKAS2 - KLA1, which contains a hexa-His tag at N-end and C-end. In rKAS1-KsA1 and KAS-KLA1 His-tag located at the C-ends.

Table 4. Oligonucleotide primers used for amplification of nucleotide posledovatelnostei encoding fragments hybrids CRA A1 and AS

Recombinant (r)proteinOligonucleotidesPosledovatel the activity (5'-3') Features* (5'-3')
1-KAS2KAS2-directGACCATGGCTCATCACC ASSASSINAS GGAGTCAGCTTTGCA(SEQ ID NO: 47)GA buffer-Ncol (including the start codon ATG)-CT-(His)6-AS nt(1992-2012)
KAS2-reverseGACTCGAGTTATTTGTC CTTATTAGTGAGTGCTTT(SEQ ID NO: 48)GA buffer-Xhol-TTA stop KAS1 (nt 2099-2075)
rKLA1IKLA1-,. directGACCATGGGTTGGQGA GACAATACGGGTTAC (SEQ ID NO: 49)GA buffer-Ncol (including the start codon ATG)-CT - A1 (nt 2946-2966)
KLA1-reverseGACTCGAGACCTCCGTT AGGCAAATCC (SEQ ID NO: 50)GA buffer-Xhol-AI (nt 3863-3845)
rKAS2-KLA1KAS2-KLA1-reverseCCGTATTGTCTCCCCAT TTGTCCTTATTAGTGAGT GCTTTC(SEQ ID NO: 51)A1 (nt2961-2946)-KAS1 (nt 2099-2075)
KAS2-KLA1-directCACTAATAAGGACAAAT GGGGAGACAATACGGG TTAC(SEQ ID NO: 52)KAS1 (nt 2084-2099)-A1 (nt 2946-2966
rKAS1-KsA1KAS1-KsA1-direct 1CATGGATCTGAGACCGC ATGGGCTGATCCACTTT TCTTGTTGGATGCCGAT(SEQ ID NO: 53) AS (nt 2025-2057)-A1 (nt 2970-2;987)
KAS1-KsA1-direct 2CCATGGCTTTGAATACC GGAGTCAGCTTTGCAAA CTATACAGCGCATGGAT CTGAGACCGCASEQ ID NO: 54)Ncol-CT-AS (nt 1989-2042)
KAS1-KsA1-reverseCTCGAGGAATGATTCGG AAAGTGTT (SEQ ID NO 55)Xhol-A1(nt 3663-3644)
r multi KAS-KLA1multi-CCATGGCTGATTATAGCNcol-CT-KAS4 (nt 1857-

direct 1TGGAATTCCCAGGTAGT CAGCTTTGCAAADTATA CA (SEQ ID NO: 56)188D)-KAS3, (nt.2001-2021)
multi-direct 2CTTTGCAAACTATACAG CGCATGGATCTGAGACC GCATGGGCTGATCCACT T (SEQ ID NO: 57)KAS3 (nt 2006-2057)
multi-direct 3ATGGGCTGATCCACTTC TGAATTCTTATTGGGGC GAGATCGGCAATATTAC (SEQ ID NO: 58)KAS3 (nt 2042-2060)-KAS5 (nt 2223-2240)-KAS6 (nt 2403-2417)
multi-direct 4GATCGGCAATATTACCC ATATTGGTGCTCATTAC GCTTGGGGAGACAATACG, (SEQ ID NO: 59)G-KAS6 (nt 2403-2435) to GCT (Ala Spicer)-A1(nt 2946-2960)
multi-reverseCTCGAGACCTCCGTTAG GCAAATCCAATGCCGGT GTTATCAGATAGTTGTC A(SEQ ID NO 60) Xho-A1 (nt 3863-3818)

* the number of sequences of nucleotides (nt) gene sequence lysine-specific cysteine proteinase room access U75366

Expression and purification of recombinant proteins

With designs pET28:KLA1 (KAS2, KAS2-LA1, KAS1-SA1, multi-KAS-KLA1) using isopropyl p-galactosidase (IPTG) induced the expression of recombinant proteins. All recombinant proteins were produced in the form of hybrid proteins labeled hexa-His, and they were purified in spaniah denaturation using cleaning systems NI-NTA (Invitrogen). Briefly, single colonies of transformed E. coli (DE3) was inoculable in 20 ml of medium Luria-Bertani (LB) containing 50 μg/ml kanamycin at 37°C over night under stirring on an orbital shaker. Then the specified inoculum was inoculable 1 l of LB medium containing 50 μg/ml kanamycin. After reaching the culture optical density of 5.0-7.0 at 600 nm (mid-logarithmic growth phase) induced the expression of protein 0.1 mm IPTG for 2 hours at 37°C under stirring at 200 rpm was Collected cells (7.5 g) and resuspendable them in denaturing binding buffer (8 M urea, 20 nm sodium phosphate, pH 8.0, 500 mm NaCl), the cells were destroyed by sonication in mode 3 × 15 s with an interval of 30 s using a cell disintegrator Branson Sonifer 250 (Branson Ultronics Corporation, Danbury, CT) micronesica on the installations 3, then was centrifuged at 39,000 g for 30 min at 4°C. Recombinante proteins were purified from the supernatant by drawing on preuranjeno agarose column of Ni-NTA, and then washed with denaturing buffer for washing (8 M urea, 20 nm sodium phosphate, pH 6.0, 500 mm NaCl) for elution is not bound peroxidase proteins. Then the column was washed with 10 volumes of binding buffer and suirable recombinant protein denaturing buffer for elution (8 M urea, 20 nm sodium phosphate, pH 6.0, 500 mm NaCl, 0.5 M imidazole). Purified protein deliberately against 2M urea-PBS and stored at -80°C.

Samples of recombinant proteins was analyzed by the method of SDS-PAGE and determined their molecular weight using ProtParam (http://au.expasv.ora/tools/protparam.html). The protein concentration in the samples was determined by the method of Bio-Rad Protein Assay using BSA (bovine serum albumin) as a standard.

Immunization and murine model of periodontitis.

