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Sepiella maindroni TNF gene and use of gene

A needleless squid and gene technology, applied in the field of genetic engineering, can solve problems such as drug residues, pollute water bodies, destroy ecological balance, etc., achieve rapid response, convenient operation, and improve immunity

Active Publication Date: 2019-11-12
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regular use of drugs may cause pathogens to develop resistance to certain drugs, and the drugs will accumulate in the squid and in the water, which will not only cause drug residues, but also pollute the water and destroy the ecological balance

Method used

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  • Sepiella maindroni TNF gene and use of gene
  • Sepiella maindroni TNF gene and use of gene
  • Sepiella maindroni TNF gene and use of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The complete ORF of the TNF gene of squid mansoni was obtained by the following method:

[0034] S1: Total RNA was extracted from the collected 8 tissue samples including spleen, pancreas, gills, heart, brain, optic lobe, liver and intestine;

[0035] (1) Take 30mg of tissue samples in a 1.5mL centrifuge tube;

[0036] (2) Add 200 μL Trizol Regent to the centrifuge tube;

[0037] (3) Add 800 μL Trizol Regent after rapid and thorough grinding with an electric grinding rod;

[0038] (4) Blow and suck the precipitate repeatedly with a pipette gun to resuspend it, and place it at room temperature for 5 minutes;

[0039] (5) Add 200 μL of chloroform, cap the centrifuge tube tightly, shake vigorously by hand for 15 seconds, and place at room temperature for 10 minutes;

[0040] (6) Centrifuge at 12000×g for 15 minutes at 4°C to separate the solution into upper, middle and lower layers;

[0041] (7) Carefully pipette the upper aqueous phase solution into another centrifuge...

Embodiment 2

[0086] Tissue-specific Distribution and Expression of TNF Gene in Squid Mansoni

[0087] Firstly, 8 tissues of spleen, pancreas, gills, heart, brain, optic lobe, liver and intestine were collected from healthy squid, extracted total RNA, synthesized cDNA by reverse transcription, and set aside;

[0088] According to the sequencing results of the TNF gene, design fluorescent quantitative PCR primers (as shown in Table 1), use the qRT-PCR method, use the GAPDH gene as an internal reference, and use the SYBR Green chimeric fluorescence method to perform real-time quantitative PCR according to the instructions of SYBR Premix Ex Taq MⅡ Amplification reaction (qRT-PCR) to analyze the tissue differential expression distribution of TNF gene in each tissue.

[0089] The 20μL amplification reaction system is as follows:

[0090]

[0091] A two-step method was used for amplification: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, extension at 60°C for 45 s, 40 cyc...

Embodiment 3

[0095] Expression of TNF Gene in Squid Mansoni at Different Developmental Stages

[0096]Squid at the larval stage, pre-spawning stage, and spawning stage were selected, and the gills, liver, and optic lobe were collected respectively, and the expression of the TNF gene in different developmental stages was analyzed according to the method in Example 2. The result is as Figure 4 , it can be seen that for the larval squid, the expression level of TNF gene in the liver is the highest; In squid mansoni, the expression of TNF gene was the highest in the liver.

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Abstract

The invention provides a sepiella maindroni TNF gene and use of the gene, and belongs to the technical field of genetic engineering. The sepiella maindroni TNF gene has a base sequence of SEQ ID NO:1,and the TNF protein encoded by the gene has an amino acid sequence of SEQ ID NO:2. According to the present invention, the sepiella maindroni TNF gene is expressed in eight tissues including spleen,pancreas, gill, heart, brain, optic lobe, liver and intestine of the sepiella maindroni, with the highest expression in the liver; the gene is sensitive to the recognition of Aeromonas hydrophila or Vibrio parahaemolyticus infection and has a swift response; and the expression vector and the encoded protein of the gene are immunopotentiators for the sepiella maindroni, and can improve the immunityof the sepiella maindroni, especially the immunity of the sepiella maindroni to Aeromonas hydrophila or Vibrio parahaemolyticus.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a TNF gene of squid mansoni and its application. Background technique [0002] Sepiella japonica belongs to the kingdom Animal, phylum Mollusca, class Cephalopoda, order Sepioidea, family Sepiidae, genus Sepiella ), which are mainly distributed in the coastal waters of the Northwest Pacific Ocean and the North Indian Ocean. As one of the most important cephalopod economic species in the coastal area of ​​the East China Sea, artificial breeding has gradually emerged. However, the losses caused by infectious diseases during the breeding process are very large, and the prevention and treatment of diseases mainly rely on drugs . Frequent use of drugs may cause pathogens to develop resistance to certain drugs, and the drugs will accumulate in the squid and in the water, which will not only cause drug residues, but also pollute the water and destroy the ecolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/28C07K14/525C12Q1/6851C12N15/85C12N5/10A61K38/17A61K48/00A61P37/04
CPCC07K14/525C12Q1/6851C12N15/85A61K48/005A61K48/0025A61P37/04A61K38/00C12Q2561/113C12Q2531/113C12Q2521/107Y02A50/30Y02A40/81
Inventor 刘慧慧霍利平葛德隆
Owner ZHEJIANG OCEAN UNIV
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