Peptide stimulating formation of specific antibodies having effect on survivin in tumour tissue

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining antigenic peptides and can be used in medicine. A biologically active peptide with the following amino acid sequence is obtained: Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg- Arg-Ala-Ile-Glu-Gln-Leu-Ala. The obtained peptide can stimulate formation of antibodies which are specific to tumour-associated survivin protein.

EFFECT: invention enables detection of survivin in tissue using an immunohistochemical technique in order to diagnose cancerous growths.

2 dwg, 1 tbl, 2 ex

 

The invention relates to the field of biochemistry, specifically to obtain peptide for immunization which it is possible to obtain antibodies that detect protein survivin, and can be used in medicine for the diagnosis of cancer.

Development of new methods of clarifying the diagnosis of neoplastic diseases is an important trend in modern Oncology. One of the most promising markers of malignant tumors is survivin. This endogenous protein from the protein family of inhibitors of apoptosis is involved in mitosis and prevents programmed cell death. Survivin virtually absent in adult healthy tissues, however, its overexpression is shown in various neoplasma person. There is evidence that the high content of survivin in cancer cells correlates with stage of disease, poor prognosis of the disease development and resistance of tumors to chemotherapy. Therefore, quantitative assessment of the content of survivin in tumors and its localization in the cell gives the possibility to select the optimal treatment regimen for cancer.

To study the content of survivin in the tissues used immunohistochemical method based on detection of the protein by antibodies to it, and the reliability of the method depends on the specificity of the used ant the phone Different antibodies to the same tumor samples give different results content of survivin in tissues and its localization in the cell [Vischioni Century, Oudejans J.J., Kruyt F.A.E. Immunmohistochemical detection of nuclear survivin in NSCLC: a comparison of commercial antibodies. // Histopathology. 2007. V.50. P.671-680]. There is therefore a need for new antibodies to survivin and searching for new immunogens by which antibodies can be obtained.

For an objective assessment of the content and localization of survivin necessary antibodies directed to different regions of the sequence of this protein. Such antibodies can be obtained only by using synthetic fragments of survivin.

Known synthetic peptides, which are used as immunogens to generate antibodies to survivin, namely: the peptide corresponding to the amino acid sequence 1-12 of survivin [http://www.abcam.com/Survivin-peptide-ab7866.html]and peptides simulating C-terminal parts of survivin person 125-142 [http://www.abcam.com/Survivin-antibody-ab47038.html] and 131-142 [http://www.abcam.com/Survivin-antibody-ab27468.html]. These peptides are useful in research practice to generate antibodies to different regions of the protein, however, the diagnostic value of antibodies to them are not as well as connect surviving only in cell cultures by the method of Western blot turns and immunocytochemical method, but not in the tissues of human tumors is A.

Known closest to the stated on the manifest properties of the synthetic peptide sequence PTLPPAWQPFLKDHRI section 4-19 of survivin [.Trieb, R.Lehner, .Stulnig, et al. Survivin expression in human osteosarcoma is a marker for survival. // EJSO. 2003. V.29. P.379-382]. Antibodies obtained by immunization of rabbits given peptide, identify survivin method of immunohistochemistry on paraffin sections of osteosarcoma, which makes them suitable for use in diagnostic purposes. The Western blot turns with these antibodies confirmed their specificity to a protein with a molecular mass of approximately 16.5 kDa, which corresponds to the mass of surviving.

The invention solves the problem of expanding the range of peptides to generate antibodies that are suitable for diagnosing cancer by detecting survivin in samples of human tumors by immunohistochemical method.

The problem is solved by peptide that stimulates the formation of specific antibodies, detecting survivin in tumor tissues, of the formula:

Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg-Arg-Ala-Ile-Glu-Gln-Leu-Ala.

The synthesis of the target product carry out solid-phase method using alkoxybenzenes polymer. Condensation is realized by one of two methods: 1) using tetrafluoroborate O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylrhodamine (TBTU); 2) using 1-hydroxybenzotriazole (NOVT) and N,N'-DIIS is propylbromide (DIPC). The accession of the next Fmoc-protected amino acid residues spend once except when the growing peptidyl polymer after the condensation reaction detect unreacted amino groups. Control over the content of unreacted amino groups peptidyl polymer is carried out using ninhydrin test. To separate the peptide from the polymer carrier and the final release use triperoxonane acid with the addition of thioanisole, phenol, ethicial, triisopropylsilane and water. The structure and homogeneity of the target product data confirm amino acid analysis, mass spectrometry and high performance liquid chromatography.

