CN109810992A

CN109810992A - It is a kind of encode lefteye flounder Extracellular ATP hydrolase CD39 cDNA full length sequence and its application

Info

Publication number: CN109810992A
Application number: CN201910101788.8A
Authority: CN
Inventors: 李硕; 孙金生; 陈晓丽; 李佳芳
Original assignee: Tianjin Normal University
Current assignee: Tianjin University; Tianjin Normal University
Priority date: 2019-02-01
Filing date: 2019-02-01
Publication date: 2019-05-28

Abstract

The invention discloses the detection method of lefteye flounder Extracellular ATP hydrolase CD39 gene and its to the adjustment effect of the fish innate immunity.The present invention is by finally obtaining the full length cDNA sequence of lefteye flounder CD39 in conjunction with RACE technology through reverse transcription and PCR amplification with the conserved regions design degenerate primer of different plant species CD39 coding sequence.Lefteye flounder CD39 albumen is a kind of important Extracellular ATP hydrolase, by control effect of extracellular ATP concentration, plays important regulative to the innate immunity that fish Extracellular ATP mediates.This research provides new evidence for the Study on Molecular Mechanism that the fish innate immunity regulates and controls, and also can establish important foundation to research and develop for the immune function controlling agents of fish CD39 albumen.

Description

It is a kind of encode lefteye flounder Extracellular ATP hydrolase CD39 cDNA full length sequence and its application

Technical field

The present invention relates to field of biology, specifically lefteye flounder Extracellular ATP hydrolase CD39 full length gene cDNA sequence and its The clone and application of the albumen of coding and the gene.

Background technique

In recent years, with the increasing living standards of the people, fish as a kind of high protein and low fat food by The more and more pro-gazes of people.Since aquaculture scale worldwide constantly expands, domestic and international aquatic products trade and water The increasingly frequent of the trans-regional exchange of seed is produced, the chance of aquiculture animal transmission of pathogen greatly increases.Further, since modern water Produce aquaculture height is intensive and high density production feature, lead to fisheries water environmental degradation and water body bacterium mass propagation, Cause cultivated animals stress reaction, body immune system is caused to be suppressed.The interaction of above-mentioned internal and external factor, leads to the world Aquaculture creature disease outburst tends to frequently in range, and Epidemic Scope tends to extensively, to bring greatly to aquaculture Economic loss.By the technical support with modern biology technology as aquaculture, economic animal disease is immunized Prevention and treatment is the important means of aquatic animal disease prevention and treatment.Therefore, fish immunology research in recent years receives more next in the world More attention.

The immune system of fish and mammal has certain conservative.In order to adapt to water body environment, fish develop out Itself distinctive immunological characteristic has the innate immunity and Acquired immune response two systems, but its acquired immune system is simultaneously Not perfect, therefore, the prior effect that plays is immunized in fish immunity system in natural (congenital).

Studies have shown that cell can under the conditions of mechanical damage, immunostimulation, meronecrosis, Apoptosis and Infected with Pathogenic Fungi etc. It is by way of exocytosis, ion channel that ATP is extracellular from being promptly released into the cell.Extracellular ATP is that one kind can activate place The potent proinflammatory disease signaling molecule of main innate immunity is activated by acting on cell membrane surface P2 purinoceptor or ATP Ion channel, trigger different downstream signal (such as Ca²⁺Signal, cyclic adenosine monophosphate signal and protein kinase signal etc.), it is formed fast Purine immune signal access, the innate immunity regulation of wide participation body.However, excessively high Extracellular ATP concentration can lure Guided cell generates excessive innate immune reaction, has an adverse effect to body.Therefore, the concentration of Extracellular ATP must be by tight Lattice control.By Extracellular nucleotidase hydrolyze Extracellular ATP, thus reduce Extracellular ATP using concentration, by the rush of Extracellular ATP Inflammatory effector maintains appropriate level, is the important channel for maintaining immunity of organism stable state.

Extracellular two triphosphopyridine nucleotides hydrolase I(ecto-nucleoside triphosphate Diphosphohydrolase, e-NTPDase), also known as CD39 is that one kind is distributed widely in the eucaryotes such as plant, animal In, molecular weight is about 70 ~ 100kD, the glycoprotein that is positioned on cell membrane.CD39 is the important composition of purine immune signal access Member, main function are hydrolysising ATP and ADP, form AMP, and the latter is hydrolyzed to adenosine by 5' Extracellular nucleotidase (CD73) again, And then anti-inflammation effect is converted by the rush inflammatory effector of Extracellular ATP, to maintain body appropriateness, effective immune response. CD39 Important regulative is risen to body innate immunity.Studies have shown that the adjustable Extracellular ATP receptor P2X7 mediation of CD39 is thin The release of born of the same parents' apoptosis and pro-inflammatory cytokine IL-1 β and IL-8；The Extracellular ATP that CD39 can also discharge TLR receptor-inducible It is hydrolyzed to adenosine rapidly, and then weakens the activation degree of macrophage；In addition, being overexpressed CD39 facilitates airway epithelial cell Pseudomonas aeruginosa is resisted to infect.

