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cDNA full-length sequence of paralichthys olivaceus mode recognition receptor TLR8 and application thereof

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

Inactive Publication Date: 2015-11-11
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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  • cDNA full-length sequence of paralichthys olivaceus mode recognition receptor TLR8 and application thereof
  • cDNA full-length sequence of paralichthys olivaceus mode recognition receptor TLR8 and application thereof
  • cDNA full-length sequence of paralichthys olivaceus mode recognition receptor TLR8 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Flounder spleen tissue isolation:

[0079] Healthy flounder (about 0.5kg) purchased on the market was kept in an aquarium at 25°C for three days, injected intraperitoneally with 400 μg of the simulated RNA virus molecule Poly(I:C), killed the flounder 2 hours later by breaking the neck, and placed it on an ultra-clean bench. Dissect and separate spleen tissue. The above steps are all aseptic operations.

Embodiment 2

[0081] Extraction and purification of total RNA:

[0082] (1) Take 100 mg of spleen tissue, cut it into pieces with scissors, add 1 mL of Trizol reagent (Invitrogen, USA) and 10 μL of heparin, grind and homogenize thoroughly in a homogenizer, then transfer to a 1.5 mL RNase-free centrifuge tube, shake and mix well, and keep at room temperature Leave it for 5 minutes to fully lyse.

[0083] (2) Centrifuge at 4° C. and 12000 rpm for 5 minutes, and transfer the supernatant into a new RNase-free centrifuge tube.

[0084] (3) Add 0.1mL 5mol / L NaCl to each tube and mix well, then add 0.3mL chloroform to each tube, shake vigorously for 15s, place at room temperature for 3min, centrifuge at 12000r / min for 15min at 4°C, and try not to inhale the middle layer In the case of aspiration, transfer the supernatant to another clean 1.5mL centrifuge tube.

[0085](4) Add isopropanol of equal volume to the obtained supernatant to each tube, mix gently, place at room temperature for 10 min,...

Embodiment 3

[0089] Cloning and sequencing of intermediate fragments:

[0090] (1) Cloning and sequencing of middle fragment 1:

[0091] (1.1) Primer design:

[0092] 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 ( Stegastespartitus ) fish Recently, primers were designed using Primer5 primer design software, using the damselfish TLR8 sequence (XM_008275651) as a template, combined with the sequence alignment results of other fishes as a correction, and designed degenerate primers for the sequence amplification of TLR8 middle fragment 1.

[0093] TLR8-F1: 5'-CCTTCCAGHCCRGACCDG-3'

[0094] TLR8-R1: 5'-GTCTTGCGRCTGTATYGG-3'

[0095] (1.2) cDNA first-strand synthesis:

[0096] With the purified flounder spleen total RNA described in Example 2 as a template, use the QIAGEN QuantitectReverseTranscription kit of QIAGEN Comp...

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Abstract

The invention discloses a cDNA full-length sequence of a paralichthys olivaceus mode recognition receptor TLR8 and a clone and detection method. The sequence has 3614 bp in total length, an open reading frame containing 3078 bp and codes 1024 amino acid. According to the cds sequence conserved region design and a primer of the proximal species TLR8, by means of inverse transcription and PCR amplification, under the combination of the RACE technique, the full-length cDNA sequence of the paralichthys olivaceus TLR8 is obtained. The TLRs family is a main 'pattern recognition receptor' for animals to identity and invade pathogens, and by sensing and identifying the relative molecular pattern of the pathogens, the natural immune system is activated. The acquisition of the gene sets a foundation for researching the gene expression regulation mechanism and immunologic functions of the fish TLR8, and molecular materials are provided for researching 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 Applications(China)
IPC IPC(8): C12N15/12C07K14/705C12Q1/68
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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