CN107557366A - A kind of Epinephelus coioides innate immunity acceptor TLR13 genes and its carrier for expression of eukaryon and application - Google Patents

Abstract

Present invention firstly discloses a kind of Epinephelus coioides innate immunity acceptorTLR13Gene and its carrier for expression of eukaryon and application.TLR13The nucleotide sequence of gene such as SEQ.ID.NO：Shown in 1,TLR13The full length cDNA sequence of gene such as SEQ.ID.NO：Shown in 2, the pair of primers sequence such as SEQ.ID.NO that is included in its sequence：Shown in 4~5, the amino acid sequence such as SEQ.ID.NO of TLR13 albumen：Shown in 3.The present invention provides two kinds containing describedTLR13The carrier for expression of eukaryon of full length gene cDNA sequence, pEGFP N3 TLR13 and pcDNA3.1 TLR13.In addition, the present invention willTLR13Gene or TLR13 albumen are applied in the research in terms of the expression of regulation inflammatory factor, identification bacteria RNA, identification vibrio parahaemolytious RNA and regulation inflammatory factor.

Description

A kind of Epinephelus coioides innate immunity acceptorTLR13Gene and its carrier for expression of eukaryon With application

Technical field

The invention belongs to technical field of biological genetic engineering, more particularly, to a kind of Epinephelus coioides innate immunity by BodyTLR13Gene and its carrier for expression of eukaryon and application.

Background technology

Toll sample acceptors（Toll-like receptor, TLR）Family is " dissident " identification and congenital immunity reaction Activation basis, belong to I type transmembrane receptor.TLRs has the TIR guarded in film（Toll/IL-1 receptor）Domain, it is main It is used to raise adaptor protein marrow sample differentiation factor in immune response（Myeloid differentiation factor 88, MyD88）With Toll-like receptor adaptor protein 1（TIR domain-containing adaptor inducing interferon-β （TRIF）/TIR domain-containing adaptor molecule （TICAM-1）, TRIF1）, Further to activate the immune response in downstream.TLRs also has the region that conservative leucine does not repeat outside film, is mainly used in knowing Not different pathogen-associated molecular patterns.

The downstream passages of TLR mediations are mainly activated by the MyD88 signal paths relied on or the TRIF signal path relied on The immune response in downstream.After specific ligand stimulation, TLR will form homodimer or heterodimer, and recruitment connects Noggin MyD88, MAL（myeloid differentiation protein 88 adapter-like）, TRIF, and TRAM （TRIF-related adaptor molecule）, a series of then kinases in activation downstream, and mediate transcription factor NF- κ B （nuclear factor-κB）, AP1（activator protein-1）Deng activation, finally cause the production of immune inflammatory factor It is raw.

The TLR of bony fish has certain difference compared with mammality in species and function.At present, in the mankind TLR1 ~ 10 are found that, TLR1 ~ 13 are found that in mouse, and in bony fish, on the basis of mammiferous, find in addition TLR18 ~ 23, a variety of distinctive TLR family members of fish such as 25,26.Research shows, the TLR2 of fish may identify virus and It is not the part of bacterium class, TLR4 does not identify that LPS, TLR5 have intracellular and two kinds of forms positioned at cell membrane, Er Qiedou Flagellin can be identified.And the special TLR22 of fish has a variety of hypotypes, can identify including lipopolysaccharides, double-stranded RNA analog （Polyinosinic-polycytidylic acid, Poly（I:C））, peptide glycan（Peptidoglycan, PGN）Etc. a variety of Different types of ligand stimulation, activate downstream immune pathway.

At present, study and find in mouse, TLR13 expression can be by cerebral cysticercosis, vesicular stomatitis virus and thin Bacterium 23S rRNA stimulation up-regulation, and it was found that mouse TLR13 can identify bacterium 23S rRNA.But in bony fish, Very few to TLR13 researchs, whether TLR13 can also play a part of identifying bacterium in bony fish is immune, and which kind of can be identified The functions such as bacterium are still not clear.

