Method for preparing nontoxic anti-anthrax vaccine

FIELD: biotechnology, vaccines.

SUBSTANCE: invention relates to producing vaccines and describes anti-anthrax vaccine that comprises mutant protein toxin from Bacillus anthracis chosen from mutant PA or mutant LF, or mutant EF or their combinations. Mutations of toxins provide the retained immunogenicity in decreasing the level of their toxicity. For the development of vaccine with reduced reactivity invention proposes recombinant DNA-construction for expression of said protein-toxins. DNA-construction comprises the expressing vector and DNA fragment comprising, in turn, genes encoding the corresponding protein-toxin (PA, LF or EF). Invention describes a method for preparing mutant proteins by using the transformed prokaryotic host. Prepared mutant protein-toxin possessing immunogenic properties and absence of toxicity is used for preparing anti-anthrax vaccine comprising one or more mutant protein-toxins and in combination with protein-toxin PA, LF or EF of wild type. Using the invention provides to develop the safety and effective vaccine against anthrax.

EFFECT: improved preparing method of vaccine.

13 cl, 2 dwg

The invention relates to recombinant DNA constructs and to a method for producing a non-toxic protivoseborainey vaccine.

DESCRIPTION of the prior art

Anthrax, a zoonotic disease, is called the gram-positive sporelike microorganismsBacillus anthracis. Humans become accidental hosts through food of animal origin, animal products and get infected from the environment containing microorganismsBacillus anthracis(P.S. Brachman, 1970, Annals. N.Y. Acad. Sci. 174, 577-582). Anthrax is one of the oldest known bacterial diseases and occurs in most parts of the world, including India. Major virulent factorsB. anthracisinclude capsules poly-D-glutamic acid and three-component toxin complex anthrax. Anthrax toxin (Leppla S.H., 1991, In Source Book of Bacterial protein toxins, pp.277-302), including protecting the antigen PA(83 kDa), lethal factor (LF-(90 kDa) and the swelling factor (EF-(KD), is a major virulence factorB. anthracis. The catalytic components of this complex, LF/EF, need RA to penetrate into the cytosol of the cell. RA in combination with LF (called lethal toxin) causes death in experimental animals (H. Smith and J. Keppie, 1954, Nature, 173, 869-870). RA in combination with EF (called edema toxin) at the Expo is imentally animals causes swelling on the skin (J.L. Stanley and Smith H., 1961, J. Gen. Environ., 26, 49-66). RA represents the receptor-binding component, which provides movement of the catalytic components, LF and EF, in target cells. After moving into the cell LF, which is metalloproteinases, breaks down some of the mitogen-activated proteinkinase kinase (MAPKK), leading to inactivation of signal transduction pathways (Duesbery N.S., et al., 1998, Science, 280, 734-737). On the other hand, EF, penetrate into the cells, is activated by calmoduline, causing an increase in intracellular levels of camp (Leppla S.H., 1982, Proc. Natl. Acad. Sci. USA, 79, 3162-3166).

In the first phase of intoxication is the binding of the RA with the surface receptors of cells (Bradley, K.A., et al., 2001, Nature, 414, 225-229). After binding to receptors on the cell surface RA is cleaved by proteases on the cell surface with the formation of fragment size 63-kDa (Klimpel R.K., et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10277-10281), which oligomerizes and associated with the LF/EF (Milne, J.C., et al., 1994, J. Biol. Chem., 269, 20607-20612). Binding of LF/EF is competitive. Then the whole complex undergoes receptor-mediated endocytosis. Acidification of endosome (Friedlander A.M., 1986, J. Biol. Chem., 261, 7123-7126) leads to the embedding of RA-oligomer in the membrane of endosome with the formation of pores (Milne, J.C. and R.J. Collier, 1993, Mol. Environ., 10, 647-653)through which LF/EF moves into the cytosol of the cell.

RA im is no four domains, which initially form antiparallel beta-strands with several short helices with less than four turns (Petosa, C., et al., 1997, Nature, 385, 833-838). Domain 1 is responsible for the binding of LF/EF during intoxication anthrax. Domain 2 is primarily beta barrel (beta barrel) and is designed to be embedded in the membrane and moving. Domain 3 is the smallest domain and is important for oligomerization of RA and, apparently, also to associate with RA LF/EF. Domain 4 is the receptor-binding domain.

