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Grass carp ifn-γ2 gene and its encoded recombinant protein and application

A protein and grass carp technology, applied in application, genetic engineering, plant genetic improvement and other directions

Active Publication Date: 2021-06-22
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these studies failed to successfully isolate and purify fish IFN protein, they all showed that fish have an antiviral response similar to that of mammalian IFN.

Method used

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  • Grass carp ifn-γ2 gene and its encoded recombinant protein and application
  • Grass carp ifn-γ2 gene and its encoded recombinant protein and application
  • Grass carp ifn-γ2 gene and its encoded recombinant protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Isolation of grass carp head kidney macrophages

[0016] Experimental fish acquisition: Six 150g grass carp were fished from the aquatic experiment base, and deeply anesthetized with MS-222 solution.

[0017] 1. Collection of Head Kidney Samples

[0018] Take blood from the tail vein and draw it clean; cut off the head and directly pick the head kidney tissue, put it in a sterilized disposable culture dish, and wash it with AIM solution for 2-3 times on the ultra-clean bench until the blood color of AIM fades , centrifuge at low temperature, and cool down to 4°C.

[0019] 2. Single Cell Suspension Preparation

[0020] The tissue obtained in the previous step was soaked in AIM solution for 30 minutes, and the cell suspension was obtained after grinding.

[0021] 3. Slowly add 3ml of cell suspension to the upper layer of Percoll continuous density gradient solution to make it layered, and centrifuge at 400g low temperature for 30min.

[0022] 4. Aspirate ab...

Embodiment 2

[0027] Example 2: Extract macrophage RNA and obtain cDNA

[0028] 1. Extraction of total RNA

[0029] ① Suck off the culture medium of the above-mentioned adherent cells, then lyse with 1mL TRIzol, then mix well in the 2-well plate, and add to the 1.5ml EP tube.

[0030] ② Add 200 μL of chloroform to the homogenate lysate in the above step, cover the centrifuge tube tightly, and shake it. After the fully emulsified solution becomes milky white, let it stand on ice for 5 minutes.

[0031] ③ Centrifuge at 12,000g for 15 minutes at 4°C.

[0032] ④Take out the centrifuge tube. At this time, the homogenate is divided into three layers, namely: a colorless supernatant, a white protein layer in the middle, and a colored lower organic phase. Absorb the supernatant and transfer it to another new 1.5ml centrifuge. tube.

[0033] ⑤ Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube up and down to mix thoroughly, and place it on ice for 5 minutes.

[003...

Embodiment 3

[0041] Example 3: Cloning and sequence analysis of grass carp IFN-γ2

[0042] 1. Primer Design

[0043] Use clone manager software to design upstream and downstream primers, and the primer sequences are as follows:

[0044] Upstream primer F1: 5'-ATGATTGCACAACACATG-3'

[0045] Downstream primer R1: 5'-CTAAGACTCCTGCCTCTT-3'

[0046] 2. PCR amplification: use grass carp head kidney macrophage cDNA as a template, and perform PCR amplification with F1 and R1 primers. The reaction system is 20 μL. The PCR reaction system is as follows:

[0047]

[0048] PCR reaction conditions: the reaction conditions are 94°C, 5min; 72°C, 30sec, 57°C, 45sec, 72°C, 10min; 35 cycles.

[0049]3. PCR product identification: After the amplification is completed, take 10 μL of the PCR product and apply it with 10×nucleic acid loading buffer, 1.0% agarose gel, 1×TAE buffer, 120V, electrophoresis for 30 minutes to observe the results, and obtain the target fragment .

[0050] Take 1 μL of the PMD-...

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Abstract

The invention discloses a grass carp IFN-γ2 gene, which has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence table. The invention also discloses a recombinant protein expressing the grass carp IFN-γ2 gene, which has the amino acid sequence shown in SEQ ID NO: 2 in the sequence table. The recombinant protein can promote the activation of grass carp head kidney macrophages and up-regulate the expression of immune-related genes in vitro. Injecting the recombinant protein into grass carp can significantly reduce the mortality rate of grass carp infected with Flavobacterium columnar, improve the immune protection rate of grass carp, regulate the immune index of grass carp, and enhance the ability of grass carp to resist bacterial infection.

Description

technical field [0001] The invention belongs to the biological field, and in particular relates to a grass carp IFN-γ2 gene, its encoded protein and its application. Background technique [0002] Grass carp is an important freshwater economic fish in my country, mainly in polyculture, but the "four diseases" and pathogenic microorganisms in the breeding process have caused certain losses to the yield and quality of grass carp. Interferon (Interferon, IFN) is one of the most important cytokines in the body. Its main biological functions are broad-spectrum antiviral activity, anti-cell proliferation and immune regulation functions. It can induce cells to produce cytokines in an antiviral state. It does not directly participate in the biological process of anti-virus, but regulates the infection of the virus by producing effector genes downstream of the target gene. It has the characteristics of multipotency, low molecular weight, and secreted type. [0003] Interferon was ori...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/57C12N15/23C12N15/70C12N1/21A61K38/21A61P37/04C12R1/19
CPCA61K38/00A61P37/04C07K14/57C12N15/70
Inventor 袁改玲周建成陈桐胡亚珍刘小玲苏建国
Owner HUAZHONG AGRI UNIV
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