Grouper is the rare aquaculture fish in China, and delicious meat is nutritious, have very high edibility and Economic value.Epinephelus coioides（Epinepheluscoioides）Belong to one of grouper species relatively conventional in cultivation, Belong to Perciformes in classification（Perciformes）, Sushi sections（Serranidae）, Epinephelinae（Epinephelinae）, lithosporic Fish category（Epinephelus）.In recent years, the development of Grouper cultivating industry is swift and violent, but is accompanied by the expansion of cultivation scale, disease Frequently outburst, seriously constrains the development of grouper industry.Because bacterium infection has a great influence to caused by Grouper cultivating, because This, the effect probed into Epinephelus coioides innate immunity acceptor TLR13 functional characteristics and its played in bacterial defenses, on the one hand The theoretical research of fish innate immunity signal path can be enriched, while can also be fish immunity in production application, is prevented The researchs such as disease provide the foundation of science and the basis of practice.

Vibrio parahaemolytious is a kind of gram negative pathogenic bacteria of halophagia, is the Aquatic farming animals such as fish, shrimp, shellfish Important pathogen.Meanwhile vibrio parahaemolytious still causes one of important pathogen of mankind's food origin disease, In Guangdong Province is China Aquatic products vibrio parahemolyticus pollutes the area of most serious.Therefore, identification and defense mechanism of the bony fish to it are understood in depth, And targetedly high-efficiency environment friendly prevention and control strategy is formulated, the present situation for improving abuse of antibiotics is strong to promote the strong of seawater fishery Kang Gaoxiao is cultivated, and while high-quality aquatic products are secured good health, reducing Human Health Risk has great significance.

The content of the invention

The technical problems to be solved of the present invention are to make up the blank of prior art, there is provided a kind of Epinephelus coioides are naturally exempted from Epidemic disease acceptorTLR13The nucleotide sequence of gene.

Another technical problem to be solved by the present invention is that provide a kind of comprising describedTLR13Full length gene cDNA sequence it is true Nuclear expression carrier.

A present invention also technical problems to be solved are to provide described in one kindTLR13Gene or TLR13 albumen are scorching in regulation Application in the expression of inflammation factor.

A present invention also technical problems to be solved are to provide described in one kindTLR13Gene or TLR13 albumen are thin in identification Application in bacterium RNA.

A present invention also technical problems to be solved are to provide described in one kindTLR13Gene or TLR13 albumen are secondary in identification Hemolysis vibrion RNA simultaneously adjusts the application in the expression of inflammatory factor.

The purpose of the present invention is achieved by the following technical programs：

The invention provides a kind of Epinephelus coioides innate immunity acceptorTLR13Gene, the nucleotide sequence of the gene is such as Shown in SEQ.ID.NO1.

Described Epinephelus coioides innate immunity acceptorTLR13The full length cDNA sequence of gene is as shown in SEQ.ID.NO2.

Described Epinephelus coioides innate immunity acceptorTLR13The amino acid sequence of the albumen of gene code is such as Shown in SEQ.ID.NO3.

The Epinephelus coioides innate immunity acceptorTLR13The protein structure of the albumen of gene code includes 1 segment signal peptide Sequence, 14 LRR domains, 1 typical TIR domains and 1 membrane spaning domain.

Present invention also offers comprising describedTLR13The carrier for expression of eukaryon of full length gene cDNA sequence.

Further, the carrier is pcDNA3.1-TLR13 or pEGFP-N3-TLR13, and wherein TLR13 refers to that right will Ask described in 2TLR13Full length gene cDNA sequence；Wherein pcDNA3.1 refers to pcDNA3.1 plasmids, and wherein pEGFP-N3 refers to PEGFP-N3 plasmids.

Present invention also offers the application of described gene or described albumen in the expression of regulation inflammatory factor.

Present invention also offers the application of described gene or described albumen in bacteria RNA is identified.

Present invention also offers described gene or described albumen in identification vibrio parahaemolytious RNA and to adjust inflammatory factor Expression in application.

Beneficial effects of the present invention：

The present invention provides the Epinephelus coioides innate immunity acceptor firstTLR13The nucleotide sequence of gene is based on the sequence Row, are additionally provided described in two kindsTLR13The carrier for expression of eukaryon pEGFP-N3-TLR13 and pcDNA3.1-TLR13 of gene, and Provide describedTLR13Gene andTLR13The albumen of gene code is in the expression of regulation inflammatory factor, identification bacteria RNA, identification Vibrio parahaemolytious RNA simultaneously adjusts the application in the expression of inflammatory factor, has prominent substantial advance.

Brief description of the drawings

Fig. 1TLR13Full length gene cDNA PCR amplification electrophoretograms.

The bacterium solution PCR checking electricity of Fig. 2 recombinant plasmids pEGFP-N3-TLR13 and pcDNA3.1-13 conversion bacillus coli DH 5 alpha Swimming figure.

