Method for obtaining preparation that contains the complex of heat shock proteins 70, 90, 96 associated with antigenous peptides

FIELD: biopharmacology, preparative biochemistry and medicine.

SUBSTANCE: the method deals with homogenizing mammalian tissues and centrifuging. It is necessary to add ammonium sulfate to supernatant that contains the complex of heat shock proteins (HSP) up to 35-65% saturation, the residue developed should be dissolved in phosphate buffer at pH 7.2 followed by chromatography upon a column with sephadex G-50, then obtained protein fraction should be exposed to the action of chromatography upon heparin-sepharose. Fractions that contain TSP 70, 90, 96 the content of which was detected with the help of corresponding antibodies should be united, concentrated and salted out due to ultrafiltration. The innovation provides more simplified and shortened terms for implementing the method without applying expensive equipment. The complexes obtained should be applied in immunotherapy of oncological diseases.

EFFECT: higher efficiency.

2 dwg, 1 tbl

 

The invention relates to the field of biopharmacology, preparative biochemistry, medicine and can be used for selection of heat shock proteins associated with antigenic peptides.

The level of technology

It is known that tumors contain antigens capable of inducing an immune response in the body [1]. Antigens are some (few) of tumors in animals and humans isolated and characterized [2]. Tumor antigens used for immunization capable of inducing an antitumor immune response that is specific to the tumor antigens which were immunized, which is associated with specific antigenic composition of each individual tumor [3]. This can be attributed to genetic differences between proteins from different individuals, and a variety of mutations that occur during neoplastic transformation of cells. Isolation and identification of antigens specific for tumor-specific immune response is a separate and complex task. Such a task at the current level cannot be solved within the framework of treatment for an individual patient. However, there is another approach to solve this problem. Induced under conditions of stress proteins (stress proteins), or heat shock proteins (HSP), or chaperones are synthesized in the tumor tissue more than the s with normal tissues. It has been shown that HSP tumor tissue ecovalence bind peptide antigens of this tissue [3]. Thus, it is not required for the induction of specific immune response to isolate and identify the specific antigens of the tumor, it is enough to distinguish from tumor tissue HSP assosiated with antigens of this fabric. It should be emphasized that HSP, through the interaction with specific receptors, stimulate the T-cell-mediated immunity, which is especially important for antitumor immunity [4, 5]. It is possible to conclude that the work in this area seems to be quite reasonable and promising. Such assosiated with antigenic peptides, proteins isolated from tumor tissue, and positive results were obtained from a regression of tumors with the introduction of such autologous vaccine. This applies to families of proteins HSP70, BTS, BTS and others [5, 6, 7]. Discovered that certain HSP form complexes with certain proteins and peptides, i.e, it is likely that the antigens to bind to the HSP70, are not associated with BTS. In addition, the activity of one HSP in respect of the specific binding protein is increased in the presence of another HSP [6, 7].

The prior art work on the methods of allocation of HSP-peptide complexes. One of them (United States Patent 5997837. Srivastava, December 7, 1999) protected method to obtain Bethshemesh complexes from tumor mammalian cells or from cells infected by viruses, bacteria or other infectious agents in the absence of ATP. Note that the selection of one individual HSP is a disadvantage of this patent is based on the task the use of exogenous introduction of this protein to enhance specific antitumor immune response. This reduces the diversity of antigens involved in the immune response of the organism, which follows from the above, the specificity of the formation of complexes of HSP-antigenic peptide. The allocation of HSP70-peptide complexes from tumor cells of mammals, proposed by the author, includes the following stages: tissue homogenization, centrifugation at 1000000g, chromatography on a column with concanavalin-And separate (ConA of sepharose), which is secreted HSP70-peptide complexes are not associated. This is not bound peroxidase material is subjected to dialysis and further chromatography using a MONOQ and FPLC system. At the final step, the material Myslivets (50-70% saturation with ammonium sulfate (NH4)2SO4). Before using derived HSP70-peptide complexes they should be in any way transferred to a solution suitable for immunization. The advantage of the proposed method is the ability to obtain a homogeneous HSP70-peptide complexes, which eliminates the risk of introduction is ecipient transforming or immunosuppressive agents, such as transformed DNA or transforming growth factor - β.