Lefteye flounder (Paralichthys olivaceus) belong to dish-shaped mesh, lefteye flounder section, Paralichthys, it is commonly called as " flatfish ", " a left side Mouthful ", it is the main marine fish in China and East Asia Region, economic value with higher.As cultivation is advised in recent years The continuous expansion of mould, epidemic disease caused by the pathogen such as virus, bacterium broken out under the conditions of intensive culture it is increased, Serious economic loss is caused to lefteye flounder aquaculture, seriously constrains the sustainable health development of lefteye flounder aquaculture.Currently, by It is less in understanding for lefteye flounder immunoregulatory molecules mechanism, cause the exploitation of related immune Prevention Technique with application by serious It restricts.

Effect of extracellular ATP is a kind of endogenous signaling molecule important, with potent immunosuppressant stimulatory function, in activation fish It plays an important role during innate immunity.Although the immune response of appropriateness removes cause of disease and maintains body stable state tool It plays an important role, but excessive innate immunity even results in individual death it will cause body injury, necrosis.Therefore, divide There is the functional gene for adjusting fish innate immunity intensity from identification, help to go deep into understanding and control Extracellular ATP is in fish Effect in the class innate immunity.

Extracellular two research of nucleoside triphosphate hydrolase 1(CD39) be Extracellular ATP immune signal circuit upstream key regulatory genes, There is close relationship with the intracorporal many inflammatory reactions of machine and immune response, can reflect the immune of fish to a certain extent Ability.The separation identification extracellular CD39 gene of lefteye flounder helps to go deep into parsing CD39 Structure and Function and its exempt from fish The association of epidemic disease response deepens that people are fine to fish and the full appreciation of complicated immune response process, thus for find diagnosis, It prevents and treats fish epidemic disease and new strategy is provided, and be the design of fish molecule breeding for disease resistance, disease resistant transgenic fish Design, fish gene vaccine design etc. important theoretical basis is provided.

Summary of the invention

The object of the present invention is to provide the cDNA sequences and cloning process of a kind of lefteye flounder Extracellular nucleotidase CD39.The lefteye flounder Extracellular ATP hydrolase CD39 gene cDNA sequence, gene cDNA overall length 1685bp, the open reading frame containing 1494bp have Nucleotide sequence shown in SEQ ID NO.1；It encodes 497 amino acid, has amino acid sequence shown in SEQ ID NO.2 Column.The invention also discloses the specific primer for detecting lefteye flounder Extracellular ATP hydrolase CD39 gene cDNA sequence, features It is:

Forward primer: 5 '-CCTTCCGACTCTACGGTAATGAC-3 ' SEQ ID NO.3

Reverse primer: 5 '-GGATCTGATATGGATACTGGACCT-3 ' SEQ ID NO.4.

More detailed description of the present invention is as follows:

The technical solution adopted by the present invention is that by designing degenerate primer with the sequence conservation CDS of different plant species CD39 gene, By RT-PCR reverse transcription and PCR amplification, the total length expressed sequence of lefteye flounder CD39 gene cDNA is obtained in conjunction with RACE technology；Lefteye flounder The total 1685bp of CD39 full length gene cDNA, including open reading frame are 1494bp, encode 497 amino acid altogether, contain 114bp's 5' non-translational region, 3' non-translational region and poly (A) tail of 77bp.These gene structure features can illustrate resulting cDNA sequence It is classified as the full length cDNA sequence of lefteye flounder CD39 gene.The protein molecular weight of lefteye flounder CD39 gene coding is 55.3KDa, isoelectric point It is 8.58, there is the sequence similarity of 82%-99% with the CD39 albumen of other species.

The present invention further discloses lefteye flounder Extracellular nucleotidase CD39 gene orders as lefteye flounder disease prevention and cure target Application in terms of gene, and in the application as lefteye flounder disease real-time fluorescence RT-PCR context of detection, there are also it to lefteye flounder day Application in terms of right immunoregulation.

The present invention further discloses lefteye flounder Extracellular nucleotidase CD39 gene cDNA sequence cloning process, it is characterized in that Including following key step:

(1) healthy Paralichthys olivaceus anatomical isolation head-kidney tissue:

Healthy Paralichthys olivaceus is chosen, is put to death after temporarily being supported 3 days in aquarium, anatomical isolation head-kidney tissue.

(2) extraction and purifying of total serum IgE:

It is extracted from lefteye flounder head-kidney using Trizol method and obtains lefteye flounder head-kidney total serum IgE.Total serum IgE purifying uses DNA enzymatic facture.

(3) cloning and sequencing of lefteye flounder CD39 gene cDNA intermediate segment:

(3.1) synthesis of the first chain of cDNA

Using the lefteye flounder head-kidney total serum IgE of purifying as template, with Oligo (dT)₁₆As reverse transcription primer, the first chain of cDNA is carried out Synthesis, obtains the first chain of cDNA, the cDNA template as following PCR amplifications.

