CN107557366B - Epinephelus coioides natural immune receptor TLR13 gene, eukaryotic expression vector and application thereof - Google Patents

Abstract

The invention discloses a Epinephelus coioides natural immune receptor for the first timeTLR13Genes, eukaryotic expression vectors thereof and application.TLR13The nucleotide sequence of the gene is shown as SEQ.ID.NO: as shown in figure 1, the first and second main bodies,TLR13the full-length cDNA sequence of the gene is shown as SEQ.ID.NO: 2, and a pair of primer sequences contained in the sequence are shown in SEQ ID No: 4-5, wherein the amino acid sequence of the TLR13 protein is shown in SEQ.ID.NO: 3, respectively. The invention provides two compositions containingTLR13Eukaryotic expression vector of gene full-length cDNA sequence, pEGFP-N3-TLR13 and pcDNA3.1-TLR 13. In addition, the invention willTLR13The gene or TLR13 protein is applied to the research of regulating the expression of inflammatory factors, identifying bacterial RNA, identifying Vibrio parahaemolyticus RNA and regulating inflammatory factors.

Description

Epinephelus coioides natural immune receptorTLR13Gene, eukaryotic expression vector and application thereof

Technical Field

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a Epinephelus coioides natural immune receptorTLR13Genes, eukaryotic expression vectors thereof and application.

Background

The Toll-like receptor (TLR) family is the activation basis for "allogenic" recognition and innate immune responses and belongs to the type i transmembrane receptor. TLRs have a TIR (Toll/IL-1 receptor) domain conserved within the membrane and are used primarily to recruit the adaptor protein myeloid differentiation factor 88 (MyD 88) and the Toll-like receptor adaptor protein 1 (TIR domain-associating adaptor-beta (TRIF)/TIR domain-associating adaptor molecule (TICAM-1), TRIF 1) in immune responses to further activate downstream immune responses. TLRs also have regions of leucine repeats that are not conserved outside the membrane, primarily for the recognition of different pathogen-associated molecular patterns.

TLR-mediated downstream pathways activate downstream immune responses primarily through either MyD 88-dependent signaling pathways or TRIF-dependent signaling pathways. When stimulated by a particular ligand, TLRs will form homodimers or heterodimers, recruit the adaptor proteins MyD88, MAL (muscle differentiation protein 88 adaptor-like), TRIF, and TRAM (tri-related adaptor molecules), then activate a series of downstream kinases, and mediate the activation of transcription factors NF- κ B (nuclear factor- κ B), AP1 (activator protein-1), etc., ultimately leading to the production of immune inflammatory factors.

TLR1 ~ 10 is found in humans, TLR1 ~ 13 is found in mice, TLR18 ~ 23, 25, 26 is found in mammals in teleost fish, etc. research shows that TLR2 of fish may recognize ligands of virus but not bacteria, TLR4 cannot recognize LPS, TLR5 has both intracellular and cell membrane forms and can recognize flagellin, and TLR22 specific to fish has various subtypes, and can recognize various ligands including lipopolysaccharide, double-stranded RNA analog (polysinic-polycystic-peptidic acid, Poly (I: C)), peptidoglycan (peptidoglycan, PGN), etc. to activate downstream immune channels.

At present, the expression of TLR13 is found to be up-regulated by stimulation of encystosis, vesicular stomatitis virus and bacterial 23S rRNA in mice, and mouse TLR13 is found to recognize bacterial 23S rRNA. However, TLR13 has been studied only rarely in teleost fish, and it is unclear whether TLR13 can also recognize bacteria in teleost fish immunity, and thus its function is able to recognize which bacteria.

The grouper is a rare aquaculture fish in China, has delicious meat quality and rich nutrition, and has high edible value and economic value. Epinephelus coioides (A) and (B)Epinepheluscoioides) Belongs to one of the common grouper species in the culture and is divided intoCategories belongs to Perciformes (Perciformes), Serranidae (Serranidae), Epinephelinae (Epinephalaea), Epinephelus (Epinephalus). In recent years, the grouper breeding industry develops rapidly, but diseases are frequently outbreaked along with the expansion of the breeding scale, and the development of the grouper industry is severely restricted. Because the bacterial infection has great influence on the grouper culture, the functional characteristics of the natural immune receptor TLR13 of the grouper obliquus and the function of the natural immune receptor TLR13 in bacterial defense are explored, so that on one hand, the theoretical research of natural immune signal pathways of fishes can be enriched, and meanwhile, scientific basis and practical basis can be provided for research of fish immunity, disease prevention and the like in production and application.