Experiments with mice were performed as described previously (3)? with the approval of the ethics Committee for experiments on animals of the University of Melbourne. 6-8 week BALB/c mice (no 12 mice per group) contained in the mikroinsulators, were immunized subcutaneously (s.c. 100 ál) 50 μg of one of the recombinant proteins, complex RgpA-Kgp or 2×109killed by formalin cells of P. gingivalis strain W50 or PBS; each antigen was emulsiable in incomplete hell is the guy's adjuvant (IFA). After 30 days, mice were performed buttonow, injection of antigen (subcutaneous injection, emulsified in IFA) and 12 days later took the blood from the retro-bulbar venous plexus. 4 days after the second immunization, mice were administered kanamycin (Sigma-Aldrich, New South Wales, Australia) at a concentration of 1 mg/ml in deionized water ad libitum for 7 days. 3 days after injection of antibiotic (2 days after phlebotomy) mice were inoculable orally 4 times every 2 days 1 × 1010live P. gingivalis W50 (25 µg) in PG buffer (50 mm Tris-HCl, 150 mm NaCl, 5 mm CaCl2and 5 mm cysteine-HCl, pH 8.0) containing 2% (weight/volume) of carboxymethyl cellulose (CMC; Sigma - Aldrich, New South Wales, Australia), the control group was injected only PG buffer containing 2% (W/V) CMC. Inoculum were prepared in an anaerobic chamber and then immediately introduced into the gingival edge of the molars of the upper jaw. Two weeks later, mice were administered an additional 4 doses (2 days 1 × 1010live cells of P. gingivalis W50 (25 μl) in PG buffer containing 2% (W/V) CMC. The number of live bacteria in each inoculum tested by culture on blood agar. Mice were fed powdered food (Barastock, Australia) and were kept in cages with mesh bottom to prevent the risk of bleeding. 4 weeks after the last dose, the mice were drawing blood from the retro-bulbar plexus of mice were scored, took the upper jaw and is elili it into two parts, one half (right) used to measure the resorption of the alveolar bone, and the other half (left) was used for PCR in real time.

The right half of the upper jaw boiled for 1 minute in deionized water, mechanically cleaned of flesh and immersed in 2% (wt/vol) potassium hydroxide (16 hours, 25°C). Then half jaw was washed twice with deionized water and immersed in 3% (weight/volume) solution of hydrogen peroxide (6 hours, 25°C). After washing half of the jaw (two times with deionized water) were stained with 0.1% (weight/volume) aqueous solution of methylene blue and took pictures buccal side of each half of the jaw with the camera Olympus DP12 installed on the microscope for dissection, using software OLYSIA BioReport version 3.2 (Olympus Australia Pty Ltd., New South Wales, Australia) to assess the horizontal bone resorption. Horizontal resorption of bone occurs in the horizontal plane perpendicular to the crest of the alveolar bone (ABC), which leads to the reduction of the height of the ridge. Each half of the upper jaw was aligned so that the images molar literal lingual tubercles each tooth was applied, and images were applied micrometer scale so that measurements could be standardized for each shot. The area from the CEM is toanalog connection to the FAA each posterior tooth was measured using the software OLYSIA BioReport version 3.2. Measurement of bone resorption was conducted in two replications one expert in the conditions of a randomized double-blind study.

Each, you can go

The definition of subclasses of antibodies using ELISA method.

To determine the answers subclasses of antibodies in the serum of mice was performed enzyme-linked immunosorbent assay (ELISA) in three replications, while wells flat-bottom polyvinyl microtiter tablet (Dynatech Laboratories, McLean, VA), the suspension is killed by formalin cells of P. gingivalis W50 in phosphate-buffered saline (PBS) (0.01 M Na2HPO4, 1.5 mm KN2RHO4, 0.15 M NaCl), pH 7.0, containing 0.1% (volume) Tween 20 (PBST) with a concentration of 5 µg/ml After removal of the coating solution in the wells was made PBST containing 2% (weight/volume) of dry milk to block uncoated plastic for one hour at room temperature. The wells were three times washed in PBST and to each well was added a serial dilution of mouse serum in PBST containing 0.5% (weight/vol) skim milk powder (SK-PBST), and incubated for 16 hours at room temperature. After that, the wells were washed six times in PBST, SK-PBST was added in a dilution of 1/2,000 goat IgG to IgM, IgA, IgG1, IgG2a, IgG2b or IgG3 mouse (Sigma, New South Wales, Australia), and left in contact for 2 hours at room temperature. The tablets were washed six times in PBST and to each well was added in a dilution of 1/5,000 to yugata horseradish peroxidase antibody rabbit, specific immunoglobulins (Sigma, New South Wales, Australia) in SK-PBST, and incubated the plates for 1 hour at room temperature. After the wells are washed six times PBST bound peroxidase antibodies was determined by adding 100 μl of substrate ABTS [0.9 mm 2,2'-Azino-bis(3-ativans-thiazolin-6) sulfonic acid in 80 mm citric acid, containing 0.005% (volume) of hydrogen peroxide, pH 4.0] in each well. The optical density was determined on the reader microtiter plates (Bio-Rad microplate reader model 450) at a wavelength of 415 nm.

Gel electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate SDS-PAGE and Western blotting.

Conducted analysis of recombinant proteins (10 μg) using the system for electrophoresis XCell surelock Mini-Cell. Recombinant cells were mixed with 20 µl of sample buffer (10% [weight/volume] SDS, 0.05% [weight/volume] bromophenol blue, 25% [volume] glycerol and 0.05% [volume] 2 - mercaptoethanol). the pH is brought to a value of 8.0 1.5 M Tris-HCI, and then the solution was heated for 5 min at 100°C. Recombinant proteins (10 µg/well) was applied onto Novex 12% (weight/volume) ready miniheli with Tris-glycine, and electrophoresis was performed at an amperage of 30-50 mA and a potential difference of 125 V using electrophoresis Nv (Nv, San Diego, CA). Proteins were visualized using 0.25% (weight/volume) of the dye Kumasi blue R250.

Analysis of epitope the sequence of peptides active site KDR-proteinase (KAS-2).

Determined the binding sites of the antibodies, peptides active site Lys-specific proteinase KAS2 (433-468 SEQ ID No: 28) with synthesis of biotinylated on M-end overlapping peptides consisting of 8 residues (indent 1, the overlap of 7 residues) to multicarinata system synthesis of peptides (Chiron Technologies, Melbourne, Australia) using standard solid-phase synthesis Protocol Biotinylated peptides (µg/ml) in 0.1 M PBS. pH 7.4 was applied to a streptavidin coated tablets over night at 4°C (Nunc, NSW Australia). After that, the wells of tablets were washed 4 times with PBST and perform the mapping of the epitope peptides by contacting tablet method of analysis by ELISA according to the manufacturer's instructions Chiron Technologies using sera of mice at a dilution of 1:1000 in 1% w/v fat-free dry milk in 0.1 M PBS, pH 7.4, containing 0.1% v/v Tween 20 (SK-PBST). After that, the wells were washed 6 times with PBST, was added in a dilution of 1/2,000 IgG goat against mouse IgG (Sigma, New South Wales, Australia) in SK-PBST and left for 2 hours at room temperature. The tablets were washed 6 times with PBST and each well was made by dilution of 1/5,000 conjugate of horseradish peroxidase with rabbit antibodies specific for immunoglobulin goat (Sigma, New South Wales, Australia) in SK PBST and incubated for 1 hour at room temperature. After that, the wells were washed 6 times with PBST, bound peroxidase antibodies was determined added the m to each well 100 μl of ABTS substrate [0.9 mm 2,2'-2,2'-azinobis(3-ethylbenzothiazoline 6) sulfonic acid in 80 mm citric acid, containing 0.005% (volume) of hydrogen peroxide, pH 4.0]. The optical density at a wavelength of 415 nm was determined on the reader microtiter plates (Bio-Rad microtiter reader tablet, model 450).

