Strain of hybrid cultivated cells of animals mus musculus, producing monoclonal antibodies, specific to peptide, which possesses apoptotic activity with respect to cancer cells

FIELD: medicine.

SUBSTANCE: described is strain of hybrid cultivated cells of animals Mus. Musculus RIM9 - producer of monoclonal antibodies, specific to peptide of human milk - lactaptin and its recombinant analogues, which possess apoptotic activity with respect to human cancer cells. Invention allows to obtain monoclonal antibodies, recognising general linear antigen determinant of recombinant and natural lactaptin.

EFFECT: possibility to elaborate sensitive and specific test-systems for detecting lactaptin, antibodies to it and for isolation of lactaptin from human milk by method of affinity chromatography.

2 tbl, 4 ex

 

The invention relates to biotechnology, namely, hybridoma technology, and represents a new strain of hybrid animal cells Mus. Musculus producing cell cultures and peritoneal cavity of syngeneic animals monoclonal antibodies (MAB) to the peptide milk man, which is a fragment of κ-casein and having apoptotic activity against cancer cells (lactation). The invention can be used in scientific research and promising to create different test systems to determine lactating, antibodies specific to him, to highlight lactating from the milk man.

Known and widespread General biological mechanism responsible for the maintenance of the constancy of the number of cells, the morphology and the culling of defective cells is programmed cell death or apoptosis. It can be induced as external factors proapoptotic cytokines, contact with the membrane of activated cytotoxic lymphocytes and natural killer cells, and intracellular events, such as the depletion of metabolites, the dividing chain of oxidative phosphorylation, accumulation prepariruetsya damage nuclear DICK and so on [1].

Physiological modulators of metabolism can act regulatory peptides (beta-to samohiny and phosphopeptide), formed by the proteolytic cleavage of casein milk man. So, caseinophosphopeptides have cytomegaloviruses effects by stimulating the activity of immune cells [2]. While casomorphine involved in ensuring the absorption of nutrients and the subsequent synthesis of hormones, immunopeptides and casacinema provide immune protection and the transfer of information between the endocrine and the nervous system.

Currently identified and described a new species-specific peptide fractions of milk protein [3], which differ in functionality from the closest analogue. The peptide is a fragment of κ-casein person length 74 amino acid residue has a molecular weight of about 8.6 kDa and installed amino acid sequence (lactation). This peptide is similar multimeric forms of alpha-lactalbumin in milk [4]that in the presence of free fatty acids of milk induce apoptotic cell death of murine lymphoblastoid leukemia L1210 and lung sarcoma person A without affecting the viability of the "non-cancer cells of the primary culture of fibroblasts of human kidney oo(HRTEC) [5], has cytotoxic activity against cancer cells in culture. However, unlike a multimeric form of alpha-lactalbumin milk man lactatin you who indicates apoptotic cancer cell death of a person, in the absence of unsaturated fatty acids of milk necessary to alpha-lactalbumin [6].

To date, monoclonal antibodies (MAB)specific to the molecule of casein milk cows: alpha-, beta-, κ-casein and their proteolytic fragments[7, 8, 9, 10].

The most closest to the claimed strain - prototype is a hybrid strain of cultured mouse cells Mus. Musculus LICR-LON 14.1 producing monoclonal antibodies specific to κ-casein milk [11].

The disadvantage of this strain is that secreted them µa interact with determinant whole molecule κ-casein, however, do not interact with a fragment of κ-casein milk man, having apoptotic activity against cancer cells.

The technical task of the present invention to provide a hybrid strain of cultured mouse cells Mus. Musculus producing monoclonal antibodies specific to the peptide milk man, having apoptotic activity against cancer cells.

The goal of the project is achieved by the proposed hybrid strain of cultured animal cells Mus musculus RIM9 producing monoclonal antibodies with high affinity for the antigenic determinant peptide having apoptotic activity about what to wear to cancerous human cells (lactation).

The strain was obtained as follows.

Mice of Balb/c mice subjected to immunization with recombinant analogue lactating isolated from Escherichia Coli. The antigen is injected into the paw pads, subcutaneously and intraperitoneally. Cells from spleen and lymph nodes immune mouse hybridized with cells of the myeloma Sp 2/0 in the presence of polyethylene glycol. For the cultivation and selection of hybrid cells using the environment adding azaserine and gipoksantina. Hybrids producers are selected using ELISA using as antigen as lactating isolated from the milk man and his recombinant analogue. The most productive clone display in popular culture and denote RIM9.