The essence of the invention is a new peptide of the formula Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg-Arg-Ala-Ile-Glu-Gln-Leu-Ala (SEQ ID NO 1), simulating areas (80-88) and (153-162) sequence of survivin 2V person obtained using standard techniques of peptide synthesis. Antibodies obtained by immunization of rabbits declare a synthetic peptide specific to survivin, as evidenced by their binding to recombinant survivin in enzyme-linked immunosorbent analysis and Western blot turns. These antibodies detect survivin in samples of human tumors, as evidenced by specific immunohistochemical reaction in the test with paraffin slice and tissue tumors of patients with the diagnosis of cancer of the bladder.

The technical result of the invention is the ability of the inventive peptide to stimulate immunization in free form, without conjugation with macromolecular carrier, antibodies specific to survivin and identifying this protein in tumor samples of human tissues by immunohistochemical method.

The invention is illustrated by the drawings.

Figure 1. The binding of the antibody to the peptide (SEQ ID NO 1) with recombinant survivin in the immunoblot. The molecular mass of 17 kDa, detected by antibodies to the peptide (I), corresponds to the molecular mass of surviving.

Figure 2. Immunohistochemical staining with antibodies to the peptide (SEQ ID NO 1) sections of urinary bladder tumors. Dark stained cell nuclei and cytoplasm, bound antibodies.

Example 1. The synthesis of the peptide Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg-Arg-Ala-Ile-Glu-Gln-Leu-Ala (SEQ ID NO 1).

Operation 1.

0.3 g alkoxybenzenes polymer with a content of Oh-groups 0.5 mmol/g of polymer is placed in a flow reactor, washed with dimethylformamide (2×10 ml), again add 10 ml of dimethylformamide and left to swell for 5-10 min, then the dimethylformamide is removed. The synthesis of the peptide are in a flow reactor at room temperature.

Step 2. Obtaining Fmoc-Ala-polymer (I).

In 4 ml of dimethylformamide is dissolved 0,494 g (1.5 mmol) Fmoc-Ala-OH·H2O add 116 μl (0.75 mmol) NN'-diisopropylcarbodiimide and 1.8 mg (0.015 mmol) of 4-dimethylaminopyridine. The solution is stirred at 0°C. After 10 min the solution was added to alkoxybenzenes the polymer in a flow reactor. The mixture was incubated at periodic stirring for 12 hours. The obtained Fmoc-Ala-polymer washed with dimethylformamide (2×10 ml), ethanol (2×10 ml) and again with dimethylformamide (2×10 ml). Unreacted amino groups acelerou a mixture of 2 ml of acetic anhydride and 2 ml of pyridine and 6 ml of dimethylformamide for 2 hours Rinse repeat.

Step 3. Obtaining Fmoc-Leu-Ala-polymer (II).

a) the Obtained Fmoc-Ala-polymer (I) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) strength of 0.159 g (0.45 mmol) of Fmoc-Leu-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again dimetilformamidom (2×10 ml).

Step 4. Obtaining Fmoc-Gln-Leu-Ala-poly the EPA (III).

(a) Fmoc-Leu-Ala-polymer (II) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,166 g (0.45 mmol) of Fmoc-Gln-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 5. Obtaining Fmoc-Glu(OBut)-Gln-Leu-Ala-polymer (IV).

(a) Fmoc-Gln-Leu-Ala-polymer (III) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,192 g (0.45 mmol) of Fmoc-Glu(OBut)-HE 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer was washed with Dimity what formamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Step 6. Obtaining Fmoc-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (V).

(a) Fmoc-Glu(OBut)-Gln-Leu-Ala-polymer (IV) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) strength of 0.159 g (0.45 mmol) of Fmoc-Ile-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

C) strength of 0.159 g (0.45 mmol) of Fmoc-Ile-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

g), the polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dim what telharmonium (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 7. Obtaining Fmoc-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (VI).

(a) Fmoc-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (V) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,148 g (0.45 mmol) of Fmoc-Ala-OH·H2O and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 8. Obtaining Fmoc-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (VII).

(a) Fmoc-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (VI) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,292 g (0.45 mmol) of Fmoc-Arg(Pbf)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-atilde is Isopropylamine and stirred at 0°C for 10 minutes The solution is added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 9. Obtaining Fmoc-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (VIII).