The statistical analysis. Data of bone resorption were processed using univariate analysis of variance (ANOVA) and T3 Dunnet test (SPSS for Windows, version 12). The antibody titers of IgA subclasses, IgM, and IgG were statistically processed using the t-test, t-test using the software SPSS (SPSS for Windows, version 12).

Example 2

Characterization and purification of recombinant proteins (KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1).

To describe the protective ability of the fragments domain adesina A1 CRA and chimeric constructs the CRA proteases and fragments domain adesina A1 KDR against infection P. gingivalis, were obtained and purified recombinant proteins: KsA1 KLA1, KAS1-KSA1 and KAS2-KLA1. Recombinant proteins (s1 and LA1) and recombinant chimeric proteins (KAS1-KsA1 and KAS2-KLA1) was purified from calf inclusion method Ni-chelate affinity chromatography and purified proteins were analyzed using SDS-PAGE (Figure 1). Each purified recombinant protein consisted of one major protein band with a molecular mass of 40, 36, 31, and 32 kDa, which corresponded to KAS2-KLA1, KLA1, KsA1 and KAS1-KsA1, and data molecular weight corresponded to the molecular mass of the recombinant protein with His-tag specific with what omashu program ProtParam. To describe the immunogenicity of recombinant proteins, KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1 used for immunization of mice that received serum used in the analysis with tablets, coated peptide KAS2 and tablets, covered with formalin killed cells of P. gingivalis (Figure 2). According to the results of the analysis it was found that the serum with recombinant chimeric proteins KAS1-KsA1 and KAS2-KLA1 recognize peptide KAS2 (Figure 2A) at the same level, and serum specific to KAS2 (conjugate KAS2-diphtheria toxin), and formalin killed cells of P. gingivalis W60 (Figure 2B). However, the serum against recombinant protein KLA1 recognized only formalin killed cells of P. gingivalis W50 (Figure 2B).

Example 3

The effect of immunization with recombinant proteins (KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1) on crestal bone loss caused by P. gingivalis, on a murine model of periodontitis.

Recombinant proteins KsA1, KLA1, KAS1-KsA1 and KAS2 - KLA1, formalin killed P. gingivalis strain W50 and complex RgpA-Kgp used to determine and compare the degree of protection from resorption of the alveolar bone caused by P. gingivalis, using a modified murine models of bone destruction of periodontal proposed by Baker et al. (4). Mice were immunized (day 0 and 30 day) recombinant proteins KsA1, KLA1, KAS1-KsA1 or KAS2-KLA1, RgpA-Kgp complex or formalin killed cells of P. gingivalis strain W50 (FK-VV50) or only the adjutant S mice is errorline entered the living cells of R. gingivalis W50. Immunization all of these recombinant antigens, complex RgpA-Kgp and cells FK-W50 protected BALB/c mice from resorption of the alveolar bone caused by infection with P. gingivalis, as these animals had a significantly (p<0.001) lower bone resorption compared with mice from the groups immunized with PBS (Figure 3). However, in mice immunized KAS2-KLA1, was less bone resorption than in mice immunized with KLA1 (p<0.01); KsA1 (p<0.001), complex RgpA-Kgp (p<0.001), cells FK-W50 (p<0.001) and mice that nothing was injected (p<0.001). There were no significant difference in the degree of bone between mice immunized KAS2-KLA1, and mice immunized S1-sA1. Moreover, in mice immunized KAS1-KsA1, observed significantly less bone resorption compared with mice that did not injected (p<0.01), and mice immunized with the complex RgpA-Kgp (p<0.05), it did not differ from that of bone resorption in mice immunized KsA1, KLA1; complex RgpA-Kgp and FK-W50.

Example 4

Answers subclasses of antibodies by immunization with recombinant proteins (KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1) on mouse models of periodontitis.

Before and after oral administration of live cells of P. gingivalis Northallerton let the blood and received the serum by centrifugation. The Figure 4 shows the reactivity of subclasses of antibodies against at item formalin cells R. gingivalis W50 for each immunogen (KsA1, KLA1, KAS1-KsA1 or KAS2-KLA1 or killed by formalin cells of P. gingivalis strain W50 (FK-W50)) on a murine model of periodontitis. All protective immunogen caused the formation of high titers of IgG antibodies to FK-W50. Moreover, each of the protective antigen caused the formation of predominantly antibody lgG1 subclass, weak antibodies lgG2a, lgG2b and lgG3, FK-50 - specific antibodies (Figure 4). Before inoculation (Figure 4A) and after inoculation (Figure 4) each immunogen was caused by the formation of predominantly antibody lgG1 subclass.

Example 5

Mapping of epitopes KAS2 (433-468).

Synthesized biotinylated overlapping peptides consisting of 8 residues (indent 1, the overlap of 7 residues), for KAS2 (433-468) and put them on the tablets covered with streptavidin. Then determined the epitope binding antibody, using sera of mice immunized with KAS1-KsA1, KAS2-KLA1 and conjugate KAS2 - diphtheria toxin (Figure 5). A twofold increase in absorbance (415 nm) compared to the background were considered positive antibody response (threshold OD). Serum recognizes the following sequence of peptides derived from SEQ ID No.28, namely KAS1-KsA1, recognize peptides 435-442, 436-443, 445-452, 446-453 and 447-454 (threshold OD=0.07, Figure 5A), while KAS2 - KLA1 recognize peptides 435-442, 447-454 and 448-455 (threshold] OD=0.07, Figure 5A). It is allows to assume, what happened registering the lowest number of epitopes, namely peptide 436-442 (VSFANYT and its variant VGFANYT), peptide 447-452 (ETAWAD and its variant ETSWAD) and peptide 448-453 (TAWADP and its variant TSWADP). Peptides that contain the epitope peptides 436-442 include GVSFANYT, GVGFANYT, VSFANYTA and VGFANYTA. Peptides that contain the epitope peptides 447-452 and/or 44&-453 include SETAWAD, SETSWAD, ETAWADP, ETSWADP, TAWADPL and TSWADPL, in particular GSETAWAD, GSETSWAD, SETAWADP, SETSWADP, ETAWADPL, ETSWADPL, TAWADPLL and TSWADPLL.

Example 6

Synthesis of peptides and RAS KAS for conjugation with protein

Peptides were synthesized manually or by using a microwave synthesis system peptides CEM Microwave. This used standard Protocol for solid-phase peptide synthesis with Fmoc. Peptides were synthesized in the form of a carboxamide using Rink resin-AM-SURE (ARTES, KY, USA). The connection is performed with activation of HBTU/HOBt using 4 EQ. Fmoc-amino acids and 6 EQ. DIPEA. Fmoc group was removed with 20% piperidine in 1M HOBVDMF.

