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

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

Active Publication Date: 2014-01-29
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 is optimized based on the preferred codons of CHO cells, and artificially synthesized methods are used to synthesize optimized fusion genes containing the signal peptide encoding hFSH protein β chain and its mature peptide, CTP and hFSH α chain mature peptide. The synthetic 756bp DNA fragment was inserted between the EcoRV restriction sites in a transfer vector such as pUC57, and the hFSH plasmid (phFSH) was obtained. DNA sequencing was used to verify the correctness of the inserted sequence.

[0065] The 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 were artificially synthesized. The obtained fusion gene fragments were inserted into the transfer vector such as PUC19 between the BamHI and EcoRI sites to...

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, and Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) with a 960μFd capacitor was used, and the electric field was set to 250V, 2~5×10 in the cuvette 7 10μg plasmid DNA linearized with PvuI was added 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 undergone preliminary screening for resistance. The westemblotting method was used to detect the expression of hFSH-Fc with anti-hFSH antibody. Using DHFR to amplify the selectable marker gene to increase the expression level of t...

Embodiment 3

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

[0071] The high-yield cell line obtained in Example 2 was firstly cultured in a culture dish with serum-free domestication, and then transferred to a shake flask for suspension domestication. During the domestication process, the medium was screened at the same time, and different components were added to observe the cell Growth status, growth trend, and biochemical indicators such as the activity of the expression product and sialic acid. The preferred cell culture conditions are: 100μM Cu added to the basal medium 2+ Add 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 domestication is successful, the cells are expanded to a sufficient amount, and the 7L bioreactor monitors the culture. When the cell density exceeds 1×10 7 The temper...

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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 in vivo is significantly prolonged, and its curative effect is better than the existing human follicle stimulating hormone. Background technique [0002] At present, the worldwide infertility rate is as high as 15%, making it the third most serious disease 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 ingredient of drugs commonly used to treat male and female infertility in the current market. The current market in my country includes human urine-derived FSH...

Claims

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Application Information

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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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