The vibrio parahaemolyticus is halophilic gram-negative pathogenic bacteria and is an important pathogenic bacterium of cultured aquatic animals such as fish, shrimp, shellfish and the like. Meanwhile, vibrio parahaemolyticus is one of the important pathogenies causing human food-borne diseases, and the Guangdong area is the area with the most serious pollution of the vibrio parahaemolyticus in aquatic products in China. Therefore, the identification and defense mechanism of teleost is deeply known, and a targeted high-efficiency environment-friendly prevention and control strategy is formulated, so that the method has important significance for improving the current situation of antibiotic abuse, powerfully promoting the healthy and high-efficiency culture of marine fishery, and reducing the human health risk while obtaining healthy high-quality aquatic products.

Disclosure of Invention

The invention aims to solve the technical problem of making up the blank of the prior art and provides a epinephelus coioides natural immune receptorTLR13The nucleotide sequence of the gene.

Another technical problem to be solved by the invention is to provide a liquid crystal display device comprising the liquid crystal display deviceTLR13Eukaryotic expression vector of gene full-length cDNA sequence.

The invention also aims to solve the technical problem of providing theTLR13Use of a gene or TLR13 protein for modulating the expression of an inflammatory factor.

The invention also aims to solve the technical problem of providing theTLR13Use of a gene or TLR13 protein for the recognition of bacterial RNA.

The invention also provides a technical problem to be solvedSubject to provide aTLR13The application of the gene or TLR13 protein in identifying Vibrio parahaemolyticus RNA and regulating the expression of inflammatory factors.

The purpose of the invention is realized by the following technical scheme:

the invention provides a Epinephelus coioides natural immunoreceptorTLR13The nucleotide sequence of the gene is shown in SEQ.ID.NO1.

The Epinephelus coioides natural immunoreceptorTLR13The full-length cDNA sequence of the gene is shown in SEQ ID.NO2.

The Epinephelus coioides natural immunoreceptorTLR13The amino acid sequence of the gene-encoded protein is shown in SEQ.ID.NO3.

The Epinephelus coioides natural immunoreceptorTLR13The protein structure of the gene-encoded protein includes 1 signal peptide sequence, 14 LRR domains, 1 typical TIR domain and 1 transmembrane domain.

The invention also provides a composition comprising the sameTLR13Eukaryotic expression vector of gene full-length cDNA sequence.

Further, the vector is pcDNA3.1-TLR13 or pEGFP-N3-TLR13, wherein TLR13 refers to claim 2TLR13The full-length cDNA sequence of the gene; wherein pcDNA3.1 refers to pcDNA3.1 plasmid, and wherein pEGFP-N3 refers to pEGFP-N3 plasmid.

The invention also provides the application of the gene or the protein in regulating the expression of inflammatory factors.

The invention also provides the application of the gene or the protein in identifying the bacterial RNA.

The invention also provides the application of the gene or the protein in identifying the Vibrio parahaemolyticus RNA and regulating the expression of inflammatory factors.

The invention has the beneficial effects that:

the invention provides the Epinephelus coioides natural immune receptor for the first timeTLR13Nucleotide sequences of genes based on said sequences, two such genes are also providedTLR13Eukaryotic expression vectors of the gene pEGFP-N3-TLR13 and pcDNA3.1-TLR13, and provides the sameTLR13Genes andTLR13the application of the gene coded protein in regulating the expression of inflammatory factors, identifying bacterial RNA, identifying Vibrio parahaemolyticus RNA and regulating the expression of inflammatory factors has prominent substantive progress.

Drawings

FIG. 1 shows a schematic view of aTLR13PCR amplification electrophoresis of full-length cDNA of the gene.

