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Recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc)

A fusion protein, pfsh-fc technology, applied in its preparation method and its application in animal husbandry, recombinant porcine follicle-stimulating hormone fusion protein field

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

So far, no recombinant pFSH product has been used in the field of veterinary medicine

Method used

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  • Recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc)
  • Recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc)
  • Recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0068] Synthesize the fusion gene containing the signal peptide encoding pFSH protein β chain and its mature peptide, CTP and pFSH α chain mature peptide by artificial synthesis, and insert the synthesized 756bp DNA fragment into the transfer vector such as EcoRV restriction enzyme in pUC57 Between the cut sites, a pFSH plasmid (ppFSH) was obtained, and the correctness of the inserted sequence was verified by DNA sequencing.

[0069] Fusion gene L encoding flexible peptide linker (Linker, detection "L") and Fc variants (vIgG2Fc, vIgG4Fc and vIgG1Fc) fragments containing BamHI (5' end) and EcoRI (3' end) restriction sites were artificially synthesized - vIgG2Fc, L-vIgG4Fc and L-vIgG1Fc. The obtained fusion gene fragments were respectively inserted between the BamHI and EcoRI sites of a transfer vector such as PUC19 to obtain plasmids containing three variants, namely pL-vIgG2...

Embodiment 2

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

[0073] The expression plasmid pCBAEA3-pFSH-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 pFSH-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 primary resistance screening. Using the method of western blotting, use anti-pFSH antibody to detect the expression of pFSH-Fc, such as Figure 8 . The use of DHFR to amplify the selectable marker...

Embodiment 3

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

[0075] 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 pFSH-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 recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc) and a preparation method thereof. The fusion protein is a dimerized fusion protein, wherein the amino acid sequence of the fusion protein sequentially comprises pFSH beta subunits, carboxy-terminal peptide (CTP), pFSH alpha subunits, peptide connectors and human IgG Fc variants from a terminal N to a terminal C. Compared with existing pFSH, the pFSH-Fc has a longer in-vivo half-life period and better efficacies. The invention further relates to application of the pFSH-Fc to preparation of medicaments in the animal breeding fields.

Description

technical field [0001] The invention relates to the fields of molecular biology and animal husbandry. More specifically, the present invention relates to recombinant porcine follicle-stimulating hormone fusion protein (pFSH-Fc), its preparation method and its application in animal husbandry. Compared with the existing follicle-stimulating hormone derived from porcine pituitary gland, the fusion protein has a longer half-life and better drug efficacy. Background technique [0002] Porcine Follicle-stimulating hormone (pFSH) is the main ingredient of drugs commonly used in the field of animal reproduction. Currently on the market, pFSH is a glycoprotein hormone extracted from the anterior lobe of the pig pituitary gland. Its molecular weight is 43KD as detected by SDS-PAGE. pFSH promotes the development of follicles in the ovaries of sows, each containing a mature egg. When these follicles mature, they secrete estrogen, which stimulates the typical resting estrous behavior o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C07K1/22C07K1/20A61K38/24A61P15/00
Inventor 侯永敏吴茂柏李屹晨雷瑶王安新邓衡露
Owner GUANGZHOU VBIO PHARM CO LTD
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