Recombinant human follicle-stimulating hormone and its preparation
A technology of human follicle-stimulating hormone and follicle-stimulating hormone, which is applied in the protein field, can solve problems such as immune risks, and achieve the effects of high expression, high purity, and high activity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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 genome DNA (Clontech company) as the template, and the amplification program is as follows: 95 ℃ 5 minutes, thirty 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...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com