FIG. 2 shows the PCR-verified electrophoresis chart of the recombinant plasmid pEGFP-N3-TLR13 and the bacterial liquid PCR-verified electrophoresis chart of Escherichia coli DH5 alpha transformed by pcDNA3.1-13.

Figure 3 effect of over-expression of TLR13 for two eukaryotic expression vectors.

Figure 4 overexpression of TLR13 upregulates mRNA transcription of various inflammatory factors.

FIG. 5 recognition of bacterial RNA and upregulation of downstream IFN- β promoter activity following overexpression of TLR 13.

FIG. 6 Effect of TLR13 knock-down on RNA-mediated production of immune factors by Vibrio parahaemolyticus.

Detailed Description

The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are raw reagent raw materials which are conventionally commercially available or commercially available, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.

Example 1 Epinephelus coioidesTLR13Cloning of genes

(1) Extraction of spleen total RNA of Epinephelus coioides

Taking healthy Epinephelus coioides, anesthetizing on ice for 3 minutes, separating spleen tissues, and extracting total RNA by using a Trizol reagent.

(2) First Strand cDNA Synthesis

Taking 1 mu g of Epinephelus coioides head and kidney total RNA sample, carrying out DNase treatment to remove genomic DNA pollution, mixing with RNA OligodT, carrying out reverse transcription, and storing the obtained product at-20 ℃ for later use.

(3) Cloning of TLR13 gene cDNA complete sequence of epinephelus coioides

Designing specific primers TLR13FullF and TLR13FullR, as shown in SEQ ID NO.4 and SEQ ID NO.5, obtaining the ORF fragment with the size of 2844 bp by amplification, obtaining the electrophoresis result as shown in figure 1, cutting gel, recovering a target band and purifying the product. The product was ligated into pTZ57R/T vector, transformed into DH 5. alpha. E.coli, and positive clones selected for sequencing. BLAST homology analysis shows that the target product is a cDNA sequence fragment of TLR13 gene. The sequences used in the PCR amplification are as follows:

example 2 construction and validation of recombinant plasmids pEGFP-N3-TLR13 and pcDNA3.1-13

According toTLR13The full-length cDNA sequence of the gene is synthesized by a pair of primers at two end sequences of the coding gene, wherein an upstream primer contains an XhoI cutting site and a sequence, a downstream primer contains a BamHI cutting site so as to containTLR13The pTZ57R/T plasmid of the coding gene is used as a template to carry out PCR amplification to obtain a single band specifically amplified, and the size of the product is about 2800 bp. The PCR amplification product was cloned to expression vector PcDNA3.1 and pEGFP-N3 to obtain a recombinant expression vector, and the PCR verification result of the recombinant plasmid is shown in FIG. 2, wherein the verification result of pEGFP-N3-TLR13 is shown in lane 1, and the verification result of PcDNA3.1-TLR13 is shown in lane 2. The primer sequences used in amplification and validation are shown in table 1 below:

example 3 overexpression Effect of recombinant plasmids pEGFP-N3-TLR13 and pcDNA3.1-13

A proper amount of 293T cells (the number of cells in each well is about 1 × 106) are inoculated into a 96-well plate, the transfection can be carried out when the cells grow to 60% ~ 80% the next day, the cells are changed into a serum-free Opti-MEM culture solution before transfection, 150 ng of recombinant plasmids pEGFP-N3-TLR13 and pcDNA3.1-13 or blank vector control of the recombinant plasmids are transfected into each well, the transfection solution is A solution (10 μ L of the Opti-MEM culture solution is added with plasmids and mixed evenly), B solution (10 μ L of the Opti-MEM culture solution is added with 0.25 μ L of lipo3000 and mixed evenly, the mixture is placed at room temperature for 5 min), the A solution and the B solution are mixed evenly to obtain the transfection solution, the transfection solution is placed at room temperature for 20min and added into corresponding transfection wells for incubation for 6 h, and finally the cells are changed into a DMEM culture solution containing 10%.

Collecting cells after 36 hours of transfection, extracting total RNA, performing reverse transcription, performing fluorescence quantitative PCR to detect the mRNA level of TLR13, adopting TLR13 RTF and TLR13 RTR as amplification primers, wherein the selected internal reference gene is EF1, and the adopted amplification primers are EF1RTF1 and EF1RTR1, analyzing the result according to the CT value obtained by detection, calculating the relative expression of TLR13 by using a 2- △△ CT method, and the experimental result is shown in figure 3. the primer sequences used in the detection are shown in the following table 2:

as can be seen from the attached figure 3, after the recombinant plasmid pEGFP-N3-TLR13 or pcDNA3.1-13 is transfected, the expression level of TLR13 mRNA in cells can be obviously improved. The result shows that the recombinant plasmid is successfully constructed, and the expression of the TLR13 can be obviously improved in cells after transfection.

Example 4 mRNA transcriptional upregulation of multiple inflammatory factors following overexpression of TLR13

The method comprises the steps of inoculating a proper amount of GS cells (the number of the cells in each well is about 4 x 106) into a 6-well plate, transfecting when the cells grow to 50% ~ 60% the next day, changing to an Opti-MEM culture solution without serum before transfection, transfecting 2000 ng pEGFP-N3-TLR13 or pEGFP-N3 blank vector control in each well, wherein the transfection solution is an A solution (50 mu L of the Opti-MEM culture solution is added with a plasmid and mixed evenly), a B solution (50 mu L of the Opti-MEM culture solution is added with 4 mu L lipo2000 and mixed evenly, the mixture is placed at room temperature for 5 min), mixing the A solution and the B solution gently to obtain a transfection solution, placing the mixture at room temperature for 20min, adding the mixture into corresponding transfection wells, incubating for 12 h, and finally changing to an L15 culture solution containing 10% fetal calf serum.

Collecting cells after 48 hours of transfection, extracting total RNA, reverse transcription, performing fluorescent quantitative PCR to detect the mRNA level of IL-6, IL-1 beta, IL-12 and TNF alpha, wherein the selected internal reference gene is EF1, the adopted amplification primers are EF1RTF1 and EF1RTR1, analyzing the result according to the CT value obtained by detection, calculating the relative expression quantity of the immune factors by using a 2- △△ CT method, and the experimental result is shown as the attached figure 4. the primer sequences used in the detection are shown as the following table 3:

as can be seen from figure 4, after TLR13 is over-expressed in GS cell cells, the expression of IL-1 beta, IL-6, IL-12 and TNF alpha, which are inflammatory factors, can be obviously improved. The results show that the method has the advantages of high yield,TLR13genes or TLR13 proteins can modulate the expression of inflammatory factors.

Example 5 recognition of bacterial 23S rRNA and Up-Regulation of downstream IFN- β promoter Activity following overexpression of TLR13

(1) Gene transfection of eukaryotic cells

A proper amount of 293T cells (the number of cells in each well is about 2 x 105) are inoculated into a 24-well plate, the cells can be transfected when the cells grow to 50% ~ 60% the next day, the cells are switched to a serum-free Opti-MEM culture solution before transfection, 700 ng pcDNA3.1-13 or pcDNA3.1 blank vector control, 200 ng PGL 3-IFN-beta-Luc report plasmid and 100 ng pRL-TK plasmid are transfected into each well, the transfection solution is an A solution (50 mu L of Opti-MEM culture solution, plasmids are added and mixed evenly), a B solution (10 mu L of Opti-MEM culture solution, 1.5 mu L lipo2000 is added and mixed evenly, and the mixture is placed at room temperature for 5 min), the A solution and the B solution are mixed evenly to obtain the transfection solution, the transfection solution is placed at room temperature for 20min and is added into corresponding transfection wells, 4 ~ 6 h, and finally the cells are switched to a DMEM culture solution containing 5% bovine serum.

(2) Reporter gene transcriptional activity assay

After 36 h of transfection, 293T cells were stimulated with gradient ORN Sa19 and its mutation Control ORN Sa19 Control, and the cells were harvested 18 h later and tested using a dual-luciferase reporter gene assay kit, the results of which are shown in FIG. 5, where the values shown are the ratio of firefly luciferase to Renilla luciferase.

As can be seen in figure 5, overexpression of TLR13 significantly increased cell activation of IFN- β promoter activity mediated upon ORN Sa19 stimulation. The results show that the method has the advantages of high yield,TLR13the gene or TLR13 protein can recognize bacterial RNA.