Resin bearing peptides and RAS KAS, was swelled in DMF and M-terminal Fmoc group was removed with 2% v/v DBU in DMF containing 2% v/v piperidine. Then N-terminal amino group was obtained with the help of a group of S-acetylmercaptosuccinic acid (ZAMA) using 5 EQ. SAMA-OPfp and 5 EQ. t. The reaction was observed by analyzing the trinitrobenzene sulfonic acid (TNS). Upon receipt of a negative response to TNBSA the resin was washed (5 × DMF, 3 × DCM and 3 ×diethyl ether). Then the resin was dried under vacuum. Separation of peptides from the resin was performed using a mixture of cleavage F:phenol:IS:EDT:water(92:2:2:2:2) within 2.5 hours, depending on the content of arginine in the peptide. After cleavage the resin was removed by filtration, and the filtrate was concentrated to a volume of approximately 1 ml in a stream of nitrogen. After deposition of the peptide products in the icy air, they were centrifuged and washed three times. Precipitation with peptides was dissolved in 5-10 ml of water containing 0.1% v/v TFA and the insoluble residues were removed by centrifugation. Peptides were purified using reversed-phase high-performance liquid chromatography (RP-HPLC) chemical groups can be used to obtain derived peptide for conjugation with proteins, they will make a reactive group such bands as Galenia group (bromine, chlorine, iodine), maleimide, Succinimidyl, hydrazine, oxime, thiol, which could then be used for connection of the received derived peptide with a protein such as KDR through his natural cysteine residues, or were derived from it by adding complementary reactive group that facilitates mechanical legirovanie for formation of a conjugate of the peptide-protein.

Conjugation of peptides SAMA with SA1. In a solution containing 10 μg/ml recombinant KA1 or other domain adhesin complex RgpA-Kgp in phosphate-saline buffet is e (0.1 M sodium phosphate, 0.9% NaCl, pH 7.4), was added 0.1 ml of 1% w/v solution of ester m-maleimido benzoyl-M-hydroxysuccinimide (MBS) in DMF. After 30 minutes remove unreacted MBS and collected the modified MBS KA1 using gel filtration using a PD10 column (Pharmacia, NSW, Australia), equilibrated with buffer for conjugation (0.1 M sodium phosphate, 5 mm EDTA; pH 6.0). Purified SAMA-peptide (1.3 mm) was dissolved in 200 ml of 6M guanidine HCl containing 0.5 M Tris; 2 mm EDTA, pH 6.0, and was dissolved in 800 μl of MilliQ water, removed the protective group in situ by adding 25 μl of 2M NH2HE (40 EQ) dissolved in MilliQ water. Collected MBS-KA1 immediately introduced into the reaction without protection SAMA-peptide and was stirred for 1 hour at room temperature. Conjugate of peptide-KA1 was separated from unreacted peptide by gel filtration using PDIO column, equilibrated IPBS pH 7.4 and liofilizirovanny. The reaction was monitored by analysis of Ellman.

Example 7

Antibodies

Polyclonal sera to recombinant proteins were obtained using subcutaneous immunization of mice with proteins. Mice were immunized in the first day, 25 μg of protein in incomplete Freund's adjuvant and 30 day 25 μg of protein in incomplete Freund's adjuvant. Immunization was performed using standard procedures. Received polyclonal serum with high titer against proteins. When necessary the STI received monoclonal antibodies directed against certain recombinant proteins using standard techniques.

Example 8

Immunization for the formation of antibodies.

6-8-week-old BALB/c mice or CD1 (outbred mice breed Swiss) 10 mice in the group were immunized subcutaneously (s.c. 100 ál) 50 μg of chimeric constructs KAS2-LA1 or antigen emulsified in incomplete Freund's adjuvant (IFA). After 30 days the mice were injected antigen (s.c. injection, emulsified in IFA) and after 12 days the mice were scored and conducted blood to obtain serum.

The definition of subclasses of antibodies by the method of ELISA. To determine the answers subclasses of antibodies in the serum of mice was performed enzyme-linked immunosorbent assay (ELISA) in triplicate using 5 µg/ml of chimeric constructs KAS2-LA1 or killed by formalin cells of P. gingivalis W50 or complex RgpA-Kgp in phosphate-buffered saline (PBS) (0.01 M Na2NRA4, 1.5 mm KH2RHO4, 0.15 M NaCl), pH 7.0, containing 0.1% (volume) Tween 20 (PBST), which was applied to the wells flat-bottom polyvinyl microtiter plates (Dynatech Laboratories, McLean, VA). After removal of the specified buffer for sorption in the wells was added PBST containing 2% (weight/vol) skimmed milk powder to block uncoated plastic for 1 h at room temperature. After that, the wells were washed 2 times with PBST, each well add ulali serial dilution of the serum of the mouse in PBST, containing 0.5% (weight/vol) skim milk (SK-PBST), and incubated for 16 CV at room temperature. After that, the wells were washed 6 times with PBST, SK-PBST was added in a dilution of 1/2,000 goat IgG to IgM IgA, lgG1, lgG2a, lgG2b, or lgG3 mouse (Sigma, New South Wales, Australia) and left to contact for 2 hours at room temperature. The tablets were washed 6 times with PBST, wells were made by dilution of 1/5,000 conjugate of horseradish peroxidase with rabbit antibodies specific for immunoglobulin goat (Sigma, New South Wales, Australia) in SK-PBST and incubated for 1 hour at room temperature. After that, the wells were washed 6 times with PBST, bound peroxidase antibodies was determined by adding to each well 100 μl of ABTS substrate [0.9 mm 2,2'-2,2'-azinobis(3-ethylbenzothiazoline-6) selifontova acid in 80 mm citric acid, containing 0.005% (volume) of hydrogen peroxide, pH 4.0]. The optical density at a wavelength of 415 nm was measured using reader microtiter plates (Bio-Rad microtiter reader tablet, model 450).

Answers subclasses of antibodies induced by immunization with the recombinant protein KAS2-KLA1 outbred mice (CD1, Swiss).

Of CD1 mice (Swiss) were immunized KAS2-LA1, letting blood and received the serum by centrifuging. The Figure 6 shows the reactivity of subclasses of antibodies to the chimeric construct KAS2-LA1, formalin killed cells of P. gingivalis W50 and complex RgpA-Kgp. Protein KAS2-LA1 calls the al production of IgG antibodies dominated lgG1, which recognize protein KAS2-LA1 and cross-interacted with formalin killed cells of P. gingivalis W50 and complex RgpA-Kgp (Figure 6). Moreover, protein KAS2-LA1 caused education is only weakly immunoreactive, lgG2a, lgG2 and lgG3 antigen-specific antibodies (Figure 6).

Example 9

Creating a structural model of the CRA and the surface defining the available sequence of the active site.

