CN108997491A - Epinephelus coioides innate immunity receptor TLR5S gene and its new application for encoding albumen - Google Patents
Epinephelus coioides innate immunity receptor TLR5S gene and its new application for encoding albumen Download PDFInfo
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- 108010015385 valyl-prolyl-proline Proteins 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses the application of Epinephelus coioides innate immunity receptor TLR5S gene and its coding albumen in identification bacterial flagellum, regulating cell in signal path and adjusting inflammatory factor.Help to understand fish body resistance pathogenic microorganism, the especially physiology course of vibrios invasion, can be fish immunity in production application, the researchs such as pre- disease prevention provide the foundation of science and the basis of practice.Can be prepared into production application has effects that resist the drug that vibrio parahemolyticus is proliferated, and also can be used as immunopotentiator application.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to Epinephelus coioides innate immunity receptor TLR5S gene
And its new application of coding albumen.
Background technique
Congenital immunity is the first line of defence that body resists external pathogenic microorganism invasion, is fish quickly and directly exempts from
Epidemic disease defence.Toll-like receptor (Toll-like receptor, TLR) as main pattern-recognition in innate immune system by
Body participates in the knowledge of a variety of pathogen-associated molecular patterns (pathogen-associated molecular patterns, PAMPs)
Not, MyD88 intracellular (myeloid differentiation primary-response protein 88) dependent form is activated to believe
Number approach or MyD88 independent form signal pathway cause specific immune response to resist the invasion of pathogenic microorganism.
In mammals, the conservative region of TLR5 specific recognition and combination gram- bacteria flagellin, passes through recruitment
MyD88 transduction signal intracellular activates NF- κ B (nuclear factor- κ B), and starting proinflammatory factor and I type interferon etc. are exempted from
The expression and mediation body inflammatory response of epidemic disease gene.At present in Fugu rubripes, rainbow trout, Atlantic salmon, channel catfish, tooth
The TLR5 of 2 seed types: transmembrane TLR5 (Membrane form of is identified in the bony fishes such as flounder, Larimichthys crocea and grass carp
TLR5, TLR5M) and soluble type TLR5 (Soluble form of TLR5, TLR5S), wherein TLR5S gene is that fish institute is peculiar
TLR family member.Prove that TLR5S can promote TLR5M to mediate in conjunction with bacterial flagellin in rainbow trout TLR5 research
NF- κ B signal pathway activated.But what kind of effect machine played after capable of identifying which kind of cell component and identification to TLR5S in fish immunity
The functions such as system regulation intracellular signaling pathways activation are still not clear.
Epinephelus coioides (Epinephelus coioides) are a kind of water warm coastal waters bottom rare fish class, fine and tender taste
It is delicious, it is rich in nutriment, there is very high economic value, Perciformes (Perciformes) , Sushi section is belonged in classification
(Serranidae), Epinephelinae (Epinephelinae), Epinephelus (Epinephelus).Along with Epinephelus coioides
The development of aquaculture, virus, bacterium and helminth fish disease are becoming increasingly rampant, the serious development for restricting Epinephelus coioides industry.Bacterium
Property fish disease due to its spread speed it is fast, lethality is high, and harmfulness is big, has worldwide caused to fishery cultivating huge
Economic loss.Wherein vibrio parahemolyticus is one of the main pathogenic bacteria of aquatic animal vibriosis, is a kind of halophagia gram
Negative bacterium brings serious economic loss to mariculture industry.Selected vibrio parahemolyticus infection can cause higher lethal
The symptom of rate and abdomen hyperemia has observation after zebra fish, black blue spot filefish and Qingdao branchiostoma inject vibrio parahemolyticus
It arrives.Vibrio parahemolyticus contains there are many PAMPs, such as LPS, RNA, flagellum, and the TLR in body can pass through identification parahemolyticas
Vibrios component, activation intracellular signaling pathways are to cause machine immune response.Intracellular signaling pathways excessive activation or it is unbalance all
The serious inflammatory reaction of body can be caused and bring damage to body, to safeguard immune homeostasis, body develops a set of complicated negative out
Adjustment and control system carries out strict monitoring to intracellular signal.
It probes into the functional character of Epinephelus coioides innate immunity receptor TLR5S and its is defendd in the congenital immunity of directed toward bacteria
The effect of middle performance helps to understand fish body resistance pathogenic microorganism, the especially physiology course of vibrios invasion, can be in production application
In be fish immunity, the researchs such as pre- disease prevention provide science foundation and practice basis.
Summary of the invention
The purpose of the present invention is to provide the new use of Epinephelus coioides innate immunity receptor TLR5S gene and its coding albumen
On the way.
