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A long-acting fusion protein with erythropoietin activity

A technology of erythropoietin and fusion protein, which is applied to the preparation of the above compounds, recombinant proteins, and long-acting fusion proteins, and can solve problems such as changing EPO and decreasing activity

Active Publication Date: 2022-02-08
北京翼方生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PEGylation may change the structure of EPO, resulting in a significant decrease in activity

Method used

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  • A long-acting fusion protein with erythropoietin activity
  • A long-acting fusion protein with erythropoietin activity
  • A long-acting fusion protein with erythropoietin activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of a vector encoding NESP-CTP-IgG1Fc:IgG1Fc' fusion protein.

[0041]The nucleic acid sequences encoding the first strand and the second strand were artificially synthesized by Nanjing GenScript Company and respectively connected to the PUC57 vector, named pUC57nesp and PUC57Fc respectively. Double-digest PUC57nesp with AvrII and Bstz17I, double-digest PUC57Fc with EcoRV and PacI, and double-digest Invitrogen pCHO1.0 expression vector with EcoRV and PacI at the same time, recover the gene fragments from the gel, and recover the digested vector with a DNA purification and recovery kit pCHO1.0, and two gene fragments nesp and Fc.

[0042] The vector pCHO1.0 recovered by enzyme digestion was connected to Fc, and the correctly connected plasmid was named pCHO-Fc, and pCHO-Fc was digested with AvrII and Bstz17I, and the DNA purification and recovery kit was directly recovered, and the same double The nesp gene connection recovered by enzyme digestion...

Embodiment 2

[0043] Example 2. Preparation and cell culture of cell lines expressing NESP-CTP-IgG1Fc: IgG1Fc' fusion protein.

[0044] The recombinant expression vector pCHO-NESP-Fc was transformed into Escherichia coli DH5α competent cells, the transformed bacteria were rejuvenated with LB liquid screening medium containing kana and then expanded to 100ml for mass culture, shaken at 37°C overnight, and the plasmid was extracted and purified. The final plasmid DNA concentration was adjusted to 1 μg / μL for electroporation to transform CHO-S cells.

[0045] One day before transfection, the host cells were divided into 0.5×10 6 The seeding density was subcultured to 20 mL of fresh medium. Cells in the logarithmic growth phase were collected in parallel on the day of electroporation, and the number of cells in each group was 1×10 7 After centrifuging to remove the medium, wash the cells with PBS, discard the supernatant, resuspend the cells in 0.8ml PBS and add them to the electroporation cu...

Embodiment 3

[0048] Example 3. Purification of fusion proteins

[0049] The solid-liquid separation is carried out by conventional centrifugation technology, and the cell components in the fermentation system are removed. The fermentation supernatant was purified using the following steps:

[0050] A purification method for purifying an Fc fusion protein according to this embodiment specifically includes the following steps:

[0051] 1) First use Mabselect SuRe LX to capture the fusion protein, including: equilibrate the chromatographic column for 5 column volumes with affinity balance solution (25mM Tris, 25mM NaCl, pH7.5); load the sample, and the retention time of the sample is 10min; complete the loading After sampling, rebalance 4 column volumes with affinity balance solution, then wash 4 column volumes with eluent (25mM Tris, 0.3M NaCl, pH7.5); wash the layer with 50mM acetate, pH4.6 The column was analyzed with 3 column volumes, the sample was eluted with 50mM acetate, pH=3.5, and...

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Abstract

The invention relates to the field of genetic engineering drugs, in particular to a long-acting fusion protein with the activity of promoting erythropoietin, which is a recombinant protein prepared by genetic engineering technology. The fusion protein consists of two chains, the first chain contains mutant NESP or its homologous sequence with human erythropoietin activity, human chorionic gonadotropin beta subunit carboxy-terminal peptide or its homologous sequence and human IgG1Fc segment or its homologous sequence and other three functional units, the second chain contains the human IgG1 Fc segment or its homologous sequence. The invention adopts the Knob into hole technology, which is beneficial for the two chains to form a heterodimer. The fusion protein has EPO activity and longer half-life. The invention also relates to the preparation method and medical application of the fusion protein.

Description

technical field [0001] The invention relates to the field of genetic engineering drugs, in particular to a long-acting fusion protein with the activity of promoting erythropoietin, which is a recombinant protein prepared by genetic engineering technology. The invention also discloses the preparation method and medical application of the above compound. Background technique [0002] Erythropoietin (EPO), also known as erythrocyte-stimulating factor and erythropoietin, belongs to the salivary glycoprotein hormone. It was first discovered in 1906. It is an endogenous compound in the human body with a molecular weight of 34,000 Daltons. amino acid composition. It is mainly derived from the kidney (a small amount from the liver) and is synthesized by the interstitial cells surrounding the cortical tubes. The natural EPO molecule contains four stable α-helical structures, which are necessary spatial conformations to maintain the biological activity of erythropoietin. The natura...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K38/18A61K47/64A61K47/68A61P7/06
CPCC07K14/505C07K14/59A61K47/64A61K47/68A61P7/06C07K2319/30C07K2319/31A61K38/00
Inventor 李世崇吴彦卓
Owner 北京翼方生物科技有限责任公司
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