Gene for expressing antibacterial peptide, antibacterial peptide and application

A technology of bacteriostatic peptides and expression vectors, which is applied in the expression of bacteriostatic peptide genes, bacteriostatic peptides and its applications, and can solve problems such as ecological pollution in water areas, eel residues, and enhanced drug resistance of pathogenic organisms

Active Publication Date: 2018-07-20
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually antibiotics are needed to control diseases, but antibiotic treatment has great disadvantages, such as residues in eels, enhanced drug resistance of pathogens, ecological pollution of waters, etc.

Method used

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  • Gene for expressing antibacterial peptide, antibacterial peptide and application
  • Gene for expressing antibacterial peptide, antibacterial peptide and application
  • Gene for expressing antibacterial peptide, antibacterial peptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The gene cloning of embodiment 1 antimicrobial peptide, expression purification and identification

[0036] 1. Design and synthesis of bacteriostatic peptide gene primers:

[0037] Referring to the Japanese eel antimicrobial peptide gene sequence with high homology to the antimicrobial peptide gene fragments screened in the subtractive library published in Genbank, the primers of the antimicrobial peptide gene were designed and evaluated by using the combination of Primer5 and Oligo7 software. Good primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0038] Table 1 The primers designed for the antibacterial peptide gene of eel

[0039]

[0040] 2. Extraction of total RNA and reverse transcription to obtain cDNA

[0041] The liver, spleen and kidney tissues of the European eel in the experimental group were collected for RNA extraction. Extract about 100 mg of tissue into a 1.5mLEP tube, add 0.2mL TRIzol reagent, first cut the tissue into pieces wi...

Embodiment 2

[0092] Example 2 Bacteriostasis test of bacteriostatic peptides on Staphylococcus aureus: the antibacterial effect of elecilin protein on Staphylococcus aureus was identified by plate inhibition test.

[0093] 1 Materials and methods

[0094] 1.1 Experimental materials:

[0095] TSA agarose plate, double antibody (streptomycin, penicillin), Staphylococcus aureus;

[0096] The eluate after 5 times of elution in the Thrombin shearing GST tag assay ( Image 6 shown)

[0097] 1.2 Experimental method: Take 200 μL of the diluted bacterial solution on each agarose plate and smear the plate. After the liquid on the surface of the plate is dry, add 10 μL of the diluted sample to the designated area of ​​each plate, and place it in a constant temperature incubator at 28°C. Observe the zone of inhibition.

[0098] 2 Experimental results

[0099] The results of the plate inhibition zone test were as follows: Figure 7 , The antibacterial test of elecilin protein to Staphylococcus au...

Embodiment 3

[0100] Embodiment 3 bacteriostatic peptide is to the MIC antibacterial test of Staphylococcus aureus:

[0101] Through the MIC experiment, different concentrations of antimicrobial peptides were mixed with Staphylococcus aureus, and the OD value of each bacterial solution was measured after culturing for 24 hours.

[0102] 1.1 Culture medium: LB medium and nutrient broth

[0103] 1.2 Samples for test:

[0104] Amp (100mg / ml, 1000×) AMP and ampicillin are normal antibiotics with strong antibacterial ability;

[0105] elecilin protein (0.5mg / ml)

[0106] 1.3 Method

[0107] 1.3.1 Sample preparation

[0108] 1.3.2 Preparation of bacterial suspension Counted by flow cytometer (BD), calculate the OD of 0.6-1.0 bacterial suspension concentration after activation and transfer once, and dilute each bacterial strain to 1×10 before the test 5 ~5×10 5 cfu / mL bacterial suspension for use.

[0109] 1.3.3 Determination of Minimum Inhibitory Concentration (MIC) Add 90 ul of bacterial...

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Abstract

The nucleotide sequence of SEQ ID NO:1 is found in European eel for the first time, and antibacterial peptide with bacteriostatic activity is obtained through genetic engineering. Experiments prove that the antibacterial peptide can effectively inhibit the activity of staphylococcus aureus; the antibacterial function is realized. The inventor builds a suppression subtractive hybridization libraryof difference expression genes in tissues before and after the European eel infected with ibrio vulnificus by a suppression subtractive hybridization technology. From the library, people find that theoccurrence frequency of the gene is high; through bacteriostasis experiments, the condition that the gene has a pathogenic bacterium inhibition effect is found; the gene can be used as an antibioticsubstitute for controlling the pathogenic bacterium growth. The inventor expresses the gene through the genetic engineering; after the treatments of purification, GST label removing and the like are performed on the product, the effect that an extract has bacteriolysis activity is found.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for expressing bacteriostatic peptide genes, bacteriostatic peptides and applications. Background technique [0002] Bacterial diseases pose a great threat to the eel aquaculture industry and often cause huge economic losses. Usually antibiotics are needed to control diseases, but antibiotic treatment has great disadvantages, such as residues in eels, enhanced drug resistance of pathogens, and ecological pollution of waters. Therefore, there is an urgent need for a suitable antibiotic substitute to control pathogenic bacteria and prevent and treat bacterial diseases. Contents of the invention [0003] The inventor provides an isolated polynucleotide having the nucleotide sequence shown in SEQ ID NO:1. [0004] SEQ ID No. 1: [0005] atgagaagtgatacacataagatgaagagctctgttggacctctgctgctgctctcccttgttgcttttgtctctgtgacattggccaggagtgtcttcacctttacagatgtccttgccgcggccactgcagacttc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/46C12N15/63A61K38/17A61P31/04
CPCA61K38/00C07K14/461
Inventor 陈叙林晨韬张丽娟李素一柯翎陈华吴唯维
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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