Above-mentioned purpose of the invention is achieved through the following technical solutions: Epinephelus coioides innate immunity receptor
The application of TLR5S gene and its coding albumen in identification bacterial flagellum, regulating cell in signal path and adjusting inflammatory factor.
The bacterium is preferably vibrio parahemolyticus.
Epinephelus coioides innate immunity receptor TLR5S gene and its coding albumen in the present invention are recognizing parahemolyticas
After the flagellum of vibrios, downstream can be adjusted by the activation of negative regulation intracellular signaling pathway with signal path in regulating cell
The expression of inflammatory factor especially balances the expression of inflammatory factor.Therefore it can be used as drug, it is secondary molten to resist
The proliferation of hemorrhagic vibrios adjusts inflammatory reaction as immunomodulator.
Therefore, the Epinephelus coioides innate immunity receptor TLR5S gene in the present invention and its coding albumen can also made
It is standby to have effects that apply in the drug for inhibiting vibrio parahemolyticus to be proliferated；It can also be used as immunomodulator application.
The nucleotide sequence such as SEQ.ID.NO:1 institute of Epinephelus coioides innate immunity receptor TLR5S gene of the present invention
Show.
The amino acid sequence of Epinephelus coioides innate immunity receptor TLR5S gene coded protein of the present invention is such as
Shown in SEQ.ID.NO:2.
In addition, the full length cDNA sequence of Epinephelus coioides innate immunity receptor TLR5S gene of the present invention is such as
Shown in SEQ.ID.NO:3.
The full length cDNA sequence overall length 2168bp of Epinephelus coioides TLR5S gene of the present invention, 5 including a 79bp
` noncoding region, the 3` noncoding region of 154bp and the open reading frame of a 1935bp.
The protein structure of Epinephelus coioides innate immunity receptor TLR5S gene coding of the present invention is by 17 LRR structures
Domain is constituted.
The invention has the following advantages that the present invention provides Epinephelus coioides innate immunity receptor TLR5S gene and its volumes
Application of the code albumen in identification bacterial flagellum, regulating cell in signal path and adjusting inflammatory factor.Help to understand fish body
Pathogenic microorganism, the especially physiology course of vibrios invasion are resisted, can be fish immunity, the research such as pre- disease prevention in production application
The foundation of science and the basis of practice are provided；And further has effects that the drug for inhibiting vibrio parahemolyticus to be proliferated in preparation
Middle application can also be used as immunomodulator application.
Detailed description of the invention
Fig. 1 is the PCR gel electrophoresis figure in the region EcTLR5S gene ORF in embodiment 1；
Fig. 2 is the gel electrophoresis figure that carrier for expression of eukaryon pcDNA4.0-TLR5S is verified in embodiment 2；
Fig. 3 is the effect of the overexpression EcTLR5S of carrier for expression of eukaryon in embodiment 3；
Fig. 4 is that EcTLR5S participates in identification vibrio parahemolyticus flagellum in embodiment 4；
Fig. 5 is the I κ B- α phosphorylation for being overexpressed EcTLR5S in embodiment 5 and vibrio parahemolyticus flagellum being inhibited to cause；
Fig. 6 is to be overexpressed the NF- κ B that EcTLR5S inhibits vibrio parahemolyticus flagellum to cause in embodiment 6 to enter core, and A figure is
The stimulation of vibrio parahemolyticus flagellum causes NF- κ B and enters nuclear model, and B figure is to be overexpressed EcTLR5S to draw vibrio parahemolyticus flagellum
The influence of the NF- κ B transcription factor core displacement process of hair；
Fig. 7 is to inhibit vibrio parahemolyticus flagellum to mediate the inflammatory factor generated after being overexpressed EcTLR5S in embodiment 7
Transcription；
Fig. 8 is the shadow for mediating the inflammatory factor generated after striking drop EcTLR5S in embodiment 8 to vibrio parahemolyticus flagellum
It rings.
Specific embodiment
Application method of the invention is further illustrated combined with specific embodiments below.Following embodiment and attached drawing are only used for showing
Example property explanation, is not considered as limiting the invention.Unless stated otherwise, reagent raw material used in following embodiments is normal
The life reagent raw material that commercially available or commercial sources obtain is advised, unless stated otherwise, method and apparatus used in following embodiments is
Method and apparatus commonly used in the art.
The clone of 1 Epinephelus coioides TLR5S gene of embodiment
(1) extraction of Epinephelus coioides head-kidney total serum IgE
Healthy Epinephelus coioides are taken, are anaesthetized 3 minutes on ice, separating head nephridial tissue, it is total to extract it using Trizol reagent
RNA。
(2) synthesis of the first chain of cDNA
1 μ g Epinephelus coioides head-kidney total serum IgE sample is taken to carry out DNA enzymatic processing to remove the pollution of genomic DNA, with RNA
OligodT mixing, carries out reverse transcription, and products therefrom is placed in -20 DEG C and saves backup.
