Anguilla japonica antibacterial peptide Cathelicidin1 gene promoter and application thereof

A promoter, antimicrobial peptide technology, applied in the field of genetic engineering, can solve problems such as missing transcription binding sites

Pending Publication Date: 2021-11-30
JIMEI UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The promoter is the key factor determining gene expression and regulation. In order to study the expression regulation mechanism of the fish Cathelicidin1 gene, we obtained the possible 5′ flank regulation of the Japanese eel Cathelicidin1 gene by comparing the open reading frame sequence of the Japanese eel Cathelicidin1 gene with the genome sequence The promoter sequence of the Japanese eel Cathelicidin1 gene was obtained after primer design and PCR cloning verification, and the analysis showed that the promoter had the transcription binding sites of C / EBPalp, NF-kappaB, GATA, GAAA, and AP1 in the reported rainbow trout Cathelicidin1 promoter However, CREBP and TFIID transcription binding sites are missing, and there are unique transcription factor binding sites such as RAP1, Sp1, AP-2alph, RAP1, USF, NF-EM5, etc., showing the species of the Japanese eel Cathelicidin1 gene promoter sequence specificity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anguilla japonica antibacterial peptide Cathelicidin1 gene promoter and application thereof
  • Anguilla japonica antibacterial peptide Cathelicidin1 gene promoter and application thereof
  • Anguilla japonica antibacterial peptide Cathelicidin1 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning of the Japanese eel antimicrobial peptide Cathelicidin1 gene promoter

[0041] 1. Using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 Kit to extract and purify the genomic DNA from the Japanese eel muscle tissue. The specific operation is as follows:

[0042] 1. Take 10 mg of Japanese eel muscle tissue and mince it with a blade, put it in a 2ml centrifuge tube, add 180 μL of Buffer GL, 20 μL of Proteinase K and 10 μL of RNase A (10 mg / mL), and warm it in a water bath at 56 ° C overnight for lysing.

[0043] 2. Add 200 μL Buffer GB and 200 μL 100% ethanol to the lysate, and mix well by pipetting. Place the SpinColumn on the Collection Tube, transfer the solution to the Spin Column, centrifuge at 12,000rpm for 2 minutes, and discard the filtrate.

[0044] 3. Add 500 μL of Buffer WA to the Spin Column, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate.

[0045] 4. Add 700 μL of Buffer WB (100% ethanol with a specified v...

Embodiment 2

[0057]Example 2 Prediction of the transcription factor binding site of the Japanese eel antimicrobial peptide Cathelicidin1 gene promoter

[0058] Log in to the online prediction software Alibaba2 (http: / / gene-regulation, com / pub / programs / alibaba2 / index.html) for the binding sites of transcription factors in the 5′ flanking region of the gene, and activate the antimicrobial peptide Cathelicidin1 gene that has been verified by cloning tests After copying the subsequence, paste it in the dialog box in fasta format, and click START to perform the prediction analysis of transcription factor binding sites. The result is as figure 1 Shown:

[0059] The main transcription factor binding sites of the Japanese eel antimicrobial peptide Cathelicidin1 gene promoter are as follows:

[0060]

Embodiment 3

[0061] Example 3 Analysis of the activity of the Japanese eel antimicrobial peptide Cathelicidin1 gene promoter

[0062] 1. Construction of the recombinant luciferase reporter gene vector pGL3-Cathelicidin1-pro containing the promoter fragment of the Japanese eel antimicrobial peptide Cathelicidin1 gene.

[0063] The Japanese eel antimicrobial peptide Cathelicidin1 gene promoter fragment was inserted into the luciferase reporter gene vector pGL3-Basic of Promega Company, so that the expression of the firefly luciferase (Luciferase) reporter gene was controlled by the Japanese eel antimicrobial peptide Cathelicidin1 gene promoter, and the obtained The recombinant vector was named pGL3-Cathelicidin1-pro. Specific steps are as follows:

[0064] Synthesize an upstream primer with a SacI restriction site:

[0065] 5'-CGAGCTCCAGTTATTTTGGCAGAGCCTTGTAGA-3' (as shown in SEQ ID NO: 5),

[0066] Downstream primers with Hind III restriction sites:

[0067] 5'-CCCAAGCTTAGTTTCAGCATGAGGG...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an anguilla japonica antibacterial peptide Cathelicidin1 gene promoter and an application thereof. According to the anguilla japonica antibacterial peptide Cathelicidin1 gene promoter, the anguilla japonica antibacterial peptide Cathelicidin1 gene promoter sequence is successfully cloned, and the anguilla japonica antibacterial peptide Cathelicidin1 gene promoter pGL3-Cathelicidin1-pro luciferase reporter plasmid is successfully constructed; and reporter gene analysis experiments prove that the anguilla japonica antibacterial peptide Cathelicidin1 gene promoter can be induced and activated by a virus simulant poly I:C, an important surface antigen LPS of gram-negative bacteria and an important pathogenic bacterium aeromonas hydrophila of aquatic animals. The invention discloses clone of an anguilla japonica antibacterial peptide Cathelicidin1 gene promoter and inducible expression analysis of strong promoter activity of the anguilla japonica antibacterial peptide Cathelicidin1 gene promoter, and provides a good experimental system for researching an expression regulation mechanism of a fish antibacterial peptide Cathelicidin1 gene, a natural immune response mechanism of fish for resisting pathogenic bacterium infection, and particularly a regulation mechanism of a fish inflammation-related NF-[kappa]B and MAPK signal path network, and therefore, conditions are created for constructing an expression vector by using the promoter to efficiently express an exogenous gene or applying the promoter to transgenic fish construction.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Japanese eel antibacterial peptide Cathelicidin1 gene promoter and application thereof. Background technique [0002] Antimicrobial peptides (AMPs) are a class of biologically active small molecule peptides, which are an important part of the body's innate immune defense system and can be effective against Gram-negative bacteria, Gram-positive bacteria, fungi, viruses and parasitic Both have good inhibitory or killing effects, and play an important role in natural immune response[Chung, C.-R., J.-H.Jhong, Z.Wang, W.Chen, Wan, Horng and T. - Y. Lee (2020). "Characterization and Identification of Natural Antimicrobial Peptides on Different Organisms." International Journal of Molecular Sciences 21: 986.]. Fish, like mammals and other higher vertebrates, have both innate and adaptive immune systems. As an important part of the innate immune system of fish, antimicrob...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/113C12N15/85A01K67/027
CPCC07K14/461C12N15/85C12N15/8509A01K67/0278A01K2227/40A01K2267/03C12N2800/106C12N2800/107Y02A50/30
Inventor 冯建军彭欣慰林鹏王艺磊陈鹏云
Owner JIMEI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap