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Antimicrobial peptide fusion protein and its preparation method and application

A technology of fusion protein and antimicrobial peptide, which is applied in the preparation of antibacterial drugs. The field of antimicrobial peptide fusion protein and its preparation can solve the problems of unsuitability for large-scale production, many bottlenecks in antimicrobial peptide technology, and microscopic cecropin antimicrobial peptide. To achieve the effect of guaranteeing the later purification and renaturation and fusion protein activity, strong activity and large expression

Active Publication Date: 2020-11-13
杭州依思康医药科技有限公司
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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, but The cecropin antibacterial peptide contained in it is very small

Method used

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  • Antimicrobial peptide fusion protein and its preparation method and application
  • Antimicrobial peptide fusion protein and its preparation method and application
  • Antimicrobial peptide fusion protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Construction of recombinant expression vector

[0040] 1. Design and synthesis of the target fragment

[0041] Obtain cecropin nucleotide sequence (as shown in SEQ ID NO.6) by NCBI database, find shark liver peptide orf-863 gene (as shown in SEQ ID NO.7) in the shark liver small peptide cDNA database of this laboratory , orf-863 shark liver peptide is a small peptide translated from the shark liver peptide orf-863 gene with an isoelectric point of 11.42 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.8) in the middle of the acid sequence to form cecropin+3GSA coding fragment+shark liver peptide orf-863 fusion gene (hereinafter referred to as orf- 863s fusion gene, as shown in SEQ ID NO.4) and shark liver peptide orf-863+3GSA coding fragment+cecropin fusion gene (hereinafter referred to as orf-863s fusion gene, ...

Embodiment 2

[0048] Example 2: Induced expression and concentration of fusion proteins

[0049] 1. Induced expression of recombinant fusion protein

[0050] After the sequencing was correct, the plasmid was extracted, and the recombinant plasmid was transformed into BL21(DE3) Escherichia coli for induced expression.

[0051] The two successfully constructed engineering bacteria were inoculated in 100mL LB culture medium, and cultured on 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

[0057] Embodiment 3: Oxford cup method detects antibacterial activity

[0058] Detection of antibacterial activity of recombinant protein orf-863 and recombinant protein orf-863s

[0059] 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 bacterial liquid 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, orf863, orf863s to the Oxford cup, Amp is a positive control and ddH2O is a negative control. After culturing overnight at 37°C, observe the size of the inhibition zone to determine the antibacterial activity...

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Abstract

The invention provides an antibacterial peptide fusion protein, which comprises an antibacterial peptide cecropin protein and an orf-863 shahepatide protein, wherein the antibacterial peptide cecropin protein is connected with the orf-863 shahepatide protein through a 3GSA flexible peptide segment. The fusion protein is a soluble recombinant protein formed through horizontal fusion of an antibacterial peptide cecropin protein coding gene, an orf-863 shahepatide protein coding gene and a 3GSA flexible peptide coding gene between the antibacterial peptide cecropin protein coding gene and the orf-863 shahepatide protein coding gene and prokaryotic expression, and the size is about 30KD. An antibacterial activity research shows that the protein has antibacterial activity, the problems that a natural antibacterial peptide is low in expression quantity and large in damage to a host, and cannot be easily obtained through a genetic engineering method are solved through a construction system, and the antibacterial peptide fusion protein has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biotechnology and pharmaceutical engineering, in particular to an antibacterial peptide fusion protein and a preparation method thereof, as well as the 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 component of natural immunity and generally consist of 20-60 amino acid residues. 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 cells, but have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61P31/00
CPCA61K38/00C07K14/43586C07K14/461C07K2319/00C12N15/70C12N2800/101
Inventor 吕正兵杜瑶吴佳许国强程丹丹舒建洪
Owner 杭州依思康医药科技有限公司
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