Gene cloning, expression and application of recombinant human fibroblast growth factor-20

A FGF-20, prokaryotic expression technology, applied in the direction of medical preparations containing active ingredients, recombinant DNA technology, microorganism-based methods, etc., to achieve the effects of reducing production costs, increasing yield and expression, and simplifying purification steps

Inactive Publication Date: 2015-01-21
WENZHOU MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a new type of growth factor, FGF-20 has few related reports at home and abroad

Method used

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  • Gene cloning, expression and application of recombinant human fibroblast growth factor-20
  • Gene cloning, expression and application of recombinant human fibroblast growth factor-20
  • Gene cloning, expression and application of recombinant human fibroblast growth factor-20

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1F

[0031] The construction of embodiment 1 FGF-20 protein high-efficiency expression engineering bacterium

[0032] According to the human FGF-20 natural sequence (accession number: BC098128.1) and amino acid sequence published in GenBank, optimize according to the codon preference of Escherichia coli, and design the coding sequence primers of rhFGF-20 without changing the amino acid sequence . The full-length nucleotide sequence of rhFGF-20 was obtained by PCR amplification. Add start codon and stop codon to the 5' end and 3' end of the preferred sequence of rhFGF-20, respectively, and introduce specific enzyme cutting sites Nde I and BamH I, and then treat rhFGF with NdeI and BamHI double enzymes respectively -20 gene and expression vector pET3a-c (or pET28a), digested at 37°C for 3h to 4h, recovered the corresponding fragments, ligated overnight at 16°C with T4 DNA ligase, and constructed a recombinant expression vector (see figure 1 , figure 2 ). The ligation product was...

Embodiment 2

[0033] The establishment of embodiment 2 FGF-20 protein high-density culture method

[0034] In the present invention, the fermentation conditions for expressing rhFGF-20 by engineered bacteria are not particularly limited. Conventional fermentation conditions in the art can be used. Usually, engineering bacteria are composed of tryptone, yeast powder, ammonium salt, etc. as nitrogen source, glucose, glycerin, etc. basal medium; and through the regulation of control parameters (pH, temperature, dissolved oxygen, stirring speed, etc.) , prolong the logarithmic growth cycle, and significantly increase the cell yield and expression level. Specifically: Inoculate the FGF-20 engineering strain into LB medium at a ratio of 1:200-300 for activation to prepare first-generation seeds, cultivate for 3h-4h, and wait for A 600 reach about 0.8-1.0, inoculate into LB medium containing phosphate buffered saline at a ratio of 1:10-20 to amplify and prepare second-generation seeds, cultivat...

Embodiment 3

[0037] The establishment of embodiment 3FGF-20 protein purification method

[0038] 1. Bacteria fragmentation

[0039] After the cells were thawed at room temperature, they were fully suspended in cell lysis buffer (20mM Tris-HCl, 2mM EDTA-2Na, 0.1M NaCl, Triton X-100, 0.2% deoxycholic acid at a ratio of 1:20 (g:mL) Sodium, pH7.5), high pressure homogenization. The crushing method is 200bar circulation once, and then 800bar homogenization for 1~2 times. After microscopic examination shows no intact E. coli in the visible range, centrifuge at 20,000rpm, 4°C, 30min, discard the supernatant, and collect the precipitate.

[0040] 2. Inclusion body cleaning

[0041] Take the above-mentioned precipitate after centrifugation, add 10 times the amount of washing buffer (20mM Tris-HCl, 2mM EDTA-2Na, 0.1M NaCl, Triton X-100, 0.2% sodium deoxycholate, pH7.5) to fully stir and wash, After 10min to 15min, centrifuge at 20000rpm at 4°C for 30min, discard the supernatant, and collect the p...

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PUM

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Abstract

The invention discloses a method for the expression and production of FGF (fibroblast growth factor)-20. By the method of bioinformatics for optimization design, full length FGF-20 nucleotide sequence is synthesized, and is connected with pET series carriers, the expression carrier connection is introduced into proper escherichia coli host cells, a high expression quantity and stable inheritance engineering strain is obtained through screening, a high density fermentation method, an inclusion body expression FGF-20 purification method and a corresponding quality detection method are established, and mass production of rhFGF-20 is possible. On the basis, clinical application of the FGF-20 in tissue repair and degenerative diseases of the nervous system and effect of the FGF-20 in occurrence, development and migration of tumors can be further explored.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to an optimized method for producing fibroblast growth factor-20 (FGF-20), an expression vector, a host, expression and purification methods and possible clinical applications used in the method. 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 in the body is small but widely distributed. Most of the FGFs members are closely related to embryonic development, tissue damage repair, neuroprotection and nutrition, tumor occurrence, development and metastasis, and have broad ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P21/02C07K1/22C07K1/18C07K1/16A61K38/18A61P17/00A61P17/02A61P25/16A61P25/28C12R1/19
Inventor 田海山李校堃姜潮马吉胜赵央郑婕
Owner WENZHOU MEDICAL UNIV
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