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Perna viridis antibacterial peptide Pernalins, and signal peptide, coding gene and application thereof

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

Active Publication Date: 2020-09-01
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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  • Perna viridis antibacterial peptide Pernalins, and signal peptide, coding gene and application thereof
  • Perna viridis antibacterial peptide Pernalins, and signal peptide, coding gene and application thereof
  • Perna viridis antibacterial peptide Pernalins, and signal peptide, coding gene and application thereof

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 invention provides a perna viridis antibacterial peptide Pernalins, and a signal peptide, coding gene and application thereof. The perna viridis antibacterial peptide Pernalins is selected from one or more of Pernalin A, Pernalin B, Pernalin C and Pernalin D, and the amino acid sequences of the Pernalin A, the Pernalin B, the Pernalin C and the Pernalin D are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 4. After perna viridis is infected with vibrio parahaemolyticus, the antibacterial peptide Pernalins in the blood lymphocytes can respond to immune defense, has a continuous immune defense effect for a long time, and maintains the immune response of an organism.

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 Applications(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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