Long-acting recombinant follicle-stimulating hormone and application thereof

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, complicated preparation process, frequent injection and administration, etc. The effect of prolonging the half-life in vivo, improving protein activity and reducing the number of injections

Active Publication Date: 2014-01-29
GUANGZHOU VBIO PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, recombinant hFSH (Human follicle-stimulating hormone, referred to as hFSH), a human drug produced by CHO cells, is on the market, but there are still the following problems: first, the expr...

Method used

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  • Long-acting recombinant follicle-stimulating hormone and application thereof
  • Long-acting recombinant follicle-stimulating hormone and application thereof
  • Long-acting recombinant follicle-stimulating hormone and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Construction of gene expression vector encoding recombinant hFSH-Fc fusion protein

[0062] 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.

[0063] The fusion gene L-vIgG2Fc encoding the flexible peptide linker (Linker, detect "L") and the Fc variant (vIgG2Fc) fragment containing BamHI (5' end) and EcoRI (3' end) restriction sites were artificially synthesized respectively. The obtained fusion gene fragments were respectively inserted into transfer vectors such as PUC19 between the BamHI and EcoRI sites to ob...

Embodiment 2

[0066] Example 2. Stable expression of recombinant hFSH-Fc fusion protein in mammalian cells

[0067] 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 capacitance of 960 μF, setting its electric field to 250 V, and 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. Using the method of western blotting, use anti-hFSH antibody to detect the expression of hFSH-Fc, such as Figure 6 . The use of DHFR to amplify the selectable marker gene...

Embodiment 3

[0068] Example 3. Production and purification of recombinant hFSH-Fc fusion protein

[0069] 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: adding 100 μM Cu2+ to the basal medium, adding 2 mM Man NAc (N-acetyl-D-aminomannose) to the feeding medium ), the 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 ...

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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 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 better efficacy than existing FSH. The invention also relates to an application of a recombinant hFSH-Fc composition to preparation of drugs in the field of animal breeding.

Description

technical field [0001] The invention relates to the fields of molecular biology and veterinary medicine. More specifically, the present invention relates to a long-acting recombinant human follicle-stimulating hormone fusion protein, its preparation method and application. The in vivo half-life of the fusion protein is significantly prolonged, and its curative effect in the field of animal reproduction is better than that of existing follicle-stimulating hormone products. Background technique [0002] Follicle-stimulating hormone (FSH) is the main ingredient of drugs commonly used in the field of animal reproduction. FSH currently on the market is mainly a biochemical variety extracted from the pituitary gland of pigs. It can promote estrus in pigs and has wider application in the fields of cattle and sheep breeding. Biochemically extracted FSH has defects such as virus contamination, limited sources of raw materials, difficult collection, low content and complicated purif...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/20C12N15/62C12N15/85A61K38/24A61K47/48A61P15/00
CPCA61K38/00A61P15/00A61P5/06C07K14/59C07K2319/00C07K2319/30C07K2319/75C12N15/79C12N2800/60
Inventor 侯永敏吴茂柏李屹晨雷瑶徐桢琦
Owner GUANGZHOU VBIO PHARM CO LTD
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