Example 6 Effect of knocking down TLR13 on RNA-mediated production of immune factors of Vibrio parahaemolyticus

(1) Extraction of Vibrio parahaemolyticus RNA

200. mu.L of Vibrio parahaemolyticus was inoculated into 35 mL of a medium and cultured for 12 hours, and the bacteria were centrifuged to extract RNA by Trizol reagent method.

(2) RNA stimulation experiment of vibrio parahaemolyticus after TLR13 knock-down

The appropriate amount of GS cells (about 1 × 104 cells per well) were inoculated into a 96-well plate, transfected the next day when the cells grew to 50% ~ 60%, and replaced by serum-free Opti-MEM medium before transfection, the siRNA sequences used were SiTLR13 (sequence: sense: 5'-GCCUAUCAGCUGUCUCCAUTT-3'; antisense: 5'-AUGGAGACAGCUGAUAGGCTT-3') and Negative Control sequence (sequence: sense: 5'-UUCUCCGAACGUGUCACGUTT-3'; antisense: 5'-AUGGAGACAGC UGAUAGGCTT-3'), the transfection medium was diluted at a concentration of 20 nM., liquid A (5 μ L of Opti-MEM medium, 0.3 μ L of siRNA was added, mixed well), liquid B (5 μ L of Opti-MEM medium, 0.4 μ L of lipo3000 was added, mixed well, and left at room temperature for 5 min), liquid A and liquid B were mixed well gently to obtain transfection medium, left at room temperature for 15 min, added to the corresponding transfection wells, the enzyme was added for 6 h, the last, after transfection with 10% bovine serum fetuses L15, hemolyzed, added with equal amount of RNA, and the RNA was extracted by PCR, and the amount of RNA was measured by reverse transcription factor (RNA) and the total RNA was measured by PCR at a concentration of 2 mg/mL, and the total RNA was measured after 2 mL.

As can be seen from figure 6, the activation of downstream inflammatory factors mediated by the RNA stimulation of Vibrio parahaemolyticus can be significantly inhibited after TLR13 is knocked down. The results show that the method has the advantages of high yield,TLR13the gene or TLR13 protein can recognize Vibrio parahaemolyticus RNA and regulate inflammatory factors.

SEQUENCE LISTING

<110> Zhongshan university

<120> Epinephelus coioides natural immune receptor TLR13 gene, eukaryotic expression vector and application thereof

<130>

<160>5

<170>PatentIn version 3.3

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<213> nucleotide sequence of TLR13 gene of Epinephelus coioides

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atcgcctgcc aaatgactgc tctgacaagt cttcatttga gtggaattag ctttgtcagt 960

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tatttggaag agctgtccga gttttccctc ggatcaataa aacagctcag acgcctggac 1080

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acaaaactag tagaactcaa tctcagccaa aatcgcattt caacactcaa gggatgtgtt 1260

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aatctttcag ttacaccatt ttttggtgaa atggatttca ttacaaaggt ttttacagga 1560

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tatactcccc tcctgacaag tcttcacata tttcaaagtg atctgcaagt cttaaaccct 1860

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tctctggatt ttctagccca ggtcaacctc tctgcactca gagtgttgac agttagagac 1980

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<213> full-length cDNA sequence of TLR13 gene of Epinephelus coioides

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cagcaagagt tcttgtcaaa aaaatgagac acatttatcc attttcccat tacctttctt 1800

