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Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc)

A fusion protein, hfsh-fc technology, applied in its preparation method and application, in the field of long-acting recombinant human follicle-stimulating hormone fusion protein, can solve the problems of low expression of recombinant hFSH, difficult purification, short half-life in vivo, etc., to reduce cell Toxic and side effects, improve biological activity, and prolong the half-life in vivo

Active Publication Date: 2014-12-03
UNICOHEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to provide a recombinant human follicle-stimulating hormone-Fc fusion protein (Human follicle-stimulating hormone-Fc fusion protein, referred to as hFSH-Fc), its preparation method and application, to solve the problems of low recombinant hFSH expression and purification Difficult and short half-life in vivo

Method used

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  • Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc)
  • Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc)
  • Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Construction of gene expression vector encoding recombinant hFSH-Fc fusion protein

[0064] The gene sequence design was optimized based on the preferred codons of CHO cells, and the optimized fusion gene containing the signal peptide encoding hFSH protein β chain and its mature peptide, CTP and hFSH α chain mature peptide was synthesized by artificial synthesis. The synthetic 756bp DNA fragment was inserted between the EcoRV restriction sites in the transfer vector such as pUC57 to obtain the hFSH plasmid (phFSH), and the correctness of the inserted sequence was verified by DNA sequencing.

[0065] A fusion gene L-vIgG2Fc encoding a flexible peptide linker (Linker, referred to as "L") and a human IgG2Fc variant (vIgG2Fc) fragment containing BamHI (5' end) and EcoRI (3' end) restriction sites was artificially synthesized. The obtained fusion gene fragments were respectively inserted between the BamHI and EcoRI sites of a transfer vector such as PUC19 to obtai...

Embodiment 2

[0068] Example 2. Stable expression of recombinant hFSH-Fc fusion protein in mammalian cells

[0069] The expression plasmid pCDNA3-hFSH-L-Fc constructed in Example 1 was transfected into DHFR enzyme-deficient CHO host cells (CHO-DHFR - ), figure 2 b shows a schematic diagram of the recombinant dimerized hFSH-Fc fusion protein. Transfection was carried out by electroporation, using a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) with a capacity of 960 μFd, setting its electric field to 250 V, and using 2 to 5 × 10 cells in the cuvette. 7 Add 10 μg of plasmid DNA linearized with PvuI to each cell. Two days after transfection, the medium was changed to a growth medium containing 100 μg / mL Zeocin resistance marker gene to obtain transfectants that had passed the initial resistance screening. The expression of hFSH-Fc was detected with anti-hFSH antibody by westemblotting method. The use of DHFR to amplify the selectable marker gene increases the expression ...

Embodiment 3

[0070] Example 3. Production and purification of recombinant hFSH-Fc fusion protein

[0071] The high-yield cell strain obtained in Example 2 was first acclimatized in a culture dish without serum, and then transferred to a shake flask for suspension acclimatization. During the acclimatization process, the medium was screened at the same time, and different components were added to observe the growth of the cells. Growth state, growth trend, and biochemical indicators such as the activity of the expressed product and sialic acid, the preferred cell culture conditions are: basal medium with 100 μM Cu 2+ , adding 2mM ManNAc (N-acetyl-D-aminomannose) to the feeding medium, this method can increase the glycosylation degree of the recombinant hFSH-Fc fusion protein, and increase the sialic acid content by about 20%. After the acclimatization is successful, the cells are expanded to a sufficient amount, and the 7L bioreactor is monitored for culture. When the cell density exceeds 1×...

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Abstract

The invention discloses a long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc) and a preparation method thereof. The recombinant hFSH-Fc is a dimerization fusion protein. An amino acid sequence of the hFSH-Fc comprises an hFSHbeta subunit, CTP (carboxy-terminal peptide), an hFSHalpha subunit, a flexible peptide linker and a human IgG (immunoglobulin G)2 Fc variant from the N terminal to the C terminal in sequence. The hFSH-Fc has longer half-life in vivo and smaller side effects than existing hFSH. The invention also relates to an application of a recombinant hFSH-Fc composition to preparation of drugs for treating and / or preventing infertility.

Description

technical field [0001] The invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to a long-acting recombinant human follicle-stimulating hormone fusion protein, its preparation method and application. The half-life of the fusion protein is significantly prolonged in vivo, and its curative effect is better than that of the existing human follicle-stimulating hormone. Background technique [0002] At present, the infertility rate in the world is as high as 15%, becoming the third major disease seriously affecting human health after cancer and cardiovascular and cerebrovascular diseases. The number of infertility in my country accounts for more than 10% of the number of reproductive women, and its incidence is on the rise. Human follicle-stimulating hormone (hFSH) is the main component of drugs commonly used to treat male and female infertility in the current market. The current market in my country includes FSH dr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/22C07K1/20C12N15/62C12N15/85A61K38/24A61K47/48A61P15/00A61P15/08
CPCA61K38/24C07K2319/30C07K14/59A61K38/00A61P15/00A61P15/08
Inventor 侯永敏李强吴茂柏廖莎张玉杰徐桢琦
Owner UNICOHEALTH CO LTD
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