The other patent - United States Patent: 5750119 "Immunotherapeutic stress protein-peptide complexes against cancer, Srivastava, May 12, 1998, which describes a method of separation of HSP70-, BTS and BTS-peptide complexes. The selection includes the destruction of cells by exposure to ultrasound and (or) mechanical homogenization in lytic buffer: 5 mm Na-phosphate buffer, pH 7.0, 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 1 mm PMSF(phenylmethanesulfonyl). The lysate was centrifuged at 1000g for 10 min and the resulting supernatant was again centrifuged at 100000g 90 minutes the Supernatant was mixed with ConA-separate, mixed with sorbent 2-3 hours, not contacting the sorbent material was separated by filtration and dialyzed against buffer (10 mm Tris-acetate, pH 7.5, 20 mm NaCl, 0.1 mm EDTA-ethylenediaminetetraacetic acid, 15 mm 2-mercaptoethanol)required for chromatography using a MONOQ and FPLC system. HSP70-peptide complexes were suirable when 20-500 mm concentration of NaCl in the same buffer. Collected immunoreactive with antibodies to HSP70) fractions were vysalivanie at 50-70% saturation (NH4)2SO4, was centrifuged at 17000 rpm. in minutes, freed from (NH4)2SO4on a column of Sephadex G25.

Selection BTS-peptide complexes was similar, only with chromatography using the-W and MONOQ FPLC system BTS-peptide complexes were suirable at 200-600 mm NaCl concentration.

Selection BTS-peptide complexes was similar to phase chromatography on ConA-sepharose. BTS-peptide complexes were barbirollis on the ConA sepharose, sorbent filled column was washed her lytic buffer was kept between 10% α-methylmagnesium and suirable BTS-peptide complexes α-methylmagnesium. Then the material was subjected to chromatography using a MONOQ and FPLC system. BTS-peptide complexes were suirable at 400-550 mm NaCl concentration. This patent is accepted as a prototype.

This method provide a homogeneous HSP70-, BTS and BTS-peptide complexes revealed by means of appropriate analytical methods: polyacrylamide gel electrophoresis (SDS page) and Western blot turns. However, the prototype method is time consuming due to mnogostadiinost selection HSP, and also includes the use of expensive equipment and reagents.

The technical result of the claimed method is a very fast (comparable with the time of treatment), including the minimum possible number of stages, without the use of expensive equipment and reagents selection of HSP70-, BTS and BTS-peptide complexes.

HSP70-, BTS and BTS-peptide complexes obtained the claimed method may be used in immunate the FIPA cancer.

Disclosure of inventions

The selection of HSP70-, BTS and BTS-peptide complexes include: tissue homogenization, the homogenate centrifugation, salting out with 35-65% saturated (NH4)2SO4followed by centrifugation or filtration, desalting on a column of Sephadex G50, chromatography on a column of heparin-separate, concentration and desalting of preparation by ultrafiltration.

Declare to us a way of separating HSP 70-, HSP 90 and HSP 96-peptide complexes allows you to select HSP 70-, HSP 90 and HSP 96-peptide complexes in a single procedure.

An example of carrying out the invention

A fragment of the tumor tissue of breast cancer patient weight of 1 g was homogenized using a disperser Miccra D-1 "Roth" with nozzle DS-8/P (Nashim consult) in 4 ml of buffer (10 mm NaHCO3, pH 7.0, 2 mm EDTA and 2 mm PMSF) at 4°C. the Homogenate was centrifuged at 35000g and 4°C for 25 min, adosados collected and HSP-peptide complexes besieged by salting out (NH4)2SO4. Collected sediment (centrifugation or filtration), formed by 35-65% saturated (NH4)2SO4and dissolved it in phosphate buffer (10 mm sodium phosphate; 2 mm MgCl2; 0,5 mm dithiothreitol; pH of 7.2). Next conducted chromatography dissolved residue on a column of Sephadex G-50 fine, equilibrated with the same buffer. When the volume was separated protein fraction from the material, molecular weight of less than 50 kDa and translated it into the buffer needed to affinity chromatography on heparin-sepharose (heparin-sepharose 6 Fast Flow, Amersham Biosciences). The protein fraction obtained after chromatography on a column of Sephadex G-50 fine, made in a column of heparin-separate. Then sequentially washed column: phosphate buffer, pH of 7.2, 2 mm MgCl2, 0,5 mm dithiotreitol; the same buffer containing 0.05 M NaCl and 0.05-0.7 M NaCl in the same buffer. Combined fractions, eluruumina phosphate buffer, pH of 7.2, 2 mm MgCl2, 0,5 mm dithiothreitol, 0,05M NaCl (containing HSP70 and BTS-peptide complexes) and eluruumina at 0.4-0.7 M NaCl concentration, which contain BTS. The combined fractions were concentrated and absoluely using ultrafiltration. Detection of HSP70, BTS and BTS in the homogenate and fractions after each stage of selection was performed using the respective antibodies by the method of the dot-blot. The relative content of each HSP was determined using the respective antibodies by serial dilution, assuming a unit maximum breeding, which is determined corresponding to the HSP, the method of the dot-blot. The degree of purification of HSP70, BTS and BTS in the fractions and the final product was analyzed by electrophoresis in SDS page under denaturing conditions in the presence of Na dodecyl sulfate (Ds-Na-PAG). For proof will naturally the tion of conformity protein bands allocated HSP carried out the Western blot turns. The amount of protein was assessed spectrophotometrically.

