Natural immune receptor TLR5S gene of Saddletail grouper and novel application of protein encoded with natural immune receptor TLR5S gene

An oblique grouper and natural immunity technology, applied in the field of molecular biology, can solve the problems of great harm, economic loss of fishery and breeding, and high lethality

Inactive Publication Date: 2018-12-14
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial fish diseases have caused huge economic losses to fish farming all over the world due to their rapid spread, high fatality rate and great harm
Among them, Vibrio parahaemolyticus is one of the main pathogenic bacteria of vibriosis in aquatic animals. It is a halophilic Gram-negative bacterium, which brings serious economic losses to the mariculture industry.

Method used

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  • Natural immune receptor TLR5S gene of Saddletail grouper and novel application of protein encoded with natural immune receptor TLR5S gene
  • Natural immune receptor TLR5S gene of Saddletail grouper and novel application of protein encoded with natural immune receptor TLR5S gene
  • Natural immune receptor TLR5S gene of Saddletail grouper and novel application of protein encoded with natural immune receptor TLR5S gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning of Example 1 TLR5S Gene of Plaque-banded Grouper

[0027] (1) Extraction of total RNA from the head and kidney of the grouper

[0028] Healthy grouper was taken, anesthetized on ice for 3 minutes, head kidney tissue was separated, and total RNA was extracted with Trizol reagent.

[0029] (2) Synthesis of the first strand of cDNA

[0030] 1 μg of total RNA sample from the head and kidney of the grouper was taken and treated with DNase to remove the contamination of genomic DNA, mixed with RNAOligodT, and subjected to reverse transcription, and the obtained product was stored at -20°C for future use.

[0031] (3) Cloning of the complete cDNA sequence of the TLR5S gene of the grouper

[0032] Design specific primers, the upstream primer is TLR5S-ORF-F: 5`-ATGTGGACGCTGGGTCTTCA-3`; the downstream primer is TLR5S-ORF-R: 5`-CTGCTGTGTGATGTCATCAG-3`, the size of the amplified ORF fragment is 1935bp, electrophoresis The result is as figure 1 As shown, the target band ...

Embodiment 2

[0034] Example 2 Construction and verification of eukaryotic expression vector pcDNA4.0-TLR5S

[0035]According to the full-length cDNA sequence of TLR5S gene, BamHI and KpnI double restriction sites were selected to design primers, in which the upstream primer was EcTLR5S-BamHI-F: 5`-CGCGGATCCGCCACCATGTGGACGCTGGGTCTTCA-3`; the downstream primer was EcTLR5S-KpnI-R: 5`- CCGCTCGAGCTGCTGTGTGATGTCATCAG-3`. Using the pGEM-T plasmid containing the TLR5S coding gene as a template, carry out PCR amplification, and the size of the specific amplification product is about 2000bp, and connect the TLR5S with restriction sites to the DNA that has been double-enzymatically digested and purified with BamHI and KpnI In the final pcDNA4.0 eukaryotic expression vector, the recombinant expression vector was obtained, and the gel electrophoresis results of the recombinant expression vector verified by PCR are shown in figure 2 , the correctness of the sequence was verified by sequencing, and the...

Embodiment 3

[0036] Example 3 Overexpression Effect of Recombinant Plasmid pcDNA4.0-TLR5S

[0037] Inoculate an appropriate amount of 293T cells into a 12-well plate, transfect when the cells grow to 70%-80%, and replace with serum-free Opti-MEM culture medium before transfection. Each well was transfected with 1500ng recombinant plasmid pcDNA4.0-TLR5S, or pcDNA4.0 blank vector control. Six hours after transfection, the medium was replaced with DMEM medium containing 5% fetal bovine serum. The cells were collected 36 hours after transfection, the total protein was extracted, and the expression of EcTLR5S fusion protein was detected by western blot. The experimental results were as follows: image 3 As shown, β-actin was used as an internal reference protein. The results showed that the recombinant plasmid was constructed successfully.

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Abstract

The invention discloses a natural immune receptor TLR5S gene of Saddletail grouper and application of a protein encoded with the natural immune receptor TLR5S gene in the aspects of identifying bacterial flagellum, regulating intracellular signal pathways and adjusting inflammatory factors. The natural immune receptor TLR5S gene has the advantages of being beneficial for knowing pathogenic microorganisms resistant to a fish body and even a physiological process of vibrio invasion and providing a scientific evidence and a practice foundation for studies on fish immunology, prevention of diseases and the like in production application. Additionally, the natural immune receptor TLR5S gene can be prepared into a drug with an effect of resisting the multiplication of vibrio patahaemolyticus inthe production application, and the natural immune receptor TLR5S gene can also be applied as an immunopotentiator.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a new application of the natural immune receptor TLR5S gene and its coded protein of the oblique-banded grouper. Background technique [0002] Innate immunity is the body's first line of defense against the invasion of foreign pathogenic microorganisms, and it is the rapid and direct immune defense of fish. Toll-like receptors (Toll-like receptors, TLRs), as the main pattern recognition receptors in the innate immune system, participate in the recognition of various pathogen-associated molecular patterns (PAMPs), activate intracellular MyD88 (myeloid differentiation primary-response protein 88)-dependent signaling pathway or MyD88-independent signaling pathway induces a specific immune response to resist the invasion of pathogenic microorganisms. [0003] In mammals, TLR5 specifically recognizes and binds to the conserved region of Gram flagellin, transduce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/12A61K38/17A61P31/04
CPCA61K38/00A61P31/04C07K14/705
Inventor 卢丹琪何良格于雪张勇林浩然
Owner SUN YAT SEN UNIV
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