(3) clone of Epinephelus coioides TLR5S gene cDNA complete sequence
Design specific primer, upstream primer TLR5S-ORF-F:5`-ATGTGGACGCTGGGTCTTCA-3`；Downstream
Primer is TLR5S-ORF-R:5`-CTGCTGTGTGATGTCATCAG-3`, and the ORF clip size expanded is 1935bp, electricity
Result of swimming is as shown in Figure 1, gel extraction purpose band and purified product.Purified product is connected to pGEM-T carrier, conversion is extremely
Positive colony sequencing is selected after DH5 α Escherichia coli.
By sequencing result it is found that the nucleotide sequence of Epinephelus coioides innate immunity receptor TLR5S gene such as
Shown in SEQ.ID.NO:1, the full length cDNA sequence such as SEQ.ID.NO:3 institute of Epinephelus coioides innate immunity receptor TLR5S gene
Show, the full length cDNA sequence overall length 2168bp of gene, the 5` noncoding region including a 79bp, the 3` non-coding of a 154bp
The open reading frame of area and a 1935bp.Analysis of nucleotide sequences is it is found that Epinephelus coioides innate immunity receptor TLR5S
The amino acid sequence of the coding albumen of gene is as shown in SEQ.ID.NO:2, and encoded protein structure is by 17 LRR structural domain structures
At.
The building and its verifying of 2 carrier for expression of eukaryon pcDNA4.0-TLR5S of embodiment
According to TLR5S full length gene cDNA sequence, BamHI and KpnI double enzyme site design primer, middle and upper reaches are selected
Primer is EcTLR5S-BamHI-F:5`-CGCGGATCCGCCACCATGTGGACGCTGGGTCTTCA-3 `；Downstream primer is
EcTLR5S-KpnI-R:5`-CCGCTCGAGCTGCTGTGTGATGTCATCAG-3`.With the pGEM- containing TLR5S encoding gene
T plasmid is template, carries out PCR amplification, obtains specific amplification products size in 2000bp or so, will have restriction enzyme site
TLR5S, which is connected to, have been used in BamHI and KpnI double digestion and pcDNA4.0 carrier for expression of eukaryon after purification, is recombinantly expressed
Carrier, the gel electrophoresis result that recombinant expression carrier is verified through PCR are shown in Fig. 2, dash forward through sequence verification correct sequence, without frameshit
Become, it was demonstrated that pcDNA4.0-TLR5S vector construction is completed.
The overexpression effect of 3 recombinant plasmid pcDNA4.0-TLR5S of embodiment
By suitable 293T cell inoculation into 12 orifice plates, when cell it is long to 70%~80% when transfect, be changed to before transfection
The Opti-MEM culture solution of serum-free.It is empty that every hole transfects 1500ng recombinant plasmid pcDNA4.0-TLR5S or pcDNA4.0 respectively
White vehicle Control.Transfection is changed to the DMEM culture solution of the fetal calf serum containing 5% after 6 hours.Transfection collected cell after 36 hours,
Total protein is extracted, western blot is carried out and detects EcTLR5S expressing fusion protein, experimental result is as shown in figure 3, wherein β-
Actin is as internal reference albumen.The result shows that construction of recombinant plasmid success.
4 EcTLR5S of embodiment participates in identification vibrio parahemolyticus flagellum
By suitable GS cell inoculation into 96 orifice plates, when cell confluency degree reaches 90% or more, some cause of diseases are used
Associated molecular pattern, such as: LPS (lipopolysaccharides), PGN (peptide glycan), Poly I:C (polyinosinic acid), the parahemolyticas arc inactivated
Bacterium, vibrio parahemolyticus flagellin stimulate cell respectively, and stimulation harvests cell after 6 hours, extract total serum IgE, and reverse transcription carries out
The mRNA of fluorescence quantitative PCR detection EcTLR5S expresses variation, and wherein EF1 is as reference gene.According to the obtained CT value of detection into
The analysis of row result, utilizes 2-△△ctMethod calculate the relative expression quantity of the above EcTLR5S, experimental result such as 4 institute of attached drawing
Show.The specific primer sequence used in detection such as the following table 1:
The primer used in 1 fluorescence quantitative PCR detection EcTLR5S of table
Figure 4, it is seen that the stimulation of vibrio parahemolyticus flagellin can cause EcTLR5S mrna expression amount
Conspicuousness up-regulation, this result prompt EcTLR5S participate in immune defense process, have the ability to vibrio parahemolyticus flagellin into
Row Immune discrimination.
