Recombinant human follicle stimulating hormone and preparation thereof

A human follicle-stimulating hormone and follicle-stimulating hormone technology, applied in the field of protein, can solve problems such as immune risk, and achieve the effects of high expression, high purity and high activity

Active Publication Date: 2013-07-31
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, Chinese Patent No. 98807811 is dedicated to stabilizing FSH by introducing disulfide bonds, while Chinese Patent No. 99808813 and No. 200480032665 introduce some mutations to improve performance and stability, but these changes in protein structure will increase the mutation The hidden danger of FSH being recognized by the body as a heterologous protein, which brings immune risks when used in vivo

Method used

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  • Recombinant human follicle stimulating hormone and preparation thereof
  • Recombinant human follicle stimulating hormone and preparation thereof
  • Recombinant human follicle stimulating hormone and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Cloning and expression of embodiment 1 rhFSH gene

[0048] 1. Cloning of the intron sequence in the human FSHα subunit gene

[0049] The inventors have found that although multiple introns in the FSHα subunit gene do not affect the final expressed FSHα subunit, they affect the expression level of the FSHα subunit, some increase expression, and some reduce expression. The intron upstream of the FSHα subunit gene can significantly increase the expression level, so this intron fragment is reserved for cloning.

[0050] Its cloning adopts conventional PCR method, amplifies with human genomic DNA (Clontech company) as the template, and the amplification procedure is as follows: 95 ℃ 5 minutes, 30 cycles (each cycle 94 ℃ 1 minute, 50 ℃ 1 minute, 72°C for 1 minute), 72°C for 10 minutes; the primers used for amplification are as follows (respectively introducing NheI and PstI restriction sites):

[0051] Primer α1: 5′AAGGTAAGT GCTAGC AAATT3'

[0052] NheI

[0053] Primer ...

Embodiment 2

[0083] Purification and identification of embodiment 2 rhFSH

[0084] Get the culture supernatant 1000ml of the engineered cell line of embodiment 1, cross the molecular weight cut-off and be the ultrafiltration membrane of 10kD, take the filtrate loading sample and use the 20mmol / L Tris-HCl (pH 8.0) equilibrium containing 0.1mol / L NaCl DEAE Sepharose FF anion-exchange chromatography column (GE Healthcare company) and collect flow-through liquid (see attached Figure 4 ); increase the salt ion concentration of the flow-through liquid collected in this step to make it reach 0.5mol / L NaCl, load the sample Phenyl Sepharose HP chromatography column (GE Healthcare company) to carry out chromatography, and use 20mmol / LTris-HCl (pH 8.0) Buffer elution, collect the peak containing FSH in the eluate detected by ELISA (see Figure 5 ); The collected eluent is loaded onto a Capto Q chromatography column (GE Healthcare company) for chromatography, and is used respectively with 20mmol / L T...

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Abstract

The invention provides high-specific-activity recombinant human follicle stimulating hormone and a preparation method thereof. In addition, the invention also provides a method for preparing an intermediate used in the method, gene, vector and cells.

Description

field of invention [0001] The invention belongs to the technical field of protein, in particular, the invention relates to a recombinant human follicle-stimulating hormone with high specific activity and a preparation method thereof. In addition, the present invention also relates to intermediates used in the preparation method. Background technique [0002] Follicle-stimulating hormone (FSH) is an important hormone that regulates reproductive-related functions in female and male mammals (including humans), and can be used to treat anovulatory syndrome, luteal phase defect, and infertility. FSH is a heterodimeric glycoprotein composed of non-covalently associated α and β subunits. Among them, both the α subunit and the β subunit are glycoproteins, each with two glycosylation sites. The presence and composition of the glycosyl moieties of FSH determine the performance and stability of its biological functions. For protein products obtained through translation, splicing, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/59C12N15/85A61K38/24A61P15/08C12N15/16C12N5/10
Inventor 金磊刘洪霞尉继征郝鹏石欣欣丁丽艳
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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