(a) Fmoc-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (VII) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,292 g (0.45 mmol) of Fmoc-Arg(Pbf)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in) 0,292 g (0.45 mmol) of Fmoc-Arg(Pbf)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

g) Polymer treatment is with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 10. Obtaining Fmoc-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (IX).

(a) Fmoc-Arg(Pbf)- Arg(Pbf)-Ala-Ile-Glu(OBut)-Glu-Leu-Ala-polymer (VIII) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,153 g (0.45 mmol) of Fmoc-Val-OH and 0,069 g (0.45 mmol) NOT dissolved in 4 ml of dimethylformamide. To the solution was added 70 μl (0.45 mmol) of DIPC and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 11. Obtaining Fmoc-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (X).

(a)Fmoc-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer(IX) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,211 g (0.45 mmol) of Fmoc-Lys(Boc)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml Dima is Informatica. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 12. Obtaining Fmoc-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer(X1).

(a) Fmoc-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (X) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) strength of 0.159 g (0.45 mmol) of Fmoc-Leu-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

C) strength of 0.159 g (0.45 mmol) of Fmoc-Leu-OH and 0,069 g (0.45 mmol) NOT dissolved in 4 ml of dimethylformamide. To the solution was added 70 μl (0.45 mmol) of DIPC and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic displacement is Ivanyi leave for 2 hours The polymer is washed with dimethylformamide (2×10 ml).

d) the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 13. Obtaining Fmoc-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer(XII).

(a) Fmoc-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XI) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,179 g (0.45 mmol) of Fmoc-Thr(But)-HE 0,069 g (0.45 mmol) NOT dissolved in 4 ml of dimethylformamide. To the solution was added 70 μl (0.45 mmol) of DIPC and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 14. Obtaining Fmoc-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer(XIII).

(a) Fmoc-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pb)-Ala-Ile-Glu(OBu t)-Gln-Leu-Ala-polymer (XII) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,173 g (0.45 mmol) of Fmoc-Ser(But)-OH and 0,069 g (0.45 mmol) NOT dissolved in 4 ml of dimethylformamide. To the solution was added 70 μl (0.45 mmol) of DIPC and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in) 0,173 g (0.45 mmol) of Fmoc-Ser(But)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

d) the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 15. Obtaining Fmoc-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XIV).

(a) Fmoc-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XIII) is treated with 10 ml of 20%solution of piperidine is dimethylformamide and 20 minutes washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,173 g (0.45 mmol) of Fmoc-Thr(But)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 16. Obtaining Fmoc-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbl)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XV).

(a) Fmoc-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbl)-Ala-Ile-Glu(OBut)-Glu-Leu-Ala-polymer (XIV) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,160 g (0.45 mmol) of Fmoc-Asn-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring OS is ablaut 2 hours The polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 17. Obtaining Fmoc-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XVI).

(a) Fmoc-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XV) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,264 g (0.45 mmol) of Fmoc-Cys(Trt)-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 18. Obtaining Fmoc-Ala-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(Bu t)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)Gln-Leu-Ala-polymer (XVII).

(a) Fmoc-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Gln(OBut)-Gln-Leu-Ala-polymer (XVI) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,148 g (0.45 mmol) of Fmoc-Ala-OH and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol). N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 19. Obtaining Fmoc-Tyr(But)-Ala-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XVIII).

(a) Fmoc-Ala-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XVII) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again digitiform the home (2×10 ml).

b) 0,207 g (0.45 mmol) of Fmoc-Tyr(But)-HE 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×10 ml).

in the Polymer is treated with a solution of 43 μl (0.45 mmol) of acetic anhydride and 77 μl (0.45 mmol) of N-ethyldiethanolamine in 4 ml of dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), ethanol (2×10 ml), again with dimethylformamide (2×10 ml).

Operation 20. Obtaining Fmoc-Ala-Tyr(But)-Ala-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbl)-Arg(Pbf)-Ala-Ile-Glu(OBut)-Gln-Leu-Ala-polymer (XIX).