This result prompt EcTLR5S can participate in the Immune discrimination process to vibrio parahemolyticus flagellin.
Embodiment 5 is overexpressed the I κ B- α phosphorylation that EcTLR5S inhibits vibrio parahemolyticus flagellum to cause
By suitable GS cell inoculation into 12 orifice plates, when cell it is long to 70%~80% when transfect, will contain before transfection
The L15 culture medium of 10% fetal calf serum is changed to the Opti-MEM culture solution of serum-free.Every hole transfects 1500ng respectively
PcDNA4.0-TLR5S expression vector or the control of pcDNA4.0 empty vectors.Transfection was changed to the tire ox blood containing 5% after 6 hours
Clear L15 culture solution.Transfection 36 hours after using 1 μ g/mL vibrio parahemolyticus flagellum stimulate cell, respectively stimulation 0,20,
40, cell is collected when 60,120,180min, extracts total protein, carries out I κ B- α phosphorus in western blot detection NF- κ B access
The horizontal variation of acidification.Experimental result such as Fig. 5, wherein β-actin is used as internal reference albumen.
I κ B- α phosphorylation is the one of the important signs that of NF- κ B Pathway Activation, and shown in Fig. 5, being overexpressed EcTLR5S can inhibit
Signal of interest molecule I κ B- α phosphorylation in NF- κ B access shows that EcTLR5S gene or EcTLR5S gene coded protein can recognize
Vibrio parahemolyticus flagellum and the NF- κ B Pathway Activation for inhibiting it to be induced.
Embodiment 6 is overexpressed the NF- κ B transcription factor that EcTLR5S inhibits vibrio parahemolyticus flagellum to cause and enters core
Building vibrio parahemolyticus flagellum stimulation initiation NF- κ B first enters nuclear model, by suitable GS cell inoculation to 8 holes
In chamber slide, when cell it is long to 40%~60% when transfect, the L15 culture medium containing 10% fetal calf serum is changed to before transfection
The Opti-MEM culture solution of serum-free.Every hole transfects 200ngpcDNA4.0-EcNF- κ B-p65 expression vector respectively, and transfection 6 is small
When after be changed to the L15 culture solution of the fetal calf serum containing 5%.It is pierced after transfection 36 hours using 1 μ g/mL vibrio parahemolyticus flagellum
Swash cell, stimulates 0,30,60,100, film-making after 140min respectively.Cell is fixed with 4% paraformaldehyde, DAPI contaminates nucleus, often
Mounting after anti-quencher is added dropwise in hole, by bimolecular confocal laser scanning microscope as a result, result is as shown in A figure in Fig. 6.
Then detection is overexpressed the NF- κ B transcription factor core displacement process that EcTLR5S causes vibrio parahemolyticus flagellum
Influence, by suitable GS cell inoculation into 8 hole chamber slides, when cell it is long to 40%~60% when transfect, before transfection
L15 culture medium containing 10% fetal calf serum is changed to the Opti-MEM culture solution of serum-free.Every hole transfects 200ng pcDNA4.0-
EcNF- κ B-p65 and 200ng pcDNA4.0-TLR5S expression vector, transfection were changed to the fetal calf serum containing 5% after 6 hours
L15 culture solution.Transfection 36 hours after using 1 μ g/mL vibrio parahemolyticus flagellum stimulate cell, respectively stimulate 0,30,60,100,
Film-making after 140min.Cell is fixed with 4% paraformaldehyde, DAPI contaminates nucleus, and mounting after anti-quencher is added dropwise in every hole, by double
Molecular laser confocal microscopy as a result, result as shown in B figure in Fig. 6.
NF- κ B subunit p65 activate be into core NF- κ B Pathway Activation one of the important signs that, from Fig. 6 A as can be seen that pair
The stimulation of hemolysis vibrion flagellum can cause NF- κ B subunit p65 and activate into core.Fig. 6 B significantly presses down after can be seen that overexpression EcTLR5S
It has made the NF- κ B subunit p65 that vibrio parahemolyticus flagellum is caused to activate into core, has shown EcTLR5S gene or EcTLR5S gene
The NF- κ B Pathway Activation that coding albumen can recognize vibrio parahemolyticus flagellum and it is inhibited to be induced.
