Long-acting recombinant human chorionic gonadotrophin-Fc fusion protein

A technology of fusion protein and amino acid, applied in the fields of molecular biology and medicine, can solve the problems of short half-life in vivo, difficult purification, low expression of recombinant HCG, etc., and achieve the effects of improving biological activity, prolonging stable protein, and reducing toxic and side effects.

Active Publication Date: 2014-07-02
UNICOHEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims to provide a recombinant human chorionic gonadotrophin-Fc fusion protein (Human chorionic gonadotrophin-Fc fusion protein, referred to as HCG-Fc) and its preparation method and application, so as to solve the problem of low expression level and difficult purification of recombinant HCG in the prior art. , the problem of short half-life in vivo

Method used

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  • Long-acting recombinant human chorionic gonadotrophin-Fc fusion protein
  • Long-acting recombinant human chorionic gonadotrophin-Fc fusion protein
  • Long-acting recombinant human chorionic gonadotrophin-Fc fusion protein

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Example 1. Construction of gene expression vectors encoding recombinant HCG-Fc fusion proteins

[0063] The gene sequence design was optimized based on the preferred codons of CHO cells, and the optimized fusion gene containing the signal peptide encoding the β chain of HCG protein and its mature peptide and the mature peptide of the HCG α chain was synthesized by artificial synthesis. The 756bp DNA fragment was inserted between the EcoRV restriction sites in the transfer vector such as pUC57 to obtain the HCG plasmid (pHCG), and the correctness of the inserted sequence was verified by DNA sequencing.

[0064] 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 transfe...

Embodiment 2

[0067] Example 2. Stable expression of recombinant HCG-Fc fusion protein in mammalian cells

[0068] The expression plasmid pCDNA3-HCG-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 HCG-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 western blotting method, use anti-HCG antibody to detect the expression of HCG-Fc, such as Figure 8 . The use of DHFR to amplify the selectable marker gene increas...

Embodiment 3

[0069] Example 3. Production and purification of recombinant HCG-Fc fusion protein

[0070] Using the high-yield cell line obtained in Example 2, firstly carry out serum-free domestication culture in a petri dish, then transfer to a shake flask for suspension domestication culture, during the domestication process, carry out the screening of the medium at the same time, add different components 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 HCG-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 densi...

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Abstract

The invention discloses a recombinant human chorionic gonadotrophin-Fc fusion protein (HCG-Fc for short) and a preparation method thereof. The HCG-Fc protein is a dimerized fusion protein, and the amino acid sequence of the fusion protein sequentially comprises an HCG beta subunit, an HCG alpha subunit, a flexible peptide joint and a human IgG Fc mutant from an N end to a C end. Compared with the existing chorionic gonadotrophin, the fusion protein has longer in-vivo half life, higher bioavailability and lower side effect. The invention also relates to an application of a recombinant HCG-Fc fusion protein composition in preparation of medicaments 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 chorionic gonadotropin fusion protein, its preparation method and application. The half-life of the fusion protein in the body is significantly prolonged, the bioavailability is significantly increased, the frequency and dose of medication are greatly reduced, and the side effects are reduced. Background technique [0002] Human chorionic gonadotrophin (HCG) is the main component of drugs commonly used in the field of human reproduction, widely used in obstetrics and gynecology and infertility treatment, the main purposes include inducing ovulation, promoting sperm development, and treating dysfunctional uterine bleeding And habitual abortion, prevention and treatment of premature birth, relief of labor pain, etc. At present, HCG on the Chinese market is mainly extracted from the urine of pregnant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C07K1/22C07K1/20A61K38/24A61K47/48A61P15/08
Inventor 侯永敏李强徐桢琦吴茂柏张玉杰王安新
Owner UNICOHEALTH CO LTD
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