gaagagtcta ctgatacaaa attatgattg gtatcatgaa atctcacctg attttctgac 1860

aggtctgaat tctttagatt actttggagc ggagaaatta tttacagagt cacctcaccc 1920

agacacattc agatatactc ccctcctgac aagtcttcac atatttcaaa gtgatctgca 1980

agtcttaaac cctgaagtgc ttcagccaat cctcaacctg caggctctcg acctttccaa 2040

aaacaagctc agatctctgg attttctagc ccaggtcaac ctctctgcac tcagagtgtt 2100

gacagttaga gacaatgaat tgaccgtgat caacgacatg gtctttcagt ctctccctgc 2160

actgacatac ctggacctga ctggtaaccc tttcacttgt aactgctcta acactggctt 2220

tatccaatgg gtgaagaaca acaaccaaac acaggttgtt aatgcctacc agtacacttg 2280

tacctttcct gtggctaaac aaggaacaaa gttgctggac tttgacgtcc agtcctgttg 2340

gatggacgtt agcttcctct gcttcatttc tagcttttgc ctgacgctga tgattctcct 2400

cacatccttc atctaccact ttctgaggtg gcagctagcc tacacctact acctcttcct 2460

ggccttcctc tacgacagca ggaagaggaa gaagggcgct cctcatcact acgatgcttt 2520

catctcctac aatgttcatg acgaggactg ggtttacaga gagatgcttc cagtgctgga 2580

gggagagcag ggctggagac tctgtctgca ccacagagac ttccaaccag gtaaacccat 2640

catagacaac ataacagacg ccatctacgg cagcaggaag accatctgtg tgatcacccg 2700

gcgttacctg cagagcgaat ggtgctccag agagatccag atggccagct tccgtctgtt 2760

tgacgagcag aaggacgtgt tgatcctgct gttcctggag gagatcccgg cctatcagct 2820

gtctccatac caccgcatga ggaagctggt gaagaggcac acctacctga gctggccgca 2880

ggccggccaa cacacaggag tcttctggca gaacgtatgg agagctctgg agacagggga 2940

agctcccatt gagaccaaca acctgctgac tggatgctga gaatatcact ggaccacagt 3000

cccaacttag agcgtctgtg gagcctcaca gaactcattt tcctcttcag gaactgaaac 3060

acacaacatt tatctgtagt ctcttgttct gctttttctt cctccatcta ctgccacttg 3120

aggagcaagt tcctccttac ccaaaatttc aggaaatgat acaaaaccaa gtctgggtat 3180

cctcaggggg gaaaggctgt aactctggta attaataatg caggacatgt cttagagatt 3240

gttgttttaa tttacagaca atattctgat tcagaaaatc tgacttgtgt tttgctgcat 3300

gttctccatc tgtaagttct agcacagcgt tgaggagtac attcttttca aattaaatct 3360

cccagtaacc tttaggtatt tttaaaacca tgtttccatt agtgaaatat tttcactcag 3420

tagcagggca tcatcgtatt ttactaaagc ttcggaattt ccgctgttag tcctgttgta 3480

cagcatgtct taaaaatgtc atacctgcac ttgcaattga aatctttgag ttcgagaaaa 3540

ttaaaatgtt ttaacgtgcc aaaaaaaaaa aaaaaaaaaa aaaaagt 3587

<210>3

<211>948

<212>PRT

<213> TLR13 protein amino acid sequence of Epinephelus coioides

<400>3

Met Ser Val Met Gly Ser Trp Pro Leu Phe Phe Leu Gln Ser Phe Leu

1 5 10 15

Ile Leu Leu Pro Ser Ala Leu Leu Pro Phe Asn Pro Leu Leu Ala Phe

20 25 30

Ser Leu Lys Asn Cys Thr Val Asp Asn Asn Val Thr Trp Val Glu Cys

35 40 45

Ala Asp Arg Asp Leu Thr Gly Val Pro Asp Asp Ile Leu Ile Thr Val

50 55 60

Val Thr Leu Asp Leu Asn Phe Asn His Ile Ser Lys Ile Asn Arg Thr

65 70 75 80

Asp Phe Ser Arg Phe Ser Lys Leu Gly Tyr Leu Lys Ile Ala Asn Asn

85 90 95

Leu Ile Ser His Val Asp Asp Gly Ala Phe Ala Glu Leu Val Glu Leu

100 105 110

Ile Glu Leu Asp Met Gly Ser Asn Asn Leu Ile Asn Leu Thr Asn Tyr

115 120 125

