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A recombinant method for production of an erythropoiesis stimulating protein

A technology of erythropoietin and red blood cells, which is applied in the field of erythropoietin, can solve the problems of short plasma half-life and limitation, and achieve the effect of reducing clearance time, increasing activity, restoring health status and quality of life

Inactive Publication Date: 2008-07-23
阿维斯塔金格兰技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bioavailability of commercially available protein therapeutics, such as EPO, is limited due to its short plasma half-life, susceptibility to protease degradation

Method used

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  • A recombinant method for production of an erythropoiesis stimulating protein
  • A recombinant method for production of an erythropoiesis stimulating protein
  • A recombinant method for production of an erythropoiesis stimulating protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Synthesis of recombinant erythropoietic-stimulating protein (NESP)

[0040] The DNA sequence encoding the highly glycosylated form of human erythropoietin was obtained by de novo synthesis method. This approach will allow better codon optimization for the particular mammalian cell to be used. In addition, synthetic DNA is used for eukaryotic / prokaryotic expression purposes, providing isolatable quantities of polypeptides exhibiting the biological properties of naturally occurring erythropoietin (EPO), as well as the in vivo and in vitro biological activities of EPO.

[0041]The nucleotide sequence encoding the erythropoietic-stimulating protein is shown in SEQ ID No.1. Nucleotide residues in the erythropoietin protein that are altered by incorporation of additional glycosylation sites compared to the naturally occurring transcript of the human gene encoding erythropoietin are highlighted in capital letters .

[0042] As part of the codon optimization proce...

Embodiment 2

[0045] Example 2: Authenticity verification of de novo synthesized cDNA encoding erythropoietic proteins

[0046] The authenticity of the original cDNA sequence (AVCIP-Nesp) and codon-optimized cDNA sequence (AVCIP-Nesp-Opt) synthesized from scratch was verified by automated DNA sequencing, and the results are shown in Figure 2 and Figure 3.

Embodiment 3

[0047] Example 3: Subcloning AVCIP-Nesp and AVCIP-Nesp-Opt cDNA into mammalian cell-specific expression vector pcDNA3.1D / V5-His

[0048] The de novo synthesized original cDNA sequence (AVCIP-Nesp) and codon-optimized cDNA sequence (AVCIP-Nesp-Opt) were subcloned into the mammalian cell-specific expression vector pcDNA3.1D / V5-His to produce ready-to-use Transfected constructs. Details of the method used are given below:

[0049] A. Reagents and Enzymes:

[0050] 1. QIAGEN Gel Extraction Kit and PCR Purification Kit

[0051] 2. pcDNA3.1D / V5-His vector DNA (Invitrogen)

[0052]

[0053] All reactions were performed according to the manufacturer's recommended procedures. For each reaction, dilute the provided 10x reaction buffer to a 1x final concentration.

[0054] B. Restriction Digestion of Vector and Insert:

[0055] method

[0056] Use the following DNA samples and restriction enzymes:

[0057]

[0058] Restriction enzyme digestion reaction:

[0059]

[0060...

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Abstract

The present invention relates to a recombinant method for the production of erythropoietin in a highly glycosylated form (5 N-linked glycosylation in total relative to the 3 N-linked glycosylation in native EPO). The increased glycosylation sites will result in a higher number of sugar chains, and a higher sialic acid content than human EPO, which will result in a longer half-life for the recombinant molecule. The present invention also relates to the construction of an expression cassette comprising a nucleic acid sequence encoding a highly glycosylated form of erythropoietin, and stable expression in host cells. The present invention also relates to optimized methods for purifying erythropoietic proteins. The recombinant EPO and its salts and functional derivatives of the present invention can contain active ingredients for increasing hematocrit in pharmaceutical compositions, and thus can be used to treat anemia and restore the health status and quality of life of patients.

Description

field of invention [0001] The present invention relates to a recombinant method for the production of a highly glycosylated form (5 N-linked glycosylation in total compared to 3 N-linked glycosylation in native EPO) of erythropoietin. The increased glycosylation sites will result in a higher number of sugar chains and a higher sialic acid content than human EPO, which will result in a longer half-life of the recombinant molecule. [0002] The invention also relates to the construction of an expression cassette comprising a nucleic acid sequence encoding a highly glycosylated form of erythropoietin, and to its stable expression in a host cell. [0003] The present invention further relates to an optimized method for purifying erythropoiesis stimulating protein. [0004] The recombinant EPO of the present invention and its salts and functional derivatives can contain active ingredients in pharmaceutical compositions for increasing hematocrit, and thus can be used to treat anemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/505
CPCA61K38/00C07K14/505A61P7/06
Inventor V·莫拉瓦拉帕特尔
Owner 阿维斯塔金格兰技术有限公司
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