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Recombinant human fallicle-stimulating hormone and preparation method thereof

A human follicle-stimulating hormone and follicle-stimulating hormone technology, applied in the protein field, can solve the problems of high cost of recombinant FSH products and unaffordable economic burden of patients

Active Publication Date: 2019-10-08
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 strength and financial reasons, recombinant FSH products are obviously more expensive than urine-derived FSH products, and the average patient cannot afford it financially

Method used

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  • Recombinant human fallicle-stimulating hormone and preparation method thereof
  • Recombinant human fallicle-stimulating hormone and preparation method thereof
  • Recombinant human fallicle-stimulating hormone and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0113] The invention provides a method for preparing recombinant follicle-stimulating hormone, comprising the following steps:

[0114] (1) Construct the eukaryotic expression vector PGM that can express FSHα subunit gene and FSHβ subunit gene simultaneously, and contain DHFR expression unit and KTPA-CMV.

[0115] (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;

[0116] (3) transfecting the transfection vector into eukaryotic cells and culturing; and

[0117] (4) The culture supernatant was successively subjected to ultrafiltration, ammonium sulfate precipitation, hydrophobic ch...

Embodiment 1

[0144] Example 1: Human FSHα subunit gene and FSHβ subunit gene sequence cloning

[0145] 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: 1. After research, it was found that the expression levels of the designed 8 FSHα subunit sequences were different, some were high, and some were low. Among them, the FSHα subunit gene SEQ ID NO: 1 has the highest expression level, so SEQ ID NO:1 was selected for cloning.

[0146] In addition, FSHβ subunit gene can express the final FSHβ subunit, but the expression level of FSHβ subunit is not high. Expression research, including SEQ ID NO: 2, after research, it was found that the expression levels of the designed 10 FSHβ subunit sequences varied, som...

Embodiment 2

[0148] Embodiment 2: the generation of PGM

[0149] 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.

[0150] Table 1 - Oligonucleotides

[0151]

[0152]

[0153] The cloned dhfr gene expression unit was double-digested (Blg II and MluI) using a conventional enzyme digestion system, and the fragments were recovered with a gel recovery kit. Carry out double ...

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Abstract

The invention provides a recombinant human fallicle-stimulating hormone and a preparation method thereof. The preparation method specifically comprises the steps of (1) operably inserting a first expression box for expressing an FSH alpha subunit and a second expression box for expressing an FSH beta subunit in a eukaryotic expression vector so as to obtain a transfection vector which can expressFSH alpha subunit protein and FSH beta subunit protein; (2) transferring the transfection vector into an eukaryotic cell so as to obtain a transformed eukaryotic cell which integrates the first expression box and the second expression box in a chromosome; (3) under the condition suitable for expression, culturing the transformed eukaryotic cell, and expressing the FSH alpha subunit and the FSH beta unit so as to obtain a fermentation product containing a recombinant fallicle-stimulating hormone; and (4) performing separation and purification on the fermentation product so as to obtain the recombinant fallicle-stimulating hormone. The method disclosed by the invention can replace a conventional human FSH use method and a conventional human FSH preparation method, and rhFSH of which the activity and the purity are notably improved is obtained.

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 a major 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 is...

Claims

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Application Information

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Patent Type & Authority Applications(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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