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Recombinant human follicle-stimulating hormone and its preparation method

A follicle-stimulating hormone, alpha subunit technology, applied in the protein field, can solve the problems of expensive recombinant FSH products and unaffordable patients

Active Publication Date: 2021-10-01
JINGZE PHARMA (HEFEI) CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to technical and financial reasons, recombinant FSH products are significantly more expensive than urine-derived FSH products, and the average patient cannot afford it financially

Method used

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  • Recombinant human follicle-stimulating hormone and its preparation method
  • Recombinant human follicle-stimulating hormone and its preparation method
  • Recombinant human follicle-stimulating hormone and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0114] The invention provides a method for preparing recombinant follicle-stimulating hormone, comprising the following steps:

[0115] (1) Construct the eukaryotic expression vector PGM that can express both FSHα subunit gene and FSHβ subunit gene, and contains DHFR expression unit and KTPA-CMV.

[0116] (2) The FSHα subunit gene and the FSHβ subunit gene are operatively inserted into PGM to obtain the transfection vector PGM / rhFSH capable of simultaneously expressing the FSHα subunit protein and the FSHβ subunit protein, wherein the FSHα subunit gene contains The cDNA sequence and the upstream intron sequence of the subunit protein, the FSHβ subunit gene includes the cDNA sequence and the upstream intron sequence encoding the FSHβ subunit protein;

[0117] (3) transfecting the transfection vector into eukaryotic cells and culturing; and

[0118] (4) The culture supernatant is sequentially subjected to ultrafiltration, ammonium sulfate precipitation, hydrophobic chromatograp...

Embodiment 1

[0145] Example 1: Human FSHα subunit gene and FSHβ subunit gene sequence cloning

[0146] The inventors found that the FSH α subunit gene can express the final FSH α subunit, but the expression level of the FSH α subunit is not high. Based on experience, 8 FSH α subunit genes were designed by introducing enzyme cleavage sites, KOZAK sequences and signal peptides The sequence was used for expression research, including SEQ ID NO: 3. After research, it was found that the expression levels of the designed 8 FSHα subunit sequences were different, some with high expression levels, and some with low expression levels. Among them, the FSHα subunit gene SEQ ID NO: 3 has the highest expression level, so SEQ ID NO:3 was selected for cloning.

[0147] In addition, the FSHβ subunit gene can express the final FSHβ subunit, but the expression level of the FSHβ subunit is not high. Based on experience, 10 FSHβ subunit gene sequences were designed for expression by introducing restriction sit...

Embodiment 2

[0149] Embodiment 2: the generation of PGM

[0150] The dhfr gene expression unit on the pSV2-dhfr vector (ATCC 37146) was cloned into the pcDNA3.1(+) vector (Invitrogen). The inventors designed and introduced the Blg II and Mlu I restriction sites, screened to obtain the sequence comprising SEQ ID NO: 5 and SEQ ID NO6, and performed routine PCR on the dhfr gene expression unit on the pSV2-dhfr vector (ATCC 37146) clone. The cloning product was confirmed by electrophoresis on 1% agarose gel (containing EB), and its length was 1.1kb, which was consistent with the prediction (see figure 1 ). The dhfr gene expression unit includes SV40 early promoter, dhfr coding region, SV40 termination and polyA signal sequence.

[0151] Table 1 - Oligonucleotides

[0152]

[0153]

[0154] The cloned dhfr gene expression unit was double-digested (Blg II and MluI) using a conventional enzyme digestion system, and the digested fragments were recovered with a gel recovery kit. Similarl...

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Abstract

The invention provides a recombinant human follicle-stimulating hormone and a preparation method thereof. Specifically, steps are included: (1) the first expression cassette for expressing FSH α subunit and the second expression cassette for expressing FSH β subunit are operatively inserted into the eukaryotic expression vector to obtain the protein capable of expressing FSH α subunit and the transfection vector of FSH beta subunit protein; (2) transforming the transfection vector into eukaryotic cells, thereby obtaining the transformed eukaryotic cells integrated with the first expression cassette and the second expression cassette in the chromosome; (3) Under conditions suitable for expression, the transformed eukaryotic cells are cultivated to express the FSHα subunit and the FSHβ subunit, thereby obtaining a fermentation product containing recombinant follicle-stimulating hormone; (4) to the described The fermentation product is separated and purified to obtain the recombinant follicle-stimulating hormone. The method of the invention can replace the use and preparation method of the existing human FSH, and obtain rhFSH with significantly improved activity and purity.

Description

technical field [0001] The invention belongs to the technical field of proteins, and in particular relates to a polypeptide with follicle hormone activity and a preparation method for the polypeptide. Background technique [0002] Low FSH concentration due to insufficient FSH synthesis and secretion is the main cause of female and male infertility. In women, this condition is characterized by anovulation or abnormal ovulation; in men, infertility may result from insufficient production of viable spermatozoa. In the diagnosis and treatment of infertility, FSH is a bioactive molecule that plays an important role. FSH plays an important role in human assisted reproductive technology. It is the main drug used to treat male and female infertility in the market. One of the drugs. [0003] FSH is a heterodimeric glycoprotein with a molecular weight of about 31KD, composed of non-covalently bonded α subunits and β subunits, of which the relative molecular weight of the α subunit i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/16C07K14/59C07K1/36C07K1/30C07K1/20C07K1/16
CPCC07K14/59C12N15/85
Inventor 彭红卫张玉晶张磊王冲叶凡俞峻红赵家跟李晓鹏俞亚波
Owner JINGZE PHARMA (HEFEI) CO LTD
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