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Production method for recombinant human fibroblast growth factor-18 and application of growth factor-18

A technology for human fibroblasts and growth factors, applied in the field of genetic engineering, can solve the problems of FGF18 being unable to stably and efficiently express protein biological activity, low protein purity, etc., and achieve the effects of improving yield, simplifying purification process, and increasing expression amount

Inactive Publication Date: 2015-12-30
WENZHOU MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, one of the objectives of the present invention is to provide a production method of recombinant human fibroblast growth factor-18 protein. The present invention uses the insect cell expression system to control the main factors affecting the expression of the protein, such as the concentration of the virus. Optimizing factors such as concentration (MOI), time of harvest (TOI) and cell state, solved the problem that FGF18 could not be expressed stably and efficiently without affecting the biological activity of its protein. The two-step separation and purification technology of the column solves the problem of low purity of FGF18 protein

Method used

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  • Production method for recombinant human fibroblast growth factor-18 and application of growth factor-18
  • Production method for recombinant human fibroblast growth factor-18 and application of growth factor-18
  • Production method for recombinant human fibroblast growth factor-18 and application of growth factor-18

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Embodiment 1

[0084] 1. Experimental method

[0085] 1.1 Construction of Bacmid-FGF18 vector

[0086]According to the human FGF-18 natural sequence (accession number: NM-003862.2) and amino acid sequence published in GenBank, its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 4, according to insect cell codon preference Considering the elimination of the hairpin structure and other secondary structures that are not conducive to expression, the primers for the coding sequence of rhFGF-18 were designed without changing the amino acid sequence. The full-length target sequence of rhFGF-18 was obtained by PCR amplification. When the full sequence of the recombinant human fibroblast growth factor-18 (rhFGF-18) gene was artificially synthesized, a BamHI restriction site was introduced at the 5' end of the gene and a HindIII restriction site was introduced at the 3' end. Then, use the following primers to perform PCR amplification using the artifi...

Embodiment 2

[0107] 3 Experimental methods

[0108] 3.1 Culture of Sf9 insect cells

[0109] 3.1.1 Recovery of Sf9 insect cells

[0110] Take out the cell cryopreservation tube from the liquid nitrogen tank, quickly put it into a 37°C water bath, and shake it constantly to heat the cells to melt evenly. After about 1 minute, add the cell cryopreservation solution to 25ml of Sf-900ⅡSFM medium in the ultra-clean workbench. Seal and place in a constant temperature shaker at 27°C, 110rpm, without CO 2 to cultivate.

[0111] 3.1.2 Suspension culture of Sf9 cells

[0112] Cells were cultured in suspension in shake flasks, and cell survival and death rates were recorded by counting with trypan blue. When the density reaches 2×10 6 cells / ml, it means that the cells are in a good growth state, which is in the logarithmic growth phase, and the cells can be expanded and cultured to keep the cell concentration at 2-5×10 5 pieces / ml.

[0113] 3.2 Bacmid-FGF18 transfected insect cells

[0114] ①...

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Abstract

The invention relates to a production method for recombinant human fibroblast growth factor-18 protein. The production method comprises the following steps: 1, expressing the fibroblast growth factor-18 protein in insect cells by using an insect baculovirus vector expression system, specifically comprising 1) obtaining recombinant baculovirus Bacmid-FGF18 (fibroblast growth factor-18), wherein the FGF18 gene is a fibroblast growth factor-18 gene and has a base sequence as shown in SEQ ID NO: 1, and 2) infecting the insect cells with the Bacmid-FGF18 recombinant baculovirus with MOI (Multiplicity of Infection) of 1 to 32, and then culturing the infected insect cells for 12 to 96 hours at the temperature of 25 to 29 DEG C to harvest cell fluid; and 2, purifying the cell fluid harvested in the step 1 to obtain the human fibroblast growth factor-18 protein. The invention also discloses application of the human fibroblast growth factor-18 protein in treating bone diseases and alopecia.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a production method and application of recombinant human fibroblast growth factor-18. Background technique [0002] FGF18, a new member of the FGF family, is a secretable protein. In 1998, Japanese scientist Ohbayashi isolated FGF18 from mouse embryos for the first time. The human FGF18 gene encodes a protein sequence consisting of 207 amino acids with a molecular mass of about 23kDa. Sequence analysis results showed that the FGF18 gene is a highly conserved sequence consisting of 207 amino acids, and the mouse FGF18 has 99.5% and 99% homology with the amino acid sequences of human and rat. Structurally, FGF18 is similar to FGF8 and FGF17, and is a member of the same subfamily with 70%-80% amino acid sequence homology. FGF18 plays an important role in the process of bone growth and development, and is also involved in the activity of cortical neurons, the survival, different...

Claims

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Application Information

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IPC IPC(8): C12N15/866C07K14/50C07K1/36C07K1/34C07K1/22C07K1/18A61K38/18A61P17/14A61P19/08
Inventor 马吉胜姜潮田海山刘敏黄金枝
Owner WENZHOU MEDICAL UNIV
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