From 1 g of tumor breast tissue obtained at 2.59 mg of purified HSP. As can be seen in figure 1, the selected proteins possess a sufficient degree of purity (5 track): impurity proteins not detected during electrophoresis in Ds-Na-PAG, if introduced into the hole 7 μg of protein and the color silver. Main strips belong HSP70, BTS IBTS, as evidenced by reaction with the corresponding antibodies in the Western blot turns (Figure 2). A minor band with a molecular mass of less than 66 kDa, belongs to the peptide derivatives of HSP70 and BTS (detected and when the Western blot turns), i.e. as an impurity.

The resulting preparation HSP investigated for the presence of cytotoxic and proliferative activity using cell lines: NGOC (acoustic Gusarova node rats. Institute of human morphology and animals, RAMS), MCF7 (breast adenocarcinoma human. Institute of Cytology, Russian Academy of Sciences, St. Petersburg), Hela-Gal (epithelial carcinoma of the cervix person. National Institute of Health, Bethesda, USA), K-562 (suspension culture cells erythromyeloleukemia line) and FAC (embryonic fibroblasts. Institute of Poliomyelitis, RAMS). Cells were cultured in 48-hole tablets at 37°C in an atmosphere of 5% CO2in medium RPMI1640 (NGOK and K-562), DMEM (Hela-Gal), Needle-MEM (MCF7 and FEC), 2 mm L-glutamine and 10% fetal calf serum, pH 7.4, in the presence of antibiotics (penicillin and streptomycin). The granulosa cells to the state of a dense monolayer (24-48 hours). The determination of the cytotoxic activity of the HSP preparation was performed by adding to the cells of 0.5-40 μg of drug HSP. After 2448 hours was determined the cytotoxicity of the drug, using the MTT-test. Cell survival was assessed as a percentage of the corresponding control (survival in a nutrient medium without study drug HSP). As follows from the data of table 1, the HSP preparation does not possess cytotoxic activity, not has it also and proliferative activity (table 1). Test for stimulation of cell proliferation was performed by adding to the cells after 20 hours of incubation with the drug HSP solution With14-thymidine (specific radioactivity 56 MCI/mm)containing 1 MCI of labeled thymidine. Time tagging was 5 hours. Compared the incorporation of labeled thymidine into experimental (drug HSP) and control samples. The results were expressed in counts/min for 106cells and in % of control. Similar results were obtained with all used cell cultures.

Thus, the claimed method allows to obtain a set of heat shock proteins 70,90 and 96 with antigenic peptides of high purity and the lowest possible cost.

is the GLA. 1.

The determination of the cytotoxic activity of the protein preparation containing the HSP complex, and the influence of this drug on DNA synthesis by incubation for 24 hours with cells K-562.
No.µg protein in the hole (0.3 ml medium)The number of live cells % of controlThe inclusion of14-thymidine, % of control*)
11,598100
23,0102114
37,5102104
*) Enable14-thymidine in control is 10290 pulse/min to 106cells.

LITERATURE

1. Kenneth O. Lloid K.O. Tumor antigens known to be immunogenic in man. In: Specific immunotherapy of cancer with vaccines, Ann. of the New York Academy of Sciences, New York 1993, V.690, pp.50-58. Ed. J-C.Bystrin, S.Ferrone, Ph.Livingston.

2. Real F.X., Mattes M.J., A.N. Houghton, H.F. Oettgen, Lloid K.O., Old L.J. Classi (unique) tumor antigens of human melanoma. Identification of a 90000 dalton cell surface glycoprotein by autologous antibody. J. Exp.Med. 1984, V.160, pp.1219-1233.

3. Heike M., Weinmann, A., Bethke K, Galle P.R. Stress protein/peptide complexes derived from autologous tumor tissue as tumor vaccines. Biochem. Pharmacology, 1999, Vol.58, pp.1381-1387.

4.Suto R., Srivastava P.K. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science, 1995. Vol.269, pp.1585-1588.

