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Anguilla japonica cytokine IL-6 gene promoter and application thereof

A technology of IL-6 and cytokines, applied in the field of genetic engineering, can solve problems such as economic losses

Active Publication Date: 2021-08-10
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Eel (Anguilla) is an important warm-water fish widely cultivated in Asian countries, and it is also one of the species with the largest single export earnings of aquaculture species in my country. However, in recent years, large-scale and high-density farming has brought eel diseases, bacterial diseases and viruses. The frequent occurrence and prevalence of herpes virus caused huge economic losses (Fan Haiping, Yang Ming, Zhang Jiaolin and Zhong Quanfu (2019). "Eel Herpes Virus Epidemiology and Control Technology." Scientific Fish Farming (08): 46-48 . Zhong Quanfu, Fan Haiping, Lin Yu and Cai Lixuan (2020). The current situation and analysis of my country's eel industry standard system. The 17th China Standardization Forum, Fuzhou, Fujian, China.)

Method used

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  • Anguilla japonica cytokine IL-6 gene promoter and application thereof
  • Anguilla japonica cytokine IL-6 gene promoter and application thereof
  • Anguilla japonica cytokine IL-6 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Cloning of the promoter of the Japanese eel cytokine IL-6 gene

[0042] 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:

[0043] 1. Take 10 mg of Japanese eel muscle tissue and chop 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 56°C water bath overnight for lysing. Add 200 μL Buffer GB and 200 μL 100% ethanol to the lysate, and mix well by pipetting.

[0044] 2. Place the Spin Column on the Collection Tube, transfer the solution to the Spin Column, centrifuge at 12,000rpm for 2 minutes, and discard the filtrate. Add 500 μL of BufferWA to the Spin Column, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate. Add 700 μL of BufferWB (with the specified volume of 100% ethanol added before) to the Spin Column al...

Embodiment 2

[0056] Example 2 Analysis of the transcription factor binding site of the Japanese eel cytokine IL-6 gene promoter

[0057] Log in to the online prediction software Alibaba2 for transcription factor binding sites in the 5′ flanking region of the gene (http: / / gene-regulation.com / pub / programs / alibaba2 / index.html)

[0058] Copy the IL-6 gene promoter sequence that has been verified by the cloning test and paste it in the dialog box in fasta format, and click START to perform the prediction analysis of the transcription factor binding site. partial results such as Figure 1 ~ Figure 2 :

[0059] The main transcription factor binding sites of the Japanese eel cytokine IL-6 gene promoter are as follows:

[0060]

[0061]

Embodiment 3

[0062] Example 3 Analysis of the activity of the Japanese eel cytokine IL-6 gene promoter

[0063] 1. Construction of the recombinant luciferase reporter gene vector pGL3-IL-6-pro containing the promoter fragment of the Japanese eel cytokine IL-6 gene.

[0064] 1. Insert the promoter fragment of the Japanese eel cytokine IL-6 gene 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 Japanese eel IL-6 gene promoter sub-control, the luciferase recombinant vector constructed was named pGL3-IL-6-pro. Specific steps are as follows:

[0065] Synthesize an upstream primer with a SacI restriction site:

[0066] 5'-GAGCTCTTGTGAATGCCTGAGTTGAAGAC-3' (SEQ ID NO: 5),

[0067] Downstream primers with HindⅢ restriction sites:

[0068] 5'-AAGCTTCCTGCCTTTAGCTCCTTACATTAA-3' (SEQ ID NO: 6).

[0069] High-fidelity enzymes from Takara GC Buffer (Mg 2+ plus) for amplification, ...

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Abstract

The invention relates to an anguilla japonica cytokine IL-6 gene promoter and application thereof. The anguilla japonica genome is compared and analyzed through a first exon sequence of an IL-6 gene open reading frame, an IL-6 gene promoter sequence is cloned by analyzing and predicting a 5' flanking region sequence of the IL-6 gene through a touchdown PCR method, and the IL-6 gene promoter pGL3-IL-6-pro luciferase reporter plasmid is successfully constructed. Experiments prove that the IL-6 gene promoter can be induced and activated by poly I: C, LPS and aeromonas hydrophila, and it is found that the important signal channel inflammation regulation factor Caspase-1 can significantly up-regulate the luciferase activity of the IL-6 gene promoter. The invention provides a good experimental system for researching the expression regulation mechanism of the anguilla japonica cytokine IL-6 gene and the regulation mechanism of important fish inflammation-related NF-kappa B, MAPK and I-type interferon signal path network, and also can efficiently express exogenous genes after constructing an expression vector by using the promoter; or the promoter is applied to construction of transgenic fish and has important theoretical and practical significance.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Japanese eel cytokine IL-6 gene promoter and application thereof. Background technique [0002] Interleukin 6 (interleukin 6, IL-6) is an important early inflammatory cytokine, which plays an important role in the inflammatory response, mainly produced by monocytes, macrophages, Th2 cells and fibroblasts. Produced (Yang Hanchun. Animal Immunology [M]. Beijing: China Agricultural University Press, 2003). The biological function of IL-6 is mainly to play an important role in mediating innate and adaptive immune responses by promoting B cell differentiation and immunoglobulin production, inducing liver cells to produce acute phase response protein, and promoting T cell maturation. Previous studies have shown that the expression level of IL-6 gene in fish is significantly increased after being stimulated by various exogenous factors such as Aeromonas hydrophila, LPS, p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/24C12N15/70C12N15/85C12N1/21C12N5/10
CPCC07K14/5412C12N15/70C12N15/85C12N5/0692C12N2800/106C12N2510/00Y02A40/81
Inventor 冯建军
Owner JIMEI UNIV
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