This work showed that the active site peptides CRA proteases are highly immunogenic and create protection at a high level of bone resorption caused by infection with P. gingivalis. To identify additional peptides active site of proteases that can be used in the vaccine, developed a model for the catalytic domain of KDR with a set of programs Orchestrar Sybyl7.3 (Figures 7). This model is based on the PDB structure 1crv RgpB protease P. gingivalis, these proteins have 23,58% steam identity and Z-score equal 25.09 (highly accurate model). When using a meta web server Meta-PPisp to determine protein-protein interaction suggested the existence of two surfaces with protein-protein interactions in the CRA: a surface for binding of the substrate (as in RgpB) and a second surface characteristic of the CRA. The main differences between the models RgpB and the CRA are the loops that limit the second vzaimozainteresovannost, and the period of 19 residues (Val526 - Phe545), which cannot be modeled in the CRA, which falls within the second contact surface. The Figure 7 shows the first model of the CRA, on which the thick ribbon denote surface available sequence around the active site of the CRA, it was found that surface-accessible surface are Asp388-Gln394, Leu421-Ala423, Ala443 - Glu447 with Ala451, Asn510-Trp513 and lle570-Gly577 with Tyr580. According to the model (Figure 6) it is obvious that along with KAS2 (A), there are three other j sequence KAS4 (Asp388 - Val395) (C), KAS5 (Asn510-Asp516) (C) and KAS6 (Ile570-Tyr580) (D), they also have sufficient length to be targeted by the vaccine. Thus, it is possible to obtain a recombinant chimeric protein that contains each of these peptides, and you can connect it 6 N-end KLA1 to get multika-KLAI, which can be used to stimulate the immune response and thus to protect from conditions or diseases caused by infection with P. gingivalis.

Example 10

The modeling process the ADR-X-proteinase for detection of immunogenic sites located on both sides from the catalytic site.

The three-dimensional structure of the ADR-X-proteinase was determined according Eichinger A, Beisel HG, Jacob U, Huber R, Medrano FJ, Banbula A, Potempa J, Travis J, Bode W. the Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinasewith a caspase-like fold. EMBO J. 1999 Oct 15; 18(20):5453-62.

Example 11

The bottom is shows an example of a composition in the form of toothpaste, containing antibodies

Ingredient% (by weight)
The dicalcium phosphate dehydrate50,0
Glycerin20,0
Sodium-karboksimetilcelljuloza1,0
Sodium lauryl sulfate1,5
Laurylsarcosine sodium0,5
Flavor1,0
Saccharin sodium0,1
Chlorhexidine gluconate0,01
Dextranase0,01
The goat serum containing specific antibodies0,2
Waterbalance

Example 12

The following is an example of a composition in the form of a toothpaste.

Ingredient%(by weight)
The dicalcium phosphate dehydrate 50,0
Sorbitol10,0
Glycerin10,0
Sodium-karboksimetilcelljuloza1,3
Sodium lauryl sulfate1,5
Laurylsarcosine sodium0,5
Flavor1,0
Saccharin sodium0,1
Monitoroff sodium0,3
Chlorhexidine gluconate0,01
Dextranase0,01
Serum ox containing specific antibodies0,2
Waterbalance

Example 13

The following is an example of a composition in the form of a toothpaste.

Ingredient% (by weight)
The dicalcium phosphate dehydrate50,0
Sorbitol 10,0
Glycerin10,0
Lauroyl diethanolamide1,0
Monolaurate sucrose2,0
Flavor1,0
Saccharin sodium0,1
Monitoroff sodium0,3
Chlorhexidine gluconate0,01
Dextranase0,01
Lg bovine milk containing specific antibodies0,2
Waterbalance

Example 14

The following is an example of a composition in the form of a toothpaste.

Ingredient% (by weight)
Sorbitol22,0
Irish moss1,0
Gitoxin sodium (50%)1,0
Gantrez19,0
Water (deionized)2,69
Monitoroff sodium0,76
Saccharin sodium0,3
The pyrophosphate2,0
Hydrated aluminum48,0
Aromatic oil0,95
Mouse monoclonal antibodies0,3
Sodium lauryl sulfate2,00

Example 15

Below is a sample of the liquid composition in the form of a toothpaste.

Ingredient% (by weight)
Polyacrylate sodium50,0
Sorbitol10,0
Glycerol20,0
Flavor1,0
Saccharin sodium0,1
Monitoroff sodium0,3
Chlorhexidine gluconate0,01
Ethanolthat 0
Lg horses, containing specific antibodies0,2
Linoleic acid0,05
Waterbalance

Example 16

The following is an example of a composition in the form of a mouthwash for mouth

Ingredient% (by weight)
Ethanol20,0
Flavor1,0
Saccharin sodium0,1
Monitoroff sodium0,3
Chlorhexidine gluconate0,01
Lauroyl diethanolamide0,3
Lg rabbit, containing specific antibodies0,2
Waterbalance

Example 17

The following is an example of a composition in the form of a mouthwash d is I the mouth.

Ingredient(by weight)
Gantrez S-972,5
Glycerin10,0
Aromatic oil0,4
Monitoroff sodium0,05
Chlorhexidine gluconate0,01
Lauroyl diethanolamide0,2
Monoclonal antibodies rabbit0,3
Waterbalance

Example 18

The following is an example of a composition in the form of candy.

Ingredient% (by weight)
Sugar75-80
Corn syrup1-20
Aromatic oil1-2
NaF0,01-005
Monoclonal antibodies rabbit 0,3
Magnesium stearate1-5
Waterbalance

Example 19

The following is an example of a composition in the form of a massage cream for the gums.

Ingredient% (by weight)
White petrolatum8,0
Propylene glycol4,0
Stearyl alcohol8,0
Polyethylene glycol 400025
Polyethylene glycol 400037,0
The sucrose monostearate0,5
Chlorhexidine gluconate0,1
Mouse monoclonal antibodies0,3
Waterbalance

Example 20

The following is an example of a composition in the form of chewing gum.

Ingredient% (by weight)
The basis of the gums30,0
Calcium carbonate2,0
Crystalline sorbitol53,0
Glycerin0,5
Aromatic oil0,1
Mouse monoclonal antibodies0,3
Waterbalance

Example 21

The following is an example of a pharmaceutical composition.

Ingredient%(by weight)
Humanized specific monoclonal antibodies10
Sterile phosphate-saline buffer90

Example 22

The following is an example of a composition in the form of periodontal gel.

Ingredient% (by weight)
Pluronic F12720,0
Stearyl alcohol8,0
Specific antibodies3,0
Colloidal silicon dioxide (Aerosil 200)1,0
Chlorhexidine gluconate0,1
Waterbalance

It should be understood that although the present invention is described in detail, examples are given solely for purposes of illustration. Other modifications variants of realization of the present invention which are obvious to the expert in the field of molecular biology, dental, diagnostics and related disciplines, are within the scope of the present invention.

References

1. McKee A. S., A. S. McDermid, A. Baskerville, A. C. Dowsett C. D. Eliwood and P. D. Marsh. 1986. Effect of hemin on the discrimination and virulence of Bacteroides gingivalis W50. Infect. Immun. 52:349-355.

2. Slots J. 1982. Importance of black-pigmented Bacteroides in human periodontal disease. Host parasite interactions in periodontal diseases. American Society for Microbiology.