The strain deposited in the special collection of cell cultures vertebrate Russian cell culture collection of the Institute of Cytology Russian Academy of Sciences (Saint-Petersburg) under the registration number RCCC (P) D.

The essence of the invention lies in the fact that as a result of the fusion of mouse myeloma line Sp-2/0 and splenocytes and lymphocytes of BALB/c mice immunized with recombinant lactation, when used as kuusega agent polyethylene glycol with a molecular weight of 1500 and subsequent cloning method of limiting dilutions are hybridoma RIM9, producing μa to the peptide milk man (lactation)with apoptotic activity p is against cancer cells. To assess the specificity produced by hybridoma antibodies using enzyme immunoassay (ELISA) using recombinant analogue lactating and Western blot turns using lactating or its recombinant counterpart. MCA, secreted by strain RIM9, allow specific identification lactating methods ELISA, Western blot turns and highlight lactatin from the milk man's method of affinity chromatography.

The final product is a monoclonal antibody of the IgG1 subclass (according to the method of radial immunodiffusion) have a high specificity to linear determinants lactating (according to the Western blot turns).

When the cultivation of the strain used culture medium IMDM with the addition of 10% fetal calf serum, 40 μg/ml gentamicin, 2.5 µg/ml amphotericin B. Cells were cultured at 37°C in an atmosphere of 100% humidity with 5% CO2. For cultivation use plastic tablets or vials with a volume of 50 ml.

Strain RIM9 characterized by the following features.

Morphological features. Cell culture consists of weakly adherent to the surface of the culture flasks rounded cells of equal size with large nuclei.

Cultural characteristics. Culture presuspension. Sowing dose of 150 thousand KL Jr. on the Time of subcultivation - 2-3 days, Crat is here sieving 1:5-1:6. The method for removing cells - shaking. The strain produces antibodies for 15 passages in culture.

Characteristics of cultivation in the body of the animal. For growing hybridomas in ascitic form cells of 106cells in the mouse injected into the abdominal cavity of mice BALB/c mice sensitized for 14-90 days vaseline oil. Growth serous tumors celebrate 8-12 day. The total yield of ascitic fluid was 5-10 ml per mouse. Strain can Perepelitsa. By the time of the Deposit he passed 2 passage on animals.

Contamination of the strain. Bacteria and fungi in culture is not detected. Control for contamination by Mycoplasma and viruses were not conducted.

Species of Mus. Musculus.

Carcinogenesis Hybridoma intraperitoneal injection causes serous tumors in mice.

The characteristic of a useful product. Monoclonal antibodies belong to the IgG class (according to ELISA method), subclass IgG1 (immunodiffusion in Waterloo), complementary to the linear antigenic determinants lactating and its recombinant counterpart (according to Western blot turns).

Biotechnological characteristics. The strain produces μa to the peptide milk man, having apoptotic activity against cancer cells (lactation). Secreted by hybridomas MCA have a titer of about 1:102in Kul the Ural fluid and a titer of 1:10 6in ascitic fluid (according to ELISA for recombinant protein) and 1:100 in culture and 1:1000 ascitic fluids (according to immunoblotting for lactating and its recombinant counterpart). The stability of the production of antibody persists for 15 passages in culture and 2 passages on animals.

Cryopreservation. Cells frozen in growth medium with 40% fetal calf serum, 10% dimethylsulfoxide at a temperature of -70°C, store in liquid nitrogen. Viability after thawing is 50-70%.