Embodiment 7 is overexpressed the transcription for inhibiting vibrio parahemolyticus flagellum to mediate the inflammatory factor generated after EcTLR5S
Suitable GS cell inoculation is incubated overnight into 96 orifice plates, when cell it is long to 70%~80% when transfect, transfect
The preceding Opti-MEM culture solution that the L15 culture medium containing 10% fetal calf serum is changed into serum-free.Every hole transfects 300ng
PcDNA4.0-TLR5S expression vector or the control of pcDNA4.0 empty vectors.Transfection was changed to the tire ox blood containing 5% after 6 hours
Clear L15 culture solution.Cell is stimulated using 1 μ g/mL vibrio parahemolyticus flagellum after transfection 36 hours, after stimulation 6 hours, harvest
Cell, extracts total serum IgE, and reverse transcription carries out the mRNA of fluorescence quantitative PCR detection immune factor IFN-γ 2, IL-6 and TNF-α
Level, wherein EF1 is as reference gene.The analysis that result is carried out according to the CT value that detection obtains, utilizes 2-△△ctMethod calculate
The relative expression quantity of the above immune factor, experimental result are as shown in Fig. 7 out.The specific primer sequence used in detection is as follows
Table 2:
The primer sequence used in 2 fluorescence quantitative PCR detection immune factor IFN-γ 2 of table, IL-6 and TNF-α
Inhibit under vibrio parahemolyticus flagellum Induced by Stimulation it can be seen from figure 7 that being overexpressed conspicuousness after EcTLR5S
Swim the expression of inflammatory factor mRNA.
Embodiment 8 strikes the influence of the inflammatory factor generated after drop EcTLR5S to the mediation of vibrio parahemolyticus flagellum
Suitable GS cell inoculation is incubated overnight into 96 orifice plates, when cell it is long to 70%~80% when transfect, transfect
The preceding Opti-MEM culture solution that the L15 culture medium containing 10% fetal calf serum is changed into serum-free.Used siRNA sequence is,
EcTLR5S-siRNA1 (sequence: sense:5`-CCAGCCUUCAAUGUUCUUUTT-3`；Antisense:5`-
AAAGAACAUUGAAGGCUGGTT-3`) EcTLR5S-siRNA2 (sequence: sense:5`-CCUUCUGUUGAGUGACAAUTT-3
`；Antisense:5`-AUUGUCACUCAACAGAAGGTT-3`) and Negative Control sequence: (sequence: sense:5
`-UUCUCCGAACGUGUCACGUTT-3`；Antisense:5 '-AUGGAGACAGC UGAUAGGCTT-3 '), diluted concentration is
20nM.When transfection, EcTLR5S-siRNA1 and EcTLR5S-siRNA2 is mixed and made into EcTLR5S-siRNA- with 1:1 ratio
mix.Transfection liquid are as follows: A liquid (0.3 μ L siRNA is added in 5 μ L of Opti-MEM culture solution, mixes), B liquid (5 μ of Opti-MEM culture solution
0.4 μ L lipo3000 is added in L, mixes, is placed at room temperature for 5min)；A liquid and B liquid are mixed gently as transfection liquid, are placed at room temperature for
15min is added in corresponding transfection hole, is incubated for 6 hours；Finally it is changed to the L15 culture solution of the fetal calf serum containing 5%.Transfection
After 36 hours, vibrio parahemolyticus flagellin, which is added, to be stimulated, and stimulating the concentration of flagellum used is 1 μ g/mL, in equal volume
PBS as control.After stimulating 6h, cell is harvested, extracts total serum IgE, reverse transcription carries out fluorescence quantitative PCR detection detection and respectively exempts from
Epidemic disease factor expression amount, experimental results are shown in figure 8.
As can be seen from Figure 8, can be increased after striking drop EcTLR5S with conspicuousness under the stimulation mediation of vibrio parahemolyticus flagellum
Swim the expression of inflammatory factor mRNA.The result shows that EcTLR5S gene or EcTLR5S gene coded protein can recognize parahemolyticas
Vibrios flagellum simultaneously adjusts inflammatory factor expression.
The present invention is not limited within the scope of above-mentioned specific embodiment, and the embodiment above is used for the purpose of can be to this
The use process of invention is described in detail, and has the production method of equal function and technical detail to also belong in the present invention
A part of appearance.In fact, those skilled in the art are according to description above, it will be able to according to respectively needing to find different tune
Perfect square case, these adjustment all should be in the scope of the claims by the appended claims herein.
