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Innate immune TLR13 (toll-like receptor 13) gene of epinephelus coioides as well as eukaryotic expression vectors and application of innate immune TLR13 gene

An oblique-banded grouper, natural immunity technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of frequent outbreaks of diseases and restricting the development of the grouper industry.

Active Publication Date: 2018-01-09
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the grouper aquaculture industry has developed rapidly, but with the expansion of the aquaculture scale, diseases have also frequently broken out, which seriously restricts the development of the grouper industry

Method used

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  • Innate immune TLR13 (toll-like receptor 13) gene of epinephelus coioides as well as eukaryotic expression vectors and application of innate immune TLR13 gene
  • Innate immune TLR13 (toll-like receptor 13) gene of epinephelus coioides as well as eukaryotic expression vectors and application of innate immune TLR13 gene
  • Innate immune TLR13 (toll-like receptor 13) gene of epinephelus coioides as well as eukaryotic expression vectors and application of innate immune TLR13 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Slope grouper TLR13 gene cloning

[0033] (1) Extraction of total RNA from spleen of grouper

[0034] The healthy grouper was taken and anesthetized on ice for 3 minutes, the spleen tissue was separated, and the total RNA was extracted with Trizol reagent.

[0035] (2) Synthesis of the first strand of cDNA

[0036] 1 μg of total RNA samples from the head and kidney of the grouper was taken for DNase treatment to remove the contamination of genomic DNA, mixed with RNAOligodT, and reverse-transcribed, and the resulting product was stored at -20°C for future use.

[0037] (3) Cloning of the complete cDNA sequence of the TLR13 gene of the grouper

[0038] Design specific primers TLR13FullF and TLR13FullR, as shown in SEQ ID NO.4 and SEQ ID NO.5, the size of the amplified ORF fragment is 2844 bp, and the electrophoresis results are shown in the attached figure 1As shown, the target band was recovered by gel cutting and the product was purified. The product...

Embodiment 2

[0039] Example 2 Construction and Verification of Recombinant Plasmids pEGFP-N3-TLR13 and pcDNA3.1-13

[0040] according to TLR13 The full-length cDNA sequence of the gene, a pair of primers are synthesized at both ends of the coding gene, the upstream primer contains the XhoI cleavage site, the sequence, and the downstream primer contains the BamHI cleavage site to contain TLR13 The pTZ57R / T plasmid encoding the gene was used as a template for PCR amplification to obtain a specific amplified single band with a product size of about 2800 bp. The PCR amplification products were cloned into the expression vectors PcDNA3.1 and pEGFP-N3 to obtain the recombinant expression vectors. The PCR verification results of the recombinant plasmids are shown in the attached figure 2 As shown, lane 1 is the verification result of pEGFP-N3-TLR13, and lane 2 is the verification result of PcDNA3.1-TLR13. The primer sequences used in amplification and verification are listed in Table 1 below: ...

Embodiment 3

[0042] Example 3 Overexpression Effect of Recombinant Plasmids pEGFP-N3-TLR13 and pcDNA3.1-13

[0043] Inoculate an appropriate amount of 293T cells (about 1×106 cells per well) into a 96-well plate, and transfect when the cells grow to 60%-80% the next day, and replace with serum-free Opti before transfection. -MEM medium. Each well was transfected with 150 ng of recombinant plasmids pEGFP-N3-TLR13, pcDNA3.1-13 or their blank vector control. Transfection solution: A solution (10 μL of Opti-MEM culture solution, add plasmid, mix well), B solution (10 μL of Opti-MEM culture solution, add 0.25 μL lipo3000, mix well, leave at room temperature for 5 minutes); mix A solution and Gently mix solution B to become the transfection solution, place it at room temperature for 20 min, add it to the corresponding transfection well, and incubate for 6 hours; finally replace it with DMEM medium containing 10% fetal bovine serum.

[0044] Cells were collected 36 hours after transfection, tot...

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Abstract

The invention discloses an innate immune TLR13 (toll-like receptor 13) gene of epinephelus coioides as well as eukaryotic expression vectors and an application of the innate immune TLR13 gene for thefirst time. The nucleotide sequence of the TLR13 gene is shown as SEQ. ID.NO:1, the full-length cDNA sequence of the TLR13 gene is shown as SEQ. ID.NO:2, sequences of a pair of primers contained on the full-length cDNA sequence are shown as SEQ. ID.NO:4-5, and the amino acid sequence of the TLR13 protein is shown as SEQ. ID.NO:3. The two eukaryotic expression vectors containing the full-length cDNA sequence of the TLR13 gene are provided, namely, pEGFP-N3-TLR13 and pcDNA3.1-TLR13. Besides, the TLR13 gene or the TLR13 protein is applied to research of regulation of inflammatory factor expression, recognition of bacterial RNA, recognition of vibrio parahaemolyticus RNA and regulation of inflammatory factors.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and more specifically relates to a natural immune receptor of the oblique-banded grouper TLR13 Genes and their eukaryotic expression vectors and applications. Background technique [0002] Toll-like receptor (Toll-like receptor, TLR) family is the basis of "alien" recognition and activation of innate immune response, belonging to type I transmembrane receptors. TLRs have a conserved TIR (Toll / IL-1 receptor) domain in the membrane, which is mainly used to recruit adapter protein myeloid differentiation factor 88 (MyD88) and Toll-like receptor adapter protein 1 (TIR domain -containing adapter inducing interferon-β (TRIF) / TIR domain-containing adapter molecule (TICAM-1), TRIF1), to further activate the downstream immune response. TLRs also have non-conserved leucine repeat regions outside the membrane, which are mainly used to recognize different pathogen-associated molecular patter...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/79C07K14/46
Inventor 卢丹琪梁瑶思周莹于雪张勇林浩然
Owner SUN YAT SEN UNIV
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