The overexpression TLR13 of two kinds of carrier for expression of eukaryon of Fig. 3 effect.

Fig. 4, which is overexpressed TLR13, makes the mRNA transcriptional upregulations of the inflammation factor.

Fig. 5 is overexpressed the identification to bacteria RNA and the up-regulation to downstream IFN-β promoter activity after TLR13.

Fig. 6 strike drop TLR13 after to vibrio parahaemolytious RNA mediation caused by immune factor influence.

Embodiment

The application process of the present invention is further illustrated with reference to specific embodiment.Following embodiments and accompanying drawing are only used for showing Example property explanation, it is impossible to be interpreted as limitation of the present invention.Unless stated otherwise, the reagent raw material used in following embodiments is normal The life reagent raw material that purchased in market or commercial sources obtain is advised, unless stated otherwise, the method and apparatus used in following embodiments is Method and apparatus commonly used in the art.

The Epinephelus coioides of embodiment 1TLR13The clone of gene

（1）The extraction of Epinephelus coioides spleen total serum IgE

Healthy Epinephelus coioides are taken, is anaesthetized 3 minutes on ice, separating spleen tissue, its total serum IgE is extracted using Trizol reagents.

（2）The synthesis of the chains of cDNA first

1 μ g Epinephelus coioides head-kidney total serum IgEs sample is taken to carry out DNA enzymatic processing to remove the pollution of genomic DNA, with RNA OligodT is mixed, and carries out reverse transcription, and products therefrom is placed in -20 DEG C and saved backup.

（3）The clone of Epinephelus coioides TLR13 gene cDNA complete sequences

Specific primer TLR13FullF and TLR13FullR are designed, as shown in SEQ ID NO.4, SEQ ID NO.5, is expanded The ORF clip sizes arrived are 2844 bp, electrophoresis result as shown in Figure 1, gel extraction purpose band and purified product.Will production Thing is connected to pTZ57R/T carriers, converts DH5 α Escherichia coli, selects positive colony sequencing.Show through BLAST homology analysis, Purpose product is really the cDNA sequence fragments of TLR13 genes.The sequence such as following table used in PCR amplifications：

Embodiment 2 recombinant plasmid pEGFP-N3-TLR13 and pcDNA3.1-13 structure and its checking

According toTLR13Full length gene cDNA sequence, in the both ends sequent synthesis pair of primers of encoding gene, sense primer contains XhoI cleavage sites, sequence, anti-sense primer contain BamHI cleavage sites, with containingTLR13The pTZ57R/T plasmids of encoding gene For template, enter performing PCR amplification, obtain the single band of specific amplified, primer size is in 2800 bp or so.PCR is expanded and produced Thing is cloned on expression vector PcDNA3.1 and pEGFP-N3, obtains recombinant expression carrier, the PCR the results of recombinant plasmid are such as Shown in accompanying drawing 2, wherein the result for pEGFP-N3-TLR13 of marker lane 1, swimming lane 2 are testing for PcDNA3.1-TLR13 Demonstrate,prove result.The primer sequence such as table 1 below used in amplification and checking：

Embodiment 3 recombinant plasmid pEGFP-N3-TLR13 and pcDNA3.1-13 overexpression effect

By appropriate 293T cells（It is about 1 × 10 6 per hole cell number）Be inoculated into 96 orifice plates, next day treat cell length to 60% ~ It can be transfected when 80%, the Opti-MEM nutrient solutions of serum-free are changed to before transfection.150 ng recombinant plasmids are transfected respectively per hole PEGFP-N3-TLR13, pcDNA3.1-13 or their empty vectors control.Transfection liquid is：A liquid（Opti-MEM nutrient solutions 10 μ L, plasmid is added, mixed）, B liquid（The μ L of Opti-MEM nutrient solutions 10,0.25 μ L lipo3000 are added, mixed, room temperature places 5 min）；A liquid and B liquid are gently mixed and place 20 min as transfection liquid, room temperature, adds in corresponding transfection hole, it is small to be incubated 6 When；Finally it is changed to the DMEM nutrient solutions containing 10% hyclone.

