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Anguilla japonica liver expression antibacterial peptide LEP2 gene promoter and application thereof

A promoter and antimicrobial peptide technology, applied in the field of genetic engineering

Pending Publication Date: 2021-11-30
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, studies on the expression of antimicrobial peptide promoters in fish livers have only been reported in golden pomfret [Liu, B., G.-D. Liu, H.-Y. Guo, K.-C. Zhu, L. Guo, N. Zhang, B.-S. Liu, S.-G. Jiang and D.-C. Zhang (2020). "Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) from golden pompano Trachinotus ovatus(Linnaeus 1758). "Fish&Shellfish Immunology 104:419-430; Research on the function and transcriptional regulation of two antimicrobial peptide genes in pomfret ovatus, master's thesis, Liu Guangdong, Tianjin Agricultural University, 2019, English paper is published as part of the master's thesis ], which only involves the study of the transcriptional activity of the promoter in human 293 cells, but whether the liver-expressed antimicrobial peptide promoter has transcriptional activity in fish cells and whether it can be induced by pathogenic microorganisms such as viruses and bacteria The mechanism of immune regulation has not been reported

Method used

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  • Anguilla japonica liver expression antibacterial peptide LEP2 gene promoter and application thereof
  • Anguilla japonica liver expression antibacterial peptide LEP2 gene promoter and application thereof
  • Anguilla japonica liver expression antibacterial peptide LEP2 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of the promoter of the antimicrobial peptide LEAP2 gene expressed in Japanese eel liver

[0043] 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:

[0044] 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.

[0045] 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.

[0046] 3. Add 500 μL of BufferWA to the Spin Column, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate.

[0047] 4. Add 700 μL of BufferWB (100% ethanol wi...

Embodiment 2

[0059] Example 2 Antimicrobial peptide LEAP2 gene promoter transcription factor binding site prediction in Japanese eel liver

[0060] Log in to the online prediction software Alibaba2 (http: / / gene-regulation.com / pub / programs / alibaba2 / index.html) for transcription factor binding sites in the 5′ flanking region of the gene, and express the antimicrobial peptide LEAP2 in the liver that has been verified by cloning tests After copying the gene promoter sequence, 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:

[0061] The main transcription factor binding sites of the antimicrobial peptide LEAP2 gene promoter expressed in the liver of Japanese eel are as follows:

[0062]

Embodiment 3

[0063] Example 3 Analysis of the activity of the antimicrobial peptide LEAP2 gene promoter expressed in Japanese eel liver

[0064] 1. Construction of the recombinant luciferase reporter gene vector pGL3-LEAP2-pro containing the promoter fragment of the antimicrobial peptide LEAP2 gene expressed in the liver of Japanese eel.

[0065] 1. Insert the promoter fragment of the antimicrobial peptide LEAP2 gene expressed in the liver of Japanese eel into the luciferase reporter gene vector pGL3-Basic of Promega Company, so that the expression of the firefly luciferase (Luciferase) reporter gene is initiated by the antimicrobial peptide LEAP2 gene expressed in the liver of Japanese eel sub-control, the constructed recombinant vector was named pGL3-LEAP2-pro. Specific steps are as follows:

[0066] 2. Synthesize the upstream primer with MluI restriction site:

[0067] 5'-CGACGCGTCCTCACAAGGTTCACCGCAAGAC-3' (such as SEQ ID NO: 5),

[0068] Downstream primers with XhoI restriction sites:...

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Abstract

The invention relates to an anguilla japonica liver expression antibacterial peptide LEP2 gene promoter and application thereof. The anguilla japonica liver expression antibacterial peptide LEP2 gene promoter sequence is successfully cloned, and the anguilla japonica liver expression antibacterial peptide LEAP2 gene promoter pGL3-LEAP2-pro luciferase reporter plasmid is successfully constructed; and experiments prove that the anguilla japonica liver expression antibacterial peptide LEAP2 gene promoter can be induced and activated by a virus simulant polyI:C, a Gram-negative bacterium important surface antigen LPS and aeromonas hydrophila. According to the invention, a good experimental system can be provided for researching an expression regulation mechanism of the anguilla japonica liver expression antibacterial peptide LEAP2 gene, a natural immune response mechanism of fish for resisting pathogenic bacterium infection, and particularly researching an important fish inflammation related NF-[kappa]B and MAPK signal path network regulation mechanism; and 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 gene promoter for expressing antimicrobial peptide LEP2 in the liver of Japanese eel and its application. 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 the 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 syste...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70C12N15/81C12N15/85C12N15/74C12N15/75C12N1/21C12N1/19A01K67/027C12R1/19C12R1/125
CPCC07K14/461C12N15/70C12N15/81C12N15/8509C12N15/746C12N15/75A01K67/0275A01K2227/40A01K2267/01
Inventor 冯建军彭欣慰林鹏王艺磊陈鹏云
Owner JIMEI UNIV
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