Erythrocyte generating characteristic-free erythropoietin with nervous protecting function and application thereof

A technology of erythropoietin and production characteristics, applied in new erythropoietin and its pharmaceutical field, can solve the problems of losing or losing the function of stimulating erythropoiesis, so as to increase safety, avoid secondary thrombosis, expand Effects of treatment groups

Inactive Publication Date: 2010-03-17
曹国栋 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that if point mutations occur in the above three regions, the EPO molecule will lose the ability to bind to the EPO receptor, thereby losing the function of stimulating red blood cell production

Method used

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  • Erythrocyte generating characteristic-free erythropoietin with nervous protecting function and application thereof
  • Erythrocyte generating characteristic-free erythropoietin with nervous protecting function and application thereof
  • Erythrocyte generating characteristic-free erythropoietin with nervous protecting function and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Site-directed mutagenesis of EPO cDNA

[0054] Human EPO cDNA was amplified from the human kidney cDNA library, and 62 new mutant EPO cDNAs were obtained by site-directed mutation by PCR method, and these mutated EPO cDNAs were loaded into eukaryotic expression vectors. The detailed steps are as follows:

[0055] 1. Amplification of human EPO cDNA

[0056] Human EPO cDNA encodes 193 amino acids. After the EPO primary protein is synthesized, the signal peptide containing 27 amino acid residues at the amino terminal becomes mature EPO after cleavage. Mature human EPO is thus 166 amino acids. Based on the above research results, a histidine tag (His-6) for isolation and purification was added to the 3' end of EPO cDNA. Human EPO cDNA containing the His-6 tag was amplified by PCR from a human kidney Marathon-ready cDNA library (Clontech, Mountain View, USA). Primers were designed according to the published sequence of human EPO cDNA (gene sequence number: NM00...

Embodiment 2

[0188] Example 2: Preparation and purification of mEPO fusion protein

[0189] The vector pcDNA-mEPO-his6 was introduced into 293 cell line with transfection reagent Lipofectamine 2000 (Invitrogen, USA). After 24 hours of transfection, subculture at 1:12, add G4 181 mg / ml (Invitrogen, USA) for culture for 2 weeks, and then select a single clone and transfer it to a 6-well plate for further culture. Take 10 μl of medium and use Western blot or ELISA to identify cell lines with high expression of mEPO after diluting 1000 times. The expression of EPO was identified by Western blot and ELISA in the supernatant, the results are shown in figure 2. After a large amount of mEPO high-expressing cell lines were amplified, the culture medium was collected and purified by Ni-NTA agarose chromatography column (QIAGEN, Valencia, USA) to recover and purify the mEPO fusion protein. Briefly, the medium was adjusted to pH 8.0 with NaOH and 5M NaCl was added to a final concentration of 300 m...

Embodiment 3

[0191] Example 3: Detecting whether mEPO has erythropoietic properties

[0192] We investigated whether these mEPOs have erythropoietic properties using in vivo and in vitro experiments.

[0193] 1. In vitro analysis of biological characteristics of mEPO erythropoiesis

[0194] 32D-EPOR cells are cell lines formed after the lymphoblastoid 32D cells derived from mouse bone marrow are stably transfected with EPO receptors. analyze. 32D-EPOR cells were seeded in six-well plates with IDEM medium (Invitrogen), about 2×10 per well 5 cells. After 6 hours of starvation (without the presence of EPO), wild-type EPO (1 U / ml) or mEPO (1 and 100 U / ml) or PBS (control) were added, and stained with placenta blue after 72 hours, the number of viable cells was counted. In the presence of wild-type EPO, 32D-EPOR cells proliferated approximately 13-fold (2.7x10 7 ), while mEPO was the same as the PBS control, only 0.1% of the cells survived, even after increasing the concentration of mEPO b...

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Abstract

The invention relates to an erythrocyte generating characteristic-free erythropoietin with a nervous protecting function and an application thereof. The erythropoietin is an allosteric variant generated by the mutation of at least one of the three areas and other 28 interspersed loci of a wild type human erythropoietin and the three areas are as follows: (1) VLQRY: No. 11-15 amino acid of the human erythropoietin; (2) TKVNFYAW: No. 44-51 amino acid of the human erythropoietin; and (3) SGLRSLTTL: No. 100-108 amino acid of the human erythropoietin. The invention has the nervous protecting function but does not have the erythrocyte generating function, prevents the formation of secondary thrombus caused by general erythropoietin and improves the safety of clinical application; the treatment crowd is expanded so that a cerebral apoplexy patient which has higher blood viscosity and can not carry out treatment by the general erythropoietin is treated; and the invention sill has the effect ofobviously improving the nervous function by being taken after 48 hours and can be injected for a long time without simulating the blood system.

Description

technical field [0001] The present invention relates to a new type of erythropoietin and its use, more specifically to provide a new type of erythropoietin with neuroprotective effect but no side effects of erythropoiesis and its use in pharmaceuticals, belonging to biological pharmaceutical field. Background technique [0002] Erythropoietin (EPO) is a growth factor synthesized and secreted by the kidneys that stimulates the production and maturation of red blood cells (erythropoiesis). It was originally believed that EPO was only related to the production and maturation of red blood cells, but recent studies have found that EPO has a strong role in promoting neuron survival (neuroprotection) in addition to its role in promoting erythropoiesis. In vitro experiments have shown that EPO can reduce or inhibit neuronal damage caused by NMDA toxicity, free radicals and other factors. A large number of animal experiments have shown that EPO injection can reduce the size of isch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/505C07K19/00A61K38/18A61P25/00A61P9/10A61P13/12
Inventor 曹国栋陈俊罗玉敏吉训明邢娟
Owner 曹国栋
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