Sequence table
<110>Zhongshan University
<120>new application of Epinephelus coioides innate immunity receptor TLR5S gene and its coding albumen
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1935
<212> DNA
<213>Epinephelus coioides (Epinephelus coioides)
<400> 1
atgtggacgc tgggtcttca ggtggctgtc atctgtgttt tcctacaggt gccaggttgt 60
ttcccatcat gcctcatcgt gaactctgta gccaactgtg cctacaagaa cctccgctca 120
gttcctcccc tccctcctca catcacccac ctgtacctgg aggggaaccg catcagtgag 180
atcaactcca actccctgtc gggcctgaag gagctgcagg agctggacct tggagggcag 240
tttgtgcgtc tcatgatcag aaacaatgcc ttcagggagc aaagacacct gaggaggctg 300
gtgctcggtg gcaacgtcca ccttcagctg gagccgcagg cttttgtggg actgtccagt 360
ttgcagaatc tctacttata tcactgttcg ctccaacagt ctatactgga ggaggactac 420
ctggagccac tcacctcttt agagactctt gacctctttg gtaacaagat aaagagactc 480
cagccttcaa tgttctttgc aaacatgact aatttgaaaa ttctgaatct caaactgaat 540
gaaattgaca aaatatgtga gtctgatctg gtaggttttc agggaaagca ctttgaggtt 600
ctgaatttag actctgttta ccttaagacc atgtttaaaa aacgcgaatg gcagaaatgt 660
gggaatcctt tcagggggat gtcctttcag acactcgacc tgtccaacaa cgggttaagc 720
gtgagtaaat cgaaacagtt gttcacagcc attaggggga ccaagatttc caatctcaaa 780
ctgtcactgg gactcatggg taaaggattt tcattcaata tttaccctga tccagacagc 840
agcatgtttg aaagcctgaa tgacagttca gtccacactc tggatctgtc taaaaacaag 900
atatttgcac tccaagaggg ggttttcagt gcactaaaag aagttgcaat cattgatgtt 960
tcccaaaaca atgtgaatca gatacacaga aatgcctttg aaggccttca gggacattta 1020
caaatgctca acctgtcaca caacctgctg ggggaaatcc attctgacac ttttgcttct 1080
ctgacaaacc tgcaggtgtt ggacttgtct tacaatcaca ttggtgttct gggtcatgac 1140
tcatttagtg aacttcccaa attaaaagta ttaaatctaa caggaaactc tctgcgagac 1200
attggcttcc ctgccttact tccgagttta gattaccttc tgttgagtga caataaactg 1260
atagactcgc cggggagaag tatccatagg tttgctgggg atattttgca tctggacatt 1320
cgggataaca gattaacaaa cctggggggt gtttataccc ttgtgactct gctggaccgc 1380
cttcagcatc tctcttatgg aggaaaccca atcaagtggt gcaatctcgg cagagaagtc 1440
aggttcattg attcgaataa tgtcacaacc ctggatcttc acggcagctc cctgcagtct 1500
gtctggtctc agtggacatg tctggatctg tttgacaatt tcggacatgt gattactctg 1560
aacttgagcc acaacgcgct gcagtctctt cctaggggca ttttcaaggg tctcacctca 1620
gttgtggaga tggacctctc atccaacagc ttgacttatc tccagcctga tgtattgccc 1680
aaaagtctca aagtactcga cctctccaac aacttcatag cctcccctga ccctgatgct 1740
tttcgctctc tcagctccct tgacctgaac atgaaccgat ttcactgtga tgcaaacctg 1800
aagagttttc tgaattgggt aaccaacacc accgtgacac tcctgacacc tgtcaaggag 1860
ctcaaatgtg aatttccatc tgatttctat aatgttcctc tgttacgatt ctctgatgac 1920
atcacacagc agtaa 1935
<210> 3
<211> 644
<212> PRT
<213>Epinephelus coioides (Epinephelus coioides)
<400> 3
Met Trp Thr Leu Gly Leu Gln Val Ala Val Ile Cys Val Phe Leu Gln
1 5 10 15
Val Pro Gly Cys Phe Pro Ser Cys Leu Ile Val Asn Ser Val Ala Asn
20 25 30
Cys Ala Tyr Lys Asn Leu Arg Ser Val Pro Pro Leu Pro Pro His Ile
35 40 45
Thr His Leu Tyr Leu Glu Gly Asn Arg Ile Ser Glu Ile Asn Ser Asn
50 55 60
Ser Leu Ser Gly Leu Lys Glu Leu Gln Glu Leu Asp Leu Gly Gly Gln
65 70 75 80
Phe Val Arg Leu Met Ile Arg Asn Asn Ala Phe Arg Glu Gln Arg His
85 90 95
Leu Arg Arg Leu Val Leu Gly Gly Asn Val His Leu Gln Leu Glu Pro
100 105 110
Gln Ala Phe Val Gly Leu Ser Ser Leu Gln Asn Leu Tyr Leu Tyr His
115 120 125
