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Horny cell growth factor mutant with high bioactivity and its preparation process and use thereof

A growth factor, keratinocyte technology, applied in biochemical equipment and methods, botanical equipment and methods, growth factors/inducing factors, etc., can solve problems such as KGF-2 mutants that have not yet been seen, and achieve broad market development and development. Application prospects, reduce scar formation, and delay the effect of skin cell aging

Inactive Publication Date: 2005-11-02
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] So far, there have been no reports of KGF-2 mutants with mutations at the 115th and 117th amino acid residues

Method used

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  • Horny cell growth factor mutant with high bioactivity and its preparation process and use thereof
  • Horny cell growth factor mutant with high bioactivity and its preparation process and use thereof
  • Horny cell growth factor mutant with high bioactivity and its preparation process and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of embodiment one recombinant human KGF-2 and KGF-2 / STEAcDNA sequence

[0041] 1. Materials

[0042] 1. Strains, vectors and cells

[0043] Both the E.coli BL21 strain and the pET17b prokaryotic expression vector were preserved in our laboratory, and the normal rat tracheal epithelial cells (RTE) were purchased from the Peking Union Medical College Cell Center.

[0044] 2. Enzymes and Reagents

[0045] Human fetal liver cDNA library was purchased from Clontech Company, BamHI, NdeI, Taq DNA polymerase, Pfu DNA polymerase and dNTP were purchased from TaKaRa Company, T4 DNA ligase and mini-plasmid extraction kit were purchased from Promega Company, DNA recovery kit Purchased from Dingguo Biotechnology Company, recombinant human FGFR2β(IIIb) / Fc was purchased from R&D Company, enzyme label plate and 96-well cell culture plate were purchased from Costar Company, TMB was purchased from AMRESCO Company, HRP-labeled secondary antibody was purchased from CHEMICON Compan...

Embodiment 2

[0052] Expression and purification of embodiment two recombinant human KGF-2 and KGF-2 / STEA

[0053] 1. Materials

[0054] Same as embodiment one.

[0055] 2. Methods and Results

[0056] Pick single-clonal colonies from pET17b / KGF-2 and pET17b / STEA-transformed E. coli BL21 plates, insert them into 50ml of ampicillin-resistant LB medium containing 0.5% glucose, and shake on a shaker at 35°C to OD 600 = 0.6-1.0. Harvest the bacteria and transfer them into 1L ampicillin-resistant RM medium, shake at 35°C to OD 600 =0.5-1.0, IPTG was added to a final concentration of 0.5 mM. After induction of expression for 5 hours, the bacteria were harvested by centrifugation at 8000 rpm at 4°C. The bacterial cell pellet was resuspended in 50 ml of 20 mM PB (pH 7.4, containing 2 mM EDTA) buffer, and ultrasonically disrupted until the solution was transparent. Centrifuge at 10,000 rpm at 4°C, take the supernatant and pass through the SP cation exchange column, and the target protein is el...

Embodiment 3

[0057] The biological activity assay of embodiment three recombinant human KGF-2 and KGF-2 / STEA

[0058] 1. Materials

[0059] Same as embodiment one.

[0060] 2. Methods and results

[0061] 1. Detection of receptor binding ability of recombinant human KGF-2 and KGF-2 / STEA

[0062] The receptor binding ability of recombinant human KGF-2 and KGF-2 / STEA was detected by competitive ELISA method, and the specific operation was as follows: the purified recombinant human KGF-2 was coated with diluent (NaCO 3 1.59g, NaHCO 3 2.93g, dilute to 1L with distilled water) to 10μg / ml, add 100μl / well to a 96-well microtiter plate, and coat at 4°C overnight; the next day, use PBS (PH 7.4, NaCl 8g, KH 2 PO 4 0.24g, Na 2 HPO 4 1.44g, KCl 0.2g, distilled water to 1L), wash 3 times, 2min / time; add 200μl 3% skimmed milk powder (3g skimmed milk powder dissolved in 100ml PBS) to each well, block at 37°C for 2h; PBS containing 0.05% Tween20) was washed 3 times, 2 min / time; 100 μl of FGFR2IIIb r...

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Abstract

The invention discloses a growth factor mutant of keratinized cell of high bioactivity, as well as the method for producing it and the usage of it. The mutant of the invention is to mutate the 115 position amino acid residue of the growth factor mutant of keratinized cell from serine residue to threonine residue, and the 117 position amino acid residue from aminoglutaric acid residue to alanine residue. It is produced by the means of point mutation process of gene engineering, from which the receptor binding force of the mutated growth factor of keratinized cell and the breeding activity of proepithelial cell are improved dramatically. It can be used for producing polypeptide drugs for promoting the wound healing of epidermis, preventing radiation injury, and curing ulcer and enteritis, as well as for improving radiotheraphy index in curing cancer.

Description

technical field [0001] The invention relates to a cell growth factor, in particular to a highly biologically active keratinocyte growth factor mutant, as well as its preparation method and use. Background technique [0002] Currently, the fibroblast growth factors (fibroblast growth factors, FGFs) superfamily has 24 members. Keratinocyte growth factor-2 (KGF-2), also called fibroblast growth factor-10 (FGF-10), is a member of the keratinocyte growth factor (KGFs) family in the FGFs superfamily. In 1997, Emoto et al. cloned for the first time the full-length 627bp human KGF-2 cDNA, encoding a single-chain polypeptide consisting of 208 amino acid residues, with a hydrophobic signal peptide sequence consisting of about 40 amino acid residues at its N-terminus. The crystal structure analysis of the complex between human KGF-2 and its receptor FGFR2IIIb showed that the main interaction sites of KGF-2 molecule with FGFR2IIIb include Leu73, Gln74, Gly75, Asp76, Arg78, Thrl14, Phe1...

Claims

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Application Information

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IPC IPC(8): C07K14/475
Inventor 王金凤徐东刚王嘉玺彭善云邹民吉蔡欣
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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