3. O'brien-Simpson, N. M., R. R. Pathirana A. Paolini Y.-Y. Chen, P. D. Veith, T. V., R. N. Pike, N. Alley and E. C. Reynolds. 2005. An immiune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced bone loss. Journal of Immunology 175:3980-3989.

4. Baker, P. J., R. T. Evans and D. C. Roopenian. 1994. Oral infection with Porphyromonas gingivalis and induced alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Arch Oral il 39:1035-1040.

1. Chimeric or hybrid protein to induce an immune response against P. gingivalis, containing the th first peptide, connected directly or through a linker to the second polypeptide, in this case:
(a) said first peptide is characterized by the site of trypsin-like enzyme from P. gingivalis selected from the group consisting of SEQ ID NOs:1, 2, 27-34 and sequences that are at least 90% identical to these sequences;
(b) the specified second polypeptide is characterized adhesives domain of P. gingivalis or a fragment specified domain selected from the group consisting of:
(i)SEQ ID NOs:35-37 and sequences that are at least 90% identical to these sequences;
(ii)SEQ ID NOs:38, 39, 46, 81 and 82,
moreover, this plot trypsin-like enzyme from P. gingivalis and specified adhesively domain of P. gingivalis or its fragment capable of inducing an immune response against P. gingivalis.

2. Chimeric or hybrid protein in p. 1, wherein said first peptide contains the sequence[V/A]NYT[A/G]HGSETAW[A/G][D/T].

3. Chimeric or hybrid protein in p. 1, wherein said first peptide is characterized by a sequence that is identical or at least 90% identical to a sequence selected from the group consisting of SEQ ID nos:27-30, and the specified second polypeptide is characterized by a sequence that is identical or at intends 90% identical to the sequence selected from the group consisting of SEQ ID NO:36-37.

4. Chimeric or hybrid protein in p. 3, wherein said first peptide is characterized by a sequence that is identical or at least 90% identical to the sequence represented by SEQ ID NO:28, and the second polypeptide is characterized by a sequence that is identical or at least 90% identical to the sequence represented by SEQ ID NO:37.

5. Chimeric or hybrid protein in p. 3, wherein said first peptide is characterized by a sequence that is identical or at least 90% identical to the sequence represented by SEQ ID NO:27, and the second polypeptide is characterized by a sequence that is identical or at least 90% identical to the sequence represented by SEQ ID NO:36.

6. Chimeric or hybrid protein under item 1, characterized in that the C-terminal residue of the specified first peptide is covalently bonded directly or through a linker, whose length is either (i) up to 15 amino acids, or (ii) is less than 5 amino acids, with (a) N-terminal residue or (b) the C-terminal residue of the second polypeptide.

7. Chimeric or hybrid protein under item 1, characterized in that the N-terminal residue of the specified first peptide kovalan is but is connected directly or through a linker, whose length is either (i) up to 15 amino acids, or (ii) is less than 5 amino acids, with (a) N-terminal residue or (b) the C-terminal residue of the specified second polypeptide.

8. Chimeric or hybrid protein to induce an immune response against P. gingivalis, containing the first peptide, connected directly or through a linker to the second peptide, thus:
(a) said first peptide is selected from the group consisting of SEQ ID NOs:1, 2, 27-34 and sequences that are at least 90% identical to these sequences;
(b) the specified second peptide selected from the group consisting of SEQ ID NOs:71, 78, 79, 83, 84, 85 and sequences that are at least 90% identical to these sequences,
moreover, these first and second peptides are able to induce an immune response against P. gingivalis.

9. Chimeric or hybrid protein under item 8, characterized in that the C-terminal residue of the specified first peptide is covalently bonded directly or through a linker, whose length is either (i) up to 15 amino acids, or (ii) is less than 5 amino acids, with (a) N-terminal residue or (b) the C-terminal residue of the specified second peptide.

10. Chimeric or hybrid protein under item 8, characterized in that the N-terminal residue of the specified first peptide is covalently bonded directly or through l is ncera, whose length is either (i) up to 15 amino acids, or (ii )is less than 5 amino acids, with (a) N-terminal residue or (b) the C-terminal residue of the specified second peptide.

11. Chimeric or hybrid protein to induce an immune response against P. gingivalis, containing at least two sequences that are identical or at least 90% identical to the sequence of the plot trypsin-like enzyme from P. gingivalis selected from the group consisting of KAS1 (SEQ ID NO:27), KAS2 (SEQ ID NO:28), KAS3 (SEQ ID NO:29), PAS1K (SEQ ID NO:30); RAS1 (SEQ ID NO:31), RAS2 (SEQ ID NO:32), RAS3 (SEQ ID NO:33) and PAS1R (SEQ ID NO:34); and at least one sequence characterized adhesives domain of P. gingivalis or its fragment, selected from:
(i) sequences of SEQ ID NOS:35-37 or sequences that are at least 90% identical to these sequences, and
(ii) sequences of SEQ ID NOS:38, 39, 46, 81 and 82, with the specified plot trypsin-like enzyme from P. gingivalis and specified adhesively domain of P. gingivalis or its fragment capable of inducing an immune response against P. gingivalis.

12. The composition for inducing an immune response against P. gingivalis, containing an effective amount of a chimeric or hybrid protein according to any one of paragraphs.1, 8 or 11.

13. The composition according to p. 12, optionally containing excipient.

14. How preventive care is IKI condition or disease, associated with P. Gingivalis in a subject, comprising introducing to a subject an effective amount of a chimeric or hybrid protein according to any one of paragraphs.1, 8 or 11.

15. The use of chimeric or hybrid protein according to any one of paragraphs.1, 8 or 11 for the detection of antibodies to P. gingivalis in a biological sample.

16. Method of reducing the frequency or severity of condition or disease associated with P. Gingivalis in a subject, comprising introducing to a subject an effective amount of a chimeric or hybrid protein according to any one of paragraphs.1, 8 or 11.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. A biomass of the gram-negative bacteria Salmonella typhi fam. Enterobacteriacea is prepared. A cell wall peptidoglycane (CWP) of the bacteria is recovered by biomass extraction in 45% aqueous phenol at temperature 70-90°C or in aqueous solution of ionic or non-ionic detergents at temperature 37-100°C. That is followed by preparative enzymatic hydrolysis for insoluble CWP cleavage with the use of lysozyme at pH 4.5 - 8.9 and temperature 10 - 37°C. Simultaneously, a pharmacologically acceptable mixture of substances is removed by dialysis from the reaction mixture with the use of semipermeable membranes for ultrafiltration with cut-off size up to 5 kDa. The mixture of substances is also recovered by means of column gel-chromatography, particularly preparative gel chromatography on Sephadex or TSK gel columns. The end product yield is 320 mg.