The invention is illustrated by the following examples of specific performance

Example 1. Obtaining strain RIM9

Mice Balb/c were immunized with recombinant lactation isolated from Escherichia Coli. The antigen was injected in the amount of 12.5 μg per mouse pads, subcutaneously and intraperitoneally according to the following scheme: the first immunization in complete Freund Freind, 2nd immunization 2 weeks in incomplete Freund Freind, 3rd - 3 weeks after the second in incomplete Freund Freind, 4th - 3 weeks after the third. 3 days after the last immunization was administered a booster dose of recombinant lactating - 12.5 µg/mouse in buffered physiological solution (SFR) intraperitoneally. 3 days after administration of the booster dose cells in the spleen and lymph nodes immune mice were killed who were editbale with cells of the myeloma Sp 2/0 (ratio 10:3) in the presence of polyethylene glycol (MV 1500). After hybridization, the cells were sown in 96-well tablets 3×105cells in the hole. For the cultivation and selection of hybrid cells used IMDM medium with addition of 10% fetal calf serum (ETS), to 5.7 µm azaserine and 20 mm gipoksantina. Hybrids producers were selected using ELISA using recombinant analogue lactating and Western blot turns, using as antigen as lactating milk man, and its recombinant counterpart. The result is a hybrid strain of cultured animal cells Mus musculus RIM9 producing monoclonal antibodies to the peptide milk man, having apoptotic activity against cancer cells (lactation). Held twice cloning the obtained hybrid method of limiting dilutions by seeding cells into the wells of 96-hole tablet and subsequent tests on the secretion of antibodies showed that the proportion of clones that retain the production of antibodies of defined specificity is 100%.

Example 2. Detection of immobilized recombinant antigen lactating.

Cells of strain is placed in a plastic bottle with the bottom area 25 cm21×106in 7 ml of IMDM medium with 10% fetal calf serum, 40 μg/ml gentamicin, 2.5 µg/ml amphotericin B. Cells were cultured for 3-4 days at 37°C in the atmosphere, sod is rzasa 5% CO 2. Culture medium with cells harvested, centrifuged 10 min at 200 g at 4°C, cells washed 1 time with saline Hanks and in the amount of 106cells in 0.5 ml SPR intraperitoneally injected to the mice Balb/c mice sensitized for 14-90 days vaseline oil. After 8-12 days of the resulting ascitic fluid was taken, centrifuged 10 min at 300 g and 4°C. the resulting supernatant cultural and ascitic fluid is used as reagents to study the specificity of the interaction of antibodies with lactation using spot ELISA. On nitrocellulose filters (pore size 0.45 µm) size 4×4 mm with microspace absorb 40 ng of purified recombinant analogue lactation in a volume of 0.2 μl. To reduce nonspecific binding, the filters are incubated for a few minutes in TBS buffer (0.02 M Tris-HCl, pH 7.5, 0.15 M NaCl) with 0.08% gelatinosum, carry on wells of 96-hole tablet and incubated for 4 hours at room temperature with 50 μl of supernatant cultural or ascitic fluid diluted in TBS buffer with 0.08% gelatinosum, 0.05% tween-20. The filters are washed with TBS buffer with 0.05% tween-20, make rabbit antibodies against mouse IgG conjugated with horseradish peroxidase diluted 1:5000 in SFR with 0.08% gelatinosum and 0.05% tween-20. After incubation (1 hour at room temperature) filters washed op is between above and have for 30 min with peroxidase substrate solution - of hydrogen peroxide in the presence of the Chromogen - 4-chloro-1-naphthol. A positive result in the samples judged by the appearance of spots, colored in dark blue color. Positive control is immune mouse serum. Negative controls - serum of unimmunized mice and cultural environment. Nonspecific sorption method is evaluated on samples where used buffer instead of serum or culture medium. The results of determination of immobilized antigen lactating by ELISA are presented in table 1.

Table 1
Breeding the culture fluidThe colour intensity of a spotThe dilution of ascitic fluidThe colour intensity of a spot
1:2++++1:10++++
1:3++++1:100++++
1:10++++1:1000++++
1:100++ 1:10000+++
1:1000-1:100000++
1:10000-1:1000000-

The data in table 1 indicate high specificity MCA produced by cells of strain RIM9 to lactatio and shows the possibility of using antibodies for detection of immobilized recombinant analogue lactating using ELISA method. Accurate determination of recombinant analogue lactating possible when diluted culture fluid 1:100, ascitic fluid of 1:100000.

Example 3. Detection of immobilized lactating method of Western blot turns.

