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Larimichthys crocea interferon IFN1 promoter, nucleic acid constructor, cell as well as preparation method and application of larimichthys crocea interferon IFN1 promoter

A nucleic acid construct and promoter technology, applied in the field of genetic engineering, can solve problems such as lack of research

Inactive Publication Date: 2017-11-07
JIMEI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the expression regulation mechanism of fish interferon IFN1 gene, especially the regulation network of cell signal transduction pathways involved in interferon IFN1 is still relatively scarce

Method used

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  • Larimichthys crocea interferon IFN1 promoter, nucleic acid constructor, cell as well as preparation method and application of larimichthys crocea interferon IFN1 promoter
  • Larimichthys crocea interferon IFN1 promoter, nucleic acid constructor, cell as well as preparation method and application of larimichthys crocea interferon IFN1 promoter
  • Larimichthys crocea interferon IFN1 promoter, nucleic acid constructor, cell as well as preparation method and application of larimichthys crocea interferon IFN1 promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the cloning of large yellow croaker interferon IFN1 gene promoter

[0037] Based on the following figure 1 process steps.

[0038] Using the large yellow croaker genome (extract the large yellow croaker genome DNA from the large yellow croaker on the market according to the normal genome extraction method) as a template, design primers, and the primer sequences are as follows;

[0039] Forward primer: -3'SEQ ID NO:2; wherein the underlined straight line is the KpnI restriction site, and the underlined wavy line is SEQ ID NO:4;

[0040] Reverse primer: -3' SEQ ID NO:3; where the underlined straight line is the Bgl II restriction site, and the underlined wavy line is SEQ ID NO:5.

[0041] Use TaKaRa ExTaq enzyme to amplify the promoter of the large yellow croaker interferon IFN1 gene by PCR. The PCR reaction system is as follows:

[0042]

[0043] The PCR reaction conditions are as follows:

[0044]

[0045] The PCR product was detected by 1% a...

Embodiment 2

[0047] Example 2: Construction of the large yellow croaker interferon IFN1 gene promoter recombinant vector pGL4-IFN1-pro

[0048] 1) Expand and cultivate the Top10 strain of the above-mentioned pMD19-T-IFN1-pro recombinant vector, extract the plasmid, and digest the recombinant plasmid with Kpn I / Bgl II, perform agarose gel electrophoresis, and cut the gel for recovery. After the IFN1promoter;

[0049] 2) Digest the pGL4.17 luciferase reporter vector (promega company) with Kpn I / Bgl II double enzymes and recover the digested vector with a gel recovery kit;

[0050] 3) Use T4 DNA ligase from TaKaRa Company to connect the double-digested IFN1 promoter sequence fragment and the vector pGL4.17 to construct the recombinant vector pGL4-IFN1-pro. The connection system is as follows:

[0051]

[0052] Reaction conditions: Incubate overnight at 16°C;

[0053] 4) Transform the above ligation product into Escherichia coli Top10 competent cells, screen positive clones and send them ...

Embodiment 3

[0055] Example 3: Dual-luciferase reporter gene detection system analyzes the activity of the large yellow croaker interferon IFN1 gene promoter

[0056] 1. Inoculate EPC cells in a 24-well cell culture plate, 2×10 per well 5 Add 500 μl of MEM medium to each cell, and culture overnight in a biochemical incubator at 25°C;

[0057] 2. Use Invitrogen’s Lipofectamine for each well of EPC cells TM 3000 transfection reagent co-transfect 100ng of recombinant vector pGL4-IFN1-pro obtained in Example 2 and 10ng Renilla luciferase control reporter gene vector pRL-TK; at the same time, co-transfect 100ng empty vector pGL4.17 and 10ng sea Renal luciferase control The EPC cells of the reporter gene carrier pRL-TK were used as the control; 3 replicates were set for each treatment. The specific steps of cell transfection are as follows:

[0058] a) Mix the vector to be transfected (100ng pGL4-IFN1-pro+10ng pRL-TK or 100ng pGL4.17+10ng pRL-TK) with 25μl Opti-MEM medium (purchased from GIB...

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Abstract

The invention discloses a larimichthys crocea interferon IFN1 promoter, a nucleic acid constructor, a vector, a cell as well as a preparation method and an application of the larimichthys crocea interferon IFN1 promoter. The nucleotide sequence of the larimichthys crocea interferon IFN1 promoter is shown in SEQ ID NO:1 or is complementary with the nucleotide sequence shown in the SEQ ID NO:1. The invention further discloses an application of the promoter, the nucleic acid constructor, the vector, the recombinant cell or fishes or mammals containing the promoter, the nucleic acid constructor, the vector or the recombinant cell in regulating target gene expression or transgenic fish.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a large yellow croaker interferon IFN1 promoter, a nucleic acid construct, a cell and a preparation method and use thereof. Background technique [0002] Interferon (IFN) is an important class of cytokines, which can initiate cell signal transduction by binding to the corresponding receptors on the cell membrane, and has a variety of biological functions, such as activating antiviral genes, participating in the body's antiviral immunity, regulating Immune response, anti-tumor, etc. (Samuel CE. Antiviral actions of interferences [J]. ClinMicrobiol Rev, 2001; 14:778-809). Studies in mammals divide IFN into three types according to gene sequence, functional characteristics and receptor types: type I interferon, mainly composed of IFN-α and IFN-β, type II interferon is IFN-γ, III The type of interferon is IFN-λ (Pestka S, Krause CD, Walter MR. Interferons, interferon-lik...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/10C12N15/85A01K67/027
CPCC07K14/461A01K67/0275A01K2227/10A01K2227/40C12N15/8509
Inventor 邹鹏飞李莹姚翠鸾孙庆学张晴
Owner JIMEI UNIV
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