Cell is collected in transfection after 36 hours, extract total serum IgE, reverse transcription, progress fluorescence quantitative PCR detection TLR13 mRNA Level, the amplimer used is TLR13 RTF and TLR13 RTR, wherein the reference gene chosen is EF1, the amplification of use Primer is EF1RTF1 and EF1RTR1.The CT values obtained according to detection carry out the analysis of result, utilize 2- △ △ ct method meter TLR13 relative expression quantity is calculated, experimental result is as shown in Figure 3.The primer sequence used in detection such as table 2 below：

, can conspicuousness from accompanying drawing 3 as can be seen that after transfection recombinant plasmid pEGFP-N3-TLR13 or pcDNA3.1-13 Improve expressions of the TLR13 mRNA in cell.As a result show, construction of recombinant plasmid success can be in cell after transfection Significantly improve TLR13 expression.

To the mRNA transcriptional upregulations of the inflammation factor after the overexpression of embodiment 4 TLR13

By appropriate GS cells（It is about 4 × 10 6 per hole cell number）Be inoculated into 6 orifice plates, next day treat cell length to 50% ~ It can be transfected when 60%, the Opti-MEM nutrient solutions of serum-free are changed to before transfection.2000 ng pEGFP-N3- are transfected per hole TLR13 or pEGFP-N3 empty vectors compare.Transfection liquid is：A liquid（The μ L of Opti-MEM nutrient solutions 50, plasmid is added, mixed It is even）, B liquid（The μ L of Opti-MEM nutrient solutions 50,4 μ L lipo2000 are added, mixed, room temperature places 5 min）；By A liquid and B liquid It is transfection liquid gently to mix, and room temperature places 20 min, adds in corresponding transfection hole, is incubated 12 hours；Finally be changed to containing The L15 nutrient solutions of 10% hyclone.

Cell is collected in transfection after 48 hours, extracted total serum IgE, reverse transcription, carried out fluorescence quantitative PCR detection IL-6, IL-1 β, IL-12 and TNF α mRNA level in-site, wherein the reference gene chosen is EF1, the amplimer used for EF1RTF1 and EF1RTR1.The CT values obtained according to detection carry out the analysis of result, and above immune factor is calculated using 2- △ △ ct method Relative expression quantity, experimental result is as shown in Figure 4.The primer sequence used in detection such as table 3 below：

IL-1 β, IL-6, IL- can be improved with conspicuousness after TLR13 is overexpressed from accompanying drawing 4 as can be seen that in GS cells 12nd, the expression of this several inflammatory factor of TNF α.As a result show,TLR13Gene or TLR13 albumen can adjust the table of inflammatory factor Reach.

Embodiment 5 is overexpressed after TLR13 identification to bacterium 23S rRNA and to the upper of downstream IFN-β promoter activity Adjust

（1）The gene transfection of eukaryotic

By appropriate 293T cells（It is about 2 × 10 5 per hole cell number）Be inoculated into 24 orifice plates, next day cell treat it is long to 50% ~ It can be transfected when 60%.The Opti-MEM nutrient solutions of serum-free are changed to before transfection.Per hole transfect 700 ng pcDNA3.1-13 or PcDNA3.1 empty vectors compare, 200 ng PGL3-IFN- β-Luc reporter plasmids and 100 ng pRL-TK plasmids.Transfection Liquid is：A liquid（The μ L of Opti-MEM nutrient solutions 50, plasmid is added, mixed）, B liquid（The μ L of Opti-MEM nutrient solutions 10, add 1.5 μ L lipo2000, mix, room temperature places 5 min）；A liquid and B liquid are gently mixed and place 20 as transfection liquid, room temperature Min, add in corresponding transfection hole, be incubated 4 ~ 6 h；Finally it is changed to the DMEM nutrient solutions containing 5% hyclone.

（2）Reporter gene transcription Activity determination

After transfecting 36 h, 293T cells are stimulated with ORN Sa19 and its mutant controls ORN the Sa19 Control of gradient concentration, Harvesting after 18 h, is detected using dual luciferase reporter gene detection kit, experimental result as shown in Figure 5, Show value is the ratio of firefly luciferase and renilla luciferase in figure.

As can be seen that TLR13 overexpression mediates when can be stimulated with conspicuousness raising cell ORN Sa19 from accompanying drawing 5 IFN-β promoter activity activation.As a result show,TLR13Gene or TLR13 albumen can recognize that bacteria RNA.

Embodiment 6 strike drop TLR13 after to vibrio parahaemolytious RNA mediation caused by immune factor influence

（1）Extract vibrio parahaemolytious RNA

200 μ L vibrio parahaemolytious are inoculated into 35 mL culture mediums and cultivate 12 h, bacterium is centrifuged, utilizes Trizol reagents Method extracting RNA.