Installed recently the crystal structure of LF shows that LF has 4 domain (Pannifer A.D., et al., 2001, Nature, 414, 229-233). Domain 1 is involved in the binding with RA. This domain has a significant homology with the N-terminal residues 1-250 EF. In fact, most of the residues in this area is quite conservative.

Of all three proteins, toxins RA is the most immunogenic and represents a significant component of the vaccine against anthrax (Gladstone G.P., 1946, Br. J. Exp. Pathol., 27, 349-418). It is noted that the protective efficacy of RA greatly increased, if this vaccine to include small amounts of LF or EF (Pezard et al., 1995, Infect. Immun., 63, 1369-1372). However, it is also the main cause of toxicogenomic and reactogenicity of such vaccines. The anthrax toxin (Leppla S.H., 1991, In Source Book of Bactrial protein toxins, pp.277-302), including protecting the antigen (PA), lethal factor (LF) and the swelling factor (EF)is theB. anthracisthe major virulent factor.

Currently used protivoseborainey vaccine produced from decapsulating avirulent strain of microorganism, known as strain stern (M. Sterne, 1939, J. Vet. Sci. Anim. Ind, 13, 307-312). In Russia and in China, the use of live spore vaccine, based on the strain of the stern. In the UK the vaccine is a culture filtrate of strain stern, precipitated by alum, and in the U.S. the vaccine consists of a adsorbed on alhydrogel of cell-free filtrate of culture decapsulating obtained without proteolysis, strain V770 isolated from diseased anthrax in cattle (P.C.B. Turnbull, 1991, Vaccine, 9, 533-539). All of these currently used vaccines against anthrax, not to mention crude vaccines have uncertain composition. They reactogenna and do not protect against all natural strains ofB. anthracis.

In U.S. patent No. 2017606 described receiving anthrax antigen by growing the microorganism in an appropriate culture medium and separating the grown microorganisms from this culture medium.

In U.S. patent No. 2151364 describes a method for protivoseborainey vaccine, which includes recip is the suspension of anthrax and its adding to the suspension a sterile solution, containing alum.

In the patent of Russia №2115433 describes a method for protivoseborainey vaccines, including live debate decapsulating strainB. anthracisand protecting the antigen fromB. anthracis.

In patent application WO No. 0002522 describes a method for protivoseborainey vaccine using non-toxic protective antigen fromB. anthracisused for the induction of an immune response that protects against anthrax.

The disadvantage of all the above patents is that they all use the culture/disputesBacillus anthracis. Bacillus anthracisan infectious microorganism and it cannot be operated without following precautions. Get the vaccine contains traces of other toxic and non-toxic proteinsBacillus anthracisthat leads to a number of side effects and reactogenicity.

These vaccines also have some residual virulence for certain types of domestic and laboratory animals. Strain stern toxigenic and in high doses is pathogenic. Therefore it is considered unsafe and not suitable for human use. Such a vaccine could cause unwanted side effects including necrosis at the site of vaccination.

For this reason, there is a need to create protivoseborainey second generation vaccine, which is not the call to the AET side effects and has a clearly defined structure.

The purpose of the present invention is to give the anthrax toxin nontoxicity without affecting its immunogenicity, in order to create a safe and effective protivoseborainey vaccine.

To achieve this goal in the present invention results from the construction of recombinant DNA comprising the expression vector and the DNA fragment comprising the genes of protective antigen (PA) wild-type lethal factor (LF) wild-type, and the swelling factor (EF) of the wild type.

The present invention also created the design of recombinant DNA, including:

expression vector and the DNA fragment comprising the genes of the mutant protective antigen (PA), mutant lethal factor (LF) or mutant edema factor (EF).

The specified vector is a prokaryotic vector such as the vector PQE 30, and the indicated expression vector contains the promoter T5 and 6X his-tag tag.

A fragment of DNA is a gene which protects the antigen c substitution by alanine at residue Phe202.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Leu203.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Pro205.

A fragment of DNA is a gene which protects the antigen from the substitution Alani the ω residue Ile207.

A fragment of DNA is a gene which protects the antigen from the substitution of alanine by the remnants of the Pro205, Trp226 and Phe236.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Phe552.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Ile574.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Phe552 and Phe554.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Ile562 and Ile574.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Leu566 and Ile574.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Phe552 and Phe554, Ile562, Leu566 and Ile574.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Phe427.

A fragment of DNA is a gene which protects the antigen with a deletion at residue Asp 425.