(a) Fmoc-Tyr(But)-Ala-Cys(Trt)-Asn-Thr(But)-Ser(But)-Thr(But)-Leu-Lys(Boc)-Val-Arg(Pbf)-Arg(Pbf)-Ala-Ile-Gln(OBut)-Gln-Leu-Ala-polymer (XVIII) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml).

b) 0,148 g (0.45 mmol) of Fmoc-Ala-OH·H2O and 0,145 g (0.45 mmol) TBTU dissolved in 4 ml of dimethylformamide. To the solution add 77 μl (0.45 mmol) of N-ethyldiethanolamine and stirred at 0°C for 10 min. the Solution was added to the polymer and at periodic stirring, leave for 2 hours, the Polymer is washed with dimethylformamide (2×0 ml).

Operation 23. Get the targeted peptide Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg-Arg-Ala-Ile-Glu-Gln-Leu-Ala (SEQ ID NO 1).

0.3 g of peptidyl-polymer (XIX) is treated with 10 ml of 20%solution of piperidine in dimethylformamide for 20 min, washed with dimethylformamide (2×10 ml), a solution of dioxane in water (2:1) (2×10 ml), again with dimethylformamide (2×10 ml), then with ethanol (2×10 ml) and dried in a current of air. The dried peptidyl-polymer is treated with 4 ml of a mixture of triperoxonane acid-thioanisole-phenol-water-acondition-triisopropylsilane in a volume ratio 81,5:5:5:5:2,5:1 within 2 hours the Polymer is filtered off, the solution evaporated, the solid residue is washed with dry diethyl ether (2×50 ml). Dissolved in 10% acetic acid and purified by preparative HPLC under the following conditions: column Phenomenex (USA) Jupiter 10µ 18 300A (250×10 mm), solvent A: 0,1%triperoxonane acid in water, solvent B is 0.1%triperoxonane acid in acetonitrile, gradient solvent And B is 20-80% for 2 h, the flow rate of eluent to 3 ml/min, the output of the peptide register at optical wavelength of 226 nm. Collected after HPLC fraction containing the target product, lyophilized.

Amino acid analysis is performed on the device Biotronik LC-3000 (Germany) after hydrolysis of the peptide mixture 6 N. HCl-TFA (2:1) for 45 min at 170°C. the Synthesized peptide has the correct amino acid composition. Mass spectrometry was carried out by what Ecodom MALDI instrument VISION 2000 (Bioanalysis, UK). According to mass spectrometry m/z 2109 for [M+H]+(2107,46 - calculated). According to analytical HPLC retention time is 23,01 min conditions: column Phenomenex (USA) Jupiter 5µ 18 300A (250×4.6 mm), solvent A: 0,1%triperoxonane acid in water, solvent B is 0.1%triperoxonane acid in acetonitrile, gradient solvent And B - 10-70% for 1 h, the flow rate of eluent 1 ml/min, the output of the peptide register at optical wavelength of 226 nm. The amino-acid analysis: Asp 1.08 (1), Thr 1.96 (2), Ser 0.83 (1), Glu 2.14 (2), Ala 3.96 (4), Val 0.91 (1), Ile 0.96 (1), Leu 1.8 (2)

Example 2. Conducting biological tests.

The resulting fusion peptide (SEQ ID NO 1) subjected to immunization of rabbits from serum by affinity chromatography allocate protopeptide antibodies that check in immunoassay analysis and immunoblot specificity to survivin and used in the study of samples of tumor tissues by immunohistochemical method.

Operation 1. Immunization of animals.

To immunize animals prepare a solution of peptide (SEQ ID NO 1) in physiological solution (0.9% NaCl) at a concentration of 1 mg/ml, which is then mixed with an equal volume of complete adjuvant's adjuvant for the first immunization or incomplete adjuvant's adjuvant for the second immunization to obtain the emulsion. Rabbits subjected to immunization twice the dose of 1 mg of peptide per animal for each immunization with an interval of 44 days. The drug is administered intramuscularly along the spine in four points.

Step 2. Getting rabbit serum and determination of the titer protivoavarijnyh antibodies.

