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Human horny cell growth factor-2 mutant and its preparation method

A technology of keratinocytes and growth factors, applied in the field of genetic engineering, can solve problems such as failure, and achieve the effects of simplified operation, simplified purification process, and reduced cost

Inactive Publication Date: 2007-08-01
杭州北斗生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current treatment method is mainly to control the development of infection with debridement, which often lasts for several months and has the possibility of failure

Method used

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  • Human horny cell growth factor-2 mutant and its preparation method
  • Human horny cell growth factor-2 mutant and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Construction of different KGF-2 mutant expression plasmids

[0059] The mature peptide of natural human KGF-2 has 171 amino acid residues. Experiments show that removing the first 26 amino acid residues at the N-terminus of KGF-2 does not affect the binding of KGF-2 to its receptor. The removal of 26 amino acid residues of KGF The complex of -2 and its receptor was also crystallized and the three-dimensional structure was resolved. In order to efficiently express KGF-2 in E. coli, it may be considered to remove part of the N-terminal amino acid residues that do not affect its activity. The nucleotide sequence encoding human KGF-2 C-terminal 145-171 amino acid residues was chemically synthesized. Due to chemical synthesis, in order to express KGF-2 in E. coli to the greatest extent, the preferred codons of E. coli are used for synthesis. In order to realize natural expression and simplify the subsequent processing technology, a start codon ATG was added to the 5'end, and a s...

Embodiment 2

[0061] Expression analysis to pick the best mutant

[0062] The constructed KGF-2 mutant was transformed into E. coli BL21(DE3) competent cells, and the transformants were screened by antibiotics. Select well-separated colonies to inoculate LB medium containing appropriate antibiotics, add IPTG to 1 mM and culture for 4 hours to induce KGF-2 mutant expression. SDS polyacrylamide gel electrophoresis was used to separate the total bacterial protein, stained with Coomassie Brilliant Blue R250, and the percentage of KGF-2 expressed in the total protein was analyzed by gel scanning. By comparison, we selected a mutant with the first Q removed from the N-terminus. If Q is removed, the amino acid residue after M is exactly A. This is very consistent with the N-terminal rule: the second amino acid residue being Q will make the expressed protein unstable, while the second amino acid residue being A will greatly stabilize the expressed protein. The selected DNA sequence and its encoded prot...

Embodiment 3

[0086] Induced expression of recombinant KGF-2 mutant

[0087] The engineered bacteria selected in Example 2 were inoculated into LB medium containing corresponding antibiotics, and cultured with shaking at 100-300 times / min for 12-14 hours. Inoculate the LB medium containing the same antibiotic at a ratio of 1:100-1:20, and continue to culture for 3-6 hours, the culture temperature is 20-37°C, and the shaking speed is 100-300 times / min. Add IPTG to a final concentration of 0.1-2mM, and continue to incubate for 3-4 hours.

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PUM

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Abstract

The invention relates to a human KGF- 2 mutant, the amino sequence is demonstrated as sequence 2 in sequence table and the gene order is the squence 1 in squence table. The invention aslo provides the expression vector and engineering bacteria containing mutant gene. The invention aslo provides the method for preparing said KGF-2 mutant, comprising: (1) culturing bacillus coli engineering bacteria at proper condition, and inserting gene order in multiple coloning point of expression vector to express human KGF-2 mutant; (2) separating and purifying the human keratinized cell growing factor -2 mutant through column chromatography.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to a human keratinocyte growth factor-2 mutant and a preparation method thereof. Background technique [0002] Keratinocyte growth factor-2 (KGF-2) is a basic protein consisting of 208 amino acid residues, which is unstable to acid and heat. KGF-2 is named for its ability to specifically stimulate the growth of epithelial cells. Because it is a member of the fibroblast growth factor family, it is also called FGF-10. Emoto et al. cloned the cDNA of human KGF-2. The 37 amino acid residues at the N-terminus of the encoded protein constitute a hydrophobic signal peptide. The mature peptide contains 171 amino acid residues (38-208aa) and has a theoretical molecular weight of 19.3kDa. Has a strong affinity (J Burn Care Rehabil 2000 Jan-Feb; 21(1Pt1); 5-9). [0003] The following is the primary structure of human KGF-2, the part enclosed by the box is the signal peptide at its N-terminus. [...

Claims

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Application Information

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IPC IPC(8): C07K14/475C12N15/12C12N15/63C12N5/10C12P21/02
Inventor 何晓艳杨柏成刘国安刘沐荣
Owner 杭州北斗生物技术有限公司
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