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Full-length cDNA sequence of flounder pattern recognition receptor tlr8 and its application

A pattern recognition receptor, flounder technology, which is applied to the full-length expression sequence of the flounder pattern recognition receptor TLR8 gene and a protein encoded by the gene. The field of cloning and detection of the gene can solve the problem that there is no immune prevention technology and issues of treatment

Inactive Publication Date: 2018-05-22
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of understanding of the immune mechanism and mechanism of flounder, there is no effective immune control technology and treatment

Method used

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  • Full-length cDNA sequence of flounder pattern recognition receptor tlr8 and its application
  • Full-length cDNA sequence of flounder pattern recognition receptor tlr8 and its application
  • Full-length cDNA sequence of flounder pattern recognition receptor tlr8 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Flounder spleen tissue isolation:

[0078] Healthy flounder (about 0.5 kg) purchased in the market was kept in an aquarium at 25°C for three days, and 400 μg of the simulated RNA virus molecule Poly(I:C) was injected intraperitoneally. After 2 hours, the flounder was killed by neck breaking. Spleen tissue was dissected and isolated on stage. The above steps are all aseptic operations.

Embodiment 2

[0080] Extraction and purification of total RNA:

[0081] (1) Take 100 mg of spleen tissue, chop it up with scissors, add 1 mL of Trizol reagent (Invitrogen, USA) and 10 μL of heparin, grind the homogenate thoroughly in a homogenizer, then transfer to a 1.5 mL RNase-free centrifuge tube and shake to mix. , placed at room temperature for 5 minutes to fully lyse.

[0082] (2) Centrifuge at 4°C and 12,000 rpm for 5 minutes, and transfer the supernatant to a new RNase-free centrifuge tube.

[0083] (3) Add 0.1 mL of 5 mol / L NaCl to each tube and mix well, then add 0.3 mL of chloroform to each tube, shake vigorously for 15 s, place at room temperature for 3 min, and centrifuge at 12,000 r / min at 4°C After 15 min, the supernatant was aspirated and transferred to another clean 1.5 mL centrifuge tube without aspirating the middle layer as much as possible.

[0084] (4) Add an equal volume of isopropanol to each tube, mix gently, place at room temperature for 10 min, centrifuge at 12,0...

Embodiment 3

[0088] Cloning and sequencing of intermediate fragments:

[0089] (1) Cloning and sequencing of middle fragment 1:

[0090] (1.1) Primer design:

[0091] Firstly, the full-length cDNA sequences and amino acid sequences of all fish TLR8 genes were downloaded from GeneBank. According to fish taxonomy, the taxonomic status of flounder was the same as that of damselfish ( Stegastes partitus ) fish Recently, the primers were designed using Primer 5 primer design software, using the damselfish fish TLR8 sequence (XM_008275651) as a template, combined with the sequence alignment results of other fishes as corrections, and designed degenerate primers for the sequence amplification of TLR8 middle fragment 1 .

[0092] TLR8-F1: 5'-CCTTCCAGHCCRGACCDG-3'

[0093] TLR8-R1: 5'-GTCTTGCGRCTGTATYGG-3'

[0094] (1.2) cDNA first-strand synthesis:

[0095] With the purified flounder spleen total RNA described in Example 2 as a template, use the QIAGEN Quantitect Reverse Transcription kit of...

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Abstract

The invention discloses a cDNA full-length sequence of a flounder pattern recognition receptor TLR8 and a cloning and detection method. The sequence is 3614 bp in full length, contains an open reading frame of 3078 bp, and encodes 1024 amino acids. In the present invention, a degenerate primer is designed based on the conserved region of the cds sequence of the closely related species TLR8, and the full-length cDNA sequence of the flounder TLR8 is finally obtained through reverse transcription and PCR amplification, combined with RACE technology. The TLRs family is the main "pattern recognition receptor" for animals to recognize invading pathogens, and activates the natural immune system by sensing and recognizing the relevant molecular patterns of pathogens. The acquisition of this gene lays the foundation for the study of the gene expression regulation mechanism and immunological function of fish TLR8, and can also provide molecular level materials for the study of fish population genetics and evolutionary genetics.

Description

[0001] The present invention is funded by Tianjin Natural Science Foundation Key Project (14JCZDJC34200) "Research on Immune Response Mechanism of Flounder TLRs Family Induced by Edwardsiella tarda". technical field [0002] The invention relates to the field of biological technology, in particular to the full-length expression sequence of the flounder pattern recognition receptor TLR8 gene and a protein encoded by it, as well as the cloning and detection methods of the gene. Background technique [0003] With the expansion of the scale of aquaculture worldwide, the trade of aquatic products at home and abroad and the cross-regional exchange of aquatic seed have become increasingly frequent, which has greatly increased the chance of spreading pathogens in aquaculture animals. At the same time, due to the intensification of modern aquaculture, high-density production methods and the deterioration of the fishery water environment, it often triggers the stress response of farmed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/705C12Q1/6883
Inventor 高虹郑津辉孙金生张洁李庆亚耿绪云潘宝平
Owner TIANJIN NORMAL UNIVERSITY
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