（2）TLR13 strikes the vibrio parahaemolytious RNA stimulation tests after drop

By appropriate GS cells（It is about 1 × 10 4 per hole cell number）Be inoculated into 96 orifice plates, next day treat cell length to 50% ~ It can be transfected when 60%, the Opti-MEM nutrient solutions of serum-free are changed to before transfection.Used siRNA sequence is siTLR13 （Sequence：sense：5’-GCCUAUCAGCUGUCUCCAUTT-3’；antisense：5’-AUGGAGACAGCUGAUAGGCTT- 3’）And Negative Control sequences：（Sequence：sense：5’-UUCUCCGAACGUGUCACGUTT-3’；antisense： 5’-AUGGAGACAGC UGAUAGGCTT-3’）, diluted concentration 20nM.Transfection liquid is：A liquid（The μ L of Opti-MEM nutrient solutions 5, 0.3 μ LsiRNA are added, are mixed）, B liquid（The μ L of Opti-MEM nutrient solutions 5,0.4 μ L lipo3000 are added, mixed, room temperature Place 5 min）；A liquid and B liquid are gently mixed and place 15 min as transfection liquid, room temperature, adds in corresponding transfection hole, incubates Educate 6 h；Finally it is changed to the L15 nutrient solutions containing 10% hyclone.After transfecting 40 h, add vibrio parahaemolytious RNA and enter to assassinate Swash, the concentration for stimulating RNA used is 2 ug/mL, the RNA RNase A of equivalent（20 mg/mL）1 h conducts are digested at 37 DEG C Control.After stimulating 6 h, harvesting, total serum IgE is extracted, reverse transcription, fluorescence quantitative PCR detection is carried out and detects each immune factor table Up to amount, experimental result is as shown in Figure 6.

Mediation is stimulated to cause as can be seen that after striking drop TLR13 vibrio parahaemolytious RNA can be suppressed with conspicuousness from accompanying drawing 6 Downstream inflammatory factor activation.As a result show,TLR13Gene or TLR13 albumen can recognize that vibrio parahaemolytious RNA and adjusts inflammation Inflammation factor.

SEQUENCE LISTING

<110>Zhongshan University

<120>A kind of Epinephelus coioides innate immunity acceptor TLR13 genes and its carrier for expression of eukaryon and application

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 2847

<212> DNA

<213>The nucleotide sequence of Epinephelus coioides TLR13 genes

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atgtcagtga tgggaagttg gcctctgttc tttctacaga gcttcctcat cttacttccg 60