Blood is withdrawn from the ear vein of rabbits after 14 days. after the second immunization. Selected blood after incubation for 1 h at 37°C by separating the cells by centrifugation at 1000 rpm for 10 min to prepare the serum. Title protivoavarijnyh antibodies is determined using enzyme-linked immunosorbent assay (ELISA). In defenestrations tablet Nunc MaxiSorp contribute 0.1 ml in each well of a solution of the peptide in 0.05 M Na-carbonate-bicarbonate buffer (pH 9.6) at a concentration of 20 μg/ml and incubated for 16 h at 4°C. Then a solution of the peptide is removed and the plate washed four times PBS1(137 mM NaCl and 2.7 mM KCl, 8 mM NaH2PO4, 1.5 mM KH2PO4, pH 7.4)containing 0.05% tween-20. Procedure four times followed by washing after each stage of incubation. In wells contribute 0.1 ml samples of serum in double dilutions starting from 1:40 or 1:1000 and incubated for 1 h at 37°C. as control use preimmune serum of rabbits. After washing added to the wells at 0.1 ml solution of antibody goat to rabbit immunoglobulins conjugated with horseradish peroxidase (IMTEK, Russia) at a dilution of 1:3000, and after incubation, the washing contribute 0.1 ml of substrate solution (0.05% hydrogen peroxide and 0.05% o-phenylenediamine in 0.05 M Na-citrate buffer, pH 5.0). The reaction is stopped by adding 0.1 ml of 10% H2SO4. For antibody titer take the negative logarithm (-lg) values of the highest dilution of serum that gives the painting more than 0.1 OE492excess staining control sera more than twice.

The titer of antibodies to the peptide described in the invention is 5.8.

Step 3. Getting affine media.

1 g of CNBr-activated sepharose 4 (GE Healthcare bio-science AB, Sweden) are suspended in 6 ml of 1 mm HCl for 15 min prior to the formation of a transparent gel and washed with 1 mm HCl (5×10 ml). 2.8 mg of peptide dissolved in 4 ml condensing buffer (0.1 M Panso3, 0.1 M NaCl, pH 8.0), add to sepharose and with periodic stirring and left for 1 h at 20°C. After washing the condensing buffer (5×3 ml) to separate add blocking buffer (0.2 M Gly, 0.1 M NaCl, pH 8.0) and incubated for 2 hours Then sepharose washed alternately acetate (0.1 M NaOCOCH3, 0.1 M NaCl, pH 4.0) and borate buffer (0.1 M Na2B4O7·10H2O, 0.1 M NaCl, pH 8.1) (5×10 ml).

Step 4. Carrying out affinity chromatography.

2 ml of serum is applied on the column with 3.5 ml of affinity media, washed with PBS2(0.15 M NaCl, 0.01 M Na2HPO4, pH 7.4), and incubated at room temperature for 40-60 minutes Then the column was washed with 35 ml of PBS2and 35 ml of eluting buffer (0.2 M process is and glycine in 0.1 M NaCl, pH 2.5). In selected fractions of the eluate pH was adjusted to 6.5-8.0 with 1M TRIS, pH 10. The protein content determined spectrophotometrically by measuring TH280. In this case it was 0.23 mg/ml Titer, affinity-purified protivoavarijnyh antibodies (5.4) is determined using an enzyme-linked immunosorbent assay as described in methods 2 methods.

Operation 5. Linking protivoavarijnyh antibodies with recombinant survivin in ELISA.

Specificity protivoavarijnyh antibodies to survivin determined using an enzyme immunoassay as described in steps 2 methodology. For sorbirovaniya on the tablet in the wells contribute instead of a solution of the peptide in 0.1 ml of recombinant survivin containing 142 and. O. obtained by expression in E. coli at a concentration of 10 mg/ml antibody Titer is 5.4.

Step 6. Linking protivoavarijnyh antibodies with recombinant survivin in Western blot turns.

Specificity protivoavarijnyh antibodies to survivin determined using Western blot turns with recombinant survivin.

Sample preparation: 5 μl of a solution of recombinant survivin in dimethyl sulfoxide to a concentration of 30 mg/ml, add 45 ál of PBS1then solubilizers buffer containing 62 mm Tris-HCl, 2% SDS, 5% 2-mercaptoethanol, 1 mm EDTA, pH 6.8. The mixture thermostatic at 100°C for 1.5 min, then add the solution of 10% glycerol and 0.01% bromophenol blue.

Electrophoresis is performed on plates of polyacrylamide gel (15% acrylamide in the separating gel and 5% in concentrating) under denaturing conditions. Proteins concentrate at an amperage of 10 mA/plate, and share - at 20 mA/plate.

