Novel high-glycosylation erythropoietin immune fusion protein

An erythropoietin and fusion protein technology, applied in the field of bioengineering, can solve the problems of inconvenience to patients, short efficacy of EPO monomer drugs, frequent drug use, etc.

Active Publication Date: 2010-10-27
BEIJING JINGYI TAIXIANG TECH DEV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, due to the short efficacy of EPO monomer drugs, the half-life of rhEPO in the human

Method used

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  • Novel high-glycosylation erythropoietin immune fusion protein
  • Novel high-glycosylation erythropoietin immune fusion protein
  • Novel high-glycosylation erythropoietin immune fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0051] Example 1: Construction of NESP-Fc immune fusion protein expression vector

[0052] 1. Optimization of NESP-Fc immune fusion protein expression gene

[0053] There are many factors that affect cell protein expression output. When genes contain a large number of rare codons, protein expression yield will be reduced. Because the fusion protein will be expressed in the cell, according to the biased codon table expressed by CHO (see Holm L. Nucleic Acids Res. 1986 Apr 11; 14(7): 3075-87), the new erythropoiesis stimulating protein (NESP) and The DNA sequence encoded by IgG2 is optimized and the amino acid residues of the encoded protein remain unchanged. For the amino acid residue sequence of neo-erythropoiesis stimulating protein (NESP), please refer to patent US20030104996, and for the amino acid residue sequence of IgG2 protein, please refer to>gi|243169|gb|AAB21082.1|. In order to ensure the high-efficiency expression of the protein, the signal peptide sequence of the fusi...

Example Embodiment

[0089] Example 2: Expression and purification of NESP-Fc immune fusion protein

[0090] 1. Transfection of NESP-Fc immune fusion protein expression vector into 293F cells

[0091] 293F (purchased from Invitrogen, Cat No. 11625-019) cells were cultured in serum-free CD293 medium (purchased from Invitrogen, Cat No. 11913-019), centrifuged before transfection to replace fresh medium, and the cell concentration was adjusted 1×10 6 Cells / ml. Taking 100ml cells as an example, DNA (250ug) and PEI (500ug, Sigma, Cat. No: 408727) were added to 1ml 293 culture medium and mixed, and then left to stand for 5 minutes. Leave it at room temperature for 8 minutes. Add the PEI / DNA suspension dropwise to the shake flask, mix gently, and place in 5% CO 2 , 37°C shaker culture (115rpm) for 5 days and collect the culture supernatant.

[0092] 2. Detection of the concentration of immune fusion protein

[0093] The sandwich ELISA method was used to detect the transient expression of the immune fusion pro...

Example Embodiment

[0098] Example 3: Confirmation of the structure of NESP-Fc immune fusion protein:

[0099] Take the purified fusion protein for protein electrophoresis and analyze the purified product.

[0100] There was no change in the molecular weight of the protein before and after dialysis. The molecular weight of the fusion protein under reducing and non-reducing conditions was 72kDa and ~200kDa, respectively. See attached Figure 5 .

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Abstract

The invention discloses novel high-glycosylation erythropoietin immune fusion protein developed by a genetic engineering method, a polynucleotide for encoding the immune fusion protein and a method for preparing and purifying the immune fusion protein. The novel high-glycosylation erythropoietin immune fusion protein is dipolymer protein NESP-Fc formed by fusing new erythropoietin stimulating protein (NESP) with human IgG2-Fc. The immune fusion protein can be expressed efficiently in mammalian cells, has a simple purification process, and is favorable for further large-scale preparation. The immune fusion protein NESP-Fc has the biological activity which is similar to that of natural EPO, has long serum half-life period, and can be used for treating anemia caused by low EPO level.

Description

Technical field: [0001] The invention relates to an immune fusion protein for anemia, in particular to a novel hyperglycosylated erythropoietin immune fusion protein, which belongs to the technical field of bioengineering. Background technique: [0002] Anemia refers to a pathological state in which the amount of hemoglobin, red blood cell count and hematocrit in unit volume of circulating blood is lower than normal. There are many reasons for anemia. It is clinically found that patients with cardiovascular disease and chronic renal disease are often accompanied by anemia. [0003] According to the statistics of the World Health Organization: about 3 billion people in the world suffer from anemia in varying degrees, and tens of millions of people die every year due to various diseases caused by anemia. The population probability of suffering from anemia in China is higher than that of Western countries. Among the people suffering from anemia, women are significantly higher ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10C12P21/02A61K38/18A61K47/48A61P7/06A61K47/42
Inventor 程虹杨思仪马金伟李桂珠刘芳杰扈艳红胡品良李先钟喻志爱林峰何丽华白先宏
Owner BEIJING JINGYI TAIXIANG TECH DEV
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