5. Janetzki, S., Blachere N.E., Srivastava P.K. Generation of tumor-specific cytotoxic T lymphocytes and memori T cells by immunization with tumor-derived heat shock protein gp96. J.Immunother. 1998, Vol.4, pp.269-77.

6. Meng Song-Dong, Song J., Rao Z., Tien, P., Gao G.F. Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides. J. of Immunol. Methods, 2002, Vol.264, pp.29-35.

7. Venoret A., Bell, G. Purification of multiple heat shock proteins from a single tumor sample. J. of Immunol. Methods, 2000. Vol. 237, pp.119-130.

8. United States Patent No. 5997837.

9. United States Patent No. 5750119.

Brief description of drawings

Figure 1.

Electrophoregram protein fractions: after homogenization (lane 1), chromatographic separation on Sephadex G50 fine (lane 2), heparin-sepharose (tracks 3-4) and the combined fractions after chromatography on heparin-sepharose, translated in saline and concentrated (track 5). The position of the protein bands was evaluated relative to the molecular weight marker (Amersham Biosciences, UK) (track 6). Electrophoresis in the Ds-Na PAG: 9% separating gel, 3% form the gel, the color silver.

Figure 2.

Study the content of the full-sized forms of HSP70 (lane 1), BTS (lane 2) and BTS (lane 3) in the pooled and concentrated fractions at the final stage of selection HSP.

Electrophoresis in the Ds-Na PAG: 9% separating gel, 3% forming a gel detection using TMB-blot".

The way to obtain a product containing a complex of heat shock proteins (HSP) 70, 90 and 96 associated with antigenic peptides, including removal of tissue from the body of a mammal, homogenization her, centrifuger the existence and separation of the supernatant, containing HSP, characterized in that to the supernatant add ammonium sulfate to 35-65% saturation, separating the precipitate, dissolve it in phosphate buffer, pH 7,2, spend chromatography on a column of Sephadex G50, equilibrated with the same buffer, collecting the protein fraction and spend chromatography on a column of heparin-separate, collect fractions containing the HSP 70, 90 and 96, combine them, and the resulting complex protein concentrate and absoluut by ultrafiltration.



 

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3 ex

FIELD: veterinary ophthalmology.

SUBSTANCE: the present innovation deals with treating infectious-inflammatory ocular diseases in cattle due to complex treatment with applying antibiotic preparation and biolan, moreover, antibiotic should be introduced into parotid lymphatic center through catheter once or twice daily at the quantity of about 8-10 ml, and biolan - at the dosage of 3-10 mg into the lower conjunctival arch of an eyeball. Such a technique due to taking into account optimal conditions of intraocular liquid circulation provides synergistic effect of the preparations introduced.

EFFECT: higher efficiency of therapy.

1 cl, 1 ex

FIELD: medicine, biomolecular pharmacology.

SUBSTANCE: invention relates to the preparation that represents the product of biological and chemical treatment of vegetable biomass or its waste by using microorganisms. The parent raw represents carbon-containing biomass of vegetable origin wherein carbohydrate moiety represents 5% of the total biomass, not less. Biomass is mixed with water with addition of lactic acid microorganisms taken as a standard ferment with the parent content 0.2-0.5% per 1 l of an aqueous mixture. The mixture is incubated at 40°C for 6 h, not less, under condition of maintaining pH value at the level 6.75 ± 0.05 by addition of ammonium hydroxide. The prepared paste-like mass can be used as the assimilable protein precursor. Also, invention involves characterization of a method for preparing the preparation that involves treatment of the parent vegetable biomass with lactic acid microorganisms and fractional addition of ammonium hydroxide to provide pH value 6.75 ± 0.05. The final product represents the mass comprising the protein assimilable precursors. Invention provides the additional source of assimilable protein precursors.

EFFECT: improved preparing method, valuable properties of preparation.

6 cl, 7 ex

FIELD: medicine, chemico-pharmaceutical industry.

SUBSTANCE: the present innovation deals with separate introduction of aqueous tincture of shelf fungus, propolis and honey for oncological patients, moreover, shelf fungus should be applied as aqueous tincture at a certain ratio, propolis should be applied as propolis butter, moreover, additionally, one should introduce ursine fat, the tincture of aspen bark, fir oil, fruit juice of viburnum or mountain ash, moreover, patients should take components separately, successively between meals in certain order and certain quantity, moreover, the constituents as tinctures and fruit juices should be prepared just before usage and propolis oil - beforehand. The above-mentioned technique favors efficient treatment of oncological patients.

EFFECT: higher efficiency of therapy.

2 cl, 2 dwg, 4 ex, 1 tbl

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