Cys Ser Leu Gln Gln Ser Ile Leu Glu Glu Asp Tyr Leu Glu Pro Leu
130 135 140
Thr Ser Leu Glu Thr Leu Asp Leu Phe Gly Asn Lys Ile Lys Arg Leu
145 150 155 160
Gln Pro Ser Met Phe Phe Ala Asn Met Thr Asn Leu Lys Ile Leu Asn
165 170 175
Leu Lys Leu Asn Glu Ile Asp Lys Ile Cys Glu Ser Asp Leu Val Gly
180 185 190
Phe Gln Gly Lys His Phe Glu Val Leu Asn Leu Asp Ser Val Tyr Leu
195 200 205
Lys Thr Met Phe Lys Lys Arg Glu Trp Gln Lys Cys Gly Asn Pro Phe
210 215 220
Arg Gly Met Ser Phe Gln Thr Leu Asp Leu Ser Asn Asn Gly Leu Ser
225 230 235 240
Val Ser Lys Ser Lys Gln Leu Phe Thr Ala Ile Arg Gly Thr Lys Ile
245 250 255
Ser Asn Leu Lys Leu Ser Leu Gly Leu Met Gly Lys Gly Phe Ser Phe
260 265 270
Asn Ile Tyr Pro Asp Pro Asp Ser Ser Met Phe Glu Ser Leu Asn Asp
275 280 285
Ser Ser Val His Thr Leu Asp Leu Ser Lys Asn Lys Ile Phe Ala Leu
290 295 300
Gln Glu Gly Val Phe Ser Ala Leu Lys Glu Val Ala Ile Ile Asp Val
305 310 315 320
Ser Gln Asn Asn Val Asn Gln Ile His Arg Asn Ala Phe Glu Gly Leu
325 330 335
Gln Gly His Leu Gln Met Leu Asn Leu Ser His Asn Leu Leu Gly Glu
340 345 350
Ile His Ser Asp Thr Phe Ala Ser Leu Thr Asn Leu Gln Val Leu Asp
355 360 365
Leu Ser Tyr Asn His Ile Gly Val Leu Gly His Asp Ser Phe Ser Glu
370 375 380
Leu Pro Lys Leu Lys Val Leu Asn Leu Thr Gly Asn Ser Leu Arg Asp
385 390 395 400
Ile Gly Phe Pro Ala Leu Leu Pro Ser Leu Asp Tyr Leu Leu Leu Ser
405 410 415
Asp Asn Lys Leu Ile Asp Ser Pro Gly Arg Ser Ile His Arg Phe Ala
420 425 430
Gly Asp Ile Leu His Leu Asp Ile Arg Asp Asn Arg Leu Thr Asn Leu
435 440 445
Gly Gly Val Tyr Thr Leu Val Thr Leu Leu Asp Arg Leu Gln His Leu
450 455 460
Ser Tyr Gly Gly Asn Pro Ile Lys Trp Cys Asn Leu Gly Arg Glu Val
465 470 475 480
Arg Phe Ile Asp Ser Asn Asn Val Thr Thr Leu Asp Leu His Gly Ser
485 490 495
Ser Leu Gln Ser Val Trp Ser Gln Trp Thr Cys Leu Asp Leu Phe Asp
500 505 510
Asn Phe Gly His Val Ile Thr Leu Asn Leu Ser His Asn Ala Leu Gln
515 520 525
Ser Leu Pro Arg Gly Ile Phe Lys Gly Leu Thr Ser Val Val Glu Met
530 535 540
Asp Leu Ser Ser Asn Ser Leu Thr Tyr Leu Gln Pro Asp Val Leu Pro
545 550 555 560
Lys Ser Leu Lys Val Leu Asp Leu Ser Asn Asn Phe Ile Ala Ser Pro
565 570 575
Asp Pro Asp Ala Phe Arg Ser Leu Ser Ser Leu Asp Leu Asn Met Asn
580 585 590
Arg Phe His Cys Asp Ala Asn Leu Lys Ser Phe Leu Asn Trp Val Thr
595 600 605
Asn Thr Thr Val Thr Leu Leu Thr Pro Val Lys Glu Leu Lys Cys Glu
610 615 620
Phe Pro Ser Asp Phe Tyr Asn Val Pro Leu Leu Arg Phe Ser Asp Asp
625 630 635 640
Ile Thr Gln Gln
<210> 3
<211> 2168
<212> DNA
<213>Epinephelus coioides (Epinephelus coioides)
<400> 3
acgcgggggg ggagaacaga gctctgatcc tggagtgttg tttgacctca tatcattgag 60
ggtttccctg gtgagcaaga tgtggacgct gggtcttcag gtggctgtca tctgtgtttt 120
cctacaggtg ccaggttgtt tcccatcatg cctcatcgtg aactctgtag ccaactgtgc 180
ctacaagaac ctccgctcag ttcctcccct ccctcctcac atcacccacc tgtacctgga 240
ggggaaccgc atcagtgaga tcaactccaa ctccctgtcg ggcctgaagg agctgcagga 300
gctggacctt ggagggcagt ttgtgcgtct catgatcaga aacaatgcct tcagggagca 360
aagacacctg aggaggctgg tgctcggtgg caacgtccac cttcagctgg agccgcaggc 420
ttttgtggga ctgtccagtt tgcagaatct ctacttatat cactgttcgc tccaacagtc 480
tatactggag gaggactacc tggagccact cacctcttta gagactcttg acctctttgg 540
taacaagata aagagactcc agccttcaat gttctttgca aacatgacta atttgaaaat 600
