Emerald mussel antimicrobial peptide pernalins, signal peptide of antimicrobial peptide, coding gene of antimicrobial peptide and application

A technology that encodes genes and antimicrobial peptides. It is applied in the fields of application, antibacterial drugs, and genetic engineering. It can solve the problems of little research on antimicrobial peptides, achieve a wide range of applications and prospects, fast response time, and maintain immune responses.

Active Publication Date: 2021-12-24
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few research contents on antimicrobial peptides in emerald mussels

Method used

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  • Emerald mussel antimicrobial peptide pernalins, signal peptide of antimicrobial peptide, coding gene of antimicrobial peptide and application
  • Emerald mussel antimicrobial peptide pernalins, signal peptide of antimicrobial peptide, coding gene of antimicrobial peptide and application
  • Emerald mussel antimicrobial peptide pernalins, signal peptide of antimicrobial peptide, coding gene of antimicrobial peptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Test the expression level difference of antimicrobial peptide Pernalins in different tissues

[0050] 1) Collection and breeding of emerald mussels: collect emerald mussels located in the culture box of Dapeng Sea Area, Shenzhen (22°34′11″N 114°31′50″E) and culture them in the laboratory. For aquaculture in seawater, use an oxygen pump for oxygenation.

[0051] 2) Tissue sampling of Emerald mussels: every 5 of Emerald mussels was divided into 4 groups for sampling. Tissues from 6 parts were collected: feet, blood lymphocytes, hepatopancreas, sclerenchyma, gills, and mantle. After collection, they were immediately frozen in liquid nitrogen and stored at -80°C for subsequent experiments.

[0052] 3) Total RNA extraction: use TRIzol reagent to extract the total RNA of emerald mussels, absorbing no more than 5×10 6 Centrifuge at 8000×g, 4°C for 2min, remove the supernatant; add appropriate amount of TRIzol, fully resuspend, let stand at room temperature for 5min...

Embodiment 2

[0061] Example 2 Changes in the expression level of antimicrobial peptide Pernalins after emerald mussels were infected with Vibrio parahaemolyticus

[0062] 1) Collection and breeding of emerald mussels: collect emerald mussels located in the culture box of Dapeng Sea Area, Shenzhen (22°34′11″N 114°31′50″E) and culture them in the laboratory. For aquaculture in seawater, use an oxygen pump for oxygenation.

[0063] 2) Cultivate Vibrio parahaemolyticus: first cultivate Vibrio parahaemolyticus to the logarithmic growth phase in 3% LB, carry out secondary activation, when growing to the logarithmic growth phase, use phosphate buffered solution (PBS, 124mM Na 2 HPO 4 , 76mM NaH 2 PO 4 ; pH 7.0) was washed three times, and the microbial concentration was adjusted to 1×10 7 cfu / mL for subsequent experiments.

[0064] 3) Set up the control group and the experimental group: every 5 emerald mussels constitute a group, and set up 4 groups, 1 control group and 3 experimental groups...

Embodiment 3

[0069] The obtaining method of embodiment 3 Emerald mussel cDNA comprises the steps:

[0070] ① After the blood lymphocytes of Emerald mussels were extracted, the total RNA of the blood lymphocytes was extracted with TRIzol reagent, and cDNA was obtained by reverse transcription;

[0071] ②Specific primers were designed according to the ORF sequence of Pernalins, using the cDNA of Jade mussel blood lymphocytes as a template, the ORF sequence of antimicrobial peptide was obtained by PCR cloning, the primer sequence is shown in Table 1, and the specific DNA fragment of the same size as Pernalins was obtained, which was sequenced The antimicrobial peptide Pernalin A gene of 267bp, the antimicrobial peptide Pernalin B gene of 174bp, the antimicrobial peptide Pernalin C gene of 270bp, and the antimicrobial peptide Pernalin D gene of 270bp were obtained. The obtained gene fragment was confirmed to be a new gene by blast comparison on the NCBI website. The amino acid sequence of the...

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Abstract

The present invention provides the antimicrobial peptide Pernalins of emerald mussel, the signal peptide of the antimicrobial peptide Pernalins, the coding gene of the antimicrobial peptide Pernalins and the application thereof, and the antimicrobial peptide Pernalins of the emerald mussel is selected from Pernalin A, Pernalin B, Pernalin C and Pernalin D One or more; the amino acid sequences of Pernalin A, Pernalin B, Pernalin C and Pernalin D are respectively shown in SEQ ID NO: 1-SEQ ID NO: 4. After emerald mussels are infected with Vibrio parahaemolyticus, the antimicrobial peptide Pernalins in blood lymphocytes can respond to immune defense, play a long-term and continuous role in immune defense, and maintain the body's immune response.

Description

technical field [0001] The invention relates to the technical field of invertebrate genetic engineering, in particular to the emerald mussel antimicrobial peptide Pernalins, the signal peptide of the antimicrobial peptide, the coding gene of the antimicrobial peptide and its application. Background technique [0002] Antimicrobial peptides (AMPs) are an important part of the natural immune system, ubiquitous in all multicellular organisms, play an extremely important role in immune defense, can directly kill pathogens, and can also be used as immune effector molecules to regulate Host immune defense system. It also has the characteristics of broad-spectrum antibacterial, high efficiency, rapid bactericidal ability, and not easy to produce drug-resistant strains, so it has become a research hotspot at home and abroad. [0003] In 1972, Swedish scientist Boman first discovered antimicrobial peptides in Drosophila, and then Steiner and others isolated an antimicrobial peptide ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C07K14/00C07K7/08A61K38/17A61P31/04
CPCC07K14/43509C07K14/00C07K7/08A61P31/04A61K38/00C07K2319/02
Inventor 李辉曾志勇王玉婷
Owner SHENZHEN UNIV
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