Elektroprenos carried out on membrane immobilon NC with pore diameter of 0.45 μm ("Millipore, USA) under the conditions: 0.025 M Na-bicarbonate buffer (20% CH3OH, 0.1% SDS, pH 9.0) at a constant current of 400 mA for 1.5 h, and then the membrane was washed with buffer A (50 mm Tris-HCl, 200 mm NaCl, 0.1% Triton X-100, pH 7.5) (6×5 min) on the rocking chair. After that, the membrane is treated with a buffer containing 5% skim milk powder (AppliChem, Germany)for 1 h, and then incubated for 16 h with affinity-purified antibodies to the peptide (SEQ ID NO 1) in the same buffer at 4°C in a dilution of 1/200. Next, the membrane was washed with buffer A (6×5 min) and re-treated with buffer containing 5% milk for 40 min and Then incubated with secondary antibodies (goat antibodies against total rabbit immunoglobulins conjugated with horseradish peroxidase, IMTEK, Russia) in buffer containing 5% milk, over 1.5 hours, the Membrane was washed with buffer A (6×5 min) and distilled water (3×3 min). For staining of the bands to the membranes add 25 ml of 50 mm Tris-HCl (pH 7.6)containing 12.5 mg of diaminobenzidine and 10 μl of a 50% solution of hydrogen peroxide. Show during the 5-10 min in the dark. The molecular mass of 17 kDa, detected by antibodies to the peptide (SEQ ID NO 1), corresponds to the molecular mass of survivin (figure 1).

Operation 7. Immunohistochemical analysis.

The specific activity of the antibodies tested method of immunohistochemistry on serial paraffin sections. The study was conducted by the method of indirect immunoperoxidase analysis.

As the material for testing using serial deparaffinizing the tissue sections infiltrative ductal breast cancer and bladder cancer man. Surgical material fixed in 10% buffered formalin. Slices deparaffinized in standard histological posting in toluene and alcohols decreasing concentration. Thrice washed in buffered saline solution (0.01 M sodium phosphate buffer containing 0.9% sodium chloride, pH 7.4) (SPR). Endogenous peroxidase inactivating a 3% solution of hydrogen peroxide in SFR for 20 min at room temperature. After triple washing in SFR conduct the procedure damascenone antigen in 0.01 M sodium citrate buffer, pH 6.0, in a water bath at 95°C for 20 minutes After completion of incubation, the slices are cooled for 20 min at room temperature and washed three times in SFR. Non-specific interactions are blocking in 3% solution of BSA in SFR for 30 min at 37°C. Solutions of antibodies g is tovat in 1% solution of bovine serum albumin (BSA) in SFR and applied to the sections for 1 hour at a dilution of 1:20. To control for nonspecific interactions on the slices, put a solution of purified rabbit IgG at a concentration of 10 μg/ml of bound peroxidase antibodies detected using goat antibody labeled with peroxidase, rabbit immunoglobulins ("DakoCytomation", P0448) in dilution 1:100, the solution which is applied to the sections for 30 minutes as Chromogen use Liquid DAB ("DakoCytomation", K3465). Immunochemical reaction is conducted at room temperature, the successive stages of application of reagents alternate with triple washing in SFR. After staining, the slices are washed in distilled water, dehydration and conclude in the resin (Bio Mount, "Bio-Optica, Italy, 05 MM). Staining analyzed by light microscope, assessing the presence of specific staining in the tumor cells.

Examined 20 samples of tumor patients with the diagnosis of bladder cancer. All samples revealed staining of the cells. We observed staining of various degrees of intensity in the cytoplasm and/or nuclei of cells in all samples of tumor tissue (table, 2). The absence of such reactions with control rabbit IgG (Jackson ImmunoResearch) suggests that the interaction is specific. To control also investigated the binding of the antibody to the peptide (SEQ ID NO 1) with samples of normal tissues. In these cases, staining was not observed.

Table
Detection of survivin in samples of bladder tumors by antibodies to the peptide (SEQ ID NO 1)
The number of samplesOnlyLocalization in the nucleusLocalization in the cytoplasm
201912

Thus, antibodies of the claimed peptide reveal survivin in all investigated samples of tumor tissues of patients diagnosed with cancer of the bladder.

In the description of the used peptide sequence SEQ ID NO 1 Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg-Arg-Ala-Ile-Glu-Gln-Leu-Ala

The peptide that stimulates the formation of specific antibodies, detecting survivin in tumor tissues, of the formula:
Ala-Tyr-Ala-Cys-Asn-Thr-Ser-Thr-Leu-Lys-Val-Arg-Arg-Ala-Ile-Glu-Gln-Leu-Ala.



 

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