(3.2) PCR amplification

It is carried out amplification reaction using nested PCR method.Using aforementioned resulting the first chain of cDNA as template, following the first primers are utilized Carry out PCR amplification:

Forward primer: 5 '-GSGATTGTYRYCHTGGTRWC-3 '

Reverse primer: 5 '-AARAYMCCRTTGAADGARCA-3 '

Amplification obtains lefteye flounder Extracellular nucleotidase CD39 gene cDNA intermediate segment and obtains intermediate segment sequence by cloning and sequencing Column.

In this step, amplification system is preferably as follows:

Amplification program parameter is preferable are as follows:

94℃ 4min；

94 DEG C of 30sec, 52 DEG C of 30sec, 72 DEG C of 1min(30 circulations)；

72℃ 5min；12 DEG C of heat preservations.

(4) cloning and sequencing at the end lefteye flounder CD39 gene cDNA 3':

According to the intermediate segment that above-mentioned steps (3) obtain, design and synthesize for the forward primer of the end 3' nest-type PRC and general Property reverse primer AP, is respectively as follows:

Forward primer: CD39-3'-F1:5'- CCAGGGAGCTCCTGCCACCT- 3'

Versatility reverse primer AP:5'- TCGAATTCGGATCCGAGCTC- 3'

Using the lefteye flounder head-kidney total serum IgE of abovementioned steps (3) purifying as template, with Oligo (dT)₁₆AP:5'- TCGAATTCGGATCCGAGCTCV(T)₁₆- 3' is reverse transcription primer, carries out the synthesis of the first chain of cDNA, obtained cDNA first Chain, the cDNA template as the end 3' nested PCR amplification.With CD39-3'-F1 and AP be positive reverse primer carry out PCR amplification (amplification System and program parameter are expanded with intermediate segment).

(5) end lefteye flounder CD39 gene cDNA 5' cloning and sequencing:

(5.1) first chain of cDNA at the end 5' is synthesized

Using the 5' reverse transcription primer carried in the 5'RACE kit of CloneTech company, reverse transcription is carried out.Reverse transcription product Remnants RNA is digested using RNase H, obtains the first chain of cDNA.

(5.2) PCR amplification

It is used for according to the intermediate segment sequence that above-mentioned steps (3) obtain using 5 software design specific reverse primers of Primer The end 5' nest-type PRC.

CD39-5 '-R1:5'- CTGCCAGTGTTGTTATCTTT -3'

First round PCR amplification is carried out by primer of general forward primer UPM and specific reverse primers CD39-5'-R1 first；So It is to draw with versatility forward primer NUP and specific reverse primers CD39-5'-R1 afterwards using first round pcr amplification product as template Object carries out the second wheel PCR amplification (amplification system is expanded with intermediate segment).

Amplification program parameter is preferable are as follows:

94℃ 4min；

94 DEG C of 30sec, 65 DEG C of 1min, 72 DEG C of 2min(30 circulations)；

72℃ 5min；12 DEG C of heat preservations.

(6) by 5' obtained by 3' terminal sequence obtained by intermediate segment sequence, step (4) obtained by above-mentioned steps (3) and step (5) Terminal sequence is spliced with software, the full length cDNA sequence of lefteye flounder CD39 gene is obtained, as described in SEQ ID No.1, and by base Because sequence derives corresponding amino acid sequence, as described in SEQ ID No.2.

The lefteye flounder CD39 full length gene total 1685bp of cDNA, including open reading frame are 1494bp, encode 497 amino altogether Acid, the 5' non-translational region containing 114bp, 3' non-translational region and poly (A) tail of 77bp.These gene structure features it may be said that Bright resulting cDNA sequence is the full length cDNA sequence of lefteye flounder CD39 gene.Lefteye flounder CD39 gene coding protein molecular weight be 55.3KDa, isoelectric point 8.58 have the sequence similarity of 82%-99% with the CD39 albumen of other species.

Amino acid sequence is as follows:

M S A Q R E M K E K N P W H T P V T I I F T V I G V I A I V A L V T V A V L Q N R P L P Q K Y K Y G I V L D A G S S H T A L Y I Y E W P A E K D N N T G R V E Q T H S C K V K G P G I S S Y A S V P R K A G E S L S E C M Q E A R Q R V P E K R H S E T P L Y L G A T A G M R L L N L E N S L A S D K V F Q A V E E A L Q K F P F S F Q G A R I L S G Q E E G A F G W V T V N Y L D D R L K Q S L E T R G A L D L G G A S T Q I S F V S D D F D G S E S P D N G V A F R L Y G N D Y N L Y T H S F L C Y G K D Q A L R M T L A K Q T Q S G P V S I S D P C F N P G Y I E T K N Y S I V Y D S P C V S N M K P Q G A P A T F T H T G K G N F S Q C Q E V V K R N F N F K Q C K Y S Q C S F N G V F Q P R L Q G P F G A F S A Y Y F V M N F L N L A D T S I P L E A V T E K L S R Y C A T P W N Q I K Q Q H P G V K L K Y L A E Y C F S G T Y I I T L L T E G Y N F T S Q N Y P N I K F I K K I K G S D A G W T L G Y M L N L T N M I P A E A P D S P P L P H A G Y V S I V T V I A I L L F V L F I L S L R P L W P R C S K Q P Q I I

