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Novel FSH-Fc fusion protein as well as preparation method and application thereof

One kind of fusion protein, another technology, application in assisted reproduction, gonadotropin, new long-acting gonadotropin and its preparation field, can solve problems such as limited application of clinical treatment

Active Publication Date: 2017-03-15
泰泽惠康生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when preparing recombinant FSH in this design, it is difficult to completely separate the by-product FSHα2 or FSHβ2 homodimer from the target product FSHαβ heterodimer, unless additional purification tags such as HIS are introduced, and the introduction of purification tags, and will limit its clinical application
Patent CN201210476665 discloses a design in which only the FSH-β subunit is fused with Fc, and the half-life of the heterodimeric FSH produced in rats is 30-40h. When preparing recombinant FSH in this design, there are two kinds of products , one is a heterodimer with the molecular form of FSHα-FSHβ-Fc2, and the other is (FSHα-FSHβ-Fc) 2 Therefore, when constructing recombinant expression cell lines and purifying products, it is also necessary to face the interference of homodimer by-products, which makes the quality control of drugs more challenging

Method used

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  • Novel FSH-Fc fusion protein as well as preparation method and application thereof
  • Novel FSH-Fc fusion protein as well as preparation method and application thereof
  • Novel FSH-Fc fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Construction of FSHα-Fch-FSHβ-Fck heterodimer expression plasmid

[0034] According to the amino acid sequence of the FSHα subunit (Gene bank No.NP_000726.1), the amino acid sequence of the human FSHβ subunit (Gene bank No.NP_000501.1) in the NCBI database, and the locked Fc shown in SEQ ID NO:1 Chain amino acid sequence (containing T148S, L150A, Y189V mutations, forming a small side chain structure) and the amino acid sequence of the button Fc chain shown in SEQ ID NO: 2 (containing T148W mutation, forming a large side chain structure), respectively artificial The coding DNA sequences of FSHα-Fch (SEQ ID NO:3) and FSHβ-Fck (SEQ ID ID NO:4) were synthesized. When synthesizing, restriction endonuclease SapI restriction endonuclease SapI restriction endonuclease restriction sites were introduced in the upstream and downstream of the coding sequence respectively.

[0035] After the coding DNA is synthesized, use the SapI restriction site to insert FSHα-Fch into...

Embodiment 2

[0037] Example 2. Expression and purification of FSHα-Fch-FSHβ-Fck heterodimeric protein

[0038] 2.1 Stable expression of FSHα-Fch-FSHβ-Fck heterodimer

[0039] CHO-K1 (ATCC, CCL-61) cells acclimatized to serum-free medium were cultured in 5×10 6 The density of cells / ml was inoculated in 30ml CD-CHO medium for culture. After 24 hours, the cell density should reach 1~2×10 6 cells / ml. Centrifuge the cell suspension at 800rpm for 5min, discard the supernatant, wash the cells twice with 50ml CD CHO medium, discard the supernatant, and resuspend in 1ml CD CHO medium. Cells and plasmids were divided into 1 × 10 7 Cells: 40 μg DNA was mixed in proportion, and electroporation transfection parameters were set: voltage 300V; capacitance 900 μF, and electric pulse was performed. Plate well 100 μL. After 24 hours, 100 μL of MSX working solution was added for pressurized screening. After the cell plate was cultured for 3 weeks, cell clones formed in the wells of the plate. Select m...

Embodiment 3

[0042] Example 3 Detection of Biological Activity of FSHα-Fch-FSHβ-Fck Heterodimer

[0043] According to Chinese Pharmacopoeia, the biological activity of FSH recombinant protein was determined by rat ovary weight gain method. HCG is used to assist the test FSH drug to stimulate immature 21-day-old female rats to trigger ovarian follicle growth and oocyte production. By measuring the weight of the ovary at the end of administration, the biological activity of the test drug can be clearly evaluated. Get 3-week-old SPF female rats with a body weight of 40-60g, divide them into 7 experimental groups according to their average body weight, set up a blank control group, high, medium and low dose positive control drug dosage groups and high, medium and low dose test drug group. The control drug Lishenbao (purchased from Livzon Pharmaceutical) was administered once a day, and the test sample FSHα-Fch-FSHβ-Fck was administered only once, and the administration method was subcutaneous...

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Abstract

The invention belongs to the field of human reproductive disease treatment and assisted reproduction and relates to a novel long-acting follicle stimulating hormone (FSH) and a preparation method and application thereof to assisted reproduction. In the invention, under the double actions of non-covalent combination between an FSH [alpha] subunit and an FSH [beta] subunit and holes type and knobs type Fc knobs-into-holes buckle structures, a stable FSH fusion protein in a form of a heterodimer is folded. The novel long-acting FSH fusion protein provided by the invention not only realizes the goals of keeping the activity of the natural FSH and introducing an Fc fragment to prolong the half-life period of the FSH, but also overcomes the defect of a production process for recombination of the FSH-Fc fusion protein caused by the use of the homologous Fc fragment, thereby having a high practical value.

Description

technical field [0001] The invention belongs to the field of human reproductive disease treatment and assisted reproduction. The present invention relates to somatotropins. More specifically, the present invention relates to a novel long-acting gonadotropin, its preparation method, and its use in assisted reproduction. Background technique [0002] Follicle Stimulating Hormone (FSH) is a glycosylated protein synthesized and secreted by basophils in the anterior pituitary gland. Natural FSH is composed of α subunit and β subunit, of which α subunit It is the same as the α subunit in other glycoprotein hormones secreted by the pituitary gland, such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), and chorionic hormone (HCG) secreted by the placenta, while the β subunit has its own Specificity is the determinant of FSH biological activity. It is known that the α subunit of FSH consists of 92-96 amino acids, and the β subunit consists of 109-115 amino acids. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/24A61P15/08
CPCA61K38/00C07K14/59C07K2319/30
Inventor 高新刘蕴慧杨懿李宏杰刘玉杰
Owner 泰泽惠康生物医药有限责任公司
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