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Fusion protein with antibacterial and repairing function and production method and application thereof

A fusion protein and fusion gene technology, applied in the field of genetic engineering, can solve the problems of degradation, safety half-life extension related fusion proteins, failure to promote wound healing, etc.

Active Publication Date: 2010-12-15
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a beneficial exploration for the development of functional protein drugs that are antibacterial and wound healing. However, after the body is cleaved by thrombin, the existing fusion proteins are easily inactivated by the body environment or degraded by proteases present at the wound site, and the active half-life is only a few seconds. Hours or less, can not achieve the desired effect of promoting wound healing
[0006] In the prior art, there are no technical reports on related fusion proteins that are beneficial to exert the comprehensive effect of antibacterial and repair functions, improve the safety of clinical medicine, and prolong the half-life

Method used

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  • Fusion protein with antibacterial and repairing function and production method and application thereof
  • Fusion protein with antibacterial and repairing function and production method and application thereof
  • Fusion protein with antibacterial and repairing function and production method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1 Transformation of LL37 and haFGF genes and construction of fusion gene LL37-rhaFGF and preparation of fusion protein LL37-rhaFGF

[0053] In this example, the fusion gene LL37-rhaFGF was expressed by using the pET expression system of Novagen, the pET32a(+) plasmid was used as the expression vector, and the host cell was Escherichia coli.

[0054] Synthesize the human antimicrobial peptide LL37-Linker gene according to the preferred codons of Escherichia coli, introduce the NcoI restriction site and the enterokinase protease restriction site at the 5′ end of the gene in sequence, remove the stop codon at the 3′ end of the LL37 gene, and add Optimized to encode a hydrophobic polypeptide of 15 amino acids (Gly 4 Ser) 3 The DNA sequence; its nucleotide sequence is shown in SEQ ID NO:3.

[0055] According to the instructions of Trizol Reagent, total RNA was extracted from human fetal brain tissue. According to the cDNA sequence of haFGF (Genebank accession numbe...

Embodiment 2

[0083] Example 2 Pharmacodynamic study of the fusion protein LL37-rhaFGF of the present invention

[0084] (1) In vitro experiment:

[0085] (1) The inhibitory effect of the fusion protein on Staphylococcus aureus, Pseudomonas aeruginosa and other bacteria was detected by agar hole infiltration method.

[0086] The purified lyophilized fusion protein was dissolved into a 2.5g / L solution with physiological saline, and added to the agar wells containing Pseudomonas aeruginosa and Staphylococcus aureus respectively, with 2.5g / L Ampicillin as Positive controls, sterile water and normal saline were used as negative controls. Place them in an incubator at 37°C for 12-16 hours, measure the size of the inhibition zone, and take pictures to record the results. Sterile distilled water and normal saline control have no inhibition zone, but Ampicillin has an inhibition zone in the two bacterial plates with a diameter of about 2.5cm; the fusion protein LL-37-rhaFGF has obvious inhibition...

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Abstract

The invention discloses a fusion protein with antibacterial and repairing function and a production method and application thereof. A fusion gene is prepared by splicing an anthropogenic antibacterial peptide gene and a cell growth factor gene through a segment of hydrophobic polypeptide joint; and the fusion protein is prepared by adopting the expression of a prokaryocyte or yeast expression system, and is encoded by the anthropogenic antibacterial peptide, the polypeptide joint and the cell growth factor gene from an N end to a C end of the fusion protein. The invention simultaneously provides application of the fusion protein to the preparation of medicaments for treating wounds, burns / scalds, cold injury, chronic ulcer or bedsore. The invention creatively fuses the two kinds of polypeptide genes together to successfully construct a fusion protein with dual function of preventing infection and accelerating the repair of the damaged tissue, improves the safety of clinical medicament simultaneously, and prolongs the half-life period of the fusion protein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a fusion protein with antibacterial and repairing functions and its production method and application, especially relates to the preparation of the fusion protein for external use in epithelial repair and wound healing. technical background [0002] Wound healing disorders are common clinical problems, including burns, frostbite, chronic ulcers, diabetic bedsores, and wound healing disorders caused by infection. Traditional wound care involves mechanical or enzymatic removal of necrotic tissue to form granulation tissue, possibly supplemented by antimicrobial therapy to avoid invasive infection. A variety of topical antimicrobial agents such as hydrogen peroxide, iodine, and antibiotics are commonly used, but the risk of toxic side effects of these agents on the stroma and nascent epidermis must be considered. Furthermore, some wounds are treatment resist...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/63C07K19/00A61K38/18A61P17/02A61P31/00A61K38/17
Inventor 朱家勇卢雪梅沈娟马艳金小宝褚夫江李小波梅寒芳吴强肖明珠石大禹刘漫宇丁静
Owner GUANGDONG PHARMA UNIV
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