A dna fragment encoding a fragment of the protein transferrin receptor strain of haemophilus (options), plasmid vector (options), recombinant protein (options), isolated and purified protein (options), immunogenic composition and method of obtaining isolated and purified protein

 

(57) Abstract:

The invention relates to genetic engineering. The DNA fragment encodes a fragment of the protein transferrin receptor strain of Haemophilus. Conservative amino acid sequence of the protein contained in the description. DNA fragments encoding proteins transferrin receptor strains DL 63 or Eagan Haemophilus influenzae type b or Netherwing strains of CANCER 12085, SB33, SB12, SB29, SB30 or SB32 Haemophilus influenzae. Amino acid sequence of the encoded protein contained in the description. Plasmid vector adapted for transformation of E. coli and includes one of the above DNA fragments. Recombinant protein is produced by E. coli transformed by one of these DNA fragments. Isolated and purified protein bp1 and bp2 with the amino acid sequence shown in the description. Immunogenic composition is made in the form of a vaccine for in vivo administration and includes isolated and purified protein TR1 or TR2 strain Haemophilus influenzae, and a pharmaceutically acceptable carrier. Receive recombinant host expressing cells to activate one of the proteins TP1 or TP2, cultivate it and destroy the cell mass, receive the cell lysate and fractionary. Conduct selective extraction and re-fracc is s, caused by Haemophilus. 12 s and 13 C.p. f-crystals, 30 tab., 7 table.

The invention relates to the molecular cloning of genes encoding the transferrin receptor, and in particular to gene cloning of transferrin receptor Haemophilus influenzae.

Reference to related application

This application is a partial continuation of concurrently pending application for U.S. patent reg. 08/175116, filed December 29, 1993, which, in turn, is a partial continuation of concurrently pending application for U.S. patent 08/148968, filed November 8, 1993.

Background of the invention

Encapsulated strains of Haemophilus influenzae type b are the main causative agents of bacterial meningitis and other invasive infections in young children. However Neopalimovsky and Netherweave bacteria H. influenzae (NTHi) is responsible for the emergence of a wide range of diseases in humans, including otitis media, epiglottitis, pneumonia and tracheobronchitis. Vaccines made based on the capsular polysaccharide of H. influenzae type b conjugated to diphtheria toxoid [Berkowitz and others, 1987. (In this application for a more complete description of the modern level of science in the area belongs to this is I information for each link is given at the end of this description, immediately before the claims. The disclosure of these works is introduced in the present description by reference)], tetanus by toxodon (Classon and others , 1989, and U.S. patent 4496538), or with the outer membrane protein of Neisseria meningitidis (Black and others, 1991), has shown its effectiveness in the treatment of meningitis induced by H. influenzae type b, however, they proved to be ineffective in the treatment of NTHi-induced diseases (Bluestone, 1982).

Inflammation of the middle ear (otitis media) is one of the most common diseases among young children; and 60-70% of all children under the age of 2 years have been reported 1-3 ear infections. Chronic inflammation of the middle ear can lead to hearing loss, speech, and cognitive abilities in children. Infections caused by H. influenzae, and about 30% of cases are caused by the occurrence of acute inflammation of the middle ear and about 60% of cases of chronic inflammation of the middle ear. Only in USA antibiotics for treatment of inflammation of the middle ear and surgery associated with this disease, such as tonsillectomy, adenoidectomy, and the introduction tympanostomy tubes, spent 1-2 billion dollars per year. In addition, many microobjectives preventive vaccines against otitis media is extremely important. Netherway strains of H. influenzae are also major pathogens responsible for causing pneumonia in the elderly, and other individuals, which is particularly susceptible to respiratory infections. In this regard, it is essential that you get these antigens from H. influenzae, which could be used as components in immunogenic preparations intended for protection against many serotypes of H. influenzae.

Iron is the main nutrient required for the growth of many bacteria. Some human pathogens such as H. influenzae, Branhamella cattaraliis, n. meningitidis, n. gonorrhocae and non-pathogenic commensal strains of neisseria can use transferrin person as the source of iron (Schryvers, 1988; Schryvers and Zee 1989; Micklsen and Sparling, 1981). The receptor for bacterial transferrin (TfR) consists of two chains, Tbp1 and Tbp2. In strains of H. influenzae, molecular weight Tbp1 is approximately 100,000, and the molecular weight bp2 ranges from 60000 to 90000 depending on the strain (Schryvers and Gray-Owen, 1992; Holland and others, 1992). The expression of transferrin receptor H. influenzae is, obviously, iron and/or hemimegalencephaly (Norton and others, 1993), and the proposed site fur-binding, as it was identified, who's iron, including TfR gene of N. meningitidis (Zegrain and others, 1993).

It was found that in other bacterial TfR-operons, promoters follow tbp2 genes and tbp1 (Zegrain and others, 1993; Wilton and others, 1993). Antibodies that block the access of transferrin receptor to its source of iron, can prevent bacterial growth. In addition, antibodies against TfR, which are opsonizing or bactericidal factor, can also provide protection from bacteria through alternative mechanisms. Thus, the transferrin receptor, its fragments, its components chains or proteins derived from this receptor, are candidates for use for the manufacture of a vaccine to protect against infections caused by H. influenzae. Mice immunized with proteins TfR n. meningitidis in Adelante's adjuvant, were found resistant to homologous antigenic stimulation, and antisera against TfR possessed bactericidal and protective action in the analysis of passive transport (Danve and others, 1993). Pigs immunized with recombinant Tbp2 A. pleuropneumoniae were found resistant to homologous antigenic stimulation, but did not show resistance to heterologous stimulation (Rossi-Campos and others, 1992). These data indicate that the vaccine is STI DNA molecules, encoding the transferrin receptor and peptides that correspond to specific parts of the transferrin receptor, as well as vectors containing such sequences, in order to use them for diagnosis, immunization and production of immunological diagnostic reagents.

Poliovirus is an enterovirus, a kind of a virus belonging to the family of Picornaviruses. There are three different serotypes of Polikarpov, and in addition, there are many strains belonging to each of these three serotypes. Virulent strains are the causative agents of paralytic polio. Attenyerevan strains, i.e., strains with reduced ability to induce paralytic disease, and inactivated virulent strains have been used as vaccines. The virus infection induces lasting protective immunity of the mucous membranes. Vaccination using vaccines based on inactivated poliovirus can also induce an immune response mediated by mucous membrane.

The structure of the poliovirus is known and is highly conservative for different strains and serotypes. Were also identified patterns not their structure these picornaviruses are closely related to the polioviruses. It was shown that it is possible to Express the foreign epitopes on the capsid of poliovirus (Murdin and others, 1992), and this experience can be extended to other picornaviruses. Epitopes that have been expressed, represented, mainly, short, well-defined, contiguous epitopes; and most of them have been expressed in antigenic site I neutralization of poliovirus (NAg1) or as b the site to other picornaviruses. This website includes yourself and the loop connecting beta-chain and C (g-loop) capsid protein VPI poliovirus. SU-loop protein VPI is an available surface loop, consisting of nine amino acids that may be substituted and which may be attached, at least twenty-five heterologous amino acids (Murdin and others , 1991). Hybrid or chimeric polioviruses expressing epitopes transferrin receptor, and cultivated to a high titer, as well as being immunogenic, could be used as a vaccine and as a means for the production of immunological reagents.

Brief description of the invention

The present invention relates to the production of purified and selected nucleic acid molecules, codereuse is now the molecules of the present invention can be used for the specific detection of strains of Haemophilus, and for the diagnosis of infections caused by Haemophilus.

Isolated and purified nukleinovokisly molecules of the present invention, such as DNA, can also be used for gene expression TfR using the techniques of recombinant DNA for cost-effective production of treated and highlighted subunits transferrin receptor, its fragments, or analogs. The transferrin receptor, its subunits, fragments, or analogs, as well as encoding molecules are nucleic acids and vectors containing these nucleic acid molecules can be used in immunogenic compositions for protection against diseases caused by Haemophilus; for the diagnosis of infections caused by Haemophilus, and also as a means for the production of immunological reagents. Monoclonal antibodies or monospecific antisera (antibodies) produced against the protein transferrin receptor produced in accordance with aspects of the present invention can be used to diagnose infections caused by Haemophilus; for the specific detection of Haemophilus (for example, in in vitro and in vivo assays); and for treatment of diseases caused by Haemophilus.

Peptides corresponding to op the operations for the treatment of diseases, caused by Haemophilus, for the diagnosis of Haemophilus infections, and also as a means of production of immunological reagents. Monoclonal antibodies or antisera generated against these peptides obtained in accordance with aspects of the present invention can be used for the diagnosis of Haemophilus infections, for the specific detection of Haemophilus (for example, in in vitro and in vivo assays) and for immunization for the treatment of diseases caused by Haemophilus.

In one of its aspects the present invention relates to purified and the selected nucleic acid molecule that encodes a protein transferrin receptor, derived from strain Haemophilus, in particular from strain H. influenzae, and more specifically from the strain of H. influenzae type b, such as strain DI63, Eagan or Minna H. influenzae type b; or Netherweave strain of H. influenzae, such as a strain of CANCER 12085, SB33, S12, S29, S30 or S32 H. influenzae; or a fragment or analog of the protein of the transferrin receptor.

In one of the preferred embodiments of the invention the nucleic acid molecule can encode either a protein Tbp1 or TBp2 protein of a strain of Haemophilus. In another preferred embodiment of the present invention the nucleic acid may coleopterist, which is conservative for bacteria that produce the protein transferrin receptor. Such conservative amino acid sequence may have an amino acid sequence contained in amino acid sequences of the peptides are shown below in tables 2 and 3, for the strain Eagan Haemophilus influenzae type b, and the corresponding peptides from other strains of H. influenzae.

In another aspect the present invention relates to purified and the selected nucleic acid molecule having a DNA sequence selected from the group comprising: (a) any of the DNA sequences shown in Fig.3, 4, 5, 6, 7, 8, 9, 10 or 11 (SEQ ID 1, 2, 3, 4, 105, 108, 110, 112, 114), or a DNA sequence complementary to any of these sequences; (b) a DNA sequence encoding one of the amino acid sequence shown in Fig.3, 4, 5, 6, 7, 8, 9, 10 or 11 (SEQ ID 5, 6, 7, 8, 9, 10, 11, 12, 106, 107, 109, 111, 113, 115), or DNA sequence, complementary sequence; and (C) a DNA sequence that hybridizes in stringent conditions with any of the DNA sequences defined in (a) and (b). Preferably, the DNA sequence defined in (C), 2">

In another aspect the present invention relates to a vector adapted for transformation of a host containing a nucleic acid molecule as defined above. Such a vector may be any vector that has the characteristics of plasmid DS-712-1-3 (ATSC, incoming number 75603) or plasmid JB-1042-7-6 (ATS, incoming number 75607).

These plasmids can be adapted for expression of the encoded transferrin receptor, its fragments or analogues in lepidosiren or delipidation form in heterologous or homologous to the host. In accordance with this, in yet another aspect the present invention relates to expressing vector adapted for transformation of a host containing a nucleic acid molecule of the present invention; and expressing the item is correctly connected to nukleinovokisly molecule to ensure the expression of host protein transferrin receptor, or its fragment, or analog. In specific embodiments, the implementation of this aspect of the present invention the nucleic acid molecule can encode mostly all protein transferrin receptor, or protein Tbp1 or Tbp2 protein of a strain of Haemophilus. Expressing the element on the Torah transferrin, or its fragment, or analog. Expressing the element can also include a portion of nucleic acid encoding the signal lipidization, for expression in host lepidosirenidae form of the protein transferrin receptor, or its fragment, or analog. Expressing plasmid may have the identifying characteristics of plasmid JB-1468-29, JB-1600-1 or JB-1424-2-4. The host may be selected, for example, from Escherchia coli, Bacillus, Haemophilus, fungi, yeast or baculovirus; this can also be used expressing system virus Semliki forest.

In another aspect the present invention relates to a transformed host containing an expression vector, as defined above. This host can be selected from JB-1476-2-1, JB-1437-4-1 and JB-1607-1-1. In addition, the present invention relates to the protein transferrin receptor, or fragment or analogue produced by a transformed host.

As explained in detail below, the receptor proteins Tbp1 and bp2 were produced separately from each other. Therefore, in still another aspect the present invention relates to isolated and purified Tbp1 protein of a strain of Haemophilus separate from protein b2 strain of Haemophilus and isolated and purified protein b is influenzae type b or Netherway strain H. influenzae.

In addition, the present invention relates to synthetic peptides corresponding to parts of the transferrin receptor. In this regard, in another aspect the present invention relates to synthetic peptide containing at least six amino acids and no more than 150 amino acids and having an amino acid sequence corresponding to only part of the protein transferrin receptor or similar protein transferrin receptor strain of bacteria. Preferred bacterial strain is a strain of Haemophilus, in particular a strain of H. influenzae, and more particularly a strain of H. influenzae type b, or Netherway strain of H. influenzae.

The peptides obtained in accordance with the present invention may contain amino acid sequence, which is conservative for bacteria that produce the protein transferrin receptor, including strains of Haemophilus. This peptide can include the amino acid sequence LEGGFYGP (SEQ ID No. 74), or a sequence LEGGFYG (SEQ ID 85). The peptides of the present invention may have an amino acid sequence selected from the sequences presented below (in table 2 or 3) for strain Eagan H. influenzae type b, and the corresponding amino is giving relates to an immunological composition, which contains at least one active component selected from at least one molecule of nucleic acid, provided in this application; at least one recombinant protein, are presented in this application; at least one purified and isolated proteins Tbp1 or b2 presented in this application; at least one synthetic peptide provided in this application; and "alive" vector presented in this application; and pharmaceutically acceptable carrier. With the introduction of the host, at least one active component produces an immune response.

Immunogenic compositions of the present invention can be manufactured in the form of a vaccine for administration in vivo in order to protect the body from diseases caused by bacterial pathogens that produce receptors for transferrin. For these purposes, the composition can be made in the form of microparticles, capsules or liposomal preparations. Alternative compositions can be made in combination with a molecule, providing targeted delivery of the composition to specific cells of the immune system or to the surface of mucous membranes. The immunogenic composition can coderay the transferrin receptor. Immunogenic compositions may also contain adjuvant.

In another aspect the present invention relates to a method of protection against infection or disease caused by Haemophilus or other bacteria that produce transferrin receptor; moreover, this method provides for the stage of introduction of a susceptible host, such as a human, an effective amount of immunogenic composition defined above.

