Method of production of endostatin mouse and man

 

The invention relates to the production of endostatin mouse and man. The essence of the invention is to provide a method for obtaining and purification of endostatin from the intracellular Taurus expressed in bacterial cells and nucleic acids encoding endostatin of full length and truncated forms of the endostatin. The technical result - expanding Arsenal of tools for the diagnosis and treatment of tumors. 3 S. and 88 C.p. f-crystals, 10 ill., 3 table.

Description text in facsimile form (see graphic part).

Claims

1. The method of producing endostatin, which includes the following stages:

(a) culturing host cells which Express the gene of endostatin;

(b) isolation of the product of expression of a specified gene;

(C) dissolving the specified gene product at high pH value;

(d) refolding the specified dissolved gene product at pH values of approximately 6-8,5 and

(d) isolation properly Packed form specified gene product.

2. The method of producing endostatin, which includes the following stages:

(a) culturing host cells which Express the gene of endostatin;

(b) fibrillization 6-8,5 and

(d) allocation properly Packed form specified gene product.

3. The method according to p. 1, which indicated the increased pH value at the stage of dissolution is approximately 9-11,5.

4. The method according to p. 3, which indicated an increased pH value is about 10-11.

5. The method according to p. 4, which indicated the increased pH value of approximately pH to 10.5.

6. The method according to p. 2, wherein the specified pH value at the stage of refolding is approximately 7,0-8,0.

7. The method according to p. 6, wherein the specified pH value at the stage of refolding approximately 7.5.

8. The method according to p. 1 or 2, in which at this stage of dissolution, if any stage is provided, the urea is present in a concentration of constituting approximately 4-10 M

9. The method according to p. 8, which at this stage of dissolution of the urea is present in a concentration of constituting approximately 6 M

10. The method according to p. 1 or 2, in which at this stage refolding of urea is present at a concentration of constituting about 0.5-5 M

11. The method according to p. 10, in which at this stage refolding of urea is present at a concentration of constituting approximately 3.5 M

12. SPO is the chloride is present in a concentration component of approximately 2-8 M

13. The method according to p. 12, in which at this stage of dissolution of guanidine hydrochloride is present at a concentration of constituting approximately 4 M

14. The method according to p. 1 or 2, in which at this stage of refolding guanidine hydrochloride is present at a concentration that constitutes about 0.2-2 M

15. The method according to p. 14, in which at this stage of refolding guanidine hydrochloride is present at a concentration that constitutes approximately 1.5 M

16. The method according to p. 1 or 2, performed in the presence of a reducing agent, able to restore the disulfide bonds to sulfhydryl groups.

17. The method according to p. 16, wherein the specified reducing agent selected from the group consisting of DTT, TOGETHER, cysteine and reduced glutathione.

18. The method according to p. 17, in which at this stage of dissolution, if any stage is provided, DTT is present in a concentration of constituting approximately 2-10 mm.

19. The method according to p. 18, in which at this stage of dissolution DTT is present in a concentration of constituting approximately 5 mm.

20. The method according to p. 17, in which at this stage of refolding DTT is present in a concentration that constitutes about 0.2-2 mm.

21. approximately 0.5 mm.

22. The method according to p. 17, in which at this stage of dissolution, if any stage is provided, restored glutathione is present in a concentration of constituting approximately 5-20 mm.

23. The method according to p. 22, in which at this stage of dissolution restored glutathione is present in a concentration of constituting approximately 10 mm.

24. The method according to p. 17, in which at this stage of refolding restored glutathione is present in a concentration of constituting approximately 1-4 mm.

25. The method according to p. 24, in which at this stage of refolding restored glutathione is present in a concentration of constituting approximately 2 mm.

26. The method according to p. 17, in which at this stage of dissolution, if any stage is provided, the cysteine is present at a concentration of constituting approximately 5-20 mm.

27. The method according to p. 26, in which at this stage of dissolution cysteine is present at a concentration that constitutes approximately 10 mm.

28. The method according to p. 17, in which at this stage of refolding cysteine is present at a concentration of constituting about 0.5-4 mm.

29. The method according to p. 28, in which at this stage of refolding cysteine is present in the oxygen the nga is the agent, can increase the change of disulfide bonds.

31. The method according to p. 30, which as specified agent selected either cystine or oxidized glutathione.

32. The method according to p. 31, in which at this stage of refolding cystine is present in a concentration that constitutes approximately 0.2 to 5 mm.

33. The method according to p. 32, which at this stage of refolding cystine is present in a concentration of constituting approximately 1 mm.

34. The method according to p. 1 or 2, in which the formation of disulfide bonds during this stage of refolding occurs due to oxidation by oxygen in the air.

35. The method according to p. 34, wherein the specified phase air oxidation is carried out for about 12-96 hours

36. The method according to p. 35, wherein the specified phase air oxidation is carried out for about 24 to 72 hours

37. The method according to p. 36, wherein the specified phase air oxidation is carried out for approximately 60 hours

38. The method according to p. 1 or 2, wherein the concentration of the specified gene product during this stage of dissolution, if any stage is provided, is about 1-20 mg/ml

39. The method according to p. 38, wherein the concentration of the indicated gene is ri where the concentration of the specified gene product during this stage of refolding rate of approximately 0.1-5 mg/ml

41. The method according to p. 40, wherein the concentration of the specified gene product during this stage of refolding is approximately 0.25 mg/ml

42. The method according to p. 1 or 2, which further provides a stage of purification of endostatin in accordance with a method selected from the group comprising ion exchange chromatography, hydrophobic chromatography and HPLC with reversed phase.

