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Horny cell growth factor mutant and its preparation method and use thereof

A growth factor, keratinocyte technology, applied in biochemical equipment and methods, botanical equipment and methods, applications, etc., can solve problems such as reports of KGF-2 mutants that have not yet been seen, and achieve broad market development and application prospects, Important commercial value, the effect of promoting proliferation

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] There are no reports of KGF-2 mutants with a mutation at the 102nd amino acid residue

Method used

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  • Horny cell growth factor mutant and its preparation method and use thereof
  • Horny cell growth factor mutant and its preparation method and use thereof
  • Horny cell growth factor mutant and its preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of embodiment one recombinant human KGF-2 and KGF-2 / K102EcDNA 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 Compa...

Embodiment 2

[0052] Expression and purification of embodiment two recombinant human KGF-2 and KGF-2 / K102E

[0053] 1. Materials

[0054] Same as embodiment one.

[0055] 2. Methods and results

[0056] Pick single-clonal colonies from pET17b / KGF-2 and pET17b / K102E transformed Escherichia 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 pr...

Embodiment 3

[0057] Example 3 Determination of Biological Activity of Recombinant Human KGF-2 and KGF-2 / K102E

[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 / K102E

[0062] The receptor binding ability of recombinant human KGF-2 and KGF-2 / K102E 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 41.44g, 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), block at 37°C for 2h; 0.05% Tween20 in PBS) and washed 3 times, 2 min / time; add 100 μl FGFR2IIIb receptor chimeri...

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Abstract

The invention discloses a keratinized cell growth factor mutant of high receptor binding force, and also the process for preparing and usage of the mutant. The mutant is prepared by the 102nd mutating amino acid residue of keratinized cell growth factor, that is lycine residue, into glutacid residue, And is prepared by point mutation technique in gene project. The receptor bonding force of mutated keratinized cell growth factor and added value activity of epithelial cell both increases evidently. The mutant can be used to prepare polypeptide pharmaceutical against radiation hurt, ulcer and enteritis, and it also can advance the radiotheraphy index in killing cancer,

Description

technical field [0001] The invention relates to a cell growth factor, in particular to a keratinocyte growth factor mutant, and also relates to 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, Thr114, Phe146, Glu154 and Arg...

Claims

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Application Information

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IPC IPC(8): C12N15/12
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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