Beneficial effect possessed by the cDNA sequence and cloning process of lefteye flounder Extracellular ATP hydrolase CD39 disclosed by the invention is:

(1) present invention is a kind of new CD39 gene order from the gene order of lefteye flounder Extracellular ATP hydrolase CD39, in fish In rare research, so that people is extended to the fish such as lefteye flounder from mammal to the research object of CD39 gene.

(2) acquisition of Ben Jiyin is not only Regulation Mechanism and CD39 albumen and the fish of researching fish CD39 gene expression The relationship of premunition lays the foundation, and by gene order can the synthesis of artificial synthesized or genetically modified organism there is bioactivity Pure protein, for further research CD39 protein structure and immunologic function based theoretical.This gene can also be fish Population Genetics and evolutionary genetics research provide molecular level material, while can carry out disease-resistant correlation and disease-resistant auxiliary The research of breeding.

(3) CD39 is a kind of important extracellular nucleotide hydrolysis enzyme, directly takes part in the innate immunity mediated to Extracellular ATP The regulation of response.Under inflammation pathological state, the expression of CD39 is raised, and influences to participate in the inflammatory of the body innate immunity The synthesis and release of medium and cell factor participate in the immune response and inflammatory reaction of body.CD39 participates in fish immunity and adjusts, The immunocompetence of fish is reflected to a certain extent, and therefore, the expression of CD39 gene can be used to refer to exempting from for fish Epidemic disease power can evaluate the activation degree of fish immunity system, by detecting the mRNA abundance of CD39 as fish disease The index of immune detection during disease prevention and treatment, and play a role as lefteye flounder disease prevention and cure target gene etc..

Detailed description of the invention

Fig. 1 is lefteye flounder CD39 gene intermediate segment electrophoresis detection result: wherein Maker band is from below to up successively are as follows: 2000、1000、750、500bp;

Fig. 2 is that lefteye flounder CD39 gene 3 ' holds electrophoresis detection result: wherein Marker band is from below to up successively are as follows: 1kb, 750, 500bp；

Fig. 3 be lefteye flounder CD39 gene 5 ' end electrophoresis detection result: wherein Marker band be followed successively by from below to up 1kb, 750, 500,250,100bp；

Fig. 4 is lefteye flounder CD39 gene real-time fluorescence RT-PCR product electrophoresis result: lefteye flounder CD39 product length 127bp；Wherein Maker band is from below to up successively are as follows: 100,250,500,750,1000,2000bp；

Fig. 5 is lefteye flounder internal reference gene β-actin real-time fluorescence RT-PCR product electrophoresis result:β- actin product length 150bp；Wherein Maker band is from below to up successively are as follows: 100,250,500,750,1000,2000bp；

Fig. 6 bacteria lipopolysaccharide stimulation lefteye flounder renal cell can make lefteye flounder CD39 expression significant up-regulation rapidly；

The infection of Fig. 7 tarda can be such that lefteye flounder CD39 gene expression significantly raises rapidly；

Fig. 8 inhibits CD39 enzymatic activity that can reduce the lefteye flounder pro-inflammatory cytokine IL-1 that Extracellular ATP inducesβGene expression；

The lefteye flounder cell activity oxygen that Fig. 9 inhibits CD39 enzymatic activity that can reduce Extracellular ATP induction generates.

Specific embodiment

Illustrate the present invention below with reference to embodiment, the scheme of embodiment described here does not limit the present invention, this field it is special Industry personnel spirit according to the invention can make improvements and change, and the such modifications and variations are regarded as this hair In bright range, the scope of the present invention and essence all have the right to require to limit；Wherein the reagent is by commercially available.

Embodiment 1

The separation of lefteye flounder head-kidney tissue:

It is temporarily supported three days for 25 DEG C in the healthy Paralichthys olivaceus (20g or so) bought in the market and aquarium, in dissecting and separate on super-clean bench Head-kidney tissue.Above step is all sterile working.

