Scylla paramamosain antibacterial peptide Scyreprocin and application thereof

A technology of Scylla simulans and antibacterial peptides, which is applied in the field of Scyreprocin antibacterial peptides, can solve the problems of product quality and safety impact, drug resistance of pathogenic microorganisms, threats to human health, etc., and achieves great application value and sterilization. Fast speed, good antibacterial effect

Active Publication Date: 2020-05-29
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of antibiotics has improved feed utilization efficiency and industrial economic benefits, and the abuse of antibiotics has also caused pathogenic microorganisms to develop drug resistance and double infection. seriously affected, threatening human health

Method used

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  • Scylla paramamosain antibacterial peptide Scyreprocin and application thereof
  • Scylla paramamosain antibacterial peptide Scyreprocin and application thereof
  • Scylla paramamosain antibacterial peptide Scyreprocin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of the antimicrobial peptide Scyreprocin from Scylla pseudocaveae

[0034] The open reading frame sequence of the antimicrobial peptide Scyreprocin is:

[0035]

[0036] The full-length cDNA sequence of Scyreprocin gene was obtained and verified by RACE technology. The open reading frame of Scyreprocin gene is 255bp (including stop codon TAA), GenBank accession number: MH488960.

[0037] According to the cDNA sequence of the Scyreprocin gene, gene-specific primers were designed for repeated verification by RACE technology (Table 1).

[0038] Table 1 Scyreprocin sequence amplification primer list

[0039]

[0040]

[0041] The non-coding sequence of Scyreprocin gene was amplified by RACE technology (taking 5′UTR amplification as an example):

[0042] Using the RACE cDNA prepared by the applicant earlier as a PCR template, the specific reaction system is as follows:

[0043] ①Outer PCR reaction:

[0044]

[0045] The reaction procedure...

Embodiment 2

[0071] Example 2 Acquisition of Scyreprocin Genetic Engineering Expression Product of Antimicrobial Peptide Scyreprocin

[0072] (a) Obtain the open reading frame sequence of Scyreprocin antimicrobial peptide Scyreprocin from Scylla pseudocarpa cDNA template, clone and recombine it into the vector pET28a, confirm the recombinant vector with correct reading code by base sequencing, and transfer it into the expression strain Escherichia coli BL21 (Escherichia coli BL21), a recombinant expression strain capable of expressing the antimicrobial peptide Scyreprocin of Scylla syringae fused with a His tag was obtained.

[0073] (b) Pick a single clone of the expression strain in liquid LB medium, culture it in a shaker flask at 180 rpm at 37°C for 10-14 hours, then transfer it to liquid LB medium containing 0.5% glucose at a ratio of 1:1000 and continue to cultivate until OD 600 = 0.3, add IPTG to make the final concentration 0.5mM to induce expression, continue to cultivate the exp...

Embodiment 3

[0077] Example 3 Determination of the minimum inhibitory concentration (MIC: minimum inhibition concentration) of the antimicrobial peptide of Scylla pseudocaveus

[0078] (a) Streak the preserved Escherichia coli, Escherichia coli MC1061, Pseudomonas stutzeri, Pseudomonas hydrophila, Staphylococcus aureus, Micrococcus lyticus, etc. on the nutrient broth plate, Vibrio riverina Stretch on the 2216E seawater broth plate and invert for 12-16 hours at the corresponding optimal temperature; Streak on the plate and culture at 28°C for 1-2 weeks; spread Aspergillus niger spores on the PDA plate and culture upside down at 28°C for 3-7d. Pick colonies from each plate and inoculate them on the corresponding medium slant, continue to culture for 10-16h for bacteria, 1-3d for fungi, and 3-7d for mold, then rinse the slant with 10mM phosphate buffer (pH7.4) , Bacteria were adjusted and diluted to OD with MH liquid medium: 10mM phosphate buffer = 2:3 mixed medium 600 =0.003; Vibrio was ad...

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Abstract

The invention discloses a scylla paramamosain antibacterial peptide Scyreprocin and an application thereof. The amino acid sequence of the scylla paramamosain antibacterial peptide Scyreprocin comprises a sequence as shown in SEQ ID NO 01. The scylla paramamosain antibacterial peptide Scyreprocin is obtained through expression and purification with a genetic engineering technique. The antibacterial peptide disclosed by the invention is broad in antimicrobial spectrum, good in antibacterial effects and high in sterilizing rate, shows enormous application value, and has favorable application inthe respect of preparation of antibacterial agents. The antibacterial peptide disclosed by the invention does not have cytotoxicity on hepatic parenchymal cells AML12 of mice, human normal liver cellsL02 and the like, and can be safely used for medication or can be used as a feed composition.

Description

technical field [0001] The invention belongs to the technical field of crustacean genetic engineering, and in particular relates to Scyreprocin, an antimicrobial peptide of Scylla pseudocaveus, and its application. Background technique [0002] In order to meet the rapid growth of the world's population and the increasing demand for food, the livestock, poultry and aquaculture industries have developed rapidly in recent years, and a large number of antibiotics have been used in industries to resist various pathogenic bacteria. The application of antibiotics has improved feed utilization efficiency and industrial economic benefits, and the abuse of antibiotics has also caused pathogenic microorganisms to develop drug resistance and double infection. be seriously affected and threaten human health. Therefore, it is imminent to find high-efficiency and green alternatives to antibiotics, which has become a research hotspot in recent years. Antimicrobial peptides that are safe ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12A61K38/17A61P31/04A61P31/10A23K20/195A23K20/147
CPCC07K14/43509A61P31/04A61P31/10A23K20/195A23K20/147A61K38/00A23V2250/55A61K38/17A61P31/00A23K50/10A23K50/75C07K2319/21A61K45/06
Inventor 王克坚杨盈陈芳奕陈炎超陈慧芸彭会
Owner XIAMEN UNIV
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