AMP (antimicrobial peptide) fusion protein as well as preparation method and application thereof

A fusion protein and antimicrobial peptide technology, which is applied in the direction of peptide/protein components, antibacterial drugs, antifungal agents, etc., can solve the problems of unsuitability for large-scale production, many technical bottlenecks of antimicrobial peptides, and high synthesis costs, etc., to ensure fusion Protein activity, guaranteed purification and refolding, and high antibacterial activity

Inactive Publication Date: 2017-05-10
ZHEJIANG YITAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the many technical bottlenecks, complicated process and expensive synthesis cost of artificially synthesized antimicrobial peptides, it is not suitable for large-scale production
A single purified product may also have hemolytic problems when used, so the cecropin antimicrobial peptides applied to humans are basically the primary mixture of cecropin and these substances induced by silkworms or tussah silkworms, and are used as health food. Contains cecropin antimicrobial peptide is minimal

Method used

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  • AMP (antimicrobial peptide) fusion protein as well as preparation method and application thereof
  • AMP (antimicrobial peptide) fusion protein as well as preparation method and application thereof
  • AMP (antimicrobial peptide) fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Construction of recombinant expression vector

[0038] 1. Design and synthesis of the target fragment

[0039] Obtain cecropin nucleotide sequence (as shown in SEQ ID NO.5) by NCBI database, find shark liver peptide orf-86 gene (as shown in SEQ ID NO.6) in the shark liver small peptide cDNA database of this laboratory , orf-86 shark liver peptide is a small peptide translated from the shark liver peptide orf-86 gene with an isoelectric point of 11 that may have potential biological activity screened from the regenerated liver cDNA library of the striped bamboo shark. Add 3 tandem fragments (Gly+Ser+Ala) coding genes (as shown in SEQ ID NO.7) in the middle of the acid sequence to form shark liver peptide orf-86+3GSA coding fragment+cecropin fusion gene (hereinafter referred to as orf- 86 fusion gene, as shown in SEQ ID NO.4), and a BamH I restriction site is added to its 5' end, and an Xho I restriction site is added to the 3' end, which is synthesized by ...

Embodiment 2

[0046] Example 2: Induced expression and concentration of fusion proteins

[0047] 1. Induced expression of recombinant fusion protein

[0048] After the sequencing was correct, the plasmid was extracted, and the recombinant plasmid was transformed into BL21(DE3) Escherichia coli for induced expression.

[0049] The two successfully constructed engineering bacteria were inoculated in 100mL LB culture medium, and cultured in a constant temperature shaker at 37°C until A 600 When the value is about 0.6, add IPTG to a final concentration of 1 mmol / L, induce at 16°C for 20 hours, collect the bacteria by centrifugation at 12,000 rpm, wash twice with PBS, and ultrasonically break the induced recombinant bacteria for 30 minutes, sonicate for 3 seconds, stop for 3 seconds, and then Centrifuge at 12000rpm for 25min, take the supernatant and precipitate respectively, and detect the existence form of the expression product by SDS-PAGE electrophoresis, the results are as follows figure...

Embodiment 3

[0055] Embodiment 3: Oxford cup method detects antibacterial activity

[0056] Detection of antibacterial activity of recombinant protein orf-86

[0057] Take the agar powder and solid medium sterilized at 121°C for 20 minutes into the ultra-clean bench, first pour a thin layer of agar powder on the glass petri dish, and then separate the activated TG1 Escherichia coli and Bacillus subtilis Add it to the solid medium whose temperature has dropped to about 50°C, and pour the solid medium containing the bacteria solution after the agar powder solidifies. After the plate is solidified, put the sterilized Oxford cup that has been burned by the flame of an alcohol lamp on the plate to form a sample hole, and add 200 μL of Amp (1mg / mL), ddH2O, orf86 to the Oxford cup, and Amp is Positive control, ddH2O as negative control. After culturing overnight at 37°C, observe the size of the inhibition zone to judge the antibacterial activity of the sample.

[0058] The results showed that ...

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Abstract

The invention provides an AMP (antimicrobial peptide) fusion protein. The AMP fusion protein comprises a cecropin protein and an orf-86 APSL (active peptide from shark liver) protein which are linked by a 3GSA flexible peptide fragment. The fusion protein is a soluble recombinant protein prokaryotically expressed after a cecropin protein coding gene, an orf-86 APSL protein coding gene and a 3GSA flexible peptide coding gene located between the cecropin protein coding gene and the orf-86 APSL protein coding gene are fused on the gene level, and is about 30 KD in size. Antimicrobial activity research proves that the fusion protein has antimicrobial activity, and a constructed system solves the problems that natural AMPs are low in expression quantity, have great damage to hosts and are difficult to obtain through genetic engineering and has wide application prospect.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and biotechnology pharmaceutical engineering, in particular to an antibacterial peptide fusion protein, a preparation method thereof, and an application of the fusion protein in the preparation of antibacterial drugs. Background technique [0002] Antimicrobial peptides (AMPs) are a class of small molecular polypeptides with antibacterial activity induced by organisms when they are attacked by pathogenic microorganisms. They are the main components of natural immunity, generally composed of 20-60 amino acid residues, and High temperature, acid resistance, good water solubility, broad antibacterial spectrum, special antibacterial mechanism, rapid sterilization and no drug resistance. Many antimicrobial peptides can not only effectively inhibit the growth of various pathogenic microorganisms such as bacteria, viruses, fungi, and protozoa at low concentrations, but can even kill tumor ce...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61P31/04A61P31/10A61P31/12
Inventor 吕正兵李倩林国栋靳洋洋陈健舒建洪
Owner ZHEJIANG YITAI BIOTECH CO LTD
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