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Platelet promoting protein and the usage thereof

a platelet and protein technology, applied in the field of biomedicine, can solve the problems of short shelf life of platelets, life-threatening low level of blood platelets, allergic reactions among recipients, etc., and achieve the effects of increasing the amount of platelets in the circulating blood, monitoring bleeding times, and stimulating significantly the formation of platelets

Inactive Publication Date: 2007-08-02
XU PEILIN +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] Also provided by the invention are derivatives of the PPP. The derivatives of the “PPP” include: [1] mutants of the PPP, provided that the mutants retain the ability of increasing the numbers of platelets and enhancing the blood clotting in vivo; [2] variants of the PPP, which, as compared with the Sequence 2, comprise one or more conservative substitutions of amino acids; one or more deletions of amino acids; or one or more additions of amino acids; [3] a carboxyl terminal-truncated form or amino terminal-truncated form of the PPP having the Sequence 2; [4] a tandem repetition of partial or complete Sequence 2; and [5] a fusion protein of the PPP having the Sequence 2 and another protein or cytokine. One of such derivatives, for example, carries additional 2-6 histidines at the N-terminus of the Sequence 2.
[0013] The purified PPP was injected into normal mice and the amount of the circulating platelets was measured and the bleeding times were monitored. The results indicated that the His-PPP stimulated significantly the formation of platelets and increased the amount of platelets in the circulating blood.

Problems solved by technology

Low level of blood platelets can be life-threatening as it is prone to a mass loss of blood.
However, like other blood products, the platelets are short in shelf life, and are easy to be contaminated with blood pathogens such as hepatitis B virus and AIDS virus, and often elicit allergenic reactions among recipients.

Method used

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  • Platelet promoting protein and the usage thereof
  • Platelet promoting protein and the usage thereof
  • Platelet promoting protein and the usage thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0028] Isolation of PPP that Interacts with the Extracellular Domain of MPL by Using a Yeast Two-Hybrid System

[0029] 1.1 Construction of a Bait Protein Plasmid pLexA-MPL-EC

[0030] The primers MPLEC-F and MPLEC-R, with EcoRI and XhoI incorporated, were synthesized based on the sequence of MPL-EC as follows:

MPLEC-F:5′-CCGGAATTCCAAGATGTCTCCTTGCTGGCATCAGA-3′;MPLEC-R:5′-CCGCTCGAGTTATCCGACCACGAGCTCCAGGG-3′∘

[0031] MPL-EC was PCR amplified with using the total human DNA as the template as indicated in FIG. 1. The PCR reaction mixture of a total 50 μl contained 1×PCR reaction buffer, 5 μM MPLEC-F, 0.5 μM MPLEC-R, 1 μg human total DNA, 2U Taq DNA polymerase (Fermentas), 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 1.5 mM MgCl2. The PCR program used was: 94° C., 5 min; then 30 cycles of 94° C., 1.5 min, 55° C., 1 min, 72° C., 2 min; with an additional of 72° C., 10 min at the end of the program. The resultant PCR product of approximately 1,450 bps long was separated by and purified from ...

example 2

Construction of PPP Expression Vector pET-28b and the Expression and Purification of PPP

[0035] PPP was cloned into His-tag containing expression vector pET-28B (Novagen) (FIG. 3). The expressed protein His-PPP carried six continuous histidine residues at the N-terminus and can be purified by affinity chromatography.

[0036] pET-28b contains multiple cloning sites. The primes PPP-F and PPP-R were designed based on the restriction sites on the vector and the cDNA sequence of PPP:

PPP-F:5′-CGGGATCCGATGGGCGGAGAGCAGGAGGAGGA-3′,containing BamHI (underlined);PPP-R:5′-CCGCTCGAGCTAGTTGAATTTAGCCTTGGAAA-3′,containing XhoI (underlined).

[0037] PPP DNA was amplified with using pB42AD-PPP DNA as the template. The PCR reaction mixture of a total 50 μl contained 1×PCR reaction buffer, 0.5 μM PPP-F, 0.5 μM PPP-R, 1 μg pB42AD-PPP DNA, 2U Taq DNA polymerase (Fermentas), 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 1.5 mM MgCl2. The PCR program used was: 94° C., 5 min; then 30 cycles of 94° C., 1 mi...

example 3

His-PPP Stimulates Platelet Formation in BALB / c Mice

[0039] The procedures of protein injection and venous platelet measurement were based on Kaushansky et al., Nature, Vol. 369, 1994, 565-568 with modifications.

[0040] The His-PPP purified as described at Example 2 was diluted into a stock solution of 10 μg / ml with PBS containing 0.1% BSA and used for the injection. Thirty normal male BALB / c mice of 6-7 week old were divided randomly into three groups of ten mice each. The first group was subcutaneously injected with 10 μg / kg His-PPP once per day for seven consecutive days; the second group was subcutaneously injected with 50 μg / kg His-PPP once per day for seven consecutive day; and the third group (control) was subcutaneously injected with PBS containing 100 μg / ml of BSA once per day for seven consecutive days. Twenty μl venous blood was collected from a small lateral cut in the tail vein on day 0, 4, 7, 10, 13, 16 and 19. The platelets were counted using of an F-820 Sysmex electr...

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Abstract

The present invention discloses a protein that has strong affinity to thrombopoietin receptor (C-MPL) and the nucleotide sequences of the protein. The protein is capable of increasing the numbers of platelets and enhancing the blood clotting in vivo and is named as platelet promoting protein (PPP). The protein and its nucleotide sequences can be used for the treatment of blood diseases including thrombocytopenia.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a bio-medicament, and more specifically relates to a protein for increasing the numbers of platelets and its applications in the treatment of blood diseases. BACKGROUND OF THE INVENTION [0002] As an important component of blood, platelets are responsible for hemostasis in response to vascular injury and involved in the repairment of injured blood vessels. Low level of blood platelets can be life-threatening as it is prone to a mass loss of blood. At the present, platelet transfusion is a top choice for treatment for patients of thrombocytopenia. However, like other blood products, the platelets are short in shelf life, and are easy to be contaminated with blood pathogens such as hepatitis B virus and AIDS virus, and often elicit allergenic reactions among recipients. [0003] Thrombopoietin (TPO) plays its role of growth factor for thrombopoiesis by binding to its receptor MPL, which is made up of three parts, MPL-EC(26-49...

Claims

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Application Information

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IPC IPC(8): A61K38/17C07K14/47C07H21/04C12P21/06
CPCA61K38/00C07K2319/21C07K14/52A61P7/00A61P7/04
Inventor XU, PEILINGE, YICHENYANG, YAN
Owner XU PEILIN
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