Met Phe Gln Gly Leu Trp Lys Leu Ile Phe Leu Ser Val Glu Lys Asn

130 135 140

His Ile Thr Tyr Ile Ser Pro Leu Ala Phe Gln Ser Leu Ile Ser Leu

145 150 155 160

Gln Thr Leu Gln Leu Thr Tyr Asn Asn Leu His Gln Ile Thr Asp Ile

165 170 175

Val Pro Ile Leu Gln Leu Pro Asn Leu Asn Asp Leu Leu Ala Asp Phe

180 185 190

Asn Ser Leu Thr Ser Phe Gln Ser Asp Asp Leu Pro Phe Asn Lys Ser

195 200 205

Asn Leu Arg Thr Leu Trp Leu Cys Ser Asp Ser Met Lys Lys Phe Ser

210 215 220

Ile Thr Arg Asp Val Phe Pro His Leu Gln Ser Ile His Leu Asn Ala

225 230 235 240

Asn Thr Gly Phe Glu Trp Asp Val Pro Asp Pro Met Phe Leu Arg Ser

245 250 255

Leu Thr Thr Leu Glu Leu Ser Val Ser Asp Asn Ser Cys Glu Met Tyr

260 265 270

Gln Val Met Leu Lys Ser Ala Glu Ser Val Gln Glu Leu Ser Leu Leu

275 280 285

Phe Trp His His Asp Arg Glu Arg Asp Leu Val Asp Ile Ala Cys Gln

290 295 300

Met Thr Ala Leu Thr Ser Leu His Leu Ser Gly Ile Ser Phe Val Ser

305 310 315 320

Ile Asn Lys Thr Phe Leu Gln Ser Cys Thr Glu Ile Thr Glu Leu Asp

325 330 335

Leu Ser Tyr Asn Tyr Leu Glu Glu Leu Ser Glu Phe Ser Leu Gly Ser

340 345 350

Ile Lys Gln Leu Arg Arg Leu Asp Leu Val Arg Asn Ser Leu Ser Arg

355 360 365

Val Pro Gln Gly Val Arg Gly Leu Ser Thr Leu Glu Ile Leu Asp Leu

370 375 380

Ser Val Asn Phe Ile Ser Glu Leu Asp Cys Ser Asp Phe Gln Asn Leu

385 390 395 400

Thr Lys Leu Val Glu Leu Asn Leu Ser Gln Asn Arg Ile Ser Thr Leu

405 410 415

Lys Gly Cys Val Phe Gln Asp Leu Asn Asp Leu Lys Val Leu Asn Val

420 425 430

Ala Glu Asn Gly Val His Leu Leu Val Asp Phe Phe Lys Leu Asn Leu

435440 445

Gln Lys Leu Glu Val Leu Asp Leu Asn Met Asn Thr Leu Met Gln Leu

450 455 460

Lys Thr Asp Asp Phe Glu Asn Met Ser Ser Leu Arg Ser Leu Tyr Leu

465 470 475 480

Glu Ser Asp Thr Phe Tyr Val Ala His Lys Gly Ala Phe Glu Gly Leu

485 490 495

Asp Asn Leu Gln Asn Leu Ser Val Thr Pro Phe Phe Gly Glu Met Asp

500 505 510

Phe Ile Thr Lys Val Phe Thr Gly Leu Gln His Leu Gln Asn Leu Lys

515 520 525

Ile His Leu Thr Ser Ser Tyr Asp Ser Lys Ser Ser Cys Gln Lys Asn

530 535 540

Glu Thr His Leu Ser Ile Phe Pro Leu Pro Phe Leu Lys Ser Leu Leu

545 550 555 560

Ile Gln Asn Tyr Asp Trp Tyr His Glu Ile Ser Pro Asp Phe Leu Thr

565 570 575

Gly Leu Asn Ser Leu Asp Tyr Phe Gly Ala Glu Lys Leu Phe Thr Glu

580 585 590

Ser Pro His Pro Asp Thr Phe Arg Tyr Thr Pro Leu Leu Thr Ser Leu

595600 605

His Ile Phe Gln Ser Asp Leu Gln Val Leu Asn Pro Glu Val Leu Gln

610 615 620

Pro Ile Leu Asn Leu Gln Ala Leu Asp Leu Ser Lys Asn Lys Leu Arg

625 630 635 640

Ser Leu Asp Phe Leu Ala Gln Val Asn Leu Ser Ala Leu Arg Val Leu

645 650 655

Thr Val Arg Asp Asn Glu Leu Thr Val Ile Asn Asp Met Val Phe Gln

660 665 670

Ser Leu Pro Ala Leu Thr Tyr Leu Asp Leu Thr Gly Asn Pro Phe Thr

675 680 685

Cys Asn Cys Ser Asn Thr Gly Phe Ile Gln Trp Val Lys Asn Asn Asn

690 695 700

Gln Thr Gln Val Val Asn Ala Tyr Gln Tyr Thr Cys Thr Phe Pro Val