Embodiment 2

Total RNAs extraction and purifying:

(1) lefteye flounder head-kidney tissue 100mg is taken, is added in homogenizer, and 1000 μ LTrizol reagents are added thereto (Invitrogen company) thoroughly grinds.If it is cell, then adds 1000 μ L Trizol in culture hole, blow and beat repeatedly, with Lytic cell.1.5mL is transferred it to without in the centrifuge tube of RNase；

(2) 4 DEG C, 12000 × g centrifugation 10min；

(3) supernatant is taken, is added in new centrifuge tube, static a few minutes, cracks nucleoprotein completely；

(4) 200 μ L chloroforms are added into supernatant, after acutely shaking 15 s, the static 2-3min of room temperature；

(5) 4 DEG C, 12000 × g, centrifugation 15min.Product is divided into three layers after centrifugation: lower layer is mutually mainly protein etc., interphase Mainly DNA, upper layer are mutually RNA；

(6) upper layer phase part (rather few not more, not encounter interphase and lower layer's phase) is extracted in a new centrifuge tube, Xiang Qi 500 μ L aqueous isopropanols of middle addition (RNA: isopropanol volume ratio ≈ 1:1), mix gently, and stand 15min at room temperature to precipitate RNA；

(7) 4 DEG C, 12000 × g centrifugation 10min, discard supernatant part, then centrifuge tube is inverted on filter paper, drain centrifuge tube；

(8) 75% ethyl alcohol of 1mL is added into centrifuge tube, soft to blow and beat, washing precipitating；

(9) 4 DEG C, 7500 × g centrifugation 6min, discard supernatant clear part, and centrifuge tube is drained in inversion on filter paper；

(10) suitable RNase free H2O is added into centrifuge tube, dissolves RNA；

(11) RNA solution is put into thermostat water bath, 55 DEG C of water-bath 5min are to accelerate to dissolve.Resulting solution, that is, total serum IgE ,- 80 DEG C of preservations.

(12) total serum IgE extracted can be further purified with DNaseI enzyme (Invitrogen company), with remaining in the RNA that goes out DNA, the specific method is as follows: in 200 μ L without in RNase centrifuge tube, 1 μ 10 × DNase of L Reaction Buffer, 1 is added μ L DNase I(1U/ μ L), 2 μ g RNA, DEPC water mends to 10 μ L volumes.It is placed at room temperature for 15min, is mixed after adding 1 μ L EDTA, 65℃ 10min.The total serum IgE of purifying can be obtained.

Embodiment 3:

The clone of lefteye flounder CD39 cDNA intermediate segment and sequencing:

Design of primers:

The full length cDNA sequence and amino acid sequence for downloading different biology CD39 from NCBI first, are selected according to biological classification Taking biology is people, mouse, rat, zebra fish, Tilapia mossambica, Dongfanghong fin globefish, Africa xenopus.With Clustal W software to sequence Conservative analyzed, the conserved region of above-mentioned species CD39 gene use 5 primer-design software of Primer, design degeneracy Primer is used for PCR amplification lefteye flounder CD39 cDNA intermediate segment sequence.

Forward primer: 5 '-GSGATTGTYRYCHTGGTRWC -3 '

Reverse primer: 5 '-AARAYMCCRTTGAADGARCA -3 '

The synthesis of the first chain of cDNA:

Using the lefteye flounder head-kidney total serum IgE of purifying described in embodiment 2 as template, the GoScriptTM of Promega company is used Reverse Transcription System kit, reverse transcription primer use Random Primer provided by kit Or Oligo (dT)₁₅Primer, using the total serum IgE of lefteye flounder head-kidney tissue or cell as templated synthesis the first chain of cDNA, reaction system For 20 μ L.

PCR amplification:

The intermediate sequence of template PCR amplifications CD39 is made of above-mentioned reverse transcription product.PCR system see the table below:

PCR reaction cycle parameter is as follows:

94℃ 4min；

94 DEG C of 30sec, 52 DEG C of 30sec, 72 DEG C of 1min(30 circulations)；

72℃ 5min；12 DEG C of heat preservations.

Take gained 5 μ L of amplified production through 1.5% non denatured Ago-Gel 90V electrophoresis 30min, ultraviolet light is examined after EB dyeing Amplified fragments are surveyed, testing result is as shown in Figure 1.

Cloning and sequencing:

The recycling and purifying of target fragment: the specific band come will be amplified and dug from agarose under ultraviolet light with clean blade Out, it is placed in 1.5mL centrifuge tube, work UNIQ-10 pillar DNA plastic recovery kit is given birth to Shanghai, according to kit recommended method Recycle the DNA fragmentation in glue.

Recombination connection: using the T- carrier PCR product Cloning Kit of precious biotech firm, the DNA fragmentation of recycling is cloned In pMD18-T carrier.Coupled reaction system total volume 10 μ L, 16 DEG C of connection 12h.

The preparation of competent cell: the E.coli DH5 α single colonie 1. newly activated from picking on LB plate is inoculated in 3mL In LB liquid medium, 37 DEG C, 200r/min shaken cultivation 12h or so, until logarithmic growth phase；By the bacteria suspension with 1:100-1: 50 ratio is inoculated in 1mL LB culture medium, 37 DEG C of shaken cultivation 2-3h to logarithmic phase.2. culture solution is transferred to centrifuge tube In, 3000r/min is centrifuged 3min.3. with the CaCl of cold 0.05mol/L₂100 μ L of solution gently suspension cell, is placed on ice 3h, competent cell suspension.