In yet another aspect the present invention relates to anticigarette or antibody against the recombinant protein; isolated and purified protein Tbp1 or bp2; to synthetic peptide or immunogenic composition.

In his next aspect the present invention relates to a live vector for delivery of transferrin receptor in the host organism containing a nucleic acid molecule as defined above. This vector may be selected from Salmonella, BCG, adenovirus, poxvirus, vaccinia virus (cowpox virus) and poliovirus. In particular, the specified vector can be poliovirus, and this nucleic acid molecule may encode a fragment of the transferrin receptor having the amino acid sequence EGGFYGP, having the identifying characteristics of plasmid rtta, rttv, RTTs or rttr ADS, 75931, 75932, 75933, 75934).

In yet another aspect the present invention relates to a strain of Haemophilus, which does not produce the protein transferrin receptor. This strain may contain the gene encoding the transferrin receptor, which is functionally blocked, for example by insertional mutagenesis. The strain of Haemophilus can be attenyerevan strain, and this attentionally strain may contain a vector for delivery of transferrin receptor.

As mentioned above, in one aspect the present invention relates to isolated and purified Tbp1 protein or protein b2 strain Haemophilus, and preferably a strain of Haemophilus influenzae; moreover, these proteins are allocated separately from each other. In accordance with this, in yet another aspect the present invention relates to a method for producing isolated and purified protein Tbp1 or b2 strain Haemophilus, involving the following stages: (a) obtaining a recombinant host expressing cells to enable or Tbp1 protein or protein b2 (but not both); (b) culturing the host cells in order to obtain cell pool; (C) DuChene first supernatant and a first precipitate, where the first supernatant, in the main, contains large amounts of soluble host proteins; (e) separating the first supernatant from the first precipitate; (f) selective extraction of the first sludge removal, basically, all soluble host proteins and membrane proteins of the owner to obtain a second supernatant and extracted sludge, containing calf inclusion; (g) separating the second supernatant from the extracted sludge; (h) the solubilization extracted sludge to obtain solubilizing extract and (i) fractionation solubilizing extract to obtain fractions containing protein Tbp1 or b2.

The cell lysate can be fractionated with the first supernatant and a first precipitate can be obtained by at least one extraction detergent.

The solubilized extract can be fractionated by gel filtration to obtain fractions containing either protein Tbp1 or protein b2, which can then be Valitova to remove at least a detergent, resulting in getting more purified Tbp1 protein solution or b2.

Brief description of drawings

For a better understanding of the present izobreteniya map two plasmid clones (pBHT1 and runt) operon transferrin receptor strain DL63 Haemophilus influenzae type b.

In Fig.1B shows a restriction map of clone S-4368-3-3, JB-901-5-3 containing genes fR from strain Eagan H. influenzae type b.

In Fig. 1C shows a restriction map of clone DS-712-1-3, containing the gene for transferrin receptor from strain MinnA H. influenzae type b.

In Fig. 1D shows a restriction map of clone JB-1042-7-6, containing the gene for transferrin receptor from Netherweave strain CANCER 12085 H. influenzae.

In Fig.2 illustrates the organization and restricion card cloned genes b1 and b2 identified strains and genetic organization of TfR-operon with two successive genes (Tbp1 and Tbp2) forming an operon under the transcriptional control of a single promoter; and in addition, the drawing shows the 3.0 KB DNA fragment of the plasmid runt used for sensing libraries TfR genes from strains of Haemophilus.

In Fig.3 shows the nucleotide sequence of the gene transferrin receptor (SEQ ID 1) and deduced amino acid sequence (SEQ ID 5 - Tbp1 and SEQ ID 6 - Tbp2) from strain DL63 H. influenzae type b. The underlined amino acid sequences correspond to peptides b1 identified by amino acid b. Putative signal sequence is 2.

In Fig.4 shows the nucleotide sequence of the gene transferrin receptor (SEQ ID 2) and deduced amino acid sequence (SEQ ID 7 - Tbp1 and SEQ ID 8 - b2) from strain Eagan H. influenzae type b. Putative -35, -10 and sequence of the binding site of the ribosome is shown by a line drawn from the top.

In Fig.5 shows the nucleotide sequence of the gene transferrin receptor (SEQ ID 3) and deduced amino acid sequence (SEQ ID 9 - Tbp1 and SEQ ID 10 - b2) from strain MinnA H. influenzae type b. Expected sequence -35, -10, and the sequence of the binding site of the ribosome are shown by lines drawn on top.

In Fig.6 shows the nucleotide sequence of the gene transferrin receptor (SEQ ID 4) and deduced amino acid sequence (SEQ ID 11 - Tbp1 and SEQ ID 12 - b2) from Netherweave strain CANCER 12085 H. influenzae. Expected sequence -35, -10, and the sequence of the binding site with the ribosome are shown by lines drawn on top.

In Fig.7 shows the nucleotide sequence of the gene transferrin receptor (SEQ ID 105) and their deduced amino acid sequence (SEQ ID 106 - Tbp1 and SEQ ID 107 - b2) from Netherweave strain SB33 H. influenzae.

In Fig.8 I (SEQ ID 109 - b2) from Netherweave strain S12 H. influenzae.

In Fig.9 shows the nucleotide sequence of the gene b2 (SEQ ID 110) and deduced amino acid sequence (SEQ ID 111 - b2) from Netherweave strain S29 H. influenzae.

In Fig. 10 shows the nucleotide sequence of the gene b2 (SEQ ID 112) and its deduced amino acid sequence (SEQ ID 113 - b2) from Netherweave strain S30 H. influenzae.

In Fig. 11 shows the nucleotide sequence of the gene b2 (SEQ ID 114) and its deduced amino acid sequence (SEQ ID 115 - b2) from Netherweave strain S32 H. influenzae.

In Fig.12A shows the nucleotide sequence of the promoter regions and the 5'-end of tbp2 genes originating from strains Eagan (SEQ ID 116), MinnA (SEQ ID 117), CANCER 12085 (SEQ ID 118) and S33 (SEQ ID No. 119). Sense primer used for amplification of genes tbp 2 using PCR, underlined (SEQ ID 120).

In Fig.12B shows the nucleotide sequence of the intergenic region and the 5'-end of genes tbp 1, derived from strains of H. influenzae, such as Eagan (SEQ ID 121), MinnA (SEQ ID 122), DL63 (SEQ ID 123), CANCER 12085 (SEQ ID 124), S12 (SEQ ID 125), SB29 (SEQ ID 126), S30 (SEQ ID 127) and S32 (SEQ ID 128). Antisense primer used for gene amplification using PCR, underlined (SEQ ID 129).

In Fig.13 prolationum strains of H. influenzae, such as: S12, S29, SB30, S32 and SB33. Track 1 - SB33; track 2 - S12, lane 3 - S29, lane 4 - SB30, track 5 - S32.

In Fig.14 illustrates a comparison of the amino acid sequence of the protein Tbp1, derived from strains of H. influenzae, such as Eagan DL63, CANCER 12085 and S33 (SEQ ID 7, 5, 11 and 106); strains of N. meningitidis, such as VW and M (SEQ ID 94 and 95); and strains of N. gonorrhoeae FA19 (SEQ ID 96).

In Fig.15 illustrates a comparison of amino acid sequences b2, derived from strains of H. influenzae, such as Eagan, DL63, CANCER 12085, S12, S29, SB30 and S32 (SEQ ID 8, 6, 12, 109, 110, 112, 114), strains of N. meningitidis, such as VW and M (SEQ ID 97 and 98), strain F19 N. gonorrhoeae and strains of Actinobacillus pleuropneumoniae, such as AR and AR (SEQ ID 99 and 100).

In Fig.16A shows the putative secondary structure of the protein Tbp1 H. influenzae, and Fig. 16B shows the putative secondary structure of the protein b2 H. influenzae.

In Fig. 17 shows the construction of plasmid JB-I468-29, which expresses in E. coli protein Tbp1, derived from strain Eagan H. influenzae type b.

In Fig. 18 shows the construction of plasmid JB-I424-2-8, which expresses in E. coli protein b2, derived from strains of H. influenzae type b.

In Fig. 19 presents pairs of oligonucleotides (SEQ ID 130, 131) used: (SEQ ID 86, 87); (SEQ ID 88, 89); (SEQ ID 90, 91) and D (SEQ ID 92, 93) used to construct plasmids expressing Tbp1 and b2.

In Fig.21 shows the construction of plasmid JB-1600-1, which expresses in E. coli protein b2, derived from strain S12 H. influenzae.

In Fig. 22 illustrates electrophoresis in SDS page with LTOs products of protein expression and protein Tbp1 b2 strain Eagan Haemophilus type b, and protein b2 Netherweave strain S12 H. influenzae E. coli. Track 1: JB-1476-2-1 (T7/Eagan Tbp1) in t0; track 2: JB-1476-2-1 at the time of induction of t=4 hours; lane 3: molecular weight markers: 200 kDa, 116 kDa, of 97.4 kDa, 66 kDa, 45 kDa and 31 kDa; lane 4: JB-1437-4-1 (T7/Eagan - Tbp2) at t0; track 5: JB-1437-4-1 at the time of induction of t=4 hours; lane 6: JB-1607-1-1 (T7/JB12-Tbp2) at t0; track 7: JB-1607-1-1 at the time of induction of t=4 hours.

In Fig. 23 illustrates the scheme of purification of proteins Tbp1 and b2, expressed in E. coli.

In Fig. 24 illustrates the frequency analysis of recombinant proteins Tbp1 and b2, treated according to the scheme illustrated in Fig.23. Lane 1 - molecular weight markers (106,80; 49,5; 32,5; 27,5 and 18.5 kDa); lane 2 - the whole lysate of E. coli cells; lane 3 - solubilization bullock; lane 4 - purified proteins Tbp1 and b2.

In Fig. 25 proillyustriroval is l) in mice.

In Fig. 26 illustrates the reactivity of antisera against rTbp1 strain Eagan against different strains of H. influenzae in Western-blotting. Lane 1: BL21/D3; track 2: S12-EDDA; lane 3: S12+EDDA; lane 4: S29-EDDA; lane 5: S29 + EDDA; lane 6: S33-EDDA; lane 7: S33+EDDA; lane 8: Eagan-EDDA; lane 9: Eagan+EDDA; lane 10: B. catarrhalis 4223-EDDA; lane 11: Century catarrhalis 4223+EDDA; lane 12: N. meningitidis 608-EDDA; lane 13: 608 N. meningitidis+EDDA; track 14: induced strain JB-1476-2-1 expressing recombinant protein Tbp1 strain Eagan; track 15: the molecular mass markers. Specific ~95 kDa bands, reacted with antisera against Tbp1, on tracks 3, 4, 5, 7, 8 and 9 correspond to the strains S12, S29, S33 and H. influenzae Eagan; ~ 110 kDa band in lanes 10 and 11 correspond to the strain 4223 Century catarrhalis; and ~80 kDa band in lanes 12 and 13 correspond 608 N. meningitidis.

In Fig.27 illustrates the reactivity of antisera against rb2 strain Eagan against different strains of H. influenzae in Western-blotting. Lane 1: molecular mass markers; lane 2: induced strain JB-1437-4-1 expressing recombinant protein b2 strain Eagan; lane 3: SB12-EDDA; lane 4: S12+EDDA; lane 5: S29-EDDA; lane 6: S29+EDDA; lane 7: S30-EDDA; lane 8: S30+EDDA; lane 9: S32-EDDA; lane 10: S33-EDDA is not reacted with antisera against b2 on tracks 3, 6, 7, 8, 13, 14 and 15, which correspond to the strains S12, S29, S30, CANCER, and Eagan.

In Fig. 28 illustrates the construction of plasmids pNH1T1KFN and pNH1T1KFP used to obtain strains of H. influenzae, which do not produce transferrin receptor.

In Fig.29 illustrates the construction of plasmids encoding chimeric polioviruses, expressing the epitope derived from a protein of the transferrin receptor, which is conservative for bacteria that produce transferrin receptor.

In Fig. 30 shows a Western blot illustrating the reactivity of antisera produced by immunization of rabbits with the chimeric polioviruses, expressing the epitope derived from a protein receptor for transferrin, which is conservative for bacteria that produce transferrin receptor. Panel a shows colored Kumasi blue diamond gel detecting the purified recombinant protein Tbp 2, derived from strain S12 H. influenzae, and expressed in E. coli (lane 1); purified protein b2, derived from strain 4223 Branhamella catarrhalis (lane 2); cell lysate from telesolutions strain 4223 Century catarrhalis (lane 3), cell lysate from strain JM109 E. coli grown in VC is ispolzovaniem pool of sera, collected on the 27th day after immunization of rabbits by injecting PUITBP2A (rabbits 40, 41 and 42). Panel C shows the results obtained for the pooled immune sera from rabbits that were found minimum specific reactivity.

Some of the above figures for site-specific restrictively endonucleases were used the following abbreviations: R - Eco RI; PS - Pst I; N - Hind III; VD - Bg1 II; Nde - Nde I; Ear - Ear I and Sau-Sau 3A I.

In Fig. 28 to indicate site-specific restrictively endonucleases were used the following abbreviations: A - ACC I; In - Bam HI; E, Eco RI; O - Xho I; N - Hind III; Ps - Pst I; V - Eco RV; X - b I; G - Qg1 II; S - Sa1-I; K - kpn I and S - SAc I.

General description of the invention

To obtain purified and selected nucleic acid, which may be a DNA molecule that contains at least part of the nucleic acid encoding the transferrin receptor, typed in accordance with the present invention may be used in any strain of Haemophilus. Such strains can be obtained from clinical sources, or from the collections of bacterial cultures, such as the American type culture collection.

In accordance with one aspect of the present Ogunnaviwo and Schryvers (1992), and while the U.S. patent 5141743, the disclosure of which is introduced into the present description by reference. Although a detailed description of this method is given in U.S. patent 5141743, however, for clarity, the following is a summary of its essence. The transferrin receptor is obtained by allocating the membrane fraction of the bacterial strain expressing the activity of binding to transferrin, and subsequent purification of transferrin receptor affine method involving successive stages prior binding of transferrin to the receptor for transferrin in the membrane fraction, solubilize membrane immobilization transferrin and separation of transferrin receptor from immobilized transferrin. Alternative receptor proteins can be isolated by the method, which is a modification of the above method, in which no preliminary binding buffer to solubilize contains a high salt concentration that allows direct selection with immobilized transferrin as described in the work Ogunnariwo and Schryvers (1992).