A fragment of DNA is a gene which protects the antigen with a deletion in residue Phe 427.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Trp346.

A fragment of DNA is a gene which protects the antigen from the substitution of the alanine at residue Leu352.

A fragment of DNA is a gene which protects the antigen from the substitution Alani the ω residue Trp346, Met350 and Leu352.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Tyr148.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Tyr149.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Ile151.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Lys153.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Asp187.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Phe190.

A fragment of DNA is a gene lethal factor with the substitution of the alanine at residue Asp187, Leu188, Leu189 and Phe190.

A fragment of DNA is a gene factor puffiness with the substitution of the alanine at residue Tyr137.

A fragment of DNA is a gene factor puffiness with the substitution of the alanine at residue Tyr138.

A fragment of DNA is a gene factor puffiness with the substitution of the alanine at residue Ile140.

A fragment of DNA is a gene factor puffiness with the substitution of the alanine at residue Lys142.

The protein encoded by the specified fragment of the DNA is expressed in a prokaryotic host. Specified prokaryotic host is a strain ofE. coli.

The protein expressed by the gene DNA-fragm the fact, represents RA wild-type LF wild-type EF wild-type and mutant variants.

In addition, the present invention describes a method for the mutant protein toxin of anthrax, including:

- getting mutated genes RA, LF and EF using different obtained using mutagenic primers, such as those shown here for the PCR reaction;

- processing the specified mutant PCR product together with native matrix by the enzyme to break down this native matrix specified PCR product;

- transformation of the indicated mutant product in E. coli;

- selection of the recombinant constructs of the transformed E. coli strain and confirm the desired mutation;

- transformation confirmed mutant constructs in the corresponding expression strain ofE. colifor expression of mutant protein and

- allocation of cleaning specified downregulation of mutant protein.

This cleaning is carried out using Ni-NTA-chromatography and/or other chromatography methods.

These clone genes in the expression vector PQE containing the T5 promoter and 6X his-tag tag.

Mutations affecting the first domain of RA remains 202, 203, 205. Mutations affect the third domain of RA remains 552, 574, 552+554, 562+574, 566+574, 552+554+562+566+574, the resulting mutant proteins, coloradotitty by oligomerization. Mutations affect the second domain of RA remains 425 and 427 in the loop 4 domain 2. These mutations disrupt the ability of RA to move. These mutations affect the second domain of RA remains 346, 352 and 346+350+352 in the loop 3 domain 2 so that RA becomes biologically inactive. These mutations affect the 1st domain LF on stock 148, 149, 151, 153, 187, 190 and 187+188+189+190, disrupting the binding of LF with RA. These mutations affect the first 250 residues EF.

Protivoseborainey vaccine comprises a protein-toxin anthrax selected from RA wild-type, or LF wild type or wild-type EF.

Protivoseborainey vaccine comprises a protein-toxin anthrax selected from RA mutant or mutant LF, or mutant EF, or combinations thereof.

Protivoseborainey vaccine comprises a protein-toxin anthrax, selected from any combination selected from RA wild-type LF wild type or wild-type EF with any one or more factors selected from RA mutant, mutant LF, or mutant EF.

The pharmaceutical composition includes an effective amount of a protein toxin of anthrax, as claimed in the present invention.

DETAILED DESCRIPTION of the PRESENT INVENTION

An ideal vaccine against anthrax should contain both RA, LF, EF, but at the same time, it must be non-toxic and safe. Dedicated cleaning Ryoko is Benante proteins of a particular composition can be used in the vaccine to minimize the reactogenicity of the vaccine. In addition, these protein toxins of anthrax can be non-toxic in the introduction of mutations that affect the biological activity of the proteins without affecting their structure or immunogenicity. These non-toxic mutant protein toxins of anthrax can be used together to create a safe, erectogenic and effective recombinant vaccine against anthrax. Thus, the first objective of the present invention is to provide a method for obtaining a safe and effective second generation vaccine against anthrax, including non-toxic protein toxins of anthrax, which was produced using site-directed mutagenesis in different functionally significant domains of these proteins toxins.

The authors of the present invention were subjected to PCR amplification genes RA, LF, EF. They cloned these genes in the expression vector pQE30 (Gupta P., et al., 1998, Infect. Immun., 66, 862-865; Gupta P., et al., 1999, Protein Expr. Purif. 16, 369-376; Kumar P., et al., 2001, Infect. Immun., 69, 6532-6536). This vector contains a promoter T5 and 6x-his-tag tag that allows you to easily clean these recombinant proteins (Figure 1).