The conversion of Plasmid DNA: 1. 10 μ L connection liquid are added in competent cell under aseptic condition, are mixed gently, on ice Place 30min.2. 42 DEG C of water-bath thermal shocks 90s, rear ice bath 2-3min.3. 800 LB liquid mediums of the μ L without Amp are added, mix Afterwards, 37 DEG C, 500r/min shaking table shaken cultivation 45min.4. after 5min centrifugation, abandoning supernatant, staying 100 μ L bacterium solution 3000r/min Bacterium solution coated plate.5. the L/mL of μ containing Amp(100 is coated on after thallus is resuspended), on the plate of X-gal and IPTG, just setting culture 30min is inverted culture after band bacterium solution is cultured base absorption completely.37 DEG C, 14h, subsequent screening positive clone.6. with sterilizing Toothpick picking white single colonie, is put into the g/mL of μ containing Amp(100) 500 μ L LB culture mediums in, 37 DEG C of shaken cultivation 6-8h.

Recombinant plasmid verifying: 1. going 2 μ L bacterium solutions is template, carries out PCR amplification, PCR amplification with original upstream and downstream primer System and amplification condition are the same, detect amplified production with 1% non denatured agarose gel electrophoresis, if product is expected big When small purpose band, otherwise it is feminine gender that this recombination bacterium colony, which is positive colony,.It adds simultaneously and the negative right of bacterium solution template is not added According to.2. take the 50 μ L of bacteria suspension for being identified as positive colony that 1mL μ containing Amp(100 g/mL is added) in LB culture medium, 180r/ After 37 DEG C of shaken cultivations to logarithmic phase, 200 μ L sterile glycerols are added in min, after mixing, -20 DEG C of preservations.3. bacterium solution sequencing commission Beijing Liuhe Huada Genomics Technology Co., Ltd carries out, and Blastx of the gained sequence on NCBI is compared, tests Demonstrate,prove whether cloned sequence is CD39 homologous sequence.

Lefteye flounder CD39 cDNA intermediate segment sequence is as follows:

GGGATTGTTGTCATGGTGTCGGTAGCAGTTTTGCAGAACAGGCCTCTCCCTCAAAAGTACAAGTATGGAATCG TGCTGGACGCTGGATCCTCCCACACAGCTCTGTATATCTACGAGTGGCCGGCAGAGAAAGATAACAACACTGGCAGA GTTGAACAGACGCATTCCTGCAAAGTCAAAGGCCCAGGTATTTCCAGCTACGCGTCTGTTCCTAGGAAAGCCGGGGA GTCTCTGAGTGAGTGCATGCAGGAGGCCAGGCAGCGAGTCCCTGAAAAGAGACACAGCGAGACCCCTCTTTACCTGG GTGCTACTGCGGGGATGAGATTACTTAATTTAGAGAACAGCTTGGCGTCAGACAAGGTCTTTCAGGCTGTGGAGGAA GCGCTGCAAAAGTTCCCCTTTTCCTTTCAGGGAGCGAGAATCCTCAGCGGCCAGGAAGAGGGTGCCTTCGGATGGGT TACGGCCAACTACTTGGATGATCGCCTCAAACAGAGCTTGGAAACCAGAGGCGCCCTCGACCTTGGTGGGGCCTCCA CTCAAATTAGCTTTGTGTCAGACGATTTTGACGGCTCCGAGTCCCCCGACAACGGCGTCGCCTTCCGACTCTACGGT AATGACTACAACCTGTACACTCACAGCTTCCTGTGTTACGGGAAAGACCAAGTATTACGGATGACGCTGGCAAAACA GACTCAGTCAGGTCCAGTATCCATATCAGATCCCTGTTTCAACCCTGGTTACATCGAGACAAAGAACTACTCCATCG TCTATGACAGCCCCTGCGTGTCCAACATGAAACCCCAGGGAGCTCCTGCCACCTTCACTCACACAGGGAAAGGAAAC TTCTCTCAGTGCCAAGAAGTCGTCAAACGCAACTTCAACTTCAAACAGTGCAAATACAGCCAGTGCTCCTTCAATGG TGTC

Embodiment 4

The cloning and sequencing that lefteye flounder CD39 cDNA 3 ' is held:

Design of primers:

It is designed and synthesized just according to obtained lefteye flounder CD39 cDNA intermediate segment sequence using 5 primer-design software of Primer To primer and general reverse primer AP for 3 ' end nest-type PRCs.