In this application, the term "transferrin receptor" is used to define a family of proteins Tbp1 and/or b is different from natural strains, for example Haemophilus. Other bacterial sources transferrin receptor include, but are not limited to species such as Neisseria, Branhamella, Pasteurella and Actinobacillus.

Some, if not all, of these bacteria contain both protein Tbp1 and b2. Under purified and selected DNA molecules containing at least a part of the nucleotide sequence encoding the transferrin receptor of the present invention includes a DNA molecule encoding functional equivalents of the transferrin receptor. In this application the first protein or peptide is a "functional analogue" of the second protein or peptide in the case if said first protein or peptide is immunologically related to the second protein or peptide and/or have the same function. The functional analog may be, for example, a fragment of the protein or its mutant, formed due to amino acid substitutions, additions or deletions.

In one of the embodiments of the present invention, the transferrin receptor isolated from the strain DL63 H. influenzae type b and purified by the methods of affinity chromatography, as described in Schryvers (1989), Ogunnariwo and Schryvers (1992) and in U.S. patent 5141743. Isolated and purified transferrin receptor is IPA b were subjected to mechanical fragmentation, then added EcoRI-linkers and designed a library of expressed sequences in ZAP. This library was skanirovali using rabbit antisera against TfR and received two positive clone (pBH1T1 and runt), which had overlapping restriction map (Fig.1A and Fig.2). These clones b, resulting in the identified two large open reading frames (Fig.2). Nucleotide sequence of the genes of receptors for transferrin Tbp1 and b2 (SEQ ID 1) from strain D63 H. influenzae and their deduced amino acid sequence (SEQ ID 5 - Tbp1 and SEQ ID 6 - b2) shown in Fig.3. Analysis of these sequences showed that the operon fR consists of two consecutive genes (Tbp1 and b2), transcribed from a single promoter (as particularly shown in Fig.2 and Fig.3). The molecular mass of the protein b2 varies depending on the strain, whereas the molecular mass of the protein Tbp1 remains largely unchanged for different strains of this bacterium, and finds some variability among different bacteria, with TfR-genes. Molecular weight Tbp1 is usually 94000-106000 and molecular weight b2 mainly varies and ranges from 58000 to 98000.

Was held on the East by CYANOGEN, transferrin receptor from strain DL63 H. influenzae. N-ends b2 blocked, and the amino acid sequence of Tbp1 identified by b, and identified peptides corresponding to Tbp1 shown in Fig. 3 (underlined). These peptide sequences are Glu Thr Gln Ser Ile Lys Asp Thr Lys Glu Ala Ile Ser Ser Glu Val Asp Thr (shown in Fig.3, SEQ ID 101) and Leu Gln Leu Asn Leu Glu Lya Lys Ile Gln Gln Asn Trp Leu Thr His Gln Ile Ala Phe (shown in Fig.3, SEQ ID 102). The signal sequence Tbp1 and the putative signal sequence b2 shown in Fig.3 double lines at the top. The putative signal sequence for Tbp1 is a Met Thr Lys Lys Pro Tyr Phe Arg Leu Ser Ile Ile Ser Cys Leu Leu Ile Ser Cys Tyr Val Lys Ala (SEQ ID 103). The putative signal sequence for b2 is a Met Lys Ser Val Pro Leu Ile Ser Gly Gly Leu Ser Phe Leu Leu Ser Ala (SEQ ID 104). Deduced amino acid sequence N-terminal region b2 showed that this protein is a lipoprotein.

From strain Eagan H. influenzae type b was obtained chromosomal DNA and generated libraries. The first library was constructed from DNA, which is partially hydrolyzed by the enzyme Sau 3A, fractionally with getting ~5-10 KB fragments and cloned into a plasmid obtained on the data in ZAP. Both libraries were probed with 5'-fragment pBHIT-clone shown in Fig.2, resulting in the obtained partial clones TfR genes of strain H. influenzae Eagan, denoted by S-4368-3-3, JB-901-5-3. Thus, in accordance with other aspects of the present invention in Fig.1B and 2 illustrate plasmid clones of the S-4368-3-3, JB-901-5-3 encoding b1 and b2 from strain Eagan H. influenzae type b. DNA sequences of genes Tbp1 and b2 (SEQ ID 2) from strain Eagan H. influenzae type b and their deduced amino acid sequence (SEQ ID 7 and 8) shown in Fig.4, where the sequence b2 is the first gene in the operon. In Fig.4 alleged

the sequence of the -35, -10 and sequence of the binding site of the ribosome are shown by lines drawn on top.

Chromosomal DNA was obtained from strain MinnA H. influenzae type b. This DNA was subjected to partial hydrolysis by the enzyme Sau 3A I was fractionally 10-20 KB fragments and cloned in the BamHI site EMBL3. The library was probed with 5'-fragment of clone pBHIT (Fig.2) and received a full size clone encoding TfR (DS-712-1-3). In accordance with additional aspects of the present invention, Fig. 1C and 2 illustrate plasmid clone DS 712-1-3 encoding Tbp1 and b2 from strain MinnA H. influenzae type b. DNA sequence Tbp1 and b is provided in Fig.5, where b2 sequence is the first in the operon. In Fig.5, the estimated sequence -35, -10 and sequence of the binding site of the ribosome shows the top lines.

Chromosomal DNA was obtained from Netherweave strain CANCER 12085 H. influenzae. DNA is partially hydrolyzed by the enzyme Sau 3A, fractionally 10-20 KB fragments and cloned into the Bam HI site MBL 3. The library was probed with fragments of clone pBHIT (Fig.2) and received a full size clone encoding TfR (JB-1042-a 7-6). Restriction map of clone JB-1042-7-6 shown in Fig.1 and 2, and the nucleotide sequence of genes b1 and b2 (SEQ ID 4) from the strain of CANCER 12085 H. influenzae and their deduced amino acid sequences shown in Fig.6 (SEQ ID 11, 12), thus the sequence b2 is the first. In Fig.6, the estimated sequence -35, -10, and the sequence of the binding site with the ribosome are shown by lines drawn on top.

Chromosomal DNA was obtained from Netherweave strain S33 H. influenzae, originating from titney environment. This DNA was subjected to partial hydrolysis by the enzyme Sau 3A I was fractionally 10-20 KB fragments and cloned into the Bam HI site EMBL 3. The library was probed with fragments of clone pBHIT (Fig.2) and received a full size clone encoding TfR (- Q ID 4) from strain SB33 H. influenzae and their deduced amino acid sequences shown in Fig.7 (SEQ ID 11, 12), thus the sequence b2 is the first. It was found that the gene b2 S33 has a deletion in one basis, which leads to a shift of the reading frame in the residue 126 and to premature processing of the obtained protein in the residue 168.

Carried out R-amplification of genes tbp 2 strains S12, S28, S32 NTHi originating from titney environment, and these genes sequenced.

The nucleotide sequence of genes tbp 2 from Netherwing strains SB12 (SEQ ID 105), S29 (SEQ ID 108), SB30 (SEQ ID 110) and S32 (SEQ ID 112). H. influenzae is shown in Fig.8, 9, 10 and 11, respectively.

It was found that all amplificatoare genes tbp 2 encode a full-sized proteins b2, suggesting that the defective gene of strain S33 is atypical.

All three strains of H. influenzae type b have identical short intergenic sequence, consisting of only 13 p. O. between tbp 2 and tbp 1, and the strains of CANCER 12085 THi have longer intergenic sequence, consisting of 27 p. O. (Fig.12).

Strain S12 has intergenic sequence 13 p. O., identical to the sequence found in strains of H. influenzae type b, and strains S29, S30 and S32 contain balewadi strains have a common conservative sequence of 13 p. O., located between their genes tbp 2 and tbp 1.

Was identified pentapeptidnogo sequence near aminobenzo Tbp1 H. influenzae (Fig. 12), which is similar to TonB-block. TonB gene of H. influenzae has been recently cloned and sequenced (Jarasic and others, 1994).

Comparison of amino acid sequences b1 from strains Eagan/MinnA DL63, CANCER 12085 and S33 H. influenzae illustrated in Fig.14. Proteins Tbp1 strains Eagan and MinnA are identical and have a length of at 912 amino acids; protein Tbp1 strain DL63 has 914 residues, the protein of the strain of CANCER 12085 has 914 residues; and protein strain S33 has 911 residues. Proteins Tbp1 H. influenzae are highly conservative and have identical sequences at 95-100%. Comparison of amino acid sequences b2 from strains Eagan/MinnA, DL63, CANCER 12085, S12, S29, S30 and S32 illustrated in Fig. 15. Proteins b2 strains Eagan and MinnA are identical and contain 660 amino acids, protein b2 strain DL63 has 644 residues, and protein strain CANCER 12085 has 654 residues. In the gene tbp 2 strain S33 has a deletion in one basis, which leads to a shift of the reading frame in the residue 126 and premature processing of the protein at residue 168. This lack of Foundation confirmed by direct sequencing of PCR-amplified chrome is ka b2 are basically, less conservative with identity only 66-70%, but there are a few short segments with conservative sequence that can be identified in Fig.15. It was found that all R-amplificatoare genes tbp 2 strains S12, S29, S30 and S32 encode a full-sized proteins b2. Among the deduced proteins observed heterogeneity of sequences and sizes; for example, protein b2 strain S12 has 648 amino acids, protein strain S29 has 631 residue protein strain S30 has 630 residues, and protein strain S32 has 631 residue.

Identified putative protein structure b1 and b2 strain Eagan (Fig. 16A and 16B). Both proteins have multiple transmembrane domains; and Tbp1 protein crosses the membrane 20 times, and the protein b2 crosses the membrane 12 times.

Three external conservative epitopes were identified in aminobenzene region b1 (DNEVTGLGK - SEQ ID 43, EQVLN/DIRDLTRID - SEQ ID 139 and 140 and GAINEIEVENVKAVEISK - SEQ ID 141) and one in the C-terminal region (GI/VYNLV/LNYRYWTWE - SEQ ID 142 and 143). Only three small conservative areas may be identified in the N-end Tbp2 proteins of human pathogens: CSS/LGGG(G)SFD - SEQ ID 75, 144 and 145; inside of these proteins: LE/SGGFY/FGR - SEQ ID 74 and 146 and in the end these proteins: VVFGAR/K - 83 and SEQ ID 8philus, allows you to subdivide Haemophilus into subgroups defined by the same amino acid sequences b2. In addition, this fact allows the rational selection of the minimum number of sequences Tbp1 and/or b2 or synthetic peptides representing epitopes common to these strains of Haemophilus, which can be used in immunogenic compositions, for example, for immunization against diseases caused by Haemophilus and other bacteria that produce transferrin receptor with sequences similar to the sequences of Tbp1 and b2 from strains of Haemophilus. Thus, for immunization against many or all strains of Haemophilus and other bacterial pathogens that produce transferrin receptor, can be used the minimum number of transferrin receptor, its analogs, fragments and/or peptides.

In addition, were compared (as shown in Fig.14 and 15) amino acid sequences of the transferrin receptor, originating from a number of bacterial pathogens (H. influenzae type b, Netherway H. influenzae, Neisseria meningitidis, Neisseria gonorrhoe and Actinobacillus (Haemophilus) pleuropneumoniae).

As a result of these analyses revealed areas of Tbp1 and b2 that are to what I'm peptides, as shown in tables 2 and 3. In particular, Tbp1 (table 1 and Fig.14) are conservative sequence DNEVTGLGK (SEQ ID: 43), EQVLNIRDLTRYDPGI (SEQ ID NO 44), EQCVLNIRDLTRYDPGISVVEQGRGASSGYSIRGMD (SEQ ID NO 45), GAINEIEYENVKAVEISKG (SEQ ID NO 46) and GALAGSV (SEQ ID NO 47). Especially conservative sequences In b2 are LBGGFYGP (SEQ ID NO 74) CSGGGSFD (SEQ ID NO 75), YVYSGL (SEQ ID NO 76). CSNLSYVKFS (SEQ ID NO 77), FLLGHRT (SEQ ID NO 78), EFNVOF (SEQ ID NO 79), NAFTGTA (SEQ ID NO 80), VNGAFYG (SEQ ID NO 81), ELGGYF (SEQ ID NO 82), VVFGAR (SEQ ID NO 83) and VVFGAK (SEQ ID NO 84) (cm. table 2 and Fig.15).

Detection conservative sequences of the transferrin receptor of a number of bacterial pathogens allows you to select the minimum number of antigens with specific amino acid sequences (including sequences such as synthetic peptides), for immunization against diseases caused by pathogens that have receptors transferring. Such pathogens, in addition to the above bacteria are other strains of Neisseria, such as Neisseria gonorrhoe and Branhamella, including Branhamella catarrhalis. These conservative amino acid sequence found in many bacterial pathogens, can produce TfR-specific antibodies, including monoclonal antibodies that recognize most, if not all, of the receptors translationstring. This anticavity "learned" the transferrin receptor in Branhamella catarrhalis. This anticavity can be used for detection and neutralization of most, if not all bacteria produce protein fR, and in addition, it can also be used for passive immunization against diseases caused by these pathogens. Thus, diagnostic tests and kits that use conservative amino acid sequence, can be a valuable tool to detect, if not all, then most of the bacteria that produce transferrin receptor.

Epitopes containing the above-mentioned amino acid sequence, can be delivered to cells of the immune system using synthetic peptides containing these sequences or by using live vectors expressing such sequences or by direct injection of nucleic acid molecules encoding amino acid sequence.

Some peptides that contain conservative amino acid sequences in proteins Tbp1 strains Eagan MinnA, DL 63 and Netherweave strain CANCER 12085 H. influenzae type b, shown in table 2. Antibodies to some of these peptides were ol the ti in proteins bp2 strains Eagan, MinnA, DL63, and Netherweave strain CANCER 12085 H. influenzae type b, shown in table 3. Antibodies to some of these peptides were produced in Guinea pigs (table 4).

For production of recombinant proteins, the coding sequences of genes b1 and b2, can be cloned into the appropriate expressing vectors. Recombinant Tbp1 and b2 were expressed in E. coli using expressing the T7 system. Tbp1 gene encoding the Mature protein Tbp1 strain Eagan, was cloned with preservation of the reading frame with the T7 promoter, generating plasmid JB-1468-29, as shown in Fig.17. With the introduction of tbp1 gene in cells BL21/DE3 and the induction of IPTG or lactose expressively protein Tbp1 strain Eagan, shown in Fig.22.

