The polypeptide antigen (hcv variants), the kit for immunoassay, immunoassay method, polynucleotide (options), polynucleotide probe containing his set, the method detection hСv, the method of obtaining blood containing hcv (options)

 

The invention relates to the field of molecular biology and immunology, and can be used in medicine, particularly in the diagnosis of hepatitis C. By screening cDNA libraries selected nucleotide sequence encoding the polypeptide of hepatitis C virus (HCV), which have antigenic properties. Fragments of the isolated nucleic acid sequences containing at least 8 nucleotides, it is proposed to use as probes for detecting polynucleotides HCV in a biological material, and for obtaining blood samples that do not contain nucleic acid of the virus. The recombinant DNA obtained antigenic polypeptides of hepatitis C virus and their fragments corresponding antigenic determinants. The application of the HCV antigens allows to detect the presence in a biological sample of antibodies against hepatitis C virus and to take blood samples that do not contain these antibodies. 13 C. and 11 C.p. f-crystals, 49 ill.

Description text in facsimile form (see drawings) T

Claims

1. Substantially purified polypeptide representing the antigen of the hepatitis C virus (HCV), and specified and represent an HCV epitope, containing at least 10 consecutive amino acids.

2. The polypeptide under item 1, containing amino acids selected from the group: 1-50, 1-84, 9-177, 1-120, 35-45, 50-100, 40-90, 65-75, 80-90, 99-120, 95-110, 100-150, 150-200, 200-250, 220-240, 245-265, 250-300, 290-330, 290-305, 300-350, 310-330, 350-400 and 400-450.

3. The polypeptide according to p. 2 containing amino acids selected from the group: 1-50, 1-84, 1-120, 35-45, 50-100, 40-90, 65-75, 80-90, 99-120, 95-110 and 100-150.

4. The polypeptide under item 1 or 2, characterized in that it further associated with a solid substrate.

5. Substantially purified polypeptide representing the antigen of the hepatitis C virus (HCV), and the specified antigen includes the amino acid sequence shown in Fig. 11, or a fragment of the polypeptide, which represents an HCV epitope containing at least 10 consecutive amino acids.

6. The polypeptide under item 5, characterized in that it further associated with a solid substrate.

7. Substantially purified polypeptide representing the antigen of the hepatitis C virus (HCV), and the specified antigen contains amino acids 2887-2955 sequence shown in Fig. 17, or a fragment of the polypeptide, which represents an HCV epitope containing at least 10 consecutive amino

9. The polypeptide under item 7 or 8, characterized in that it further associated with a solid substrate.

10. The selected polypeptide containing the epitope of polyprotein hepatitis C virus (HCV), characterized in that it contains at least 10 consecutive amino acids of the region from amino acids amino acids 450 to 2850 sequence shown in Fig. 17, and is selected from the group: 450-500, 475-495, 500-550, 511-690, 515-550, 550-600, 550-625, 575-605, 600-650, 600-625, 635-665, 650-700, 645-680, 700-750, 700-725, 725-775, 770-790, 750-800, 800-815, 850-875, 800-850, 920-990, 850-900, 920-945, 940-965, 950-1000, 1000-1060, 1000-1050, 1025-1040, 1075-1175, 1050-1200, 1070-1100, 1100-1130, 1195-1250, 1200-1225, 1225-1250, 1250-1300, 1260-1310, 1260-1280, 1300-1350, 1310-1340, 1345-1405, 1350-1400, 1365-1380, 1380-1405, 1400-1450, 1450-1500, 1475-1515, 1475-1500, 1500-1550, 1515-1550, 1550-1600, 1570-1590, 1595-1610, 1590-1650, 1610-1645, 1650-1690, 1685-1770, 1690-1720, 1720-1745, 1745-1770, 1750-1800, 1775-1810, 1795-1850, 1850-1900, 1900-1950, 1900-1920, 1920-1940, 1949-2124, 1950-2000, 1950-1985, 2000-2050, 2020-2045, 2045-2100, 2045-2070, 2070-2100, 2100-2150, 2150-2200, 2200-2325, 2250-2330, 2265-2280, 2280-2290, 2300-2350, 2350-2400, 2345-2415, 2345-2375, 2348-2464, 2370-2410, 2400-2450, 2400-2425, 2415-2450, 2445-2500, 2371-2502, 2500-2550, 2505-2540, 2550-2600, 2560-2580, 2600-2650, 2620-2650, 2650-2700, 2655-2670, 2670-2700, 2700-2750, 2750-2800, 2755-2780, 2780-2830, 2785-2810, 2810-2825 and 2800-2850, and the specified polypeptide is not 1266-2124, 1472-1930, 1192-1457, 1569-1805, 1950-2124, 1266-1428, 2054-2223, 1472-1630, 1678-1805, 1569-1703, 1689-1805, 1022-1136, 1916-2020, 1793-1930, 1122-1214, 1403-1483, 1688-1735, 451-2886, 626-2886, 717-2486, 451-904, 1560-1934, 819-987, 2691-2841, moliterno associated with the solid substrate.

