Immunomimetic peptide of chemical cancerogens with specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene

FIELD: medicine.

SUBSTANCE: peptide is obtained from phage peptide library including set of static peptides with length of 12 aminoacid residues, by affine selection of phage clones containing peptide capable of specific linking to antibodies of benzo[α]pyrene and benzo[α]anthracene. Peptide displays specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene and features molecular weight of 1.3 kDa and registered aminoacid sequence LHLPHHDGVGWG encoded by nucleotide sequence SEQ ID NO:1.

EFFECT: application in medicine as a base for peptide medicine development for immunologic prevention of malignant tumours of humans.

4 dwg, 3 ex

 

The invention relates to biotechnology, genetic and protein engineering, specifically to the peptide with the ability to interact specifically with antibodies against benzo[α]pyrene and Benz[α]anthracene.

Chemical carcinogens, being of low molecular weight substances, not able to induce an immune response. At the same time, immunization of animals with conjugates of the protein with the carcinogen as hapten leads to the appearance of specific antibodies and a significant inhibition of carcinogenesis [1, 2]. However, the presence of carcinogenic substances in the composition of such conjugates excludes their use as vaccines, as a carcinogen retains the ability to induce tumors.

The most closest to the claimed peptide-immunometric by function - prototype - is a monoclonal antiidiotypic antibody with internal immunological" benzo[α]pyrene, with the ability to interact specifically with antibodies against benzo[α]pyrene [3]. Known antibody was prepared as follows. Conjugate benzo[α]pyrene were immunized rabbit and received 1 antibodies specific to benzo[α]pyrene. Antibodies 1 were immunized mice received hybridoma producing monoclonal antiidiotypic 2 antibodies, specific antibodies 1. Monoclonal antiadiotipiceskih 2 antibodies immunodeficiency who were seravalli rats then they were introduced benzo[α]pyrene. As a result, the rats were observed inhibition of tumors induced by benzo[α]pyrene, these tumors grew slower, and the life expectancy of rats was increased compared with control (without prior immunization with monoclonal antiadiotipiceskih antibodies 2). Thus was shown the principal possibility of inhibition of carcinogenesis by immunization of animal protein molecules that mimic the immunogenic properties of conjugates of carcinogen-protein and does not contain carcinogenic as hapten.

However antiidiotypic antibodies have drawbacks that prevent their use as anti-carcinogenic vaccines, and hybridoma technology has limitations for its use in order to receive any vaccines. When immunization alien immunoglobulins can be formed side individule and antihistimine antibodies that can lead to allergic and autoimmune complications, and hybridoma technology is characterized by high complexity and costs.

One way of overcoming these problems is to use peptides that mimic the immunogenic properties of conjugates of carcinogen-protein. A fundamentally new approach to the creation of anticarcinogenic vaccines is to use f the debt peptide libraries. It is possible to obtain peptides-immunogenetic chemical carcinogens that do not contain any carcinogen that can induce a tumor or related antigenic determinants that can cause adverse pathological complications.

An object of the invention is to obtain peptide-immunogenetic chemical carcinogens, specifically interacting with antibodies against benzo[α]pyrene and Benz[α]anthracene, and the determination of its amino acid sequence.

The inventive peptide derived from ragovoy peptide library containing a set of statistical peptides with a length of 12 amino acid residues (S.A.), by affinity selection of those phage clones that contain the peptide can specifically bind with antibodies against benzo[α]pyrene and Benz[α]anthracene. The peptide library displayed on the surface of phages in the composition of the minor envelope protein pIII [4].

Amino acid composition of the peptide-immunogenetic, located on the surface of phage particles was determined by sequencing of phage DNA using a modified method of Sanger [5] encodes a peptide region. The inventive peptide has a molecular mass of about 1.3 kDa and amino acid sequence LHLPHHDGVGWG encoded by the nucleotide sequence of SEQ ID NO: 1, are presented in figure 1.

With electrovanne of ragovoy peptide library peptide with the amino acid sequence LHLPHHDGVGWG has the ability to specifically interact with antibodies against benzo[α]pyrene and Benz[α]anthracene in vitro, and to induce the formation of antibodies against benzo[α]pyrene in vivo.

Thus, for the first time obtained a peptide specifically interacts with antibodies against benzo[α]pyrene and Benz[α]anthracene in vitro and is able to induce the formation of antibodies against benzo[α]pyrene in vivo.

The invention is illustrated by the following specific examples of its implementation.

Example 1. Way for affinity selection of peptides that specifically interact with antibodies against benzo[α]pyrene and Benz[α]anthracene.

The conjugates of benzo[α]pyrene and Benz[α]anthracene with BSA and the yeast gexokinaza synthesized by the method of covalent binding of the aldehyde group of the hapten with the amine groups of carrier protein [6].