Tbp2 gene encoding the Mature protein b2, was cloned with preservation of the reading frame with the T7 promoter, generating plasmid JB-1424-2-8, as shown in Fig.18. With the introduction of this gene in E. coli cells, and induction, as indicated above, ekspressiruetsya protein b2 shown in Fig.22.

Tbp2 gene from NTHI strain S12 was amplified using polymerase chain reaction (PCR). Received amplificatory DNA contained the authentic signal sequence b2 H. influenzae before last, what was b cloned in expressing system RT-7, as shown in Fig.21. When introducing the resulting plasmid (JB-1600-1) in cells BL21/LE3 E. coli and induction expressively protein b2 S12, as shown in Fig.22.

Recombinant proteins b1 and b2 produced in E. coli as a calf inclusion were purified in accordance with the scheme shown in Fig.23. Purified proteins had a purity of at least about 70%, as shown in Fig. 24. Studies of immunogenicity was performed on mice using purified recombinant proteins Tbp1 and b2. Both proteins introduced in doses of 3-10 μg, has produced a good immune response in mice (Fig.25).

Antisera produced against recombinant proteins Tbp1 or b2 originating from one of the strains of H. influenzae, gave cross-react with other strains, indicating the possibility of their use as a valuable diagnostic reagents (Fig.26 and 27).

Plasmids pUH1T1KFN and pUH1TKKFP shown in Fig.28, contain a selective marker of resistance to the antibiotic, operon cloned in the transferrin receptor, and were designed for local inactivation feathered transferrin receptor. These plasmids were used for transformation of H. in the ore 19. Such strains can be used as a negative control (since they do not produce TfR) in in vivo and in vitro assays and diagnostics. These strains, in all probability, will be also attenuirovannogo when their in vivo growth and can be used as live vaccines for immunization against diseases caused by Haemophilus.

As discussed above, the epitopes of the proteins of the transferrin receptor can be delivered to cells of the immune system through the use of live vectors expressing such amino acid sequences, and such lively vector can be a provirus. In Fig.29 illustrates the design of hybrid polioviruses, expressing the epitope of the protein transferrin receptor, including conservative epitope from b2 LFCCFYGP (SEQ ID No. 74). These viruses are recognized by antibodies produced against a peptide containing amino acid sequence LECCFYGP (SEQ ID No. 74) (table 5), suggesting that these viruses Express the antigenic sequence in a recognizable form. V12 and V12 were also neutralized by rabbit antisera against tbp2 strain DL63 H. influenzae, which indicates that at least two virus expressively serial is neutralized all viruses, that pointed to the fact that changes in the antigenic region I of neutralizing poliovirus does not have a significant impact on other antigenic region of these viruses. In addition, rabbit anticavity produced by immunization of rabbits with poliovirus chimeras PV1TBP2A or V12 recognized the peptide containing the amino acid sequence LEGGFYGP (SEQ ID No. 74). This suggests that sequence, expressed chimeras PV1TB2A and PV1TBP2B are immunogenic and produce antibodies that can recognize the sequence.

In Fig.30 panel a shows the LTO-SDS page gel detecting the purified recombinant Tbp2 from strain J12 H. influenzae and expressed in E. coli (lane 1); tbp2 from strain 4223 Branhamella catarrhalis (lane 2); cell lysate from telesolutions strain 4223 Century catarrhalis (lane 3), cell lysate from telesolutions strain JM109 E. coli (lane 4) and cell lysate from strain JM109 E. coli cultured in gelatinisation conditions (lane 5). Panel b shows the results of Western blotting replica from the gel using a pool of sera collected from rabbits immunized with V12. There was observed a strong reaction with purified communicating a bench of E. coli appreciable reaction was observed (lanes 4 and 5). Panel C shows the results obtained for the pooled sera from the same rabbit prior to immunization, which was found minimum specific reactivity. The results showed that PV1TBP2A has the ability to induce antisera specific for proteins that bind to transferrin and derived from H. influenzae and B. catarrhalis, and that these antisera can distinguish Century catarrhalis from E. coli, which does not Express the equivalent protein.

Purified and isolated DNA molecule containing at least a portion encoding the transferrin receptor strain Haemophilus, typed as described in the present description, can be successfully used as:

- nukleinovokisly probes for the specific identification of strains of Haemophilus in vitro or in vivo;

products encoded DNA molecules, can be used as diagnostic reagents, antigens for producing Haemophilus-specific antisera, for vaccination against diseases caused by Haemophilus species, and, for example, to detect infections caused by Haemophilus;

peptides corresponding to parts of the transferrin receptor, typed as described in this application can be successfully Ivantoc, for vaccination against diseases caused by Haemophilus species, and, for example, to detect infections caused by Haemophilus.

The transferrin receptor, encoded nukleinovokisly molecules of the present invention, fragments and analogues, and peptides containing the sequence corresponding to the parts of the transferrin receptor, which is conservative for different isolates of Haemophilus and other bacteria that produce transferrin receptor, can be used to diagnose diseases caused by any bacterial strain that produces the transferrin receptor, or for immunization against these diseases. In particular, peptides containing the sequence FYG are conservative in proteins transferrin receptor of many bacterial pathogens that produce transferrin receptor, and therefore can be successfully used for the diagnosis and immunization against diseases caused by bacteria that produce transferrin receptor. Such bacteria include, but are not limited to, strains of Haemophilus, Neisseria including N. meningitidis and N. gonorrhoeae) and Branhamella (including Century catarrhalis).

For every person it is obvious that Rica; the treatment of infections such as Haemophilus infection, and infections other bacterial pathogens that produce transferrin receptor; and production of immunological reagents. Below is more detailed, but not comprehensive discussion of the applications of the present invention.

1. Receipt and use of vaccines

Immunogenic compositions, suitable for use as vaccines, can be obtained using immunogenic transferrin receptor, its analogs, fragments and/or peptides described in this application. This vaccine produces an immune response through the formation of antibodies, including antibodies against the transferrin receptor and opsonizing or bactericidal antibodies. Regardless of whether vaccinated individual antigenic stimulation by the bacterium Haemophilus or other bacteria that produce transferrin receptor, antibodies bind to the transferrin receptor, preventing thereby the access of bacteria to the source of iron required for their viability. In addition, opsonizing or bactericidal antibodies against TfR can also protect the body through alternative mechanisms.

<230 and 4596792, introduced in the present description by reference. Immunogenic compositions containing vaccines can be manufactured in the form of an injectable liquid solutions or emulsions. For this purpose, the transferrin receptor, its analogs and fragments and/or peptides can be mixed with pharmaceutically acceptable excipients compatible with the transferrin receptor, fragments, analogues or peptides. Such excipients include water, saline, glycerol, ethanol, dextrose, and combinations thereof. In addition, immunogenic compositions and vaccines may contain additives such as wetting or emulsifying agents, pH-regulating tabularasa agents, or an adjuvant to enhance the effectiveness of vaccines. Immunogenic compositions and vaccines can be introduced parenterally by subcutaneous or intramuscular injection. Alternative immunogenic compositions prepared in accordance with the present invention, can be manufactured and introduced in such a way that they will produce an immune response on the surface of mucous membranes. For example, the immunogenic composition may be deposited on the surface of mucous membranes, for example, through the nose or oral (intragastric). And what positions to specific cells of the immune system or to the surfaces of the mucous membranes. Some of these molecules, delivering, is the strain B12 and fragments of bacterial toxins described in WO 92/17167 (Biotech Australia Pty. Ltd.), as well as monoclonal antibodies, are described in U.S. patent 5194254 (Barber and others). An alternative may be preferred, other methods of administration, such as suppositories and oral compositions. In the case of suppositories in the composition can be included binders and carriers such as polyalkyleneglycol or triglycerides. Oral compositions may contain initiators, such as pharmaceutical grades of saccharin, cellulose and magnesium carbonate. These compositions can be made in the form of solutions, suspensions, tablets, pills, capsules, drugs slow release drugs, or powders that contain 10-95% of the transferrin receptor, fragment, analogue and/or peptides.

The vaccine is usually administered in a way appropriate to specifically enter the dosage form, and in such quantities, as calculated, are therapeutically effective, protective and immunogenic. Enter the amount depends on the particular individual being treated, clutchy immune response. The exact amount of active ingredient required for the introduction should define the attending physician. However, a suitable dose can be easily determined by any expert and is of the order of a few micrograms transferrin receptor, its analogue, fragment and/or peptides. Suitable regimes for initial implementation and booster doses may also vary, but usually the first to enter the initial dose, and then a booster dose. Doses of the vaccine may also vary from the method of administration and the weight of the patient.

The nucleic acid molecules encoding the transferrin receptor of the present invention can also be used directly for immunization by direct injection of DNA, for example by injection for genetic immunization, either by constructing a live vector, such as Salmonella BCG, adenovirus, poxvirus, vaccinia virus or poliovirus. Discussion of some of live vectors, which were used to transfer heterologous antigens to the immune system, is given in O Hagan (1992). Methods of direct DNA introduction individuals for genetic immunization are described, for example, Ulmer and others, 1993.

The use of peptides in vivo can, first of all,eriod half-life in serum and/or tissues, and/or sufficient immunogenicity. Such chemically modified peptides are referred to in the present description "peptide analogs". The term "peptide analog" refers to any functional chemical equivalent peptide, notable for its increased stability and/or efficacy and immunogenicity in vivo - or in vitro-implementation of the present invention. The term "peptide analog" refers to any amino acid-derived peptides described in this application. Peptide analogs of the present description, get methods, which include (but are not limited to: modifications to side chains, the introduction of unnatural amino acids and/or their derivatives during peptide synthesis and the use of cross-linking agents; and other methods which impose appropriate restrictions on the peptides or their analogues.

Examples of modifications of the side chains discussed in this application can serve as a modification of amino groups, such as reductive alkylation by reaction with an aldehyde and subsequent recovery using NaBH4; amidation using methylacetamide; acetylation and with the PP 2,4,6-trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic acid anhydride and anhydride tetrahydrophthalic acid and peridomiciliary lysine using pyridoxal-5-phosphate followed by reduction using NaBH4.

Guanidinium arginine residues can be modified by education products geterotsiklicheskikh with such reagents as 2,3-butandione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by activation of carbodiimide through education about alliteratively with subsequent derivatization, for example, to obtain the corresponding amide.

Sulfhydryl groups may be modified by methods such as karboksimetilirovaniya using Vodokanal acid or iodoacetamide; oxidation of permorming acid to cysteine acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide; maleic acid anhydride or other substituted maledom; formation of mercurial derivatives using 4-chlorobenzoate mercury, 4-chlorofluoromethanes acid chloride of finalstate, 2-chloridate-4-NITROPHENOL and other compounds and carbamoyl, for example, by oxidation using N-bromosuccinimide; or by alkylation of indole ring with 2-hydroxy-5-nitrobenzylamine or sulphonylchloride. Triazinone residues can be modified by nitration with tetranitromethane with obtaining the derived 3-nitrotyrosine.

Modification of the imidazole ring of his-tag residue may be carried out by alkylation using derivatives Vodokanal acid or by N-carbamaxepine using diethylpyrocarbonate.

Examples of the introduction of unnatural amino acids and derivatives during peptide synthesis include but are not limited to, the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentane acid, 6-aminohexanoic acid, t-butylglycol, Norvaline, phenylglycine, ornithine, zakrasin, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-tierralinda and/or D-isomers of amino acids.

Immunogenicity can be significantly increased by introducing antigens with adjuvants normally used in the form of 0.05 to 1.0% solution in phosphate buffered saline solution. Adjuvants usilivaut by holding antigen near the site of the injection, thereby, the effect of "depot", all contribute to a slow, prolonged release of antigen in cells of the immune system. An adjuvant can also attract cells of the immune system to "depot" of the antigen and stimulate these cells to produce an immune response.

For the past many years immunostimulatory agents or adjuvants are used to enhance the immune response of the host, for example, to the introduction of the vaccine. Internal adjuvant, such as lipopolysaccharides, usually are components of inactivated or attenuating bacteria that are used as vaccines. External adjuvants are immunomodulators, which usually ecovalence contact with antigens, and forming composition, contributes to the enhancement of the host immune response. Thus, adjuvants have been identified as compounds which increase the immune response to parenteral introduced antigens. Some of these adjuvants are toxic and can cause unwanted side effects, which makes them unacceptable for use in the treatment of humans and many animals. Actually in the vaccines for administration to humans and animals as adjuvant is installed, that aluminum is effective for enhancing the humoral response to toxoid diphtheria and tetanus, and most recently, Aluma was stimulated HBsAg vaccine. However, although the effectiveness of Aluma is a well-established fact, but nevertheless, this efficiency has its limitations. For example, aluminum is ineffective for vaccination against flu and inappropriately produces cellular immune response. Antibodies produced austinwoman antigens in mice are mainly IgG1 isotype and may be insufficient to generate immunity in response to the introduction of some vaccine agents.

A strong immune response to the introduction of antigens can produce external adjuvants wide range. Such adjuvantly are saponins, conjugated with antigens membrane proteins (immunostimulating complexes); polymers-pluronic with mineral oil, killed mycobacteria in combination with mineral oil; complete adjuvant's adjuvant, bacterial products, such as muramyldipeptide (MDP) and lipopolysaccharide (LPS) and lipid a, and liposomes.

To efficiently induce humoral immune response (HIP) and cellular immunity (CM1) immunogen is their inflammatory processes (full beta-blockers, F), cytolysis (saponins and polymers-pluronic), pyrogenic reactions, arthritis and uveitis anterior choroid (LPS and MDP). Although F is an excellent adjuvant and is widely used in various studies, but due to its toxicity it was not licensed for use in vaccines for administration to humans or animals.

Desirable characteristics of an ideal adjuvants are:

(1) lack of toxicity;

(2) the ability to stimulate long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) the ability to produce both humoral and cellular immunity in response to the introduction of antigens in different ways, if necessary;

(5) synergy with other adjuvantly;

(6) the ability to selectively interacting with populations of antigen presenting cells (APC);

(7) the ability to specific induction of the corresponding cell TN1 or TN2-specific immune response;

(8) ability to selectively increase the levels of the corresponding isotype antibodies (for example, IgA) against antigens.

In U.S. patent 4855283 issued Lockhoff, etc. 8 Ave N-glycosylamine, N-glycosylation and N-glycosylceramide, each of which is substituted in the sugar residue of the amino acid), which can be used as immune modulators or adjuvants. For example, Lockhoff and others (1991) showed that N-glycolipid analogues detecting structural similarities with natural glycolipids such as glycosphingolipids and glycopyrrolate able to produce a strong immune response to the vaccine against herpes simplex virus, and pseudorabies vaccine. Some glycolipids were synthesized from long chain alkylamines followed and fatty acids that are directly related to sugars via the anomeric carbon atom, in order to simulate the natural functions of lipid residues.

