Human erythropoietin Fc fusion protein with high bioactivity

A fusion protein, biologically active technology, applied in the fields of molecular biology and medicine, can solve the problems of prolonging EPO derivatives and having no half-life

Inactive Publication Date: 2006-01-04
PHARMAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are no satisfactory EPO derivatives with significantly prolonged half-life so far.

Method used

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  • Human erythropoietin Fc fusion protein with high bioactivity
  • Human erythropoietin Fc fusion protein with high bioactivity
  • Human erythropoietin Fc fusion protein with high bioactivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Construction of the gene encoding the HuEPO-L-vFc fusion protein

[0025] 1.1 Construction of encoding HuEPO-L-vFc γ2 fusion protein gene

[0026] Fusion proteins are assembled from several DNA fragments. To obtain genes encoding the leader peptide and mature protein of human EPO, a human kidney cDNA library (obtained from Invitrogen, Carlsbad, CA) was used as a template for polymerase chain reaction (PCR). To facilitate cloning, SEQ ID NO: 1 introducing a restriction enzyme endonuclease site (HindIII) was used as a primer for the 5' oligonucleotide. Table 1 lists the oligonucleotide sequences used to clone the HuEPO-L-vFc fusion protein.

[0027] SEQ ID NO: 1

5'-cccaagcttggcgcggagatgggggtgca-3'

SEQ ID NO: 2

5'-cggatccgtcccctgtcctgcaggcct-3'

SEQ ID NO: 3

5'-gagcgcaaatgttgtgtcga-3'

SEQ ID NO: 4

5'-ggaattctcatttacccggagacaggga-3'

SEQ ID NO: 5

5'-tggttttctcgatggaggctgggaggcct-3'

SEQ ID NO: 6

...

Embodiment 2

[0041] Example 2. Expression of Fusion Proteins in Transfected Cell Lines

[0042] The recombinant pEFP1, pEFP2 or pEFP4 expression vector plasmids were transfected into mammalian host cell lines to express the HuEPO-L-vFc fusion protein. For stable high-level expression, a preferred host cell strain is a CHO cell deficient in the DHFR enzyme (see, eg, US Patent No. 4,818,679). A preferred transfection method is electroporation. Other methods can also be used, including calcium phosphate co-precipitation, lipofection, and protoplast fusion. In electroporation, 2-5 × 10 7 Add 10 μg of plasmid DNA linearized with BspCI to each cell. Two days after transfection, the medium was changed to growth medium containing 0.18 mg / ml G418. Transfectants resistant to selected drugs were tested for secretion of the fusion protein using an anti-human IgG Fc ELISA. Quantification of the expressed fusion protein can also be performed by ELISA using an anti-HuEPO assay. Wells producing high...

Embodiment 3

[0045] Example 3. Purification and characterization of fusion proteins

[0046] The conditioned medium containing the fusion protein was titrated to pH 7 to 8 with 1 N NaOH and filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a Prosep A column equilibrated with phosphate buffered saline (PBS). After the fusion protein is bound to Prosep A, discard the flow-through fraction. The column was washed with PBS until the OD value at 280nm was below 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer, pH 3.75. with 0.4 volumes of 1M K 2 HPO 4 For neutralization, fractions containing purified protein were pooled and dialyzed against PBS. The solution was then filtered through a 0.22 micron nitrocellulose filter and stored at 4°C. Under non-reducing conditions, the molecular weight of the purified HuEPO-L-vFc protein ranged from 120 to 140 kDa as determined by SDS-PAGE. Under reducing conditions, the purified protein migrates...

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Abstract

The present invention discloses one kind of Fc fusion protein of human EPO with similar or even higher bioactivity than rHuEPO. These kind of HuEPO-L-vFc fusion protein contains human EPO, flexible peptide joint with 20 or less amino acids and human IgG Fc variant, which has no lytic property and shows little bad Fc-mediating side effect. The present invention discloses also the method of preparing or producing this kind of fusion protein with high expression level. This kind of HuEPO-L-vFc fusion protein exhibits the effects of prolonging serum half-time, increasing bioactivity and improving dynamic performance and effect of medicine.

Description

technical field [0001] The present invention relates to the fields of molecular biology and medicine. More specifically, it relates to an Fc fusion protein of human erythropoietin with high biological activity, its preparation method and application. Background technique [0002] Erythropoietin (EPO) is a 30.4 kilodalton (kDa) glycoprotein hormone that promotes the proliferation of erythroid progenitor cells and maintains their differentiation into mature erythrocytes (see, e.g., Krantz, Blood, 77:419-434 , 1991). EPO is produced in the kidneys of adults and the liver of fetuses. In adults, EPO is primarily produced by kidney cells in response to hypoxia or anemia and circulates in the bloodstream. EPO targets a specific receptor of 66 kDa (EPO-Rc) almost exclusively on the surface of myeloid erythroid progenitor cells. Upon binding to EPO, the receptor is activated and undergoes homodimerization followed by tyrosine phosphorylation. Subsequently, a series of intracellu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/08A61K38/22A61P7/06
Inventor 金宜慧孙乃超周若芸
Owner PHARMAB
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