To obtain monoclonal antibodies against benzo[α]pyrene mice of BALB/c mice subjected to immunization with a conjugate of benzo[α]pyrene-BSA. Hybridoma get through merge cells, mouse myeloma Sp2/0 and immune splenocytes of the mouse, according to the Protocol described by Kohler and Milstein [7]. The obtained monoclonal antibodies cross-react with the conjugates of benzo[α]pyrene-BSA and Benz[α]anthracene-BSA.

To obtain polyclonal antibodies rabbits subjected to immunization with a conjugate of benzo[α]pyrene-BSA. From produce antisera monospecific antibodies against benzo[α]pyrene and Benz[α]anthracene using affinity chromatography on columns α]pyrene-glucokinase-Sepharose 4B and Benz[α]anthracene-glucokinase-Sepharose 4V.

The obtained monoclonal antibodies against benzo[α]pyrene and Benz[α]anthracene dissolved in phosphate-buffered saline, containing 137 mm NaCl; of 2.68 mm KCl; 8,1 mm Na2HPO4·12H2O; 1,47 mm KN2PO4to a final concentration of 10 μg/ml of the resulting solution of antibodies sensibiliser sterile polystyrene Petri dishes. The nonspecific binding sites blocked with 0.5%solution of BSA in phosphate-buffered saline. The original phage peptide library, diluted in phosphate-buffered saline to a final concentration of 2·1011CFU/ml (kolonialismus units/ml), incubated with the immobilized antibody for one hour at room temperature. Then Petri dishes washed with phosphate-saline buffer containing Tween 20. The elution of bound peroxidase antibody bacteriophages carried out with a solution of glycine buffer (0.2 M Glycine-HCl pH 2.2, 0.1% BSA) for 10 min with gentle rocking. Elyuirovaniya fraction is neutralized with 150 μl of 1 M Tris-HCl pH 9.1 and determine the titer of the phage. The resulting population of bacteriophages containing contacting a monoclonal antibody against benzo[α]pyrene and Benz[α]anthracene peptides, amplified by infection of cells of E. coli JM103 [10]. Isolated phages and spend the second and third rounds of affinity selection as described above.

After the third round of selection receive individual phage to onii, which is used for producing single-stranded DNA and phage ELISA. Sequencing of the DNA insert carried out according to the modified method of Sanger [5].

Example 2. The study of peptide-immunogenetic-specific binding of antibodies against benzo[α]pyrene and Benz[α]anthracene in vitro.

To characterize the ability of peptide-immunogenetic specifically bind to a monoclonal antibody against benzo[α]pyrene and Benz[α]anthracene spend ELISA with serial dilution of phage exhibiting on its surface a peptide-immunogenetic from 1011up to 106CFU/well. In wells of polystyrene 96-well plates absorb monoclonal antibodies against benzo[α]pyrene and Benz[α]anthracene, diluted to a final concentration of 5 μg/ml in phosphate-buffered saline. The nonspecific binding sites blocked with 0.5%solution of BSA in phosphate-buffered saline. After that, the wells are incubated with the analyzed clones of phage diluted in phosphate-buffered saline containing 0.05% Tween 20, 0.5% BSA. Bound peroxidase bacteriophages, exhibiting on its surface a peptide-immunogenetic, reveal peroxidase-conjugated rabbit antibodies against total proteins M13mp8. The absorption solution in the wells was measured at a wavelength of 350 nm. To control for nonspecific binding of the use of amplified phage M13m8. The results of the specific binding of peptide-immunogenetic in the composition of the pIII protein of the phage with monoclonal antibodies against benzo[α]pyrene and Benz[α]anthracene presented in figure 2, where the abscissa axis indicate the dilution of the phage, and the ordinate axis is the optical density (OD 350); curve 1 - binding M13mp8 with monoclonal antibodies against benzo[α]pyrene and Benz[α]anthracene; curve 2 - binding peptide-immunogenetic in the composition of the pIII protein of the phage with monoclonal antibodies against benzo[α]pyrene and Benz[α]anthracene.

Using Western blot analysis examined the binding of peptide-immunogenetic in the composition of the pIII protein onselectionchange clone with monoclonal and monospecific polyclonal antibodies against benzo[α]pyrene and Benz[α]anthracene and monospecific polyclonal antibodies against benzo[α]anthracene. After electrophoretic separation of recombinant and wild phage into its constituent proteins in 15%SDS page with sodium dodecyl sulfate spend their transfer to nitrocellulose membrane, which was then incubated with monoclonal antibodies and monospecific polyclonal antibodies against benzo[α]pyrene and Benz[α]anthracene (5 µg/ml). Contacting the target peptide antibodies is detected using peroxidase conjugates of antibodies against total IgG mouse or rabbit followed their krasivyi the m The Western blot turns specific binding of peptide-immunogenetic presented on figure 3, where I is the binding of monoclonal antibodies against benzo[α]pyrene and Benz[α]anthracene, II - the binding of monospecific polyclonal antibodies against benzo[α]pyrene, III - the binding of monospecific polyclonal antibodies against benzo[α]anthracene; 0 - linking pIII protein of native phage M13mp8 with antibodies against benzo[α]pyrene and Benz[α]anthracene; 1 - binding peptide-immunogenetic in the composition of the pIII protein with antibodies against benzo[α]pyrene and Benz[α]anthracene.