In U.S. patent 4258029 issued Moloney, transferred to its legal successor, and entered into the present description by reference, States that the hydrochloride octadecylsilane (REL) acts as adjuvant with the introduction of it in the form of a complex with tetanus toxoid (TT) vaccine and formalin inactivated vaccine against polio virus type I, II and III. In addition, Nixon-George and others (1990) States that octadecylamine esters of aromatic amino acids, assotsiirovannaya Century

Lipidization synthetic peptides was also used to enhance the immunogenicity of these peptides. So, for example, Wiesmuller (1989) describes peptides having a sequence homologous to the protein of FMD virus, and associated with adjuvant three-palmityl-S-glyceryl-containerterminal, which is a synthetic analogue of the N-terminal part of the lipoprotein, derived from gram-negative bacteria. In addition, Deres and others (1989) describes in vivo-premirovanii virousspecificakih cytotoxic T lymphocytes with synthetic lipopeptide vaccine, which contains synthetic peptides originating from nucleoprotein influenza virus, by linking to them from lipopeptides N-palmityl-S-[2,3]-bis(palmitate)-(2S)-propyl-[R]-cysteine (TRS).

2. The immunoassays

The transferrin receptor, its analogs and fragments and/or peptides of the present invention can be used as immunogens and antigens in immunoassays including enzyme-linked immunosorbent assay (ELISA), PIA and other non-enzymatic assays for binding with the antibody, or other known procedures for detection of antibodies against bacteria, Haemophilus, TfR and/or peptides. In ELISA-analysis of the receptor transferri the spine, for example on the surface capable of binding proteins or peptides, such as polystyrene wall tablet for micrometrology. After washing to remove not fully adsorbed transferrin receptor, its analogs, fragments and/or peptides, non-specific protein, such as a solution of bovine serum albumin (BSA) or casein, which is known to be antigenically neutral with the test sample, can be associated with the specified selected surface. This allows you to block the sites of nonspecific adsorption on immobilsarda surface and thus allows to reduce the background arising from nonspecific binding of the antisera to the surface. Preferably, the selected peptides were derived from conserved regions shown in table 2 or 3, which improves the probability of detection of cross-species except for those occasions when you need to discover one particular bacterial species. In this case, choose a polypeptide that is unique to fR this particular species. Typically, the peptides consist of 12 residues or more, and preferably from 14-30 residues. However, it should be noted that the mixture is PE is. what it refers to those instances when a mixture of peptides from conserved regions and/or nonconservative fields used for protection from perekrestnolistnaja microorganisms, and/or for diagnostics. In this case, the mixture of peptide immunogens usually called the drug "cocktail" for use as a vaccine or diagnostic agent.

The above immobilizers surface is then subjected to contact with a sample, such as investigational clinical or biological materials in a manner conducive to the formation of immune complex (antigen/antibody). For this purpose, the sample is diluted with diluents such as BSA, bovine gamma globulin (BGG) and/or saline solution, phosphate buffered (PBS)/tween. After that, the sample is incubated for 2-4 hours at a temperature of about 25-37oC. After incubation contacted with the sample surface is washed to remove materials not bound to immunocomplexes. Washing procedure includes washing with a solution such as S/twin or borate buffer.

After the formation of specific immune complexes between the test sample and the bound transferrin receptor, its counterpart, fragmento the immunocomplex can be determined by impact on this immunocomplex the second antibody, specific to the first antibody. If the sample comes from the person, then the second antibody is an antibody specific for human immunoglobulins, mainly IgG. For specific detection of the second antibody may be associated activity, such as enzymatic activity, which will produce, for example, the development of color after incubating with an appropriate chromogenic substrate. Quantitative evaluation can then be carried out by measuring the generated color using, for example, a spectrophotometer operating in the visible range.

3. The use of sequences as hybridization probes

The nucleotide sequence of the present invention containing the gene sequence of the transferrin receptor, allow for the identification and cloning of transferrin receptor, derived from any species of Haemophilus and other bacteria with genes transferrin receptor.

Due to its ability to selective formation of duplex molecules with complementary areas of other genes fR, the nucleotide sequence containing the genes for the receptor transfe the probe in relation to the other TfR-genes depending on its application can be used in a variety of hybridization conditions. To achieve a high degree of selectivity employ relatively stringent conditions to form duplexes, namely conditions with the use of low concentrations of salt and/or high temperatures, such as 0.02 M To 0.15 M NaCl at temperatures of about 50-70oC. In some cases, hybridization is carried out in less severe conditions, for example, using 0.15 M To 0.9 M salt, at temperatures of about 20oC-55oC. hybridization Conditions can be made more rigid by adding increasing amounts of formamide to destabilize the hybrid duplex. Thus, hybridization conditions can easily change depending on the results you want to obtain. However, in General, the temperature of hybridization in the presence of 50% formamide 42oFor probes that 95-100% are homologous to the target fragment; and 37oFor probes having 95% homology; and the 32oFor probes having 85-90% homology.

In clinical diagnostic use nukleinovokisly sequence TfR genes of the present invention can be used in combination with appropriate means such as a label, to assess hybridization. Specialists WPI is to avidin/Biotin, who are able to produce the detected signal. In some diagnostic options for implementation of the present invention instead of a radioactive label can be used enzyme tag such as urease, alkaline phosphatase or peroxidase. In the case of enzyme tags to identify specific hybridization with samples containing the gene sequence TfR can be used colorimetric substrates indicators that are visible to the human eye and detected spectrophotometrically.

Nukleinovokisly sequence TfR genes of the present invention can be used as hybridization probes under hybridization in solution and solid-phase hybridization. In embodiments providing for the implementation of solid-phase procedures, analyzed the DNA (or RNA) from a sample, such as clinical samples, including body fluids and exudates (e.g., serum, amniotic fluid, the effusion of the middle ear cavity, sputum, bronchoalveolar wash liquid) or even fabric, adsorb or bind any other way with the selected matrix or surface. Then this fixed is containing a series of nukleinovokisly sequence of genes fR or their fragments, under appropriate conditions. The selection of the suitable conditions of hybridization depends on the specific circumstances that require compliance with certain criteria, for example on the content of G+C, type nucleic acid target, source of nucleic acid, size of hybridization probe. After hybridization washing the surface to remove nonspecific related molecules probes conducted an assessment of specific hybridization or even take her to the quantitative assessment using the tag. Regarding the choice of peptides, preferred are part nukleinovokisly sequence, which is conservative for Haemophilus species, for example nukleinovokisly sequences encoding conservative peptide sequence shown in Fig. 8, 9, 13 and 14 and in tables 2 and 3. The selected probe may be at least 18 p. O., and its length may vary from about 30 p. O. 90 p. O.

4. Gene expression of transferrin receptor

Plasmid vectors containing replicon and regulatory sequences derived from species compatible with the host cell, can be used for gene expression of transferrin receptor in Express is providing phenotypic selection in transformed cells. For example, E. coli can be transformed with the use of RvR that contain genes for resistance to ampicillin and tetracycline, which provides easy identification of transformed cells. Plasmid RvR, or other microbial plasmid or phage must also contain, or should be modified so that they contain promoters that can be used by the host-cell for expression of its own proteins.

In addition, phage vectors containing replicon and regulatory sequences that are compatible with the host-cell, can be used as transformation vectors in relation to these owners. For example, in the lambda phage GEMTM-II can be used in the manufacture of recombinant phage vectors for transformation of host cells such as E. coli LE392.

Promoters commonly used in the construction of recombinant DNA, are promotor system-lactamase (penitsillinazy) and lactose (Chang and others, 1978; Itakura and others, 1977; Goeddel and others, 1979; Goeddel and others, 1960), and other microbial promoters such as the promoter, the T7 system (U.S. patent 4952496). Details related to the nucleotide sequences of promoters, avlee these promoters from genes. The selection of a particular promoter, mainly depends on the desired results. Hosts that can be used for gene expression of transferrin receptor, their analogs, fragments or variants, are E. coli, Bacillus sp. , Haemophilus, fungi, yeast or baculovirus expressing the system.

In accordance with the present invention, the preferred methods of obtaining protein is recombinant methods as the natural TfR protein, purified from the culture of Haemophilus species, may contain trace amounts of toxic materials or other impurities. In order to avoid this, you can use recombinante produced protein in heterologous systems, which can be separated from the host so as to minimize the presence of impurities in the purified material. Particularly preferred hosts for the expression of the desired gene are gram-positive bacteria, which do not have LPS, and therefore do not contain endotoxins. Such hosts are species of Bacillus, which can be, in particular, used for producing pyrogen free and no transferrin receptor, its fragments or analogs. In addition, recombinant methods of production allow recip the proteins, present in Haemophilus.

Biological deposits

Some plasmids which contain, at least, the part encoding the transferrin receptor from strains of Haemophilus influenzae and which are considered in this application have been deposited with the American type culture collection (ATSS) (in b, PCs Maryland, USA) in accordance with the Budapest Treaty prior to the filing of this application. Samples of the deposited plasmids will be made publicly available after the grant of a patent based on this application for a U.S. patent. The present invention is not limited to these deposited plasmids, and these plasmids are considered only as illustrations of the present invention. Any b or similar plasmid encoding the same or b antigens described in this application, is also in the scope of the present invention (see tab.I).

Strains of Haemophilus

Hybrid strain Eagan was obtained from Connaught Laboratories Limited, 1755, Stteles Ave, W, Willowdate, Ontario, Canada M2R 3T4.

Hybrid strain MinnA was obtained from the collection of Dr. Robert Munson, Department of Microbiology and Immunology, Washington University School of Medicine, Children's Hospital, St. Louis, Missouri 63110.

Hybrid strain DL63 was obtained from the collection

Dr. Eric Hansen, the collection of Dr. Robert Munson (see above).

Strains S12, 29, 30, 32, and 33 were obtained from the collection of Dr. Stephen Barenkamp, Department of Pediatrics, School of Medicine, Saint Louis University Medical Centre, St. Louis, Missouri 63104.

Examples

In the above description of the present invention is disclosed only in General terms. For a better understanding of the invention presented below are specific examples. These examples are given solely for illustrative purposes and should not be construed as a limitation of the scope of the invention. Changes in species and replacement b considered as conditions that can occur in this particular case or that, as expected, are appropriate in this case. Although in the present description are used in concrete terms, they have only a descriptive value and are not restrictive.

In the present invention are methods of molecular genetics, protein biochemistry, immunology and technology fermentation, however, the detailed descriptions in this application is not included, since they are widely reported in the scientific literature and are well known to any expert in this field.

Example 1

This example describes obtaining chromosomal DNA from strains DL 63, Eagan, Mpace brain extract, as described Harkness and others, 1992.

A. Extraction of chromosomal DNA from strain DL63 Haemophilus influenzae type b

Chromosomal DNA was prepared as follows. 250 ml culture besieged by centrifugation on the rotor J14 Beckman at 8000 rpm for 15 minutes. The precipitate after centrifugation were washed in 200 ml 50 mm Tris-Hcl (pH 8.0), centrifuged as described above, resuspendable 12.5 ml of 50 mm Tris-Hcl and 50 mm edtk (pH 8.0) and frozen at -20oBack To the frozen cell precipitate after centrifugation was added 1.25 ml of 10 mg/ml lysozyme solution in 0.25 M Tris-Hcl (pH 8.0). The precipitate after centrifugation were thawed and incubated on ice for 45 minutes. After this was added 2.5 ml of a 1 mg/ml proteinase K in 0.5% LTOs, 0.4 M ADGC, 50 mm Tris-Hcl (pH 7.5), and the resulting mixture is incubated at a temperature of 50oC for one hour, stirring occasionally. The lysate was extracted once with 15 ml of phenol, buffered by Tricom, and then to precipitate DNA was added 1.5 ml of 3 M sodium acetate and 30 ml of ethanol. DNA was wound on a glass rod, and then was dissolved in 12.5 ml of 50 mm Tris-Hcl and 1 mm edtk (pH 7.5) at 0.2 mg/ml RNase And swing by during the night. After that, the sample once the EC is s-Hcl and 1 mm ADGC (pH 7.5), and then kept at 4oC.

C. Extraction of chromosomal DNA from strain Eagan Haemophilus influenzae type b

50 ml of culture besieged by centrifugation, the precipitate resuspendable in 25 ml of TE (10 mm Tris, 1 mm edtk (pH 7.5) and 25 ml of b used to obtain chromosomal DNA. Each b was added to 0.6 ml of 10% sarkosyl and 0.15 ml of 20 mg/ml proteinase K, and the samples were incubated for one hour at a temperature of 37oC. the Lysate once were extracted with phenol, saturated Tricom, and then three times were extracted with a mixture of chloroform and isoamyl alcohol (24:1). The aqueous phase was combined to a final volume of 7 ml To precipitate DNA was added 0.7 ml of 3 M sodium acetate (pH 5,2) and 4.3 ml of isopropanol, after which DNA was wound off, washed with 70% ethanol, dried and resuspendable in 1 ml of water.

C. Extraction of chromosomal DNA from strains Eagan MinnA CANCER 12085 SK33 and Haemophilus influenzae

Cells were besieged from 50 ml of culture by centrifugation at 5000 rpm for 15-20 minutes at a temperature of 4oC. Cellular precipitate after centrifugation resuspendable in 10 ml of TE (10 mm Tris-HCl, 1 mm edtk (pH 7.5), and pronase and LTOs were added to final concentrations of 500 μg/ml and 1%, respectively. Sample incu who at one time were extracted with phenol, saturated Tris, once the mixture is saturated with tricom of phenol and chloroform (1:1) and once with chloroform. The final aqueous phase were dialyzed for 24 hours against 500 ml of 1 M sodium chloride at a temperature of 4oWith one-time replacement of the buffer, and then within 24 hours against 2500 ml TE at a temperature of 4oWith one-time replacement of the buffer. The final dialysate distributed on b for future use.

Example 2

This example describes the obtaining of chromosome libraries.