tctgaatctc aaactgaatg aaattgacaa aatatgtgag tctgatctgg taggttttca 660
gggaaagcac tttgaggttc tgaatttaga ctctgtttac cttaagacca tgtttaaaaa 720
acgcgaatgg cagaaatgtg ggaatccttt cagggggatg tcctttcaga cactcgacct 780
gtccaacaac gggttaagcg tgagtaaatc gaaacagttg ttcacagcca ttagggggac 840
caagatttcc aatctcaaac tgtcactggg actcatgggt aaaggatttt cattcaatat 900
ttaccctgat ccagacagca gcatgtttga aagcctgaat gacagttcag tccacactct 960
ggatctgtct aaaaacaaga tatttgcact ccaagagggg gttttcagtg cactaaaaga 1020
agttgcaatc attgatgttt cccaaaacaa tgtgaatcag atacacagaa atgcctttga 1080
aggccttcag ggacatttac aaatgctcaa cctgtcacac aacctgctgg gggaaatcca 1140
ttctgacact tttgcttctc tgacaaacct gcaggtgttg gacttgtctt acaatcacat 1200
tggtgttctg ggtcatgact catttagtga acttcccaaa ttaaaagtat taaatctaac 1260
aggaaactct ctgcgagaca ttggcttccc tgccttactt ccgagtttag attaccttct 1320
gttgagtgac aataaactga tagactcgcc ggggagaagt atccataggt ttgctgggga 1380
tattttgcat ctggacattc gggataacag attaacaaac ctggggggtg tttataccct 1440
tgtgactctg ctggaccgcc ttcagcatct ctcttatgga ggaaacccaa tcaagtggtg 1500
caatctcggc agagaagtca ggttcattga ttcgaataat gtcacaaccc tggatcttca 1560
cggcagctcc ctgcagtctg tctggtctca gtggacatgt ctggatctgt ttgacaattt 1620
cggacatgtg attactctga acttgagcca caacgcgctg cagtctcttc ctaggggcat 1680
tttcaagggt ctcacctcag ttgtggagat ggacctctca tccaacagct tgacttatct 1740
ccagcctgat gtattgccca aaagtctcaa agtactcgac ctctccaaca acttcatagc 1800
ctcccctgac cctgatgctt ttcgctctct cagctccctt gacctgaaca tgaaccgatt 1860
tcactgtgat gcaaacctga agagttttct gaattgggta accaacacca ccgtgacact 1920
cctgacacct gtcaaggagc tcaaatgtga atttccatct gatttctata atgttcctct 1980
gttacgattc tctgatgaca tcacacagca gtaaagtagc aactaaaagc caacacataa 2040
cggataatgt acagcgagtg ggccattatt gagaaataaa cctgcagagg ggtcttgaat 2100
caccatttct gtacataatc ccacttgtta catggccaac taatcaagtc aataatttga 2160
cacaaaat 2168
Claims (4)
1. Epinephelus coioides innate immunity receptor TLR5S gene and its coding albumen are believed in identification bacterial flagellum, regulating cell
Number access and adjust the application in inflammatory factor.
2. application according to claim 1, it is characterized in that: the bacterium is vibrio parahemolyticus.
3. application according to claim 1 or 2, it is characterized in that: the Epinephelus coioides innate immunity receptor TLR5S gene
Nucleotide sequence as shown in SEQ.ID.NO:1.
4. application according to claim 3, it is characterized in that: the Epinephelus coioides innate immunity receptor TLR5S gene is compiled
The amino acid sequence of code albumen is as shown in SEQ.ID.NO:2.
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Cited By (2)
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| CN109943651A (en) * | 2019-02-28 | 2019-06-28 | 中山大学 | Epinephelus coioides innate immunity receptor TLR5M gene and its new application for encoding albumen |
| CN112007143A (en) * | 2020-08-19 | 2020-12-01 | 中山大学 | Application of recombinant vibrio parahaemolyticus flagellin in improving immunity of fish |
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| CN112007143B (en) * | 2020-08-19 | 2023-09-22 | 中山大学 | Application of recombinant vibrio parahaemolyticus flagellin in improving fish immunity |
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