EFFECT: method enables producing the pharmacologically acceptable mixture of substances containing the following ingredients: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (GMtri); β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (GMtetra); and GMtetra dimer (diGMtetra) wherein a bond of monomer residues of GMtetra is enabled by a carboxyl group of terminal D-alanine of one GMtetra residue and ω-aminogroup of meso-diaminopimelic acid of the other GMtetra residue with the coupled tetrapeptide resides positioned in various polysaccharide chains.

4 cl, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: claimed is biopolymer with expressed immunostimulating effect, consisting of highly molecular fragment of peptidoglican of cell wall of Gram-negative bacteria, whose repeating link (RL) represents tetrasaccharide 4-O-{4-O-[4-O-(N-acetyl-β-D-glucosaminyl)-N-acetyl-β-D-muramyl]-N-acetyl-β-D-glucosaminyl}-N-acetyl-D-muramic acid, to which on carboxyl groups of both residues of muramic acid bound is tetrapeptide residue: N-(L-alanyl-D-isoglutaminyl-meso-diaminopimelonyl-D-alanine). Binding between RL is perfomed due to octapeptide bridges (dwg.1), number of RL constituting from 5 to 20 or their combination. Also claimed are method of biopolymer obtaining, based on it pharmaceutical immunostimulating composition and its application in methods of stimulating immune system of mammals and non-specific protection against bacterial infections.

EFFECT: possibility of application in immunopharmacology, in particular, for creation of medications of supporting therapy, intended for enhancing immune resistance of organism.

11 cl, 2 dwg, 2 tbl, 5 ex

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary medicine. Currently, for differentiating nonspecific tuberculin reactions in cattle, dried purified tuberculin is used for mammals and "КАМ" with considering the sensitisation pattern by the reaction intensity. A common complex allergen is produced by protein settling from M scrofulaceum No. 12-C and M intracellulare No. 13-H strain cultures with added allergen produced from Corynebacterium xerosis N1911, in amount 1350 units of activity. The presence of coryneformic bacteria allergen in the "КАМ" composition improves the efficacy of a simultaneous dried purified tuberculin test for mammals in differentiating the nonspecific coryneformic bacteria reactions.

EFFECT: use of the declared allergen allows to prevent unreasonable slaughter, as well as further diagnostic finding expenses.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biochemistry, in particular to a monoclonal human antibody, specific to alpha-toxin of S. aureus. The claimed invention additionally relates to pharmaceutical compositions for treatment of prevention of the abscess formation in an organ, which contains at least one antibody or one nucleic acid, which codes the said antibody.

EFFECT: invention makes it possible to extend an assortment of antibodies, specific to alpha-toxin of S aureus.

23 cl, 7 dwg, 4 tbl, 6 ex

FIELD: medicine, pharmaceutics.

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

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

19 cl, 9 dwg, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology, molecular biology and genetic engineering. There are presented an immunogenic composition containing a mixture of staphylococcal proteins, and comprising a staphylococcal protein binding an extracellular component, and a staphylococcal transport protein, or the staphylococcal protein binding the extracellular component, and a staphylococcal virulence regulator or a toxin, or the staphylococcal transport protein and the staphylococcal virulence regulator or the toxin. There are also presented vaccines, methods of treating, using and methods for preparing a staphylococcus vaccine.

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

23 cl, 8 tbl, 7 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention discloses composition for production of antibodies SNAP-25, capable of binding with epitope, which contains C-end (carboxylic) on residue P1 of cleaved bond of cleavage site BoNT/A of SNAP-25 cleavage product, which contains carrier, flexible linker, containing at least three amino acids, and SNAP-25 antigen, for which amino acid sequence is given. Described are versions of antibodies α-SNAP-25 with said capability and their application in method of detecting BoNT/A activity with application of cells from stable cell line, sensitive to BoNT/A intoxication, and method of determining immunoresistance to BoNT/A in mammal with application of tested animal sample and cells of stable cell lime, whose cells are sensitive to BoNT/A intoxication.

EFFECT: application of invention ensures reliability, simplicity and makes it possible to exclude necessity of animal tests from botulinum toxin analyses.

15 cl, 11 dwg, 14 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. There are presented versions (aCt1 and aCt2) of one-domain antibodies specifically binding the Chlamydia trachomatis antigen. There are described versions of the method of inhibiting an infection caused by Chlamydia wherein the method involves the preparation of elementary bodies C.trachomatis by a therapeutically effective amount of the nanoantibody aCt1 or aCt2 before being attached to infected target cells.

EFFECT: use of the invention provides the antibodies to detect and block the infections Chlamydia trachomatis that can find application in medicine.

6 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: there are presented antibodies specifically binding the lipid-associated antigen of M.hominis with the characterised amino acid and nucleotide sequences, as well as a method of treating a Mycoplasma M.hominis infection involving administering to said mammal a therapeutically effective amount of the nanoantibodies.

EFFECT: invention can find further application in treating the mycoplasma infection.

4 cl, 2 tbl, 6 ex, 5 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a method for preparing an anthrax diagnostic serum by hyperimmunisation of ox producers with an antigen of the strain Bacillus anthracis M-71. Hyperimmunisation is conducted in increasing doses: first subcutaneous introduction of the antigen 100-120 mln microbial cells together with saponin 2.5-3 mcg; 12-14 days later, the antigen is introduced intracutaneously in a dose of 2.5-3.0 bln microbial cells together with saponin 2.5-3 mcg; 6-7 days later, every 3-4 days, the antigen is introduced intravenously 12-14 times in increasing doses 10.0 to 210 bln. microbial cells. It is followed by blood sampling, keeping at temperature 37-38°C for 2-3 hours, placing in a fridge at 2-8°C for 3-5 days, separating serum. The prepared serum is preserved in 5-7% carbolic acid in isotonic solution in ratio (9-10):1 respectively. The ready serum is sterile and has an AR titre min. 1:1000, a CFT titre min. 1:20. A diagnostic set for anthrax diagnosis comprises the antigen of the strain Bacillus anthracis M-71, the anthrax diagnostic serum and healthy bovine's native serum.

EFFECT: invention provides higher specific activity of the serum and diagnostic effectiveness for an anthrax agent.

5 cl, 1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a number of polynucleotides and polypeptides β-hemolytic streptococci, in particular polypeptides and polynucleotides of Streptococcus pyogenes, and based on them immunogenic compositions, used for prevention or reduction of symptoms of colonisation or infection, caused by β-hemolytic streptococci. Immunogenic composition (versions) contains mixture of two or more polypeptides, encoded by sequence of nucleic acid (NA), which has, at least, 90% identity of sequence of NA, selected from group, consisting of peptidase C5a (SCP), open reading frame (OPC)554, OPC 1218, OPC 1358 and OPC 2459. One of versions of immunogenic composition contains polypeptide SCP, polypeptide peptidylpropylisomerase and, at least, one other polypeptide. Also disclosed are methods of protecting susceptible mammal against colonisation or infection, caused by β-hemolytic streptococcus, by immunisation of immunogenic composition by invention.

