Mutant human epidermal growth factor gene, protein, preparation methods for mutant human epidermal growth factor gene and protein, and application of mutant human epidermal growth factor gene and protein

An epidermal growth factor and mutant technology, applied in the field of genetic engineering, can solve the problems of difficult reorganization method, unclear structure and function relationship, etc., and achieve the effect of improving solubility

Inactive Publication Date: 2012-10-10
SUN YAT SEN UNIV
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  • Abstract
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Problems solved by technology

[0004] After analyzing the molecular characteristics of EGF, we found that: 1) The three-dimensional structure of EGF is relatively clear, but the relationship between its structure and function is not clear; 2) The EGF molecule is very small, the nucleic acid sequence is only 159bp, and the protein level is only 53 amino acids, difficult to perform traditional shuffling methods

Method used

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  • Mutant human epidermal growth factor gene, protein, preparation methods for mutant human epidermal growth factor gene and protein, and application of mutant human epidermal growth factor gene and protein
  • Mutant human epidermal growth factor gene, protein, preparation methods for mutant human epidermal growth factor gene and protein, and application of mutant human epidermal growth factor gene and protein
  • Mutant human epidermal growth factor gene, protein, preparation methods for mutant human epidermal growth factor gene and protein, and application of mutant human epidermal growth factor gene and protein

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Embodiment 1

[0046] The natural human epidermal growth factor has 53 amino acids, and the natural porcine epidermal growth factor has 53 amino acids, and the homology between the two is 84%. We obtained the coding nucleotide sequence of mutant EGF with high EGFR affinity by DNA directed evolution method and phage display method. See SEQ ID NO:1 and SEQ ID NO:2 for its DNA sequence and amino acid coding sequence. A new mutant human recombinant epidermal growth factor protein with high EGFR affinity is obtained by fusing the sequence with pET32a(+) vector. The specific experimental steps are as follows:

[0047] 1. Construction of directed evolution library of mutant human recombinant epidermal growth factor gene.

[0048] 1) Take the nucleotide sequence of human epidermal growth factor (hEGF) (Gene ID: 1950, SEQ ID NO: 7) and porcine epidermal growth factor (pEGF) nucleotide sequence (Gene ID: 397083, SEQ ID NO: 8) respectively ) as a template, using primers 1, 2, 3, 4 for error-prone P...

Embodiment 2

[0105] Embodiment 2MTT method detects the proliferative activity of mutant protein

[0106] 1) Cultivate NTH3T3 cells until they are in good condition and the coverage reaches 75%, digest and centrifuge, resuspend, and count the cells.

[0107] 2) According to the result of cell counting, resuspend the cells with complete medium to an appropriate concentration (to ensure that the number of cells per well of the 96-well plate is 1000), and add 100 μL of cell suspension to each well of the 96-well plate, in which the first column does not add cells as blank control. 37°C, 5% CO 2 Incubate for 4-6 hours.

[0108] 3) After observing the cell attachment with an inverted microscope, carefully aspirate the medium, replace it with a serum-free medium, and continue to 37 ℃, 5% CO 2 Starve the culture for 4 hours.

[0109] 4) Carefully suck out the serum-free medium, add the experimental drugs (hEGF, EGF / E40V) diluted in DMEM containing 0.2FBS% according to the concentration of 0....

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Abstract

The invention discloses a mutant human epidermal growth factor (hEGF) gene, protein, preparation methods for the mutant human epidermal growth factor gene and the protein, and application of the mutant human epidermal growth factor gene and the protein, and belongs to the technical field of genetic engineering. The sequences of the mutant human epidermal growth factor gene and the protein are shown as SEQ ID NO:1 and SEQ ID NO:2 respectively. Deoxyribonucleic acid (DNA) of an hEGF and a porcine epidermal growth factor (pEGF) are subjected to directed molecular evolution to construct an EGF directed evolution library by a method of the combination of error-prone polymerase chain reaction (PCR) and stagger extension process-PCR, and then the mutant human epidermal growth factor gene is screened. According to the sequence, A at 119th position of a nucleotide sequence of a wild EGF is mutated into T; Glu at 40th position of amino acid encoded by the wild EGF is mutated into Val; and the function of mutated protein is the same as that of wild EGF protein, the specific activity of the mutated protein is 20 percent higher than that of the wild EGF protein, the mutated protein can be used for preparing a medicine or a diagnostic reagent for preventing epidermal growth factor-related diseases, the using amount is reduced, and the side effect of the mutated protein is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a mutant human epidermal growth factor gene, protein, preparation method and application thereof. Background technique [0002] Human epidermal growth factor (hEGF), discovered in 1962, is a monomeric polypeptide consisting of 53 amino acid residues. It has been confirmed that hEGF is a key wound healing factor, through the combination with human epidermal growth factor receptor (EGFR), it can have a strong mitogenic effect on epithelial cells and mesenchymal cells; promote skin, cornea, lung Proliferation and differentiation of tracheal and tracheal epithelial tissues; excellent repair function for epidermal damage and ulcer; clinically, it also has significant curative effect on repair and regeneration of surface damage of intestine, stomach, liver and other organs. [0003] In summary, hEGF has broad application prospects in medical treatment, beauty and skin care...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/63C07K14/485C12Q1/68A61K48/00A61K38/18A61K8/60A61P17/02A61Q19/00
Inventor 张擎刘昕邹尚利
Owner SUN YAT SEN UNIV
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