CD39-3’-F2: 5' –CCAGGGAGCTCCTGCCACCT - 3'

AP:5'-TCGAATTCGGATCCGAGCTC- 3'

Oligo(dT)₁₆AP:5'- TCGAATTCGGATCCGAGCTC (T)₁₆- 3'

PCR amplification:

Using the lefteye flounder head-kidney total serum IgE of purifying described in embodiment 2 as template, with Oligo (dT)₁₆AP:5'- TCGAATTCGGATCCGAGCTC(T)₁₆- 3' is reverse transcription primer, carries out the first chain of cDNA and synthesizes, obtained the first chain of cDNA, As 3 ' end nested PCR amplifications template, using CD39-3 '-F2 and AP be upstream and downstream primer progress PCR amplification (amplification system and Program parameter is expanded with intermediate segment).

Gained amplified production is taken to detect through 1% agarose gel electrophoresis, as a result as shown in Figure 3.

Cloning and sequencing and recombinant plasmid verifying:

The sequencing approach of cloning and sequencing and recombinant plasmid the verifying intermediate segment referring to described in embodiment 3.

3 ' terminal sequences of lefteye flounder CD39 gene cDNA are as follows:

CCAGGGAGCTCCTGCCACCTTCACTCACACAGGGAAAGGAAACTTCTCTCAGTGCCAAGAAGTCGTCAAACGC AACTTCAACTTCAAACAGTGCAAATACAGCCAGTGCTCTTTCAATGGGGTCTTCCAGCCACGGTTGCAGGGGCCATT TGGGGCCTTCTCTGCATACTACTTTGTGATGAACTTCCTCAATCTGACAGACACATCCATCCCTCTTGAAGCTGTCA CTGAGAAGCTATCACGCTACTGTGCTACCCCTTGGAACCAGATAAAGCAGCAGCATCCAGGAGTGAAACTAAAATAT CTGGCCGAGTATTGTTTCTCTGGCACGTACATCATCACCCTGCTGACAGAAGGATACAACTTCACATCACAGAACTA CCCCAACATAAAATTCATCAAGAAGATAAAAGGCAGTGACGCAGGCTGGACGCTGGGCTACATGTTGAACCTGACCA ACATGATTCCAGCCGAGGCCCCTGACTCCCCGCCTCTGCCCCACGCTGGCTACGTCTCTATTGTCACTGTCATCGCA ATACTGCTCTTCGTCCTCTTCATCCTCAGCCTGCGTCCCCTCTGGCCCCGGTGCTCCAAACAACCACAGATCATATA AAATATACGCACACACACAAACATATATGATGTATGTTTGAGTAAAGTTGGTGAAGGGAAAAAAAAAAAAAAAAAAA A

Embodiment 5

The cloning and sequencing that lefteye flounder CD39 gene cDNA 5 ' is held:

Design of primers:

Reverse primer CD39-5 '-is designed using 5 primer-design software of Primer according to obtained CD39 intermediate segment sequence R1 is carried out reverse transcription using the reverse transcription primer that 5 ' RACE reverse transcription reagent box of Clontech provides, is equipped with using kit 5 ' end upstream primers carry out 5 ' end nest-type PRCs.

CD39-5 '-R1:5'-CTGCCAGTGTTGTTATCTTT- 3'

The synthesis of the first chain of cDNA:

Using the lefteye flounder head-kidney total serum IgE of purifying described in embodiment 2 as template, carried out using the reverse transcription primer that kit provides anti- Transcription synthesis the first chain of cDNA, reverse transcription product digest remnants RNA using RNase H, obtain first chain of cDNA at 5 ' ends.

PCR amplification:

First round PCR amplification is carried out as upstream and downstream primer using the primer UPM and CD39-5 '-R1 that kit provides first；Then with First round pcr amplification product is template, carries out second as upstream and downstream primer using the primer NUP and CD39-5 '-R1 that kit provides Take turns PCR amplification.

Amplification system and program parameter are as follows:

PCR system see the table below:

PCR reaction cycle parameter is as follows:

94℃ 4min；

94 DEG C of 30sec, 72 DEG C of 3min(5 circulations)；

94 DEG C of 30sec, 65 DEG C of 30sec, 72 DEG C of 3min(5 circulations)；

94 DEG C of 30sec, 63 DEG C of 30sec, 72 DEG C of 3min(25 circulations)；

72℃ 5min；12 DEG C of heat preservations.

Gained amplified production is taken to detect through 1% agarose gel electrophoresis, as a result as shown in Figure 4.

Cloning and sequencing and recombinant plasmid verifying:

5 ' terminal sequences of lefteye flounder CD39 gene cDNA are as follows:

TGAGAGAGTGAAAGAGGGGGAGACAGAAAGAGAGAGAGCGAGAGAGAGGGGGGGCAAAGAATAGAAAGAGCAT TGTGTTGCTGCTGAGAAAGAACCCATGTCCGCACAAAGAGAGATGAAAGAGAAGAACCCCTGGCACACGCCAGTGAC CATCATCTTCACTGTGATTGGTGTCATAGCGATTGTTGCCTTGGTGACGGTAGCAGTTTTGCAGAACAGGCCTCTCC CCCAAAAGTACAAGTATGGAATCGTGCTGGACGCTGGATCCTCCCACACAGCTCTGTATATCTACGAGTGGCCGGCA GAGAAAGATAACAACACTGGCAG

Embodiment 6:

Lefteye flounder CD39 full length cDNA sequence:

Lefteye flounder CD39 full length cDNA sequence is obtained using the EditSeq software editing in DNAStar software package, finds open reading Frame translates into amino acid sequence, as described in SEQ No.2.The protein molecular weight of lefteye flounder CD39 cDNA coding is 55.3KDa, Isoelectric point is 8.58.

