Production method for recombinant human fibroblast growth factor-17 protein

A technology of human fibroblasts and growth factors, applied in the field of genetic engineering, can solve the problems of low production concentration and purity of FGF-17 protein, and achieve the effects of optimizing culture temperature and increasing purity and concentration

Inactive Publication Date: 2016-03-23
WENZHOU MEDICAL UNIV +1
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Therefore, one of the objects of the present invention is to provide a production method of recombinant human fib

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Production method for recombinant human fibroblast growth factor-17 protein
  • Production method for recombinant human fibroblast growth factor-17 protein
  • Production method for recombinant human fibroblast growth factor-17 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1F

[0071] The construction of embodiment 1 FGF-17 protein high-efficiency expression engineering bacterium

[0072] According to the human FGF17 natural sequence (accession number: AY358869.1) and amino acid sequence published in GenBank, optimize according to the codon preference of Escherichia coli, under the condition of not changing the amino acid sequence, design the coding sequence primer of rhFGF-17, and Introduce specific enzyme cutting sites NdeI and BamHI. The nucleotide sequence of rhFGF-17 was obtained by PCR amplification, and the resulting product was ligated with a T vector (pEASYT1simplevector) at 16°C, and the ligated product was double-digested with NdeI and BamHI at 37°C for 4 hours, and the rhFGF-17 gene fragment was recovered. , then treated with NdeI and BamHI double enzyme digestion and the resulting expression vector pET3a gene fragment was ligated overnight at 16°C with SolutionI ligase to construct a recombinant expression vector (see figure 1 , figur...

Embodiment 2F

[0073] The establishment of embodiment 2FGF-17 albumen higher density culture method

[0074] In the present invention, tryptone, yeast powder, etc. are used as nitrogen sources, glucose, etc. are used as carbon sources, and phosphate is used as a buffer system, supplemented by sodium chloride as a base medium; and by controlling conditions (pH, temperature, dissolving Oxygen, rotation number, etc.) significantly improved the cell yield and expression level. Specifically: Inoculate the FGF-17 engineering strain into LB medium at a ratio of 1:50-100 to obtain the first-generation bacteria, cultivate for 8h-12h, and wait for A 600 When it reaches about 1.2-2.0, it is inoculated into LB medium containing phosphate buffered saline at a ratio of 1:100-200 for cultivation, and cultured for 2h-4h. 600 When the temperature reaches 0.6-0.8, the temperature is 16°C and 37°C, the final concentration of the inducer (IPTG) is 0.4mM and 1mM, the time is 16-30h and 4-6h, and the rotation sp...

Embodiment 3F

[0077] The establishment of embodiment 3FGF-17 protein purification method

[0078] 1. Treatment of inclusion bodies

[0079] (1) Bacterial fragmentation

[0080]After repeated freezing and thawing, the cells were fully suspended in cell lysis buffer (20mM Tris-HCl, 1mM EDTA-2Na, 0.2MNaCl, 1% TritonX-100, 0.2% sodium deoxycholate) at a ratio of 1:25 (g:mL) ,pH7.5), carry out low-temperature ultrasonic crushing, power 300w, vibration frequency output 40%, ultrasonic 5s off 5s, ultrasonic 10min, after microscopic examination of E. After 25 min, discard the supernatant and collect the precipitate.

[0081] (2) Inclusion body cleaning

[0082] Take the above centrifuged precipitate, add 20 times the amount of washing buffer (20mM Tris-HCl, 1mMEDTA-2Na, 0.2MNaCl, 1% TritonX-100, pH7.5) and stir thoroughly for washing, after 30min, centrifuge at 20000rpm, 4°C, 25min , discard the supernatant, and collect the precipitate. Take the precipitate again, add 20 times the amount of wa...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a production method for recombinant human fibroblast growth factor-17 protein. The production method at least comprises the following steps: step 1, performing recombination and amplification on human fibroblast growth factor-17 protein FGF-17 with a natural sequence in accordance with preference of colibacillus; step 2, connecting the recombinant rhFGF-17 gene to a colibacillus expression plasmid to form a recombinant expression vector, and transferring the recombinant expression vector to enable the recombinant expression vector to enter colibacillus host bacteria to obtain FGF-17 protein expression engineering bacteria; step 3, cultivating the expression engineering bacteria and then purifying, wherein in the purifying process, a cell lysis buffer solution comprises the following concentrations of components: 45-55 mM TrisTris, 280-320 mM sodium chloride (NaCl), 1.5-2.5 mM ethylenediamine tetraacetic acid (EDTA), 0.1-0.3 M cane sugar, 0.8-1.2% of polyethylene glycol octylphenyl ether TritonX-100 in percentage by volume, 0.1-0.3% of sodium deoxycholate in percentage by volume, 5-10% of glycerol in percentage by volume, and 0.8-1.2% of phenyl methyl sulfonyl fluoride (PMSF) in percentage by volume. In the production method provided by the invention, the concentration and the purity of the FGF-17 protein are high.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a production method of recombinant human fibroblast growth factor-17. Background technique [0002] Fibroblast growth factor (Fibroblast Growth Factor, FGF) is a kind of polypeptide cell growth factor widely present in various tissues. At present, 23 members have been found, the molecular weight is 16kD ~ 34kD, and its central region contains a high degree of homology (30% ~70%) of about 120 amino acid sequences, has a wide range of biological activities on various types of cells derived from mesoderm and neuroectoderm. The content of FGFs is small but widely distributed in the body. Each factor regulates a series of developmental processes, including brain development, limb differentiation and trunk formation. Most FGFs members are closely related to embryonic development, tissue damage repair, tumor occurrence, development and metastasis, etc., and have considerable applicat...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/70C07K14/50
CPCC07K14/50C07K1/22C07K1/30C12N15/70C12N1/205C12R2001/19
Inventor 姜潮马吉胜田海山刘敏吴美玉
Owner WENZHOU MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap