Biological preparation for regulating ovarian function as well as preparation method and application of biological preparation

A biological preparation and ovarian function technology, applied in the field of gene recombination, can solve the problems of no treatment or health care system, anti-Müllerian hormone yolk antibody, and inability to take oral administration, etc., to achieve targeted treatment and health care, and solve High cost problem, the effect of prolonging the half-life

Pending Publication Date: 2021-11-05
安徽中起生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, relevant institutions have developed recombinant anti-Müllerian hormone and follicle stimulating hormone, but they cannot be taken orally
And there is no anti-Müllerian hormone yolk antibody, follicle stimulating hormone yolk antibody preparation technology
There is also no treatment or healthcare system composed of recombinant anti-Müllerian hormone and anti-Mullerian hormone antibody, follicle stimulating hormone and follicle stimulating hormone antibody

Method used

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  • Biological preparation for regulating ovarian function as well as preparation method and application of biological preparation
  • Biological preparation for regulating ovarian function as well as preparation method and application of biological preparation
  • Biological preparation for regulating ovarian function as well as preparation method and application of biological preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of recombinant anti-Müllerian hormone

[0035] (1) Acquisition of the gene sequence of recombinant anti-Müllerian hormone:

[0036] Referring to the GENBANK accession number NM_000479.5, the amino acid sequence of human anti-Mullerian hormone was taken, and the TAT transduction sequence was connected to the N-terminal, and the reverse translation was the target gene. The target gene sequence was sent to Huada Gene Synthesis to obtain the pYES2 / CT plasmid containing the target gene, and the sequence was qualified, and the target gene sequence as shown in SEQ ID NO:1 was obtained.

[0037] (2) Construction of engineering bacteria:

[0038] The plasmid was introduced into Invscv1 Saccharomyces cerevisiae by the PEG / LiCl method, spread on the ampicillin SD-Ura plate, and incubated at 30°C for 72 hours, and the colony grown on the SD-Ura plate was picked for preservation and sent to Huada Gene for sequencing The target gene was identified, and the se...

Embodiment 2

[0042] Example 2: PEGylation of recombinant anti-Müllerian hormone

[0043] Vacuum freeze-dry the purified target protein AMH to obtain a powder.

[0044] Prepare the reaction buffer system, the buffer system can choose acetate buffer solution with pH 4.0, 5.0, and phosphate buffer solution with pH 6.0, 7.0, 8.0.

[0045] Take the lyophilized AMH and mPEG-SPA in a molar ratio of 1:2 to 1:12, and stir in the reaction buffer system for 2h to 12h at room temperature in the dark using a magnetic stirrer. Dialyzed in 20 volumes of PBS buffer for 12 hours. 0.22 μm sterile filtration treatment, the processed sample is the PEGylated target protein, which is denoted as mPEG-AMH.

[0046] Adjust the concentration of AMH and mPEG-AMH to 1 mg / mL, use abcam company mouse anti-AMH as the primary antibody (Cat. No. ab239491), and use abcam company goat anti-mouse IgG (HRP) as the secondary antibody (Cat. No. ab6789) for WB experiment verification. The result is as figure 1 As shown, there ...

Embodiment 3

[0047] Example 3: Preparation of recombinant follicle stimulating hormone

[0048] (1) Acquisition of the gene sequence of recombinant follicle stimulating hormone:

[0049] Referring to the amino acid sequences of GENBANK accession number AAA52476.1 and GENBANK accession number ACM91588.1, a flexible linker is used in the middle, and a TAT transduction sequence is connected at the N-terminal, and the reverse translation is the target gene. The target gene sequence was sent to Huada Gene Synthesis to obtain the pYES2 / CT plasmid containing the target gene, and the sequence was qualified, and the target gene sequence as shown in SEQ ID NO:2 was obtained.

[0050] (2) Construction of engineering bacteria:

[0051] The plasmid was introduced into Invscv1 Saccharomyces cerevisiae by the PEG / LiCl method, spread on the ampicillin SD-Ura plate, and cultured at a constant temperature of 30°C for 72 hours, and the colony grown on the SD-Ura plate was picked for preservation and sent to...

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Abstract

The invention discloses a biological preparation for regulating ovarian functions as well as a preparation method and application of the biological preparation. The biological preparation is an oral preparation composed of one or more of recombinant anti-mullerian hormone, PEGylated recombinant anti-mullerian hormone, recombinant follicle-stimulating hormone, PEGylated recombinant follicle-stimulating hormone, anti-mullerian hormone egg yolk antibody (referring to Immunoglobulin of yolk) and follicle-stimulating hormone egg yolk antibody. In the biological preparation, TAT-induced peptide can promote the intestinal absorption of the protein, the PEGylated recombinant anti-mullerian hormone and follicle-stimulating hormone can prolong the half-life period, the two recombinant proteins are respectively used for immunizing animals to obtain products containing the two protein antibodies, and the products can be directly eaten and can also be prepared into granules, tablet products and the like. The recombinant anti-mullerian hormone and the egg yolk antibody product thereof, and the recombinant follicle-stimulating hormone and the egg yolk antibody product thereof form a system capable of adjusting the ovarian function, can be used for clinical treatment of ovarian dysfunction and daily health care, and is low in cost and convenient for clinical and daily health care use.

Description

technical field [0001] The invention relates to the technical field of gene recombination, in particular to a biological preparation for regulating ovarian function and its preparation method and application. Background technique [0002] There are many factors affecting ovarian dysfunction, and the most influential ones from the perspective of hormones are anti-Müllerian hormone and follicle-stimulating hormone. Anti-Müllerian hormone (AMH) is a glycoprotein belonging to the transforming growth factor β superfamily (transforming growth factor-beta, TGF-β) member, with a molecular weight of 60kD. In the process of sexual differentiation, AMH can promote the degeneration of Mullerian ducts and make mesonephric ducts develop into male reproductive organs. In female animals, the lack of anti-Müllerian hormone makes Mullerian ducts evolve into uterus and fallopian tubes. In adult women, AMH inhibits the recruitment of primordial follicles and the development of antral follicles...

Claims

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Application Information

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IPC IPC(8): A61K38/24A61K38/22A61K39/395A61K47/60A61K47/64A61P15/08C07K14/59C07K14/575C07K16/26C07K16/02C07K19/00C07K1/107C12N15/81C12N15/16
CPCA61K38/24A61K38/22A61K39/3955A61K47/60A61K47/645A61K9/0053A61P15/08C07K14/59C07K14/575C07K16/26C07K16/02C12N15/81C07K2319/10C07K2317/11A61K2300/00
Inventor 符修乐卞传忠
Owner 安徽中起生物科技有限公司
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