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New erythrocyte-stimulating factor analogues

A technology for erythropoietin and analogs, applied in the field of erythropoietin analogs or their variants, recombinant plasmids and host cells, and can solve problems such as decreased in vitro activity

Active Publication Date: 2009-01-07
SHENYANG SUNSHINE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, another study showed that if the asparagine or serine residues as glycosylation sites were mutated to remove N-linked or O-linked oligosaccharide chains individually or together, it would make the mammalian cells The in vitro activity of mutant proteins produced in the

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Construction of Human Erythropoietin Analog DNA Fragment

[0047] There are three natural N-glycosylation sites in the amino acid sequence of human erythropoietin, which are respectively located at positions 24, 38 and 83. The positions that promote erythropoietin to produce additional glycosylation sites can be 2, 3, 4, 28, 30, 88 (see figure 1 ).

[0048] Combination between mutation groups for the above positions, such as constructing [Asn 2 Gly 3 Thr 4 ], [Asn 28 Thr 30 ], [Asn 88 Gly 89 Ser 90 ], adding N-glycosylation sites at positions 2, 28, and 88, and enhancing the glycosylation at this position by changing the amino acid sequence of the middle position or the position before the glycosylation site.

[0049] For primers additionally adding N-glycosylation site mutations, refer to SEQ ID NOS: 2-85. Using these primers, the erythropoietin analogue DNA fragment after mutation can be obtained by performing polymerase chain reaction (PCR) with ...

Embodiment 2

[0133] Example 2: Asn 2 Gly 3 Thr 4 Preparation of EPO

[0134] A. Construction, screening and cultivation of engineered cells

[0135] The plasmid pEC4-N1 constructed according to the method of Example 1 was transfected into CHO cells. The host cells were purchased from the American Type Culture Collection (ATCC) and were dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO dhfr-). Culture the cells in a 100mm culture dish, and when the cells grow to 60-95% full, rinse the cells with serum-free medium, add a transfection mixture composed of 5ml serum-free medium, 10μg pEC4-N1, and 60μg lipofectamine , cultivated at 37°C for 4 hours, aspirated the medium, added MEM medium containing 5% fetal calf serum, and cultivated overnight at 37°C. Then add MTX to the culture medium and continue to culture for 10-14 days until resistant clones appear. The cultured cells of the resistant clone were digested with trypsin, the MTX concentration was increased, and the resis...

Embodiment 3

[0144] Example 3: Asn 3 Phe 4 Ser 5 Preparation of EPO

[0145] A. Construction, screening and cultivation of engineered cells

[0146] The plasmid pEC4-N2 constructed according to the method of Example 1 was transfected into CHO cells. The host cells were purchased from the American Type Culture Collection (ATCC) and were dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO dhfr-). Culture the cells in a 100mm culture dish, and when the cells grow to 60-95% full, rinse the cells with serum-free medium, add a transfection mixture composed of 5ml serum-free medium, 10μg pEC4-N2, and 60μg lipoFectamin , cultivated at 37°C for 4 hours, aspirated the medium, added MEM medium containing 5% fetal calf serum, and cultivated overnight at 37°C. Then add MTX to the culture medium and continue to culture for 10-14 days until resistant clones appear. The cultured cells of the resistant clone were digested with trypsin, the MTX concentration was increased, and the resis...

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Abstract

The invention relates to an erythropoietin analog with at least one extra glycosylation sites or a variant thereof. The invention also relates to a DNA sequence for coding the erythropoietin analog or the variant thereof, and a recombinant plasmid and a host cell for expressing the analog or the variant thereof.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an erythropoietin analog or a variant thereof with at least one additional glycosylation site. The present invention also relates to the DNA sequence encoding the erythropoietin analog or its variant, as well as the recombinant plasmid and host cell expressing the analog or its variant. Background of the invention [0002] Erythropoietin (EPO) is the first hematopoietic growth factor discovered and used clinically. As early as 1906, Carnot et al. found that after blood loss in rabbits, a substance that could act on the hematopoietic system to accelerate red blood cell production would be produced in the peripheral blood, thus proposing that there was a humoral factor that regulated hematopoiesis in a feedback manner. After a lapse of more than 30 years, this view was confirmed, and this factor was named erythropoietin. Erythropoietin, also known as hemat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/505C12N15/12C12N15/79A61K38/18A61P7/06
Inventor 娄竞耿建玲张杰侯绪凤赵会林陆军苏冬梅胡金东
Owner SHENYANG SUNSHINE PHARMA
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