Conditions sverkhekspressiya these genes using the above recombinant plasmids from strains ofE. coliwere optimized by the authors of the present invention (Chauhan V., et al., 2001, Bochem. Biophys. Res. Commun., 283, 308-315).

Using the above recombinant plasmid, the authors of this method was introduced mutations in these genes to create the expressed recombinant proteins defective in their biological function, thereby making them non-toxic. The present invention includes the expression and secretion clearance of these mutant proteins from strains ofE. coli. In addition, it includes a full description of the selected purification of mutant proteins with point defect that makes them non-toxic.

MUTATIONS INTRODUCED IN the protective ANTIGEN AS PART of the PRESENT INVENTION

1.Mutations that make RA defective for binding to LF/EF. The authors of the present invention has introduced a series of mutations in the 1st domain RA. Among the introduced mutations mutations at residues 202, 203, 205, 207 and 205+226+236 proved to be defective for binding to LF.

2.Mutations that do RA for defective oligomerization. The authors of the present invention was introduced mutations in the 3rd domain of RA. Mutations in residues 552, 574, 552+554, 562+574, 566+574, 552+554+562+566+574 give mutant proteins that were defective in oligomerization.

3.Mutations that make RA defective to move. The authors of the present invention was introduced mutations at residues 425 and 427 in the loop 4 domain 2. These mutations disrupt the ability of RA to move.

4.Mutations that delautre defective for embedding/translocation . The authors of the present invention have found that the introduction of mutations at residues 346, 352 and 346+350+352 in the loop 3 domain 2 RA becomes biologically inactive. These mutant proteins are able to bind with receptors on the cell surface, becoming proteoliticeski active with the formation of oligomers and communicating with the LF. Biological inactivation of these mutant proteins may be due to defective embed/translocation.

MUTATIONS INTRODUCED INTO LETHAL FACTOR, AS PART of the PRESENT INVENTION

Mutations that make LF defective for binding to RA. The authors of the method was introduced mutations in the 1st LF domain. They found that the mutation of residues 148, 149, 151, 153, 187, 190 and 187+188+189+190 disrupts the binding of LF with RA.

MUTATIONS INTRODUCED INTO the SWELLING FACTOR AS PART of the PRESENT INVENTION

Mutations that make EF defective for binding to RA. The authors of the method has introduced a series of mutations in the first 250 residues of EF. It is established that the mutation at residues 137, 138, 140 and 142 radically disrupts the binding of EF with RA.

After expression and excretion clearance of mutant proteins evaluated for their biological activity.

The authors of the present invention found that the above mutants RA at joint Appendix C LF wild type are non-toxic for J774.1 cells. Also mutants LF, at joint Appendix C RA wild the IPA, be non-toxic for J774.1 cells. Similarly, when the joint Appendix C RA wild-type and mutants of EF is unable to produce cyclic amp-toxicity in cells Cho (table 2).

Dedicated cleaning mutant protein is analyzed for its biological activity by testing:

- The ability of RA to bind to receptor sites on the cell surface,

- The ability of RA to communicate with LF or EF,

- The ability of RA to oligomerization,

- The ability of the PA oligomer embedded in the membrane

- The ability of RA to move LF or EF into the cytosol,

- Ability lethal toxin to destroy cell line macrophages, such as RAW264.7 and J774A.1,

- Ability toxin edema extending SNO-cells.

STUDIES ON IMMUNIZATION

Protecting the antigen in accordance with its name is a highly immunogenic protein. In fact, it is a necessary component of the vaccine against anthrax. Immunization with recombinant RA wild type produces high titers of antibodies to RA and provides protection against lethal infection of Guinea pigs with anthrax. It was also noted that the mutant RA is as immunogenic as RA wild-type, and could easily be replaced in the vaccine RA wild-type (Singh et al., 1998, Infect. Immun. 66, 3447-3448). Studies on immunization of svidetel who are also on the essential contribution of LF/EF in immune defense. Based on these results the authors present invention has created a recombinant vaccine against anthrax, which includes mutants of all three of the anthrax toxins.

Recombinant vaccine based on the anthrax toxin created by the authors of the present invention has the following advantages:

1. The described method does not require the use of culturesB. anthracis(at any stage). Therefore, this method is safe, cost-effective and does not require the use of sophisticated equipment.

