Production method of recombinant human fibroblast growth factor-17

A technology of human fibroblasts and growth factors, which is 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: 2019-07-26
WENZHOU MEDICAL UNIV +1
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  • 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 fibroblast growth factor-17 protein, which solves the problem of low production concentration and purity of FGF-17 protein;

Method used

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  • Production method of recombinant human fibroblast growth factor-17
  • Production method of recombinant human fibroblast growth factor-17
  • Production method of recombinant human fibroblast growth factor-17

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of FGF-17 Protein High Expression Engineering Bacteria

[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 the T vector (pEASY T1 simple vector) at 16°C, and the ligated product was digested with NdeI and BamHI at 37°C for 4 hours, and rhFGF-17 was recovered. 17 gene fragments, then treated with NdeI and BamHI double enzyme digestion and recovered expression vector pET3a gene fragments were ligated overnight at 16°C with Solution I ligase to construct recombinant expression vectors (see figure 1 , figure 2 ). The ligation pro...

Embodiment 2

[0073] Example 2 Establishment of a higher density culture method for FGF-17 protein

[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 3

[0077] Example 3 Establishment of FGF-17 Protein Purification Method

[0078] 1. Treatment of inclusion bodies

[0079] (1) Bacterial fragmentation

[0080] After freezing and thawing the cells repeatedly, they were fully suspended in cell lysis buffer (20mM Tris-HCl, 1mM EDTA-2Na, 0.2M NaCl, 1% Triton X-100, 0.2% Sodium oxycholate, pH7.5), perform low-temperature ultrasonic crushing, power 300w, vibration frequency output 40%, ultrasonic 5s off 5s, ultrasonic 10min, microscopic examination of E. , 4°C, 25min, discard the supernatant, and collect the precipitate.

[0081] (2) Inclusion body cleaning

[0082] Take the precipitate after centrifugation, add 20 times the amount of washing buffer (20mM Tris-HCl, 1mM EDTA-2Na, 0.2M NaCl, 1% Triton X-100, pH 7.5) to fully stir and wash, after 30min, centrifuge at 20000rpm, 4°C, 25min, discard the supernatant, and collect the precipitate. Then take the precipitate, add 20 times the amount of washing buffer (20mM Tris-HCl, 1mM EDTA-...

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Abstract

The present invention provides a method for producing recombinant human fibroblast growth factor-17 protein, at least including the following steps: step 1, recombining natural sequence human fibroblast growth factor-17 protein FGF-17 according to the preference of Escherichia coli and amplification; step 2, connecting the recombined rhFGF‑17 gene to the E. coli expression plasmid to form a recombinant expression vector, and then transforming the recombinant expression vector into the E. coli host bacterium to obtain FGF‑17 protein expression engineering bacteria; and steps 3. Cultivate the expression engineering bacteria, and then purify; wherein, in the purification process, the cell lysis buffer of the following concentration components is used: 45-55mM tris Tris, 280-320mM sodium chloride NaCl, 1.5-2.5mM ethylenediaminetetraacetic acid DTA, 0.1-0.3M sucrose, and 0.8-1.2% volume percentage of polyethylene glycol octyl phenyl ether TritonX-100, 0.1-0.3% Sodium oxycholate, 5%-10% glycerin, 0.8-1.2% phenylmethylsulfonyl fluoride PMSF. The concentration and purity of the FGF‑17 protein produced by the production method 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. So far, 23 members have been found, with a molecular weight of 16kD ~ 34kD, and its central region contains a high degree of homology ( 30%-70%) of about 120 amino acid sequences, and 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 appli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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