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, frequent injection and administration, complicated preparation process, etc. No immunogenicity, reduce the number of injections, and improve the effect of protein activity

Active Publication Date: 2014-02-05
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 expression level of recombinant hFSH produced by the existing method is too low, and the preparation process is complicated. The cost is too high; secondly, its half-life is short, requiring frequent injections

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

[0067] Example 1. Construction of gene expression vector encoding recombinant hFSH-Fc fusion protein

[0068] 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.

[0069] The fusion gene L-vIgG4Fc encoding flexible peptide linker (Linker, detection "L") and Fc variant (vIgG4Fc and vIgG1Fc) fragments containing BamHI (5' end) and EcoRI (3' end) restriction sites were artificially synthesized respectively And L-vIgG1Fc. The obtained fusion gene fragments were inserted into transfer vectors such as PUC19 between the BamHI...

Embodiment 2

[0072] Example 2. Stable expression of recombinant hFSH-Fc fusion protein in mammalian cells

[0073] 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 method, using Gene Pulser Ehectroporator (Bio-Rad Laboratories, Hercules, CA) with 960μFd capacitor, setting the electric field to 250V, and 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 100μg / mL Zeocin resistance marker gene to obtain transfectants that had been screened for resistance. Use western blotting to detect the expression of hFSH-Fc with anti-hFSH antibody, such as Figure 7 . Using DHFR to amplify the selectable marker gene to increase the expression level of the recombinant ...

Embodiment 3

[0074] Example 3. Production and purification of recombinant hFSH-Fc fusion protein

[0075] 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 expressed product and sialic acid. The preferred cell culture conditions are: adding 100μM Cu2+ to the basal medium, and 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 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...

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Abstract

The invention discloses a long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (referred to as hFSH-Fc) and a preparation method thereof. The hFSH-Fc protein is dimerization fusion protein, the amino acid sequence of the protein sequentially comprises an hFSH beta subunit, CTP, a hFSH alpha subunit, a flexible peptide joint and a human IgG Fc variant from the N end to the C end, and the in vivo half-life period of the protein is longer than that of the existing human follicle-stimulating hormone, so that the efficacy is better. The invention also relates to a use of a recombinant hFSH-Fc fusion protein composition in preparation of drugs in the animal breeding field.

Description

Technical field [0001] The invention relates to the fields of molecular biology and veterinary medicine. More specifically, the present invention relates to 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 in the field of animal reproduction is better than 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. The currently marketed FSH is mainly a biochemical species extracted from the pituitary gland of pigs, which can be used for early estrus and simultaneous estrus of young sows, and non-estrous females overdue. Pigs promote estrus, and they are also widely used in cattle and sheep breeding. The biochemically extracted FSH has defects such as virus contamination, limited sourc...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C07K1/22C07K1/20A61K38/24A61K47/48A61P15/00
Inventor 侯永敏雷瑶李屹晨
Owner GUANGZHOU VBIO PHARM CO LTD
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