43. The method according to p. 1 or 2, wherein the specified gene endostatin consists of DNA that encodes an animal other than human endostatin

44. The method according to p. 1 or 2, wherein the specified gene endostatin selected from the group of sequences consisting of SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8.

45. The method according to p. 1 or 2, wherein the specified endostatin gene consists of DNA that encodes endostatin person.

46. The method according to p. 1 or 2, wherein the specified endostatin gene consists of SEQ ID NO: 9.

47. The method of producing endostatin from intracellular cells, isolated from bacteria, which includes the following stages:

(a) culturing host cells which Express the gene of endostatin;

(b) isolation of the product of expression of a specified gene;

(C) dissolving the specified gene product at high pH value;

(g) reaviling Packed form specified gene product.

48. The method according to p. 47, which indicated an increased pH value at the stage of dissolution is approximately from 9.0 to 11.5.

49. The method according to p. 48, which indicated an increased pH value is about 10-11.

50. The method according to p. 49, wherein the specified high pH value of about 10.5.

51. The method according to p. 47, wherein the specified pH value at the stage of refolding is approximately 7,0-8,0.

52. The method according to p. 51, wherein the specified pH value at the stage of refolding approximately 7.5.

53. The method according to p. 47, which at this stage of dissolution of the urea is present in a concentration of constituting approximately 4-10 M

54. The method according to p. 53, which at this stage of dissolution of the urea is present in a concentration of constituting approximately 6 M

55. The method according to p. 47, which at this stage refolding of urea is present at a concentration of constituting about 0.5-5 M

56. The method according to p. 55, which at this stage refolding of urea is present at a concentration of constituting approximately 3.5 M

57. The method according to p. 47, which at this stage of dissolution of guanidine hydrochloride is present at a concentration of, for example the d is present in a concentration component of approximately 4 M

59. The method according to p. 47, which at this stage of refolding guanidine hydrochloride is present at a concentration that constitutes about 0.2-2 M

60. The method according to p. 59, which at this stage of refolding guanidine hydrochloride is present at a concentration that constitutes approximately 1.5 M

61. The method according to p. 47, performed in the presence of a reducing agent, able to restore the disulfide bonds to sulfhydryl groups.

62. The method according to p. 61, wherein the specified reducing agent selected from the group consisting of DTT, TOGETHER, cysteine and reduced glutathione.

63. The method according to p. 62, which at this stage of dissolution DTT is present in a concentration of constituting approximately 2-10 mm.

64. The method according to p. 63, which at this stage of dissolution DTT is present in a concentration of constituting approximately 5 mm.

65. The method according to p. 62, which at this stage of refolding DTT is present in a concentration that constitutes about 0.2-2 mm.

66. The method according to p. 65, which at this stage of refolding DTT is present in a concentration of constituting approximately 0.5 mm.

67. The method according to p. 62, which at this stage of dissolution waste which at this stage of dissolution restored glutathione is present in a concentration component of approximately 10 mm.

69. The method according to p. 62, which at this stage of refolding restored glutathione is present in a concentration of constituting approximately 1-4 mm.

70. The method according to p. 69, which at this stage of refolding restored glutathione is present in a concentration of constituting approximately 2 mm.

71. The method according to p. 62, which at this stage of dissolution cysteine is present at a concentration of constituting approximately 5-20 mm.

72. The method according to p. 71, which at this stage of dissolution cysteine is present at a concentration that constitutes approximately 10 mm.

73. The method according to p. 62, which at this stage of refolding cysteine is present at a concentration of constituting about 0.5-4 mm.

74. The method according to p. 73, which at this stage of refolding cysteine is present at a concentration of constituting approximately 1 mm.

75. The method according to p. 47, in which during this stage of refolding agent is present, can increase the change of disulfide bonds.

76. The method according to p. 75, where as the specified agent selected either cystine or oxidized glutathione.

77. The method according to p. 76, which at this stage of refolding is asanoi stage of refolding cystine is present in a concentration component of approximately 1 mm.

79. The method according to p. 47, in which the formation of disulfide bonds during this stage of refolding occurs due to oxidation by oxygen in the air.

80. The method according to p. 79, wherein the specified phase air oxidation is carried out for about 12-96 hours

81. The method according to p. 80, at which the specified stage air oxidation is carried out for about 24 to 72 hours

82. The method according to p. 81, wherein the specified phase air oxidation is carried out for approximately 60 hours

83. The method according to p. 47, wherein the concentration of the specified gene product during this stage of dissolution is about 1-20 mg/ml

84. The method according to p. 83, wherein the concentration of the specified gene product during this stage of dissolution is approximately 2.5 mg/ml

85. The method according to p. 47, wherein the concentration of the specified gene product during this stage of refolding rate of approximately 0.1-5 mg/ml

86. The method according to p. 85, wherein the concentration of the specified gene product during this stage of refolding is approximately 0.25 mg/ml

87. The method according to p. 47, which further provides the stage PTS the Oia, hydrophobic chromatography and HPLC with reversed phase.

88. The method according to p. 47, wherein the specified endostatin gene consists of DNA that encodes an animal other than human endostatin

89. The method according to p. 47, wherein the specified endostatin gene selected from the group of sequences consisting of SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8.

90. The method according to p. 47, wherein the specified endostatin gene consists of DNA that encodes endostatin person.

91. The method according to p. 47, wherein the specified endostatin gene consists of SEQ ID NO: 9.

 

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