A. Library DL63-ZAP H. influenzae

100 μg of chromosomal DNA DL63 H. influenzae in THOSE subjected to mechanical fragmentation by passing through a 1-ml syringe with needle size 25. Fragmented DNA was a small mistake on the ends by adding water to a final volume of 405 μl, 45 ál 10S1 nuclease buffer (2 M zinc chloride, 500 mm NaOAc (pH 4.5), 10 mm zinc sulfate and 5% glycerol) and 1.7 μl of nuclease S1 at a concentration of 100 u/μl, followed by incubation for 15 minutes at a temperature of 37oC. the Sample once were extracted with a mixture of phenol and chloroform and once with chloroform, and then to precipitate DNA was added 1 ml of ethanol. The sample was incubated on ice for 10 minutes or over night at a temperature of Li 70% ethanol and dried off. EcoRI sites in DNA sequences has metilirovanie using standard procedures. This motivated DNA was added to 5 ál of mm MgCl2, 8 μl of dNTP mixture (2.5 mm each dATP, dCTP, dGTP and dTTP) and 4 μl of fragment maple at a concentration of 5 u/ál. The resulting mixture was incubated for 30 minutes at a temperature of 12oC. After adding 450 µl S (0.1 M NaCl, 10 mm Tris-HCl, 1 mm etc) (pH 8.0) and the mixture once were extracted with a mixture of phenol and chloroform and once with chloroform, and then to precipitate DNA was added 1 ml of ethanol. The sample was incubated on ice for 10 minutes or overnight at -20oC. the DNA was collected by centrifugation in microcentrifuge for 30 minutes, washed with 70% ethanol and dried off.

DNA resuspendable in 7 μl of TE and to the solution was added 14 μl of phosphorylated EcoRI-linkers (200 ng/ml), 3 ál 10x digirolamo buffer, 3 μl 10 mm ATP and 3 μl of DNA T4 ligase (4 u/μl). The sample was incubated over night at a temperature of 4oC, and then incubated for 10 minutes at a temperature of 68oFor inactivation of the ligase. To the mixture was added 218 μl of water, 45 ál 10x universal buffer and 7 μl of EcoRI at a concentration of 30 u/ál. After incubation for was polutoragodovaly size on the gradient of sucrose, collecting fractions containing DNA size 6-10 KB. Combined DNA precipitated with ethanol and resuspendable in 5 μl of TE buffer. 200 ng DNA insert ligated in 2-3 days at a temperature of 4oWith 1 μg of vector ZAP II in the final volume of 5 ál. Mixture for ligation was packaged using Gigapack II Gold (Stratagene) and were seeded in NZY-Cup with cells SUPE E. coli. Then the library was titrated, amplified and stored at a temperature of 4oWith 0.3% chloroform.

Century Library Eagan - pUC H. influenzae

Chromosomal DNA derived from a strain of H. influenzae Eagan method described in example 1C, hydrolyzed by the enzyme Sau3A for 2, 5 and 10 minutes, and the samples were subjected to electrophoresis on a preparative agarose gel. Gel slices containing DNA fragments of length 3-10 KB, cut out, and DNA was extracted using standard procedures of freezing and thawing. DNA plasmid of Fig 8:2 (figure 8, additional sites restrictively enzymes Bg1 II and Xba 1 multiple cloning) hydrolyzed by enzymes BamHI and Bg1 II, and then was dephosphorylated using calf alkaline phosphatase (CAP). DNA fragments strain of H. influenzae Eagan ligated into pUC, and this mixture was used to transform cells of E. coli JM109.

C. Library Eagan and enzyme EcoRI, and then fractionally size on preparative agarose gel. Gel slices corresponding to DNA fragments of length 7-23 KB, cut out, and DNA was subjected to electroelution during the night in the dialysis tube containing 3 ml of TAE (40 mm Tris-acetate, 1 mm etc), in the 14th Century Then DNA was twice besieged and resuspendable in the water, and then ligated overnight with EcoRI-hydrolyzed DNA ZAP II. Mixture for ligation was packaged with a set Gigapack II (Stratagene) and was passively XLI-Blue E. coli. The resulting library was titrated, amplified and stored at a temperature of 4oWith 0.3% chloroform.

D. Library L 3

Chromosomal DNA of strain MinnA H. influenzae (10 µg) was obtained by the method described in example 1C, hydrolyzed by the enzyme Sau3A I (40 units) for 2, 4 and 6 minutes, and then was fractionally size on the gradient of 10-30% sucrose in TNE buffer (20 mm Tris-HCl, 5 mm NaCl, 1 mm edtk (pH 8)). Fractions containing DNA fragments of length more than 5 KB were collected and precipitated. In the second experiment, the chromosomal DNA (2.6 mg) is hydrolyzed by the enzyme Sau3A I (4 units) for 1, 2, and 3 minutes, and then was fractionally by size by electrophoresis on agarose gel. Gel slices containing DNA fragments with a length of 10-20 KB, cut, and D is smeru DNA from each experiment were combined for ligation with the BamHI-branches EMBL 3 (Promega). Mixture for ligation was packaged using the kit Gigapack packaging, and then passively in cells of E. coli LE392. The resulting library was titrated, amplified and stored at a temperature of 4oIn the presence of 0.3% chloroform.

Chromosomal DNA of strains of CANCER 12085 or S33 H. influenzae obtained by the method described in example 1C, hydrolyzed by the enzyme Sau3A 1 (0.5 units/10 μg DNA) at a temperature of 37oC for 15 minutes, then was fractionally by size by electrophoresis on agarose gel. Gel slices corresponding to DNA fragments of length 15-23 KB, cut out, and DNA was subjected to electroelution during the night in the dialysis tube containing 3 ml of TAE, with 14 C. the resulting DNA was twice besieged and resuspendable in the water, and then ligated overnight with BamHI-branches EMBL3 (Promega). Mixture for ligation was Packed with set for in vitro packaging of Lambda (Amersham) according to the supplier's instructions and was passively in cells of E. coli NM539. The library was titrated, amplified and stored at a temperature of 4oIn the presence of 0.3% chloroform.

Example 3

This example describes the screening of libraries.

A. Library of expressed sequences DL the CSO 20 ml RTf supersnow column prepared in accordance with the manufacturer's instructions for binding of protein ligands with brachionichthyidae Separate (Sigma). The resulting matrix is washed with 3 column volumes of 50 mm Tris-HCl, 1 M NaCl, 6 M guanidine/Hcl (pH 8.0) to remove ecovalence linked solid-phase transferrin person. Then the column was balanced 50 mm Tris-HCl (pH 8.0), and associated hTf loaded with iron using 1 ml of 10 mg/ml FeCl3in buffer containing 100 mm of sodium citrate and 100 mm sodium bicarbonate, after which the column was balanced by two column volumes of 50 mm Tris-HCl (pH 8,6) and 1 M NaCl (pH 8.0). Whole bacterial membranes (300 mg whole protein) was obtained from a strain DL63 H. influenzae grown on medium that does not contain iron, as described above (Schryvers et al., 1989). Membranes were diluted to 2 mg/ml in 50 mm Tris-HCl, 1 M NaCl (pH 8.0) and was solubilizers by adding add to 15 mm and Sarkosyl NL 97 to 1.5%. After centrifugation for one hour at 40000g supernatant was applied to the hTf-column, and the column was washed with ten column volumes of 50 mm Tris-HCl, 1 M NaCl, 10 mm etc and 0.5% Sarkosyl (pH 8.0). Receptor proteins were suirable using 2 M GnHCl in the same buffer, and polyinosine faction intensively were dialyzed against 25 mm ammoniumbicarbonate buffer (5 substitutions buffer) was liofilizovane and kept at -20oC. Selected proteins used for biogas produced is x procedures. To do this, rabbits were immunized by 3 injections subcutaneous injections at intervals of two weeks using the complete adjuvant's adjuvant for the first injection and incomplete adjuvant's adjuvant for subsequent injections.

Library DL63-ZAP were sown on cells SUPE E. coli, and plaques were transferred to nitrocellulose membranes, pre-soaked in 10 mm IPTG to induce expression under the control of the promoter of the gene LacZ pBluescript. Filters were blocked using 0.5% separated milk in 50 mm Tris-HCl, 150 mm NaCl (pH 7.5), and then probed polyclonal antisera against TfR and goat anti-rabbit immunoglobulins (IgG), conjugated with horseradish peroxidase. Plaque purified three stages of screening and recombinant plasmids pBluescript (pBH1T1 and runt; Fig.1A and 12) were isolated by in vivo treatments cut (Short et al., 1988).

Century Library expressionand sequences Eagan MinnA and CANCER 12085

(i) Screening the library Eagan - pUC H. influenzae

Application of colonies on nitrocellulose was performed using standard procedures, and the filters probed with 5'-runt probe gene transferrin receptor, shown in Fig.2. The probe was subjected to tagging digoxi is whether datamotion on the nitrocellulose, and then carried out a second round of screening using a similar 5'-runt probe. Putative clones in the second cycle were analyzed by mapping restriction enzyme, and clone S-4368-3-3 (Fig. 1B, Fig. 2) were selected for sequence analysis.

(ii) Screening the library Eagan - ZAP H. influenzae

Library phage were sown using standard procedures in a Cup with LB-medium and 0.7% agarose layer in which cells were XLI Blue (Stratagene). Plaques were applied to nitrocellulose using standard procedures, and the filters were annealed at a temperature of 80oC for 2 hours in vacuum for fixing DNA. 5'-runt-probe gene transferrin receptor (Fig. 2) were labeled with digoxigenin, and the pre-filters were hybridisable for 4 hours at a temperature of 42oWith, and then hybridisable with labeled probe overnight at 42oC. Filters were washed at a temperature of 68oFrom and after autoradiography several plaques were selected for the second cycle of screening. In vivo procedure cut fahmideh DNA of the alleged clonings of the second cycle was carried out in accordance with the instructions using the ZAP system (Promega). An example are the four clone JB-901-5-3 (Fig. In agobuy DNA was purified from 500 ml of culture. The DNA insert was cut out by hydrolysis enzyme Xba I and cloned into pUC 8:2 (Fig 8, containing additional Bg1 II and Xba I sites in the multiple cloning site), and then hydrolyzed by the enzyme Xba I and dephosphorylated. Clone JB-911-3-2 (Fig. 17) contains the 3'half fR-operon of strain H. influenzae Eagan.

(iii) Screening of libraries EMBL3

Library of strain MinnA H. influenzae were sown in NZCYM-cups with LE392 cells using as a coating of 0.7% agarose in NZCYM. The deposition of plaques on nitrocellulose filters was performed using standard procedures, and the filters were processed and probed with 5' runt probe (Fig. 2) labeled with digoxigenin. Estimated plaques were sown and were subjected to the second and third cycles of screening using similar procedures. Phage DNA was obtained from 500 ml of culture using standard procedures, and then the DNA insert was cut out by hydrolysis enzyme Sa1 I and cloned in pUC, resulting received clone DS-712-1-3 (figures 1C and 2).

Library of a strain of CANCER 12085 H. influenzae were sown in NZCYM-cups with LE392 cells using a coverage of 0.7% agarose in NZCYM. Then plaques were applied to the nitrocellulose, the filters were processed and probed with digoxigenine similar procedures. Phage DNA was obtained from 500 ml of culture using standard procedures, and the DNA insert was cut out by hydrolysis enzyme Sa1 I, cloned in pUC and got the clone JB-1042-7-6 (Fig.1 and 2).

Library of strain W H. influenzae were sown on cells S392 in NZCYM-Cup covered with a top layer of 0.7% agarose in NZCYM. Plaques were applied to the nitrocellulose, the filters were processed and probed deoxyadenosine 5'-runt-probe (Fig. 2). Estimated plaques planned and subjected to the second cycle of screening using similar procedures. Phage DNA was obtained from 500 ml of culture using standard techniques, and the DNA insert was cut out by hydrolysis enzyme Sa1 I and cloned in pUC, resulting received clone JB-1031-2-9 (Fig. 2).

Example 4

This example describes the sequencing b1 and b2-TfR genes of the operon.

Plasmid DNA from clones pBHIT 1, pBHIT 2, S-4368-3-3, JB-901-5-3, DS-712-1-3, JB-I042-7-6 and JB-1031-2-9 was obtained using standard techniques. Oligonucleotide primers for sequencing long 17-25 bases were synthesized using a DNA synthesizer (380 V, the abi model) and purified by chromatography using OPC cassettes received from the company Applied Biosystems, Inc. and used in accordance with the IRB And, model the abi) and the terminal of the dye in accordance with the manufacturer's instructions. The sequence TfR-operon of strain DL63 shown in Fig.3, the sequence TfR-operon of strain Eagan shown in Fig.4, the sequence fR-operon of strain MinnA shown in Fig.5, the sequence f-operon of strain CANCER 12085 shown in Fig.6, and the sequence TfR-operon of strain SB33 shown in Fig.7.

Example 5

This example illustrates PCR amplification of genes tbp 2 Netherway strains S12, S29, SB30 and S32 H. influenzae.

Chromosomal DNA Netherway strains S12, S29, S30 and S32 H. influenzae were obtained as described above. Genes TfR designed in the form of an operon containing a gene tbp 2 followed by tbp gene 1 (see Fig.12A and 12B). Synthesized oligonucleotides to the 5'-end of the coding sequence of tbp 2 and a reverse primer to the 5'-end of the coding sequence of tbp 1.

These primers had the sequence: GGGG the GG (SEQ ID 120) corresponding MKSVPLISGS (SEQ ID 147) from the leader sequence of Tbp2; and GGCTTTTTTAGTG (SEQ ID 137), which is a reverse primer leader sequence Shuttle (SEQ ID No. 138) Tbp1 and part of the intergenic sequence (Fig.12A and 12B). PCR-amp is a 100 μl reaction mixture contained 5 ng of chromosomal DNA, 1 μg of each primer, 5 u amplitaq DNA polymerase (Perkin Elmer Cetus), and 0.5 mm dNTP (Perkin Elmer Cetus). Polymerase chain reaction was performed under the following conditions: 25 cycles at 94oC for 1.0 minute; at 45oC for 2.0 minutes and at 72oC for 1.5 minutes. Specific 2 KB fragments amplified for each sample (Fig. 13). DNA S33 used as a positive control (lane 1). Chromosomal DNA used for amplification of gene b2 meet track 1 to S33, track 2 for S12, track 3 for S29, track 4 for S30 and track 5 for S32. These fragments were cloned in the vector of the TA cloning (Invitrogen), and were determined their nucleotide sequences. Nukleinovokisly sequence b2 of strains S12 (SEQ ID 108), V (SEQ ID 110), S30 (SEQ ID 112) and S32 (SEQ ID 114) shown in Fig.8, 9, 10 and 11, respectively.

