Production method and application of recombinant human non-glycosylated erythropoietin and polyethylene glycol modified product thereof

A technology of erythropoietin and polyethylene glycol, which is applied in the field of medicine and biology, can solve the problems of protein and polypeptide drugs in vitro drug efficacy reduction, affinity reduction, drug efficacy reduction, etc., to achieve easy purification and identification, half-life extension, and protein activity. small effect

Inactive Publication Date: 2012-07-18
GUANGZHOU JINAN BIOMEDICINE RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the physical and chemical properties of proteins and polypeptides coupled with PEG will change in spatial conformation, steric hindrance, electrostatic binding, hydrophobicity, etc., and the affinity for connecting with receptors is often reduced due to changes in these physical and chemical properties, resulting in Reduced in vitro efficacy of protein and peptide drugs after PEG modification
[0006] It can be seen that protein drugs are easily degraded by proteases in vivo, and PEG-modified protein drugs can prolong their half-life, but their in vitro efficacy generally decreases

Method used

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  • Production method and application of recombinant human non-glycosylated erythropoietin and polyethylene glycol modified product thereof
  • Production method and application of recombinant human non-glycosylated erythropoietin and polyethylene glycol modified product thereof
  • Production method and application of recombinant human non-glycosylated erythropoietin and polyethylene glycol modified product thereof

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Embodiment 1

[0044] High-efficiency expression and purification of human erythropoietin (rh-ngEPO)

[0045] 1. Preparation of SUMO-rh-ngEPO gene

[0046]The mRNA was extracted from human fetal liver cells by conventional methods, and the cDNA (579bp) of EPO was amplified by RT-PCR; then, using the cDNA of pET-3C-SUMO and EPO as templates, primers were designed to amplify SUMO and EPO respectively The gene and the intermediate 15-peptide linker gene were recovered from the gel and used as a common template for recombination PCR to amplify the SUMO-rh-ngEPO gene fragment, and Nde I and BamH I restriction sites were introduced into the upstream and downstream respectively. The reaction products were identified by 1% agarose gel electrophoresis. Gel recovery and purification of the PCR product SUMO-rh-ngEPO. The gel recovery step was carried out according to the instructions of the OMEGA gel recovery kit.

[0047] 1.1 Primer design:

[0048] Primers were designed according to the SUMO ...

Embodiment 2

[0123] PEGylation of non-glycosylated human erythropoietin 1

[0124] 1) PEG modification: In pH=6.0, 50 mmol / L phosphate buffer, rh-ngEPO protein concentration is 5.0 mg / ml, and 20kDa monomethoxypolyethylene glycol propionaldehyde is added at a molar ratio of 10:1 (mPEG-ALD), react at 25°C for 12 hours, and add glycine to terminate the reaction.

[0125] 2) Separation and purification: use Superdex 200 molecular sieve column chromatography, use pH 7.0, 50mmol / L PB containing 150 mmol / L NaCl as the mobile phase, collect the second elution peak, and obtain mPEG- ALD-20kDa-L-rhEPO.

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Abstract

The invention discloses a high-efficiency recombinant expressing and purifying method of human non-glycosylated erythropoietin. According to the high-efficiency recombinant expressing and purifying method, an h-ngEPO (human-non-glycosylated Erythropoietin) and a molecular chaperone SUMO (Small Ubiquitin-Related Modifier) sequence to construct a novel SUMO-rh-ngEPO fusion gene expression vector; the vector is transferred into host bacteria, thereby establishing engineering bacteria; the engineering bacteria are cultured, the SUMO-rh-ngEPO fusion gene is inductively expressed and an SUMO part is cut off, so that an rh-ngEPO protein is obtained. According to the high-efficiency recombinant expressing and purifying method disclosed by the invention, the soluble expression and large-scale purification of the rh-ngEPO protein in cytoplasm are realized and the obtained rh-ngEPO protein is high in activity. The invention also provides a polyethylene glycol modifying method of the human non-glycosylated erythropoietin. The half-life period of the polyethylene glycol-modified human non-glycosylated erythropoietin obtained by the modifying method is remarkably prolonged and the erythropoietin multiplication activity of the polyethylene glycol-modified human non-glycosylated erythropoietin is remarkably improved; and the modifying method can be widely applied to preparation of erythropoietin generation medicaments.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to a high-efficiency expression and purification method of non-glycosylated human erythropoietin and a method for modifying non-glycosylated human erythropoietin with polyethylene glycol. Background technique [0002] Human erythropoietin (hEPO) is a major regulator of human erythropoiesis. It mainly regulates the production of red blood cells by promoting the survival, proliferation and differentiation of erythroid progenitor cells in the bone marrow and inhibiting apoptosis. It is mainly used clinically to increase the number of red blood cells, for anemia caused by chronic renal failure, anemia in AIDS patients, aplastic anemia, anemia in patients with rheumatoid arthritis, anemia in patients with myelodysplastic syndrome, sickle cell anemia, Anemia caused by malignant tumors or chemotherapy, anemia after blood loss, tissue dissection, premature infants,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/63C07K14/505C07K17/08A61K38/18A61K47/48A61P7/06
Inventor 王一飞赵振岭利奕成任哲
Owner GUANGZHOU JINAN BIOMEDICINE RES & DEV CENT
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