tctgccctgc ttccttttaa ccccttgctg gctttctcac taaaaaactg cactgttgac 120

aacaatgtga cttgggtaga atgcgcagat cgtgacctca ctggggttcc tgatgacatt 180

ctcataactg tggtaactct agatctcaac ttcaatcaca tctcaaagat aaacaggaca 240

gatttcagcc gtttttcaaa gcttgggtat ttaaaaattg caaataactt gatttcacat 300

gtagatgatg gagcttttgc agagttggtg gagttaatag agcttgacat gggttcaaac 360

aacctcataa acctgacaaa ctatatgttt cagggcctgt ggaagttaat ttttttgtca 420

gtggagaaaa accatatcac atacatctcc cctctggcct ttcagtccct gatcagctta 480

cagacgttac agctgacata taacaaccta catcaaataa ccgacattgt gcccatctta 540

caactaccaa acttgaatga tctgctcgct gattttaaca gtctcacttc ctttcagtca 600

gatgacctgc cttttaataa atcaaacctc aggacactat ggctatgtag tgattcaatg 660

aaaaagttca gcattacaag agacgttttt ccccatcttc agtctatcca tttaaatgct 720

aacactggct ttgagtggga tgtaccagac ccaatgtttc tgaggagcct gactactttg 780

gagttatctg tatctgacaa tagttgtgag atgtaccaag tgatgctgaa gagtgccgag 840

tcagtgcagg aactgtcact cctcttttgg caccatgacc gagaaagaga tctggtagac 900

atcgcctgcc aaatgactgc tctgacaagt cttcatttga gtggaattag ctttgtcagt 960

ataaacaaga catttcttca atcttgcact gaaattactg agcttgattt atcatataat 1020

tatttggaag agctgtccga gttttccctc ggatcaataa aacagctcag acgcctggac 1080

ttagtgagaa actccctgtc tagagtgcca cagggtgtca gaggcctctc cacacttgaa 1140

atcctagatc tgagtgtgaa tttcatcagt gagttagact gctctgactt tcaaaatttg 1200

acaaaactag tagaactcaa tctcagccaa aatcgcattt caacactcaa gggatgtgtt 1260

tttcaagatc tgaatgattt gaaagtcctg aacgttgcag aaaatggagt tcatttactt 1320

gttgatttct tcaaattgaa tttacagaaa ctagaagttt tggatttgaa catgaatact 1380

ttgatgcagc tcaaaacaga tgactttgag aatatgtctt ccctcaggtc tctgtattta 1440

gaatcagata cattttacgt tgcccataag ggggcttttg aaggactgga caatcttcaa 1500

aatctttcag ttacaccatt ttttggtgaa atggatttca ttacaaaggt ttttacagga 1560

ctgcaacacc tacaaaacct caaaatacat ctcacgtctt catatgacag caagagttct 1620

tgtcaaaaaa atgagacaca tttatccatt ttcccattac ctttcttgaa gagtctactg 1680

atacaaaatt atgattggta tcatgaaatc tcacctgatt ttctgacagg tctgaattct 1740

ttagattact ttggagcgga gaaattattt acagagtcac ctcacccaga cacattcaga 1800

tatactcccc tcctgacaag tcttcacata tttcaaagtg atctgcaagt cttaaaccct 1860

gaagtgcttc agccaatcct caacctgcag gctctcgacc tttccaaaaa caagctcaga 1920

tctctggatt ttctagccca ggtcaacctc tctgcactca gagtgttgac agttagagac 1980

aatgaattga ccgtgatcaa cgacatggtc tttcagtctc tccctgcact gacatacctg 2040

gacctgactg gtaacccttt cacttgtaac tgctctaaca ctggctttat ccaatgggtg 2100

aagaacaaca accaaacaca ggttgttaat gcctaccagt acacttgtac ctttcctgtg 2160

gctaaacaag gaacaaagtt gctggacttt gacgtccagt cctgttggat ggacgttagc 2220

ttcctctgct tcatttctag cttttgcctg acgctgatga ttctcctcac atccttcatc 2280

taccactttc tgaggtggca gctagcctac acctactacc tcttcctggc cttcctctac 2340

gacagcagga agaggaagaa gggcgctcct catcactacg atgctttcat ctcctacaat 2400

gttcatgacg aggactgggt ttacagagag atgcttccag tgctggaggg agagcagggc 2460

tggagactct gtctgcacca cagagacttc caaccaggta aacccatcat agacaacata 2520

acagacgcca tctacggcag caggaagacc atctgtgtga tcacccggcg ttacctgcag 2580

agcgaatggt gctccagaga gatccagatg gccagcttcc gtctgtttga cgagcagaag 2640

gacgtgttga tcctgctgtt cctggaggag atcccggcct atcagctgtc tccataccac 2700

cgcatgagga agctggtgaa gaggcacacc tacctgagct ggccgcaggc cggccaacac 2760

acaggagtct tctggcagaa cgtatggaga gctctggaga caggggaagc tcccattgag 2820

accaacaacc tgctgactgg atgctga 2847

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<212> DNA

<213>The full length cDNA sequence of Epinephelus coioides TLR13 genes

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acgcgggggg ggccttagtc tgacagtctg aagaggagca ccaacctctc atttcccata 60