2. Created by the authors of the present invention, the vaccine has a clearly defined structure and therefore does not have any variability from batch to batch.

3. In the here described invention uses a dedicated cleaning anthrax protein-toxin. As a result of the anthrax vaccine of the second generation is not reactive and may not cause any side effect unlike previous vaccines.

4. In addition, the present invention includes a non-toxic mutant proteins, which when introduced (separately or in combination) do not cause any toxicogenetic or pathogenicity, which is due to the use of existing vaccines.

5. Therefore, the invention described here is safe and suitable for use on the animal/human.

YOUNG IS a great SUMMARY of EXPERIMENTAL METHODS

Site-directed mutagenesis protein toxins anthrax

For introduction of the desired mutations in the protein-toxins of anthrax use of complementary mutagenic primers (reference Table 1) to amplify the genes of wild-type toxins anthrax (RA or LF or EF). In PCR reaction using high-precision DNA polymerasePfu. Within a few cycles on the amplifier amplified both full-chain plasmid DNA, receiving mutant plasmid with staggered gaps on opposite chains (Figure 2). The results of amplification controlled by agarose gel electrophoresis of the obtained PCR products. The amplification product is treated withDpn,I, which specifically cleaves fully methylated G^me6ATC-sequence. This cleavage reaction is carried out in a 20 µl reaction volume with 100 ng of the amplified product, 2 ál 10XDnpI reaction buffer and 0.1 Units.Dpn,I. AfterDpn,I-processingDpn,I-resistant molecules that are rich in the required specimens, collected by transforming the DNA into an appropriate strain ofE. coli. Mutations confirmed by sequencing of the above structures using set for circular DNA sequencing (Perkin Elmer).

Expression and secretion clearance of mutant proteins, toxins Siberian language is s

Proven designs transform the expression in the strains ofE. coliexpressing T5-RNA polymerase. Transformed cells are grown on the environment Luria (LB)containing 100 μg/ml ampicillin and 25 μg/ml kanamycin, at 37°With, before OP₆₀₀0,8. Then carry out the induction with 0.5 mm IPTG and continue incubation at 37°C for 3-4 hours. Cells are harvested by centrifugation at 6000 rpm for 10 minutes. Then centrifuged cells are lysed. Protein profile analyzed by SDS-electrophoresis in SDS page and Western blotting. Mutant RA secrete proteins purification using metal-chelate Ni-NTA-chromatography on affinity and other chromatographic methods (Kumar P., et al., 2001, Infect. Immun., 69, 6532-6536; Gupta P., et al., 1998, Infect. Immun., 66, 862-865; Gupta P., et al., 1999, Protein Expr. Purif. 16, 369-376). Selected clearance of mutant proteins analyzed by SDS-electrophoresis in SDS page and Western blotting and evaluated using the method of Bradford. To store the selected cleanup proteins cialiswhat against 50 mm HEPES and stored in the form of aliquots at -70°C.

Cell culture

Macrophagecolony cell line J774A.1 support in medium RPMI 1640 containing 10% V / V heat inactivated FCS, 25 mm HEPES, 100 Units/ml penicillin and 200 μg/ml streptomycin in a humid environment with 5% CO₂at 37°C.

Cho cells under eribaum in EMEM medium, containing 10% V / V heat inactivated FCS, 25 mm HEPES, 100 Units/ml penicillin and 200 μg/ml streptomycin in a humid environment with 5% CO₂at 37°C.

To study the biological activity of RA wild-type or mutant proteins with different concentrations of these proteins add together with LF (mcg/ml) to cells J774A.1 placed in 96-well plates. Incubated for 3 hours at 37°With, and then determine viable cells (Bhatnagar et al., 1989, Infect. Immun., 57, 2107-2114) using dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) (Bhatnagar R., et al., 1999, Cell Signal., 11, 111-116). MTT dissolved in RPMI, poured in each well to a final concentration of 0.5 mg/ml and incubated for a further 45 min at 37°for absorption and oxidation of the dye in viable cells. This environment will change by 0.5% (wt./about.) nitrilotriacetate (SDS), 25 mm HCl in 90% isopropyl alcohol and the plate is shaken on the device for shaking and mixing. Read the absorbance at 540 nm using a spectrophotometer for microplates (BIORAD).

In a similar way to study the biological activity of LF wild-type or mutant proteins with different concentrations of these proteins add together with RA (1 μg/ml) to cells J774A.1 placed in 96-well plates. Incubated for 3 hours at 37°C, after which determine life is sposobnye cells using MTT dye, as described in detail above.

