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Protein with thrombopoiesis substance activity

A protein and platelet technology, applied in the field of new proteins, can solve the problems of small content, failure to complete separation, biochemical and biological identification and characterization

Inactive Publication Date: 2004-06-16
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] Thus, as mentioned above, conditional activities that stimulate megakaryopoiesis and thrombopoiesis have been found in biological samples from thrombocytopenic patients and animals, but because they are present in very small amounts in natural sources such as blood and urine, they are not The isolation, biochemical and biological characterization and characterization of these factors have not yet been completed

Method used

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  • Protein with thrombopoiesis substance activity
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  • Protein with thrombopoiesis substance activity

Examples

Experimental program
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Effect test

Embodiment 20

[0189] The human liver cDNA library (hTPO-F1) constructed in Example 20 was divided into 3 small libraries (library numbers 1-3#). PCR was performed using the plasmid DNA prepared from each small pool as a template and using the synthesized primers. As a result, when the plasmid DNA prepared from the 3# library was used, a DNA fragment with the expected size was amplified. The 3# small pool was then divided into sub-pools, each containing 15,000 transformants, and screened by PCR as described above. As a result, amplification of DNA with the expected size was found in 6 of the 90 small pools. When these positive small pools were divided into subpools, each containing 1,000 clones, plasmid DNA was extracted in the same manner and PCR was performed, no DNA amplification was observed. This is considered to be due to the low recovery rate of plasmid DNA caused by the weaker growth of the clone of interest than other clones. Therefore, the original 3# pool was plated in 100 LB p...

Embodiment 18

[0198] The results of Examples 18 and 22 show that human TPO protein can still exhibit its biological activity even after removing the 3 amino acids at its C-terminus. Therefore, deletion derivative experiments were performed for further analysis of the biologically active fractions. A series of expression plasmids were prepared by PCR using the DNA of the plasmid clone pHT1-231 obtained in Example 18 as a template and synthetic oligonucleotides as primers.

[0199] The resulting expression plasmid contains the DNA encoding the human TPO deletion derivative, which lacks the C-terminal region of the TPO protein, that is, the deletion derivative encodes amino acid sequences at positions 1-211, 1-191, 1-171 and 1-163. When plasmid DNA was prepared from individual clones and transfected into COS1 cells, TPO activity was detected in each culture supernatant (Example 27).

[0200] Design a series of derivatives with deletion of C-terminal amino acid residues to position 151 and oth...

Embodiment 1-1

[0286] Purification induced by administration of antiplatelet antibodies

[0287] Rat TPO in plasma of thrombocytopenic rats

[0288] Preparation of plasma from rats with thrombocytopenia induced by administration of antiplatelet antibody

[0289] TRP was prepared as a purified source from approximately 1,000 rats according to the method described in the aforementioned Reference Example A Rat Megakaryocyte Progenitor Cell (CFU-MK) Assay System.

[0290] Purification of rat TPO from TRP

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Abstract

The present invention relates to thrombopoietin (TPO) polypeptides having the biological activity of specifically stimulating or increasing platelet production comprising the amino acid sequence 1-332 of SEQ ID NO: 6 or a derivative thereof, DNA molecules encoding TPO polypeptides, processes for production of the polypeptides, antibodies specifically immunoreactive with the polypeptides, pharmaceutical compositions comprising the polypeptides, and methods for using the polypeptides in treatment of platelet disorders such as thrombocytopenia.

Description

[0001] This application is a continuation-in-part of the now pending U.S. Patent Application No. unknown filed on January 31, 1995, the latter application being the now pending U.S. Patent Application No. 08 filed on December 22, 1994 / 361,811, which is a continuation-in-part of the now-pending U.S. Patent Application No. 08 / 320,300, filed October 11, 1994, which is a now-pending July 20, 1994 A continuation-in-part of U.S. Patent Application No. 08 / 278,083 filed April 1, 1994, which is a continuation-in-part of now-abandoned U.S. Patent Application No. 08 / 221,020, filed April 1, 1994, a now-discontinued A continuation-in-part of abandoned US Patent Application No. 08 / 212,164, filed March 14, 1994. field of invention [0002] The present invention relates to a novel protein having the activity of stimulating or increasing platelet production or enhancing the proliferation and differentiation of megakaryocyte progenitor cells in a specific manner in vivo, a DNA sequence encodin...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61KA61K38/00A61K38/17A61K38/18A61K38/19A61K38/22A61K38/36A61K39/395A61PA61P7/00A61P7/02A61P7/04A61P9/00A61P43/00C07KC07K14/435C07K14/47C07K14/475C07K14/52C07K14/575C07K14/745C07K16/18C07K16/22C07K16/24C07K16/26C07K16/36C12NC12N1/21C12N5/10C12N15/11C12N15/12C12N15/19C12N15/62C12N15/63C12P21/02C12R1/19C12R1/91G01N33/53
Inventor 宫崎洋加藤尚志大上钦也岩松明彦赤崛弘典黑木良太清水敏之武藤隆则
Owner KYOWA HAKKO KIRIN CO LTD
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