acagttcatg tagaatgagc cggagcatca tttggagtct gaggtcaaac atctggctgt 120

tttgaagatc aacatgtcag tgatgggaag ttggcctctg ttctttctac agagcttcct 180

catcttactt ccgtctgccc tgcttccttt taaccccttg ctggctttct cactaaaaaa 240

ctgcactgtt gacaacaatg tgacttgggt agaatgcgca gatcgtgacc tcactggggt 300

tcctgatgac attctcataa ctgtggtaac tctagatctc aacttcaatc acatctcaaa 360

gataaacagg acagatttca gccgtttttc aaagcttggg tatttaaaaa ttgcaaataa 420

cttgatttca catgtagatg atggagcttt tgcagagttg gtggagttaa tagagcttga 480

catgggttca aacaacctca taaacctgac aaactatatg tttcagggcc tgtggaagtt 540

aatttttttg tcagtggaga aaaaccatat cacatacatc tcccctctgg cctttcagtc 600

cctgatcagc ttacagacgt tacagctgac atataacaac ctacatcaaa taaccgacat 660

tgtgcccatc ttacaactac caaacttgaa tgatctgctc gctgatttta acagtctcac 720

ttcctttcag tcagatgacc tgccttttaa taaatcaaac ctcaggacac tatggctatg 780

tagtgattca atgaaaaagt tcagcattac aagagacgtt tttccccatc ttcagtctat 840

ccatttaaat gctaacactg gctttgagtg ggatgtacca gacccaatgt ttctgaggag 900

cctgactact ttggagttat ctgtatctga caatagttgt gagatgtacc aagtgatgct 960

gaagagtgcc gagtcagtgc aggaactgtc actcctcttt tggcaccatg accgagaaag 1020

agatctggta gacatcgcct gccaaatgac tgctctgaca agtcttcatt tgagtggaat 1080

tagctttgtc agtataaaca agacatttct tcaatcttgc actgaaatta ctgagcttga 1140

tttatcatat aattatttgg aagagctgtc cgagttttcc ctcggatcaa taaaacagct 1200

cagacgcctg gacttagtga gaaactccct gtctagagtg ccacagggtg tcagaggcct 1260

ctccacactt gaaatcctag atctgagtgt gaatttcatc agtgagttag actgctctga 1320

ctttcaaaat ttgacaaaac tagtagaact caatctcagc caaaatcgca tttcaacact 1380

caagggatgt gtttttcaag atctgaatga tttgaaagtc ctgaacgttg cagaaaatgg 1440

agttcattta cttgttgatt tcttcaaatt gaatttacag aaactagaag ttttggattt 1500

gaacatgaat actttgatgc agctcaaaac agatgacttt gagaatatgt cttccctcag 1560

gtctctgtat ttagaatcag atacatttta cgttgcccat aagggggctt ttgaaggact 1620

ggacaatctt caaaatcttt cagttacacc attttttggt gaaatggatt tcattacaaa 1680

ggtttttaca ggactgcaac acctacaaaa cctcaaaata catctcacgt cttcatatga 1740

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>Epinephelus coioides TLR13 protein amino acid sequences

<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

435 440 445

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

595 600 605

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

915 920 925

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 sequences

<400> 4

atgtcagtgatgggaagtt 19

<210> 5

<211> 16

<212> DNA

<213>TLR13FullR primer sequences

<400> 5

cctcaacgctgtgcta 16

Claims (10)

1. A kind of 1. Epinephelus coioides innate immunity acceptorTLR13Gene, it is characterised in that the nucleotide sequence of the gene is such as SEQ.ID.NO：Shown in 1.
2. 2. Epinephelus coioides innate immunity acceptor according to claim 1TLR13Gene, it is characterised in that the gene Full length cDNA sequence such as SEQ.ID.NO：Shown in 2.
3. 3. Epinephelus coioides innate immunity acceptor according to claim 1TLR13The albumen of gene code, its feature exist In the amino acid sequence such as SEQ.ID.NO of the albumen：Shown in 3.
4. 4. albumen according to claim 3, it is characterised in that the protein structure include 1 segment signal peptide sequence, 14 LRR domains, 1 typical TIR domains and 1 membrane spaning domain.
5. 5. Epinephelus coioides innate immunity acceptor according to claim 1TLR13Gene, it is characterised in that the gene Full length cDNA sequence in include 1 pair of primer sequence, the primer sequence is respectively such as SEQ.ID.NO：4~SEQ.ID.NO：5 institutes Show.
6. 6. comprising described in claim 2TLR13The carrier for expression of eukaryon of full length gene cDNA sequence.
7. 7. carrier according to claim 6, it is characterised in that the carrier is pcDNA3.1-TLR13 or pEGFP-N3- TLR13, wherein TLR13 refer to described in claim 2TLR13Full length gene cDNA sequence；Wherein pcDNA3.1 refers to PcDNA3.1 plasmids, wherein pEGFP-N3 refer to pEGFP-N3 plasmids.
8. 8. the answering in the expression of regulation inflammatory factor of the albumen described in gene or claim 3 described in claim 1 or 2 With.
9. 9. application of the albumen described in gene or claim 3 in bacteria RNA is identified described in claim 1 or 2.
10. 10. the albumen described in gene or claim 3 described in claim 1 or 2 in identification vibrio parahaemolytious RNA and adjusts inflammation Application in the expression of inflammation factor.