To study the biological activity of EF wild-type or mutant proteins with different concentrations of these proteins add together with RA (1 μg/ml) to Cho cells are placed in 96-well plates. Incubated for 3 hours at 37°C, after which the microscope is used to determine the lengthening of the cell. Determine the increased level of camp in cells during the processing of the toxin (Kumar P., et al., 2001, Infect. Immun., 69, 6532-6536) using a cAMP EIA kit from Amersham Pharmacia.

In the following experiments investigate how mutations affect the biological activity of the mutant protein toxins of anthrax.

Binding of RA receptors on the cell surface

The J774A.1 cells grown in 24-hole tablets to merge before incubation with 1 µg/ml RA wild-type or mutant proteins at 4°C for 2 hours. Then the grown cells are washed with cold RPMI medium, dissolved in SDS-lyse buffer and subjected to SDS-electroblotting in PAG. The resulting blot shown using antibodies to RA for analysis of binding of RA wild-type or mutant protein to receptors on the cell surface.

Proteolytic cleavage of RA and mutant proteins in solution

RA wild-type and mutant proteins are tested for sensitivity to cleavage by trypsin. Protein (1.0 mg/ml) cu is irout with 1 μg/ml trypsin for 30 minutes at room temperature in 25 mm HERES, 1 mm CaCl₂, 0.5 mm EDTA pH 7.5. The cleavage reaction is stopped by adding PMSF at a concentration of 1 mm. For SDS-electrophoresis in SDS page the samples boiled for 5 minutes in SDS-sample buffer and separated SDS-electrophoresis in 12%SDS page.

Linking RA LF on the surface of cells

Cells J774A.1 twice washed with RPMI and then incubated with 1 μg/ml RA wild-type or mutant protein at 4°C for 3 hours. Then these cells are washed with cold RPMI to remove unbound protein. Next, the washed cells are incubated with LF (1.0 microgram/ml) for 3 hours and then washed with cold RPMI to remove unbound LF. These cells are dissolved in SDS-lyse buffer and subjected to SDS-electrophoresis in SDS page for electromotively. The resulting blot shown using antibodies to LF for analysis of binding of RA wild-type or mutant protein with LF.

Oligomerization of RA in solution

When the proteolytic cleavage of RA oligomerized with the formation of heptameron. To study the ability of RA wild-type and mutant proteins to form oligomers these proteins (1 mg/ml) digested with trypsin for 30 minutes at 25°C. Split samples are transferred into pH 5.0 after adding 1 M Tris pH 5.0 to a final concentration of 100 mm and boiled for 5 minutes in SDS-sample buffer (0,0625 M Tris-Cl, 125% SDS, 2,5% β-mercaptoethanol and 5% glycerol, pH 6.8) before loading in 3-12%gradient gel. Exercise silver staining for detection of the formation of oligomers.

Binding of LF/EF RA associated with the cell surface

The J774A.1 cells washed with cold RPMI and then incubated with 1 μg/ml RA wild type at 4°C for 3 hours. These cells are again washed with cold RPMI to remove unbound protein. Then add LF/EF wild-type or mutant proteins (1.0 microgram/ml) and continue incubation for 3 hours. Then incubated the cells washed with cold RPMI to remove unbound LF/EF. Later, these cells are dissolved in SDS-lyse buffer and subjected to SDS-electrophoresis in SDS page for electromotively. The resulting blot shown using antibodies to LF/EF to study the binding of LF/EF RA, contacting the cell surface.

TABLE 1

TABLE 2: CHARACTERISTICS of MUTANTS

1. Recombinant DNA construct for expression of the protein toxin of Bacillus anthracis, including:

a) expressing vector,

b) a DNA fragment comprising the genes encoding the mutant protects the antigen (PA) or mutant lethal factor (LF), or mutant swelling factor (EF), where

i) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Phe202 replaced by alanine,

ii) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Leu203 replaced by alanine,

iii) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Rho replaced by alanine,

iv) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Ile207 replaced by alanine,

v) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Rho, Thr and Phe236 replaced by alanine,

vi) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Phe552 replaced by alanine,

vi) the specified fragment of DNA is a gene encoding the RA, in the amino acid sequence of which Ile574 replaced by alanine,

viii) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Phe552 and Phe554 replaced by alanine,

ix) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Ile562 and Ile574 replaced by alanine,

x) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Leu566 and Ile574 replaced by alanine,

xi) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Phe552 and Phe554, Ile562, Leu566 and Ile574 replaced by alanine,

xii) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Phe427 replaced by alanine,

xiii) the specified fragment of DNA is a gene that encodes a RA with a deletion at residue Asp 425 in amino acid sequence,

xiv) the specified fragment of DNA is a gene that encodes a RA-deletion in residue Phe 427 in amino acid sequence,

xv) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Thr replaced by alanine,

xvi) specified DNA fragment of t is made by a gene encoding the RA, in the amino acid sequence of which Leu352 replaced by alanine,

xvii) the specified fragment of DNA is a gene that encodes a RA amino acid sequence which Trp346, Met350 and Leu352 replaced by alanine,

xviii) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which Tyr148 replaced by alanine,

xix) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which Tyr 149 replaced by alanine,

XX) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which Ile 151 is replaced by alanine,

xxi) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which Lys 153 replaced by alanine,

xxii) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which Asp 187 replaced by alanine,

xxiii) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which 190 Phe is replaced by alanine,

xxiv) the specified fragment of DNA is a gene that encodes a LF, in the amino acid sequence of which Asp 187, Leu 188, Leu 189 and 190 Phe is replaced by alanine,

xxv) the specified DNA fragment represented yet a gene encoding EF amino acid sequence which Tyr 137 replaced by alanine,

xxvi) the specified fragment of DNA is a gene encoding EF amino acid sequence which Tyr 138 is replaced by alanine,

xxvii) the specified fragment of DNA is a gene encoding EF, in the amino acid sequence of which 140 Ile replaced by alanine,

xxviii) the specified fragment of DNA is a gene encoding EF amino acid sequence which Lys 142 is replaced by alanine.

2. The recombinant DNA construct according to claim 1, in which the specified expression vector is a prokaryotic vector.

3. The recombinant DNA construct according to claim 2, in which the specified prokaryotic vector represents PQE 30.

4. The recombinant DNA construct according to claim 3, in which the specified expression vector contains T5-promoter and 6X his-tag tag.

5. The method of obtaining mutagenically proteins PA, LF and EF toxin of anthrax, where the method includes the following stages:

a) obtaining a mutant gene encoding a mutant protein toxins PA, LF and EF, using different complementary mutagenic primers for PCR

b) processing the indicated mutant PCR product together with native matrix obtained in stage (a), the enzyme that is assalaam this native matrix specified PCR product,

c) transformation of the indicated mutant product of prokaryotic host,

d) isolation of recombinant DNA constructs described in claims 1 to 4, from a transformed prokaryotic host and confirm the presence of the desired mutation,

e) transformation confirmed mutant constructs in the corresponding prokaryotic host for expression of mutant protein, and

f) purification of the specified expressing the mutant protein.

6. The method according to claim 5, in which the prokaryotic host is E. coli strain.

7. The method according to claim 5, wherein said enzyme is a DpnI enzyme that specifically cleaves fully methylated G^me6ATC-sequence.

8. The method according to claim 5, in which the specified clone genes in the expression vector PQE containing T5-promoters and 6X his-tag tag.

9. The method according to claim 5, in which the specified cleaning carried out using Ni-NTA chromatography and/or other chromatographic techniques.

10. The method according to claim 5, in which

a) mutations affect the first domain of RA remains 202, 203 and 205,

b) mutations affect the third domain of RA remains 552, 574, 552+554,562+574,566+574 and 552+554+562+566+574,

c) mutations affect the second domain of RA remains 425 and 427 loop 4 domain 2,

(d) mutations affect the second d is Myung-RA on the remaining 346, 352 and 346+350+352 in the loop 3 domain 2,

(e) mutations affect the 1st LF domain at residues 148, 149, 151, 153, and 187,190 187+188+189+190,

f) mutations affect the 1st 250 residues EF.

11. Mutant protein toxin of Bacillus anthracis, has immunogenic properties expressed by the recombinant DNA construct according to claim 1 and obtained by the method according to claim 5.

12. Protivoseborainey vaccine comprising a mutant protein toxin of Bacillus anthracis, selected from RA mutant or mutant LF, or mutant EF, or combinations thereof.

13. Protivoseborainey vaccine comprising one or more mutant protein toxins of Bacillus anthracis, characterized in claim 11, in combination with a protein toxin of Bacillus anthracis PA, LF or EF wild-type.