Example 3. The study of the obtained peptide-immunogenetic on the ability to induce the formation of antibodies against benzo[α]pyrene in vivo.

Three groups of ICR mice subjected to immunization intraperitoneally three different antigens (conjugate benzo[α]pyrene-BSA, native phage M13mp8 and peptide-immunogenetics in the composition of the pIII protein of the phage) three times every 2 weeks. The first immunization hold antigen in the mixture with complete adjuvant-blockers, subsequent antigen with incomplete adjuvant's adjuvant. The serum of the mice tested in ELISA for the presence of specific antibodies in 3 days after the final immunization. For this purpose, the wells of polystyrene tablets sensibiliser conjugate benzo[α]pyrene-BSA (5 μg/ml)diluted in podslashennoyi water. Block and incu is irout with samples of blood sera of immunized mice. Bound peroxidase antibodies reveal peroxidase-conjugated rabbit antibodies against total mouse Ig. The results of ELISA of serum antibodies to benzo[α]pyrene are presented in figure 4, where the abscissa axis is the serum dilution, and the ordinate axis is the optical density (OD 350); curve 1 - immunization native phage M13mp8 (negative control); curve 2 - immunization with peptide-immunogenetics in the composition of the pIII protein of the phage; curve 3 - immunization benzo[α]pyrene-BSA (positive control).

Thus, the specific peptide-immunogenetic chemical carcinogens, specifically interacting with antibodies against benzo[α]pyrene and Benz[α]anthracene and is able to induce the formation of antibodies against benzo[α]pyrene after immunization in vivo. Such peptide-immunogenetic can be successfully used as a hapten-specific component in the method of immunoassay of antibodies specific to chemical carcinogens. Replacement conjugate carcinogen-protein to the peptide-immunogenetic eliminates carcinogenic potential threat in the production and use of analytical test systems. On the basis of peptide immunogenetical you can also create complexes with known immunomodulatory agents (adjuvants)that have been approved for clinical use in order to enhance the immune response against chemical Kahn is erogeny (formation of antibodies against chemical carcinogens), i.e. for prophylaxis of malignant tumors in humans.

Sources of information

1. Curtis G.L., Ryan W.Z., Stenback F. Antibody stimulation of benzo[α]pyrene carcinogenesis. Cancer Lett. 1978, v.4: p.223-228.

2. Moolten F.L., Schreiber Century, Rizzone A. Protection of mice against 7.12-dimethylbenz(a)antracene-induced skin tumors by immunization with a fluorinated asnalog of carcinogen. Cancer Res. 1981, v.41: p.452-459.

3. Chagnaud J.L., Faiderbe S., Geffard M. Effects of a monoclonal anti-idiotypie antybody, internal image of benzo(α)pyrene, on rat sarcomas. Acad. Sci. Paris, Sciences de la vie. 1993, v.316: p.1266-1269.

4. Scott J.K., Smith G.P. Searching for peptide ligands with an an epitope library. Science. 1990, v.249: p.386-390.

5. Kretz, K., W. Callen, V. Hedden Cycle sequencing. Methods of Enzimology. V.154: p.527-536.

6. RF patent 2141114 "a method of obtaining a conjugate of the hapten-protein", CL G01N 33/50, authors: Kostenko M.V., Glushkov A., publ. 10.11.1999.

7. Kohler g, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. Nature. 256: p.495-497.

8. Glushkov A.N., Kostyanko M.V., Anosova T.P., Polenok E.G., Mun S.A., Anosov M.P., Cherno S.V. Immunological images of polycyclic aromatic hydrocarbons. Experimental neology. 2006. V.28: p.93-176.

9. Brian K.Kay, Jeremy Kasanov, Montarop Yamabhai. Screening phage-displayed combinatorial peptide libraries. Methods. 2001. V.24: p.240-246.

Peptide-immunogenetic chemical carcinogens derived from ragovoy peptide library having a molecular weight of about 1.3 kDa, with the ability to interact specifically with antibodies against benzo[α]pyrene and Benz[α]anthracene having the amino acid sequence LHLPHHDGVGWG encoded by the nucleotide sequence of SEQ ID NO: 1.



 

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