Example 6

This example illustrates the comparison of amino acid sequences of the transferrin and identification of potentially available epitopes of proteins transferrin receptor by analysis of the secondary structure.

In Fig.14 shows a comparison of amino acid sequences Tbp1 from strain Eagan and strain DL63 H. influenzae type b; Netherway strains Racte this analysis identified areas of Tbp1, which are conservative for all of these bacteria.

In Fig.15 shows a comparison of amino acid sequences b2 from strains Eagan and DL63 H. influenzae type b; Netherway strains of CANCER 12085, SB12, S29, S30 and S32 H. influenzae strains VW and M N. meningitidis, strain FA19 N. gonorrhoe and strains 205 and 37 of Actinobacillus (Haemophilus) pleuropneumoniae (Gerlach and others , 1992). As a result of this analysis revealed areas of b2 that are conservative for all of these bacteria.

Analysis of secondary structure of proteins was performed using algorithms Chou &Fasman (1978), and charts hydrophilicity/hydrophobicity built using the algorithm Norr (1986). These values were obtained based on the average values of heptapeptide "Windows", and on the basis of these values built schedule for the mid-point of each fragment. In Fig.16A illustrates the predicted secondary structure of Tbp1 from strain Eagan H. influenzae type b, and Fig. 16B illustrates the predicted secondary structure b2 from strain Eagan H. influenzae type b. Predicted secondary structure is shown in Fig.16A and 16B were obtained using the procedures described above. However, the authors of the present invention have not yet been able to verify whether consistent patterns, depicted is how many different bacteria were identified by comparative analysis of the primary structures of the sequences it is shown in Fig.14 and 15, respectively. Some of these conservative epitopes are:

TBP1 DNEVTGLGK SEQ ID NO 43

TBP1 EQVLNRLTRIDPGI SEQ ID NO 44

TBP1 GAINEIEYENVKAVEISKG SEQ ID NO 45

TBP1 GALAGSV SEQ ID NO 46

TBP2 LEGGVYGP SEQ ID NO 74

TBP2 KSGGGSFD SEQ ID NO 75

TBP2 YVYSGL SEQ ID NO 76

TBP2 CCSNLSYVKFG SEQ ID NO 77

TBP2 FLLGHRT SEQ ID NO 78

TBP2 EFNVDF SEQ ID NO 79

TBP2 NAFTGTA SEQ ID NO 80

TBP2 VNGAFYG SEQ ID NO 81

TBP2 LEGGYF SEQ ID NO 82

TBP2 VVFGAR SEQ ID NO 83

In addition, together with the predicted secondary structures were identified four conservative accessible epitope on Tbp1 and two epitope on Tb2. These epitopes are:

Top1 DNEVTGLGK SEQ ID NO 43

Top1 EQVLN/DIRDLTRID SEQ ID NO 139 and 140

Top1 GAINEIEYENVKAVEISK SEQ ID NO 141

Top1 GI/VYNLF/LNIRYVTWE SEQ ID NO 142 and 143

Top2 CS/LGGG(G)SFD SEQ ID NO 75, 144 and 145

Top2 LE/SGGFY/FGP SEQ ID NO 74 and 146

Proteins, polypeptides or peptides containing the above-mentioned conservative amino acid sequence, are particularly valuable for use as the detecting means in the diagnosis, as well as immunogens to detect diseases or to protect against diseases caused by bacteria, producing the protein of the transferrin receptor. For immunization of the above amino acid sequences can be predstavley as Salmonella, BCG, adenovirus, poxvirus, virus, cowpox virus or poliovirus.

Example 7

This example illustrates the construction of plasmid JB-1468-29, which expresses a protein b1 strain Eagan in E. coli.

Plasmid S-4368-3-3 (Fig. 1B and 2) and AT-911-3-2 (Fig.17) contain 5'- and 3'of the gene tbp 1 strain Eagan, respectively. In Fig.17 illustrates a circuit design for plasmid JB-1468-29. Oligonucleotide sequences used for construction of plasmid JB-1468-29 shown in Fig. 20 (SEQ ID 86 and 87). Plasmid JB-1468-29 was introduced in the strain BL 21/DE3 E. coli by electroporation to produce strain JB-1476-2-1.

JB-1476-2-1 was cultured in YM medium and induced using IPTG in accordance with a standard Protocol. To obtain Tbp1 in order to study the immunogenicity and other studies, the strain JB-1476-2-1 were cultured over night in NZCYM medium containing 3% glucose. Then to fresh medium NZXYM that does not contain glucose, was added 1:40-inoculate, and the culture was grown to A578=0,3 (optical density). After this was added lactose (1%), and the culture was induced for 4 hours. Analysis of whole cell lysates for JB-1476-2-1, carried out by electrophoresis in SDS page with LTOs shown ry molecular mass: 200 kDa, 116 kDa, of 97.4 kDa, 66 kDa, 45 kDa and 31 kDa; lane 4: JB-1437-4-1 (T7/Eagan - b2) at t0; track 5: JB-1437-4-1 at the time of induction of t=4 hours; lane 6: JB-1607-1-1 (T7/SB12 - b2) at t0; track 7: JB-1607-1-1 at the time of induction of t=4 hours.

Example 8

This example illustrates the construction of plasmid JB-1424-2-8, which expresses a protein b2 strain Eagan from E. coli.

In Fig.18 shows a plasmid S-4368-3-3, which contains a gene tbp 2 from strain Eagan H. influenzae type b. In Fig.18 illustrates a plasmid JB-1424-2-8, and Fig. 19 shows the oligonucleotides used. Plasmid JB-1424-2-8 was introduced into the strain BL 21/SE3 E. coli by electroporation to produce strain JB-1437-4-1 E. coli. After induction IPTG or lactose strain JB-1476-2-1 E. coli expressed protein b2, as shown in Fig.22. Track 1: JB-1476-2-1 (T7/Eagan-Tbp1) at t0; track 2: JB-1476-1-1 at the time of inducing t=4 hours; lane 3: molecular weight markers: 200 kDa, 116 kDa, of 97.4 kDa, 66 kDa, 45 kDa and 31 kDa; lane 4: JB-1437-4-1 (T7/Eagan-b2) at t0; track 5: J-1437-4-1 at the time of inducing t=4 hours; lane 6: JB-1607-1-1 (T7/ AV-Tor) at t0; track 7: JB-1607-1-1 at the time of inducing t=4 hours.

Example 9

This example illustrates the construction of plasmids that encode l is used to construct plasmids having a leader sequence lipoprotein, derived from the E. coli lpp (SEQ ID 88 and 89), rlpB E. coli (SEQ ID 90 and 91) and pa1 E. coli (SEQ ID 92 and 93) and located in front of b2 shown in Fig.20. Constructed plasmids and strains produced these plasmids are illustrated in table 1 (see below).

Example 10

This example illustrates the construction of plasmid JB-1600-1, which expresses b2 from strain S12 E. coli.

Plasmid DS-1047-1-2 (Fig.21) contains PCR amplificatory gene tbp 2 S12. Gene tbp 2 cut as Nde1-R-restriction fragment and inserted into expressing vector RT-7, resulting in the obtained plasmid J-1600-1. After electroporation of this plasmid into cells BL21/DE3 E. coli was obtained strain JB-1607-1-1, which expresses b2 S12. After induction IPTG or lactose expressed b2 S12, as shown in Fig.22. Track 1: JB-1476-2-1 (T7/Eagan-Tbp1) at t0; track 2: 'SV-1476-2-1 at the time of inducing t= 4 hours; lane 3: molecular markers of mass: 200 kDa, 116 kDa, of 97.4 kDa, 66 kDa, 45 kDa and 31 kDa; lane 4: JB-1437-4-1 (T7/ Eagan-b2) at t0; track 5: JB-1437/4-1 at the time of inducing t=4 hours; lane 6: J-1607-1-1 (T7/S12-b2) at t0; track 7: JB-1607-1-1 at the time of inducing t=4 hours.

Example 11bp1 and b2 shown in Fig.23. Both of these recombinant protein expressibility as Taurus inclusion in E. coli and purification procedures are the same. Cells from 500 ml of the culture obtained as described in example 7 for Tbp1 in example 8 for b2, resuspendable in 50 ml of 50 mm Tris-HCl, pH 8.0, and was destroyed by ultrasound (310 minutes, the duty cycle of 70%. The extract was centrifuged at 20000g for 30 minutes and the resulting supernatant, which contained more than 95% of the soluble proteins of E. coli, is discarded.

The remaining sediment (Fig. 23, RRT1again were extracted in 50 ml of 50 mm Tris, pH 8.0, containing 0.5% Triton X-100 and 10 mm EDTA. After centrifugation at 20 000g for 30 minutes the supernatant, containing residual amounts of soluble proteins and most of the membrane proteins was discarded. After extraction, as described above, the precipitate contained bullock inclusions (Fig. 23, RRT2). Proteins Tbp1 and Tbp2 was solubilizers in 50 mm Tris (pH 8.0) containing 0.1% LTOs and 5 mm DTT. After centrifugation of the resulting supernatant was again purified on a column for gel filtration with Superdex 200, equilibrated in 50 mm Tris, pH 8.0, containing 0.1% LTOs and 5 mm DTT. Fractions were analyzed by electrophoresis in SDS page with LTOs, and those fractions that contained the cleansing is 20 000g for 30 minutes. Soluble protein remaining after treatment in these conditions, and purified Tbp1 and b2 kept at -20oC.

Analysis by electrophoresis in SDS page with LTOs described in terms of cleaning, as shown in Fig.24. Lane 1 - molecular weight markers(106, 80, 49,5, 32,5, 27,5, 18,5 kDa); lane 2: cell lysates of E. coli; lane 3: solubilization bullock; lane 4: purified Tbp1 or b2.

Example 12

In this example, illustrated study of the immunogenicity of recombinant proteins Tbp1 and b2 conducted on mice.

Groups of five mice Balb/C subcutaneously (s,C) were injected with (on days 1, 29 and 43) purified protein rTBp1 or rTBp2 (1 μg - 10 μg) obtained as described in example 11, in the presence of AlPO4(1.5 mg per dose). Blood samples for analysis of antibody titers against rivers. b1 and against rivers. b2 (by method IA) were taken at days 14, 28, 42 and 56. Studies on the immunogenicity illustrated in Fig.35.

Example 13

In this example, illustrated by enzyme-linked immunosorbent assay (l) to determine the level of antibodies against rivers. Tbp1 and against rivers. b2 in mouse sera.

The titers of antibodies to rivers. Tbp1 and rivers. b2 were determined by the method basically described Panezzuti is as described in example 11, and kept for 16 hours at room temperature, then blocked with 0.1% (wt./about.) S in S. Sera were serially diluted, added to the wells and incubated for 1 hour at room temperature. Ofinnocence fragments F(ab')2goat artemisinin immunoglobulins IgG (Fc-specific) conjugated with horseradish peroxidase was used as the second antibody. The reaction was shown using tetramethylbenzidine (TMB/N2ABOUT2), and the optical density was measured at 450 nm (using 540 nm as a reference wavelength) using a spectrophotometer to read the microplate (tablet reader) Flow Multiscan MCC. Reactive titer of the antisera was determined as the reciprocal dilution at which the observed two-fold increase in optical density compared with the optical density obtained with the sample of non-immune serum.

Example 14

This example illustrates the cross-reactivity of antisera against b1 produced by immunization with recombinant Tbp1 Eagan with different strains of H. influenzae.

Cell lysates of strains of H. influenzae cultured in BHI-medium containing NAD and heme (Harkness and others, 1992)EDDA, were isolated poemi Guinea pigs against Tbp1, produced in response to the introduction of purified recombinant protein b1 Eagan (Fig.26). Lane 1: BL21/DE3; track 2: S12-EDDA; lane 3: S12+EDDA; lane 4: S29-EDDA; lane 5: S29+EDDA; lane 6: S33-EDDA; lane 7: S33+EDDA; lane 8: Eagan-EDDA; lane 9: Eagan+EDDA; lane 10: B. catarrhalis 4223-EDDA; lane 11: B. meningitidis 4223+EDDA; lane 12: N. meningitidis 608-EDDA; lane 13: 608 N. meningitidis+EDDA; track 14: induced JB-1476-2-1 expressing recombinant Tbp1 Eagan; track 15: molecular weight markers. Specific 95 kDa bands, reacted with antisera against Tbp1, on tracks 3, 4, 5, 7, 8 and 9 correspond to the strains JB12, S29, S33 and Eagan; 100 kDa band in lanes 10 and 11 correspond to the strain 4223 Century catarrhalis; 80 kDa band in lanes 12 and 13 correspond to the strain 608 N. meningitidis.

Example 15

This example illustrates the cross-reactivity of antisera against b2 produced by immunization with recombinant b2 Eagan, with different strains of H. influenzae.

Cell lysates of strains of H. influenzae, cultivated in BHI environments, which were added NAD and heme (Harkness and others, 1992) EDDA, was isolated by electrophoresis on SDS page with LTOs, then transferred to nitrocellulose membrane and probed with antisera Guinea pigs against b2 produced is; is orozca 2: induced strain JB-1437-4-1 expressing recombinant protein b2 Eagan; lane 3: J12-EDDA; lane 4: S12+EDDA; lane 5: S29-EDDA; lane 6: SB29+EDDA; lane 7: S30-EDDA; lane 8: S30+EDDA; lane 9: S12-EDDA; lane

10: S33-EDDA; lane 11: S33+EDDA; lane 12: CANCER-EDDA; lane 13: CANCER+EDDA; lane 14: Eagan-EDDA; lane 15: Eagan+EDDA. Specific 60-70 kDa band reacted with antisera against b2 on the tracks 3, 6, 7, 8, 13, 14 and 15, which correspond to the strains SB12, SB29, S30, CANCER, and Eagan.

Example 16

This example illustrates the synthesis of synthetic peptides corresponding conservative areas in b2 and b1.

The deduced amino acid sequence of Tbp1 and b2 compared, as shown in Fig. 14 and 15, respectively. The result of this comparison were identified area conservative amino acid sequences of the transferrin receptor (see above) and, as shown in tables 2 and 3, were synthesized peptides containing part of the transferrin receptor. Such synthesis can be carried out by expression in an appropriate host recombinant vectors containing nucleic acid encoding these peptides, or by standard peptide synthesis.