To identify lactating or its recombinant counterpart spend electrophoresis of samples in a polyacrylamide gel under denaturing conditions, followed by elektroprenos antigens on nitrocellulose membrane. The nitrocellulose after transfer block 0,08% gelatinosum in SFR, cut into strips and spend ELISA as described in example 2. The presence of antibodies to determine the presence and intensity of bands required molecular weight. The titers of antibodies, secreted by strain RIM9, which identifies lactating its recombinant analogue of the method of Western blot turns, presented in table 2.

Table 2
AntigenΜa (strain RIM9)Polyclonal antibodies (LAWSUIT mouse)
RYIAG
The ratio of credits
Lactation1:1001:10001:1000
Recombinant lactation1:1001:10001:1000
QL - culture liquid, IA - immune ascitic fluid, ACTION - immune serum.

The data are shown in table 2, indicate a high specificity of the MAB produced by cells of strain RIM9 to lactatio, and show the possibility of applying ICA to identify as natural lactating and its recombinant counterpart. Determination lactating possible if the dilution of the culture liquid to 1:100, ascitic fluid up to 1:1000, the number of recombinant analogue lactating in the band is 1 microgram per track, W is Reena 2 mm.

Example 4. The use of MCA strain RIM9 to highlight lactating from the milk man.

Skim human milk was applied on the column, a 1×1 cm with a specific monoclonal antibody strain RIM9, immobilized Enrichment on sepharose 4B, in the amount of 12 mg per 1 ml of sediment gel, which is pre-balanced 150 mm NaCl, 200 mm Tris-HCl buffer, pH 7.5. Not bound peroxidase proteins were suirable buffer used for equilibration of the column. The elution of bound peroxidase with affinity sorbent peptide was performed with 100 mM glycine-HCl buffer pH of 2.5. The pH value in the fractions collected during elution with acidic buffer, brought up to 7-7 .5 with 1 M Tris-HCl buffer pH 8.0. Identification of the peptide was performed by Western blot turns, as described in example 2. According to the results of Western blot turns number lactatin isolated from 20 ml of milk man, was about 10 mg.

Thus, the obtained hybrid strain of cultured animal cells Mus musculus RIM9 is a producer of monoclonal antibodies specific to the peptide milk man, having apoptotic activity against cancer cells (lactation). Monoclonal antibodies produced by the claimed strain, recognize linear antigenic determinant lactating and its recombinant counterpart and can be used for development on the basis of their case is valid and specific test systems to determine lactating, antibodies to it and to highlight lactating from the milk man's method of affinity chromatography.

Sources of information

1. Samuilov V.D., Aleskin AV, Lagunova E.M. Biochemistry. 2000. T. VIP. S-1046.

2. Meisel H., FitzGerald R.J. Curr. Pharm. Des. 2003. V.9. P.1289-1295.

3. Nakapila V.V., Semenov D.V., Potapenko MO, Kuligina E.V., Keith UA, Romanova I.V., Richter, VA "Lactatin - protein of human milk induces apoptosis in adenocarcinoma cells MCF-7," Doklady Akademii nauk, 2008, t, No. 2, SCR-271.

4. Ramakrishnan Century, Boeggeman, E., P.K. Qasba, et al. Biochem. Biophys. Res. Commun. 2002. V.291(5). P.1113-1118.

5. Hakansson, A., Andreasson, J., Zhivotovsky C., et. al. Exp. Cell Res. 1999. V.246(2). P.451-460.

6. Svensson M., Hakansson, A., Mossberg A.-K., et. al. PNAS. 2000. V.97(8). P.4221-4226.

7. Kuzmanoff, K. M., Andresen, J., Beattie .W. J-Dairy-Sci. 1991 Mar; 74(3):803-10.

8. Zhi-Feng Kun, .Cunningham-Rundles. Hybridoma., 1989, 8(2): 223-230.

9. Kuzmanoff, K.M., Andresen, J.W., Beattie, C.W. J-Dairy-Sci.

10. US Patent 5846732, December 8,1998.

11. Earl HM, McIlhinney RA. Mol Immunol. 1985,22(8):981-91.

The hybrid strain of cultured animal cells Mus. Musculus RIM9 deposited in the culture Collection of cells of vertebrates, Institute of Cytology Russian Academy of Sciences under the registration number RCCC(P) D producing monoclonal antibodies specific to the peptide milk man - lactatio and its recombinant analogs having apoptotic activity against cancer cells.



 

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