12. The kit immunoassay for detection of antibodies against HCV containing the polypeptide or polypeptide fragment described in any of paragraphs. 1-11, in a suitable container.

13. The method of immunoassay for detection of antibodies against hepatitis C virus (HCV) in which stage of incubating a biological sample suspected of containing antibodies against HCV, with a polypeptide or polypeptide fragment described in any of paragraphs. 1-11, under conditions that allow formation complex polypeptide-antibody, followed by detection of the complex, the presence of which indicates the presence of antibodies against HCV in the sample.

14. Immunoassay on p. 13, wherein the biological sample is blood, serum or plasma of man.

15. Polynucleotide encoding antigen of the hepatitis C virus (HCV) having nucleotide sequence corresponding to the genomic sequence of HCV or sequence of cDNA containing nucleotides from -319 until 1348 sequence shown in Fig. 17, or a fragment of polynucleotide containing at least 8 consecutive nucleotides.

16. Polynucleotide under item 15, which is On the corresponding genomic sequence of HCV or cDNA sequence, containing nucleotides from 8659 up 8866 sequence shown in Fig. 17, or a fragment of polynucleotide containing at least 8 consecutive nucleotides.

18. Polynucleotide under item 17, which represents DNA.

19. Polynucleotide probe for the detection of HCV nucleic acid in a sample, comprising 8 or more consecutive nucleotides of the sequence of HCV cDNA and consists of nucleotides from -319 until 1348 or from 8659 up 8866 sequence shown in Fig. 17.

20. Polynucleotide probe under item 19, which is labeled.

21. Kit for analyzing samples for the presence of polynucleotides HCV containing polynucleotide probe under item 19 or 20 and a suitable carrier.

22. The method of detection of HCV nucleic acid in the sample, including interaction of the polynucleotide probe, as described in paragraph 19 or 20, with the sample, which presumably contains the nucleic acid of HCV, with subsequent detection of the educated thus polynucleotide duplex, the presence of which indicates the presence of HCV nucleic acid in the sample.

23. The method of obtaining blood that does not contain antibodies pratical against HCV; the specified incubation of the sample with the polypeptide or fragments of the polypeptides described in any of paragraphs. 1-11, under conditions that allow formation complex polypeptide/fragment of a polypeptide-antibody against HCV; detection formed such complexes, the presence of which indicates the content of antibodies against HCV in a specified pattern; preservation of blood in the sample which was not detected complex polypeptide/fragment of a polypeptide-antibody against HCV.

24. The method of obtaining blood that does not contain polynucleotide HCV, which includes the following stages: obtaining a blood sample, which assumes the presence of polynucleotides HCV; the specified incubation of the sample with a polynucleotide probe, as described in paragraph 19 or 20, under conditions that allow to form a polynucleotide duplex between the specified probe and any polynucleotide HCV presence are expected in the sample; the detection of any educated so polynucleotide duplex, the presence of which indicates the presence in the specified sample polynucleotides HCV; and preservation of blood in the sample which was not detected polynucleotide duplex between the probe and the

 

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