705 710 715 720

Ala Lys Gln Gly Thr Lys Leu Leu Asp Phe Asp Val Gln Ser Cys Trp

725 730 735

Met Asp Val Ser Phe Leu Cys Phe Ile Ser Ser Phe Cys Leu Thr Leu

740 745 750

Met Ile Leu Leu Thr Ser Phe Ile Tyr His Phe Leu Arg Trp Gln Leu

755 760 765

Ala Tyr Thr Tyr Tyr Leu Phe Leu Ala Phe Leu Tyr Asp Ser Arg Lys

770 775 780

Arg Lys Lys Gly Ala Pro His His Tyr Asp Ala Phe Ile Ser Tyr Asn

785 790 795 800

Val His Asp Glu Asp Trp Val Tyr Arg Glu Met Leu Pro Val Leu Glu

805 810 815

Gly Glu Gln Gly Trp Arg Leu Cys Leu His His Arg Asp Phe Gln Pro

820 825 830

Gly Lys Pro Ile Ile Asp Asn Ile Thr Asp Ala Ile Tyr Gly Ser Arg

835 840 845

Lys Thr Ile Cys Val Ile Thr Arg Arg Tyr Leu Gln Ser Glu Trp Cys

850 855 860

Ser Arg Glu Ile Gln Met Ala Ser Phe Arg Leu Phe Asp Glu Gln Lys

865 870 875 880

Asp Val Leu Ile Leu Leu Phe Leu Glu Glu Ile Pro Ala Tyr Gln Leu

885 890 895

Ser Pro Tyr His Arg Met Arg Lys Leu Val Lys Arg His Thr Tyr Leu

900 905 910

Ser Trp Pro Gln Ala Gly Gln His Thr Gly Val Phe Trp Gln Asn Val

915 920925

Trp Arg Ala Leu Glu Thr Gly Glu Ala Pro Ile Glu Thr Asn Asn Leu

930 935 940

Leu Thr Gly Cys

945

<210>4

<211>19

<212>DNA

<213> TLR13FullF primer sequence

<400>4

atgtcagtgatgggaagtt 19

<210>5

<211>16

<212>DNA

<213> TLR13FullR primer sequence

<400>5

cctcaacgctgtgcta 16

Claims (8)

1. The epinephelus coioides natural immune receptor TLR13 gene is characterized in that the nucleotide sequence of the gene is shown in SEQ ID no: 1 is shown.

2. The epinephelus coioides natural immune receptor TLR13 gene full-length cDNA is characterized in that the gene full-length cDNA sequence is shown in SEQ ID no: 2, respectively.

3. A protein coded by a TLR13 gene of a Epinephelus coioides natural immune receptor, which is characterized in that the amino acid sequence of the protein is shown in SEQ ID no: 3, respectively.

4. A primer sequence for amplifying the full-length cDNA of a natural immune receptor TLR13 gene of Epinephelus coioides is characterized in that the primer sequence is shown as SEQ ID NO: 4-SEQ.ID.NO: 5, respectively.

5. A eukaryotic expression vector comprising the full-length cDNA of the TLR13 gene of claim 2.

6. Use of the gene of claim 1 or the full-length cDNA of the TLR13 gene of claim 2 or the protein of claim 3 in the preparation of a medicament for modulating the expression of an inflammatory factor.

7. Use of the gene of claim 1, the full-length cDNA of the TLR13 gene of claim 2, or the protein of claim 3 in the preparation of a medicament for identifying bacterial RNA.

8. Use of the gene of claim 1 or the full-length cDNA of the TLR13 gene of claim 2 or the protein of claim 3 in the preparation of a medicament for recognizing RNA of Vibrio parahaemolyticus and modulating the expression of inflammatory factors.

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