EFFECT: invention provides immunogenic compositions and methods for prevention or reduction of symptoms of infections, caused by β-hemolytic streptococci of group A, B, C and G, as well as ensures immunity to wide spectrum of bacteria BHS.

41 cl, 16 dwg, 2 tbl, 3 ex

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

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

FIELD: biotechnologies.

SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.

EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.

9 cl, 7 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents an extracted polypeptide, including a sulfonylurea-reactive repressor, which is specifically bound with a polynucleotide, containing an operator sequence, where binding is adjusted by the compound of sulfonylurea and where the specified polypeptide contains an amino acid sequence, which is at least by 90% identical to the sequence SEQ ID NO:1233. The invention also relates to a polynucleotide that codes polypeptide, and also to a cell transformed by polynucleotide. Also the method is proposed to regulate transcription of target polynucleotide in a cell using the proposed polynucleotide.

EFFECT: improved characteristics of a compound.

31 cl, 29 tbl, 7 ex, 11 dwg

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is a polypeptide, obtained from strain SA-01 Thermus scotoductus, responsible for the reduction of uranium (VI) in a uranium (VI) source to uranium (IV), where the polypeptide includes an amino acid sequence SEQ ID NO:1. Also claimed are a separated molecule of nucleic acid, coding the polypeptide and including a nucleotide sequence SEQ ID NO:2, an expression vector, including the said molecules of nucleic acid, a host cell, including the said vector. Also claimed are a method of obtaining the said polypeptide and its application in the reduction of uranium (VI) in the source of uranium (VI) to uranium (IV).

EFFECT: group of inventions provides a high degree of reduction of uranium (VI) to uranium (IV).

9 cl, 10 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: protein causing an Yersinia pestis cell autoagglutination property and located on a surface of the Yersinia pestis cell is recovered, and the presence thereof is correlated with an ability of bacteria to the autoagglutinaiton growth in liquid nutrient media and with increased hydrophobic nature of the cells. Molecular weight of the protein makes 17.485 kDa, the protein content is 80%, and the neutral carbohydrate content is 6%. A method for producing the protein involves growing the strain of Y.pestis KM 1279 on 1.5% agar LB; the grown culture is washed off with cold buffered saline and dried with acetone. The cells are washed off and deposited by centrifugation in the cold; the cell deposition is suspended in NaOH (pH 9.0), kept in agitation at 26°C for two hours, and the cells are separated by centrifugation (8000 rpm). The supernatant is filtered through the membrane; the filtrate is added with neutralised ammonium sulphate to 25% saturation, kept to form a deposition to be separated by centrifugation and made free from ammonium sulphate by dialysis. The dialysed solution is added with HCl to pH 4.6, incubated to form a deposition, centrifuged; the deposition is dissolved in 5 mM NaOH. The protein is deposited by acetone, kept in the cold, centrifuged; and the deposition is lyophilised.

EFFECT: protein is used for studying its protective properties, for creating on its basis vaccination preparations and assessing its involvement in realisation of the virulent properties of the plague agent.

2 cl, 1 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method for plasmid DNA condensation provides mixing plasmid DNA with a condensing agent in the molar ratio of 1:10. A condensing agent is presented by recombinant DNA-binding Hu-protein Acholeplasma laidlawii. The invention may be used in research studies and for the purpose of gene-therapy agent delivery into the eukaryotic cells.

EFFECT: method provides higher effectiveness of eukaryotic cell transfection by plasmid DNA.

5 dwg

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: medicine.

SUBSTANCE: recombinant plasmid DNA pTB323 under the invention coding the hybrid polypeptide glutathione-8-transferase (GST) and a shorter version of the protein MPT64 (rΔMPT64), has an average molecular weight 3.6 MDa, size 5574 base pairs, consists of: a) EcoRI-BamHI-fragment of the vector plasmid pGEX-2T of size 4938 base pairs containing the β-lactamase gene inducing tac-promotor, the internal gene Iaclg coding the lactose operone repressor protein, a glutathione-5-transferase gene fragment from S. japonicum with a multiple sites of gene cloning (MSC) in 3'-terminal part of this gene and a nucleotide sequence coding a thrombine proteolysis site and found in front of the MSC; b) EcoRI-BamHI-fragment of 636 base pairs containing a truncated gene MPT64 flanked by EcoRI and BamHI restriction endonuclease sites and prepared by amplification of the gene-related fragment with genome DNA M. tuberculosis; c) a genetic marker - β-lactamase gene determining resistance of pTB323 plasmid transformed cells E. coli to the antibiotic ampicillin; d) unique restriction sites: BamHI - 930/934, EcoRI ~ 1566/1570. The recombinant bacterial strain Escherichia coli BL21/pTB323 - producer of hybrid polypeptide GST-ΔMPT64 with the properties of the mycobacterial antigen ΔMPT64 is deposited in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector, No. B-1028. The recombinant polypeptide GST-ΔMPT64 produced by the recombinant strain under the invention contains as a carrier protein the N-terminal polypeptide fragment glutathione-S-transferase S.j. (226 amino acid residues, 26.31 kDa) and has a complete amino acid sequence (431 amino acid residues, 48.76 kDa) presented in the description.

EFFECT: using the invention enables developing the high-purity polypeptide in the preparation amounts with the preserved immunogenic properties and provided separation of the target protein from the amino acid sequence of the carrier protein for studying of the immunogenic properties of the target protein.

3 cl, 4 dwg, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: microorganism, which codes antigens and protein toxins, contains a first component, which is at least one nucleotide sequence coding at least at least one complete or partial antigen of at least one wild-type or mutated protein, a second component which is at least one nucleotide sequence coding for at least one protein toxin and/or at least one protein toxin subunit, a third component consisting of at least a first subcomponent which is at least one nucleotide sequence coding at least one transport system which enables the expression of the first and second components on the outer surface of the microorganism and/or enables the secretion of the expression products of the first and second components, and/or coding at least one signal sequence which enables the secretion of the expression products of the first and second components, and/or optionally, from the second subcomponent, which is at least one nucleotide sequence coding at least one protein for lysing the microorganism in the cytosol of mammalian cells and for intracellularly releasing plasmids or expression vectors, which are contained in the lysed microorganism; and a fourth component which is at least one nucleotide sequence for at least one activation sequence for the expression of one or more of the first, second and third components, wherein said activation sequence can be activated in the microorganism and/or is tissue cell-specific, tumour cell-specific, macrophage-specific, dendrite-specific, lymphocyte-specific, function-specific or non-cell-specific, wherein any of the first, second, third or fourth components, present in the microorganism more than once, are independently identical or different, wherein the first and second components are different from each other. Also disclosed are a medicinal agent for stimulating immune response and a pharmaceutical composition based on said microorganism, methods of obtaining said microorganism, the corresponding expression plasmid and expression vector for obtaining said microorganism.

EFFECT: invention enables to obtain novel antitumour vaccines, which induce a strong systemic cellular immune system response, which increases effectiveness of antitumour therapy.

21 cl, 22 dwg, 8 ex

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