Corot is in terms of their share capital, recommended by the manufacturer, after which the peptides were tsalala from the polymer substrate using hydrofluoric acid (HF). These peptides were purified using reversed-phase liquid chromatography high resolution (OF GHWR) prepreparation column from Vydac C4 (130 cm) using a gradient of 15-55% acetonitrile in 0.1% trifluorobutane acid (TFA), exercising for 40 minutes, with a flow rate of 2 ml/min All synthetic peptides used in biochemical and immunological studies, had a purity of more than 95%, which was confirmed by analytical GHUR. The analysis of amino acids was carried out using the Waters Pico-Tag and this analysis showed good agreement with the results obtained for this composition, in theory.

Example 17

In this analysis illustrates the immunogenicity of synthetic peptides with the use of experimental animals.

Guinea pigs were immunized by intramuscular injection of 100 μg of peptide, obtained as described in example 16, and emulsified in complete Freund's adjuvant (day 0), after which through 14 (day +14) and 28 (day +28) days has introduced a booster dose using the same number of peptide emulgirovannie assay (ELISA) to determine the antibody titers. For this purpose holes for micrometrology (Nunc-Immunoplate, Nunc, Denmark) were senzibilizirani 500 nanogram any of the specifically studied peptides in 50 μl of buffer for sensitization of the surfaces (15 mm Na2CO3, 35 mm NaHCO3pH to 9.6) for 16 hours at room temperature. Then the plates were blocked with 0.1% (wt./about.) BSA in phosphate buffered saline (PBS) for 30 minutes at room temperature. Antisera were subjected to serial dilution, after which was added to the wells and incubated for 1 hour at room temperature. After removal of the antisera tablets five times washed with PBS containing 0.1% (wt. /about. ) Twin and 0.1% (wt./about.) The BSA. F(ab')2fragments from the goat immunoglobuline IgG directed against Guinea pigs and conjugated with horseradish peroxidase (Jackson Measurement Research Labs Inc., PA), diluted with a buffer for washing and added to the tablets for micrometrology. After one-hour incubation at room temperature tablets five times washed with buffer for washing. These tablets showed using the substrate tetramethylbenzidine (TMB) in N2ABOUT2(ADL, Toronto), then the reaction was stopped by adding N2SO4and measured the optical density p is acid residues, used in these ELISA-assays as a negative control. These analyses were carried out in a triple duplicate, and the reaction titer of each of the antisera was determined as the dilution corresponding to twice the increase in optical density compared with the optical density obtained for the negative control. Antisera produced in Guinea pigs, are monospecific for peptide used for immunization. Titers of sera obtained after immunization are shown in table 4.

The peptides of the present invention contain a single copy of any of the peptides listed in tables 2 and 3, or many copies of their analogues. In addition, the peptide may contain many different peptides selected from the peptides listed in tables 2 and 3 or their equivalents, and include appropriate molecules media. In this case, preferably, for the production of antibodies were used peptides from conserved regions, as in this case, the analysis immunovative or on the binding of another type can then be used to detect several species of Haemophilus. Therefore, in tables 2 and 3 give other conservative region of the transferrin receptor on neznam treatment.

Example 18

This example describes the ability of the antisera produced against peptides corresponding conservative areas of the transferrin receptor, recognizes the transferrin receptor Branhamella catarrhalis.

Guinea pigs were immunized with a peptide corresponding to conservative areas of the transferrin receptor, and as a result of this immunization antisera were obtained as described in example 17. Cell extract Branhamella catarrhalis were subjected to Western blot turns with particpations anticorodal, which specifically recognizes the transferrin receptor from this bacterium. Antipeptide serum produced in Guinea pigs immunized with N-terminal peptide b2 and peptide TR-25, specifically recognize the protein b2 from Branhamella catarrhalis and recombinant protein expressed plasmid clone runt in E. coli. Clone runt expressed a shortened version b2 beginning with amino acids 80 (i.e., NKKFYSG-SEQ ID 105). Therefore, protein b2 from runt can be recognized only by antibodies produced against a second epitope LEGGFYGP (TR-25). This analysis showed that peptides corresponding to konservativnym sequences of the transferrin receptor, can be specname, they can be used as components in immunogenic compositions, including vaccines, for producing an immune response against the transferrin receptor and protection against diseases caused by these bacteria.

Serum from rabbits were tested using ELISA for specificity to the peptide having the sequence LEGGFYGP (SEQ ID No. 74), or to b2 strain DL63 H. influenzae. ELISA-plates were senzibilizirani peptide or protein, and then blocked with 5% separated milk. Serial twofold dilution of the serum in phosphate buffered saline, 0.05% of the Twin-50 and 1% dry milk and incubated on the plates for two hours at 37oS, after which the tablets washed five times in phosphate buffered saline containing 0.05% tween-20. Washed tablets probed donkey anti-rabbit immunoglobulins IgG, conjugated with horseradish peroxidase (HRPO) for 30 minutes at room temperature, and then washed five times in phosphate buffered saline containing 0.05% tween-20. To each well for 30 minutes at room temperature in the dark was added R-substrate and the color development was stopped by adding 50 μl 1 M surpriser 19

This example illustrates the production of strains of H. influenzae, which does not Express the transferrin receptor.

2,55 - EcoRI-fragment insertion of pBH1T1 was subcloned into the EcoRI site UC4, which led to the destruction of cluster resistance to kanamycin (Kan), TA 903 of this vector (pUH1T1; Fig.28). This stage of sublimirovanny helped to facilitate subsequent insertions or HincII or Pst1 fragment U4 containing cap cluster, Hind III or Pst1 site pUH1T1 (both of which are unique sites in this design) for producing pUH1T1KFH and pUH1T1KFP (Fig.28). After hydrolysis by the enzyme EcoRI to remove the unwanted gene sequences of these constructs were introduced into the genome of H. influenzae wild type by transformation by using M-IV, as described previously (Barcak and others, 1991), and transformants were selected on agar BHINH containing 20 μg/ml kanamycin.

Example 20

This example illustrates the construction of polioviruses, expressing the epitope transferrin receptor.

cDNA clone having a base 1175-2956 genome of the Mahoney strain of poliovirus type I (V1-M), cut restricteduse enzymes PVL I and Hind III. These enzymes cut the fragment containing the base 2754-2786 that encode the four-digit code used, for example, figure 1095, refers to amino acid 95 capsid protein VP1. New hybrid cDNA clones were constructed by replacing the cut fragment synthetic oligonucleotides encoding amino acids protein b2 H. influenzae. These new hybrid cDNA clones cut restricteduse enzymes Nhe1 and SnaB1, which cut hybrid fragment containing the DNA sequence of the transferrin receptor, of the grounds 2471-2956 poliovirus. cDNA clone, for example 7LD or RTSS, whole genome V1-M cut by enzymes Nhe I and SnaB1 to cut fragment from bases 2471-2956 poliovirus. Then, this fragment was replaced with a hybrid fragment containing the DNA sequence of the transferrin receptor to produce hybrid cDNA clone genome PV1-M with bases 2754-2786, replaced the bases encoding the hybrid all-loop comprising amino acids transferrin receptor, as shown in Fig.29.

Plasmid pT7XLD and clones originating from pT7XLD, such as RTSS contain a promoter sequence for RNA polymerase T7 5'-end cDNA PV1-M RNA transcripts cDNA V1-M, including any bases encoding amino acids transferrin receptor were obtained using RNA-polymer is e viable hybrid virus marked V12, V12, PV1TBP2C and V1TBP2D. As a result of transfection rtcsu the transcripts were received wild poliovirus type indicated PV1XLD (Fig.29).

Antigenic characteristics V12, V12, V12 and PV1TBP2D illustrated in table 5. All these hybrids were neutralized by antisera Guinea pigs produced against a peptide containing the sequence LFCG-FYGP (SEQ ID No. 74), suggesting that these viruses expressed the antigenic sequence in a recognizable form. For producing these antisera female Guinea pigs were immunized 1 M 500 cells / ml volume containing 200 μg of peptide dissolved in the aluminum phosphate (3 mg/ml). Animals were immunized on days: 1, 14, 28 and 42, and took the blood on days 0, 28, 42 and 56. Serum was obtained from blood collected on day 56, PV1TBP2A and PV1TBP2B were also neutralized by rabbit antisera against b2 strain DL63 H. influenzae, testified that at least these two viruses expressed sequence and form recognized for antibodies generated against the indicated protein. All viruses were neutralized by antisera against PV1, which indicates that changes in antigenic site neutral the example 21

This example illustrates the use of poliovirus hybrids to induce anti-b2 antisera with high titer.

Rabbits were inoculable sCl purified PV1TBP2A (rabbits 40, 41, 42). It should be noted that though the viruses were "alive", but polioviruses in rabbits is not replicated, and any observed response was, essentially, a response to an inactivated antigen. On day 1 of the rabbits were inoculable 1 microgram of virus in complete Freund's adjuvant by subcutaneous injection and back, on the 14th day, the rabbits were injected re-injection (subcutaneously, in the back) 1 μg of virus in incomplete Freund's adjuvant. Blood samples were taken on day 0 (non-immune serum) and on day 27. The dose of virus for inoculation was 2,5108B. about.e, was determined from the values AND260corresponding to approximately 3,01011the virions. This value is equivalent to 0.5 PM of a virus or 30 PM epitope LEGGFYG (SEQ ID No. 74), because each virion expresses 60 copies of the epitope (table.6).

Summary descriptions

Thus, the present invention relates to purified and selected DNA molecules containing genes transferrin receptor; the sequences of these genes transferrin receptor, CTSA to peptides, the relevant sections of the transferrin receptor. These genes, DNA sequences, recombinant proteins and peptides can be used for diagnosis, immunization, and manufacture of diagnostic and immunological reagents. To prevent diseases caused by bacterial pathogens that produce transferrin receptor, can be derived vaccine, made on the basis of expressed recombinant proteins b1 and/or b2, their fragments or peptides derived from these sequences. It is possible modifications not beyond the scope of the present invention.

1. A DNA fragment encoding a fragment of the protein transferrin receptor strain Haemophilus having conservative amino acid sequence, which is conservative for bacteria that produce the protein transferrin receptor, and corresponds to the sequence SEQ ID NO: 74 or SEQ ID NO: 85.

2. The DNA fragment encoding the proteins of the transferrin receptor strain Haemophilus with amino acid sequences shown in Fig. 14 and 15, having the nucleotide sequence shown in Fig. 3, 4, 6, 7, 8, 9, 10 or 11 for strains DL63 or Eagan Haemophilus influenzae type b, is aptara transferrin strains of Haemophilus influenzae with the amino acid sequence, presented on Fig. 14 or 15 for proteins bp1 or bp2 respectively.

3. The DNA fragment under item 2, characterized in that it is embedded in expressing vector with specific identifying characteristics of plasmid JB-1424-2-8 (ATSS 75937), plasmid JB-1600-1 (ATSS 75935) or plasmid JB 1468-29 ADS 75936).

4. The DNA fragment under item 3, characterized in that it encodes a protein transferrin receptor strain of Haemophilus.

5. The DNA fragment under item 3, characterized in that it encodes a protein bp1 or protein bp2 strain of Haemophilus.

6. The DNA fragment under item 3, wherein expressing the elements represent the nucleotide portion encoding a leader sequence for secretion from the host protein transferrin receptor, or its fragment.

7. The DNA fragment under item 3, wherein expressing the elements represent the nucleotide portion encoding the signal lipidization for expression of the owner lepidosirenidae form of the protein transferrin receptor, or its fragment.

8. Plasmid vector adapted for transformation of E. Li, comprising a DNA fragment according to p. 2.

9. The DNA fragment encoding the protein of the transferrin receptor strain Minn A Haemophilus influenzae type In NUU in Fig. 5, or a fragment of DNA encoding proteins bp1 or bp2 shown in Fig. 5.

10. Plasmid vector adapted for transformation of E. Li, comprising the DNA fragment under item 9.

11. Recombinant protein transferrin receptor produced by transformed E. Li, comprising the DNA fragment under item 1.

12. Recombinant protein transferrin receptor produced by transformed E. Li, comprising a DNA fragment according to p. 2.

13. Recombinant protein, transferrin receptor, produced by transformed E. Li, comprising the DNA fragment under item 9.

14. Isolated and purified protein TVR strains of Haemophilus influenzae with the amino acid sequence shown in Fig. 14.

15. Protein under item 14, characterized in that said strain of Haemophilus is a strain of Haemophilus influenzae type b or Netherway strain of Haemophilus influenzae.

16. Isolated and purified protein TVR strain of Haemophilus influenzae, shown in Fig. 15.

17. Protein under item 16, characterized in that said strain of Haemophilus is a strain of Haemophilus influenzae type b or Netherway strain of Haemophilus influenzae.

18. Immunogenic composition, made in the form of a vaccine for in vivo administration in order to protect from Bo the least one active component, selected from the group comprising isolated and purified protein bp1 strains of Haemophilus influenzae by p. 14; isolated and purified protein bp2 strains of Haemophilus influenzae by p. 16, and its pharmaceutically acceptable carrier, and at least one of these active ingredients produces an immune response when administered to the host.

19. Immunogenic composition under item 18, further comprising adjuvant.

20. The method of obtaining isolated and purified protein bp1 or bp2 strain of Haemophilus in PP. 14 or 16, respectively, comprising the following stages: (a) obtaining a recombinant host expressing cells to activate one of the proteins bp1 or bp2; (b) culturing the specified host from receiving cell mass; (c) the destruction of cell mass with obtaining a cell lysate; (d) fractionation of cell lysate with the first supernatant and a first precipitate and a first supernatant comprises, essentially, most of the soluble proteins of the owner; (e) the Department specified the first supernatant from the specified first precipitate; (f) selective extraction of the first sludge removal, essentially all soluble host proteins and membrane proteins of the owner to obtain a second supernatant and knogo extracted sludge; (h) solubilisation extracted sediment was getting solubilizing extract and (i) fractionation solubilizing extract to obtain fractions containing protein bp1 or protein bp2.

21. The method according to p. 20, characterized in that the specified cell lysate fractionary by centrifugation.

22. The method according to p. 21, characterized in that the phase selective extraction of the first sludge includes the extraction of at least one detergent.

23. The method according to p. 22, characterized in that the solubilized extract is subjected to fractionation by gel filtration to obtain fractions containing protein bp1 or protein bp2.

24. The method according to p. 23, characterized in that it includes subsequent dialysis of the fractions containing protein bp1 or bp2, in order to remove at least one of the specified detergent with obtaining further purified protein solution bp1 or bp2.

25. The method according to p. 20, characterized in that said strain of Haemophilus is a strain of Haemophilus influenzae.

 

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