The lefteye flounder CD39 full length gene total 1685bp of cDNA, including open reading frame are 1494bp, encode 497 amino altogether Acid, the 5' non-translational region containing 114bp, 3' non-translational region and poly (A) tail of 77bp.These gene structure features it may be said that Bright resulting cDNA sequence is the full length cDNA sequence of lefteye flounder CD39 gene, and sequence is as follows:

TGAGAGAGTGAAAGAGGGGGAGACAGAAAGAGAGAGAGCGAGAGAGAGGGGGGGCAAAGAATAGAAAGAGCAT TGTGTTGCTGCTGAGAAAGAACCCATGTCCGCACAAAGAGAGATGAAAGAGAAGAACCCCTGGCACACGCCAGTGAC CATCATCTTCACTGTGATTGGTGTCATAGCGATTGTTGCCTTGGTGACGGTAGCAGTTTTGCAGAACAGGCCTCTCC CCCAAAAGTACAAGTATGGAATCGTGCTGGACGCTGGATCCTCCCACACAGCTCTGTATATCTACGAGTGGCCGGCA GAGAAAGATAACAACACTGGCAGAGTTGAACAGACGCATTCCTGCAAAGTCAAAGGCCCAGGTATTTCCAGCTACGC GTCTGTTCCTAGGAAAGCCGGGGAGTCTCTGAGTGAGTGCATGCAGGAGGCCAGGCAGCGAGTCCCTGAAAAGAGAC ACAGCGAGACCCCTCTTTACCTGGGTGCTACTGCGGGGATGAGATTACTTAATTTAGAGAACAGCTTGGCGTCAGAC AAGGTCTTTCAGGCTGTGGAGGAAGCGCTGCAAAAGTTCCCCTTTTCCTTTCAGGGAGCGAGAATCCTCAGCGGCCA GGAAGAGGGTGCCTTCGGATGGGTTACGGTCAACTACTTGGATGATCGCCTCAAACAGAGCTTGGAAACCAGAGGCG CCCTCGACCTTGGTGGGGCCTCCACTCAAATTAGCTTTGTGTCAGACGATTTTGACGGCTCCGAGTCCCCCGACAAC GGCGTCGCCTTCCGACTCTACGGTAATGACTACAACCTGTACACTCACAGCTTCCTGTGTTACGGGAAAGACCAAGC ATTACGGATGACGCTGGCAAAACAGACTCAGTCAGGTCCAGTATCCATATCAGATCCCTGTTTCAACCCTGGTTACA TCGAGACAAAGAACTACTCCATCGTCTATGACAGCCCCTGCGTGTCCAACATGAAACCCCAGGGAGCTCCTGCCACC TTCACTCACACAGGGAAAGGAAACTTCTCTCAGTGCCAAGAAGTCGTCAAACGCAACTTCAACTTCAAACAGTGCAA ATACAGCCAGTGCTCTTTCAATGGGGTCTTCCAGCCACGGTTGCAGGGGCCATTTGGGGCCTTCTCTGCATACTACT TTGTGATGAACTTCCTCAATCTGGCAGACACATCCATCCCTCTTGAAGCTGTCACTGAGAAGCTATCACGCTACTGT GCTACCCCTTGGAACCAGATAAAGCAGCAGCATCCAGGAGTGAAACTAAAATATCTGGCCGAGTATTGTTTCTCTGG CACGTACATCATCACCCTGCTGACAGAAGGATACAACTTCACATCACAGAACTACCCCAACATAAAATTCATCAAGA AGATAAAAGGCAGTGACGCAGGCTGGACGCTGGGCTACATGTTGAACCTGACCAACATGATTCCAGCCGAGGCCCCT GACTCCCCGCCTCTGCCCCACGCTGGCTACGTCTCTATTGTCACTGTCATCGCAATACTGCTCTTCGTCCTCTTCAT CCTCAGCCTGCGTCCCCTCTGGCCCCGGTGCTCCAAACAACCACAGATCATATAAAATATACGCACACACACAAACA TATATGATGTATGTTTGAGTAAAGTTGGTGAAGGGAAAAAAAAAAAAAAAAAAAA

Application example:

Embodiment 1

Lefteye flounder Extracellular ATP hydrolase CD39 cDNA sequence detects lefteye flounder disease prevention and cure side as real-time fluorescence RT-PCR in preparation The application in face.Mainly detected using the real-time fluorescence RT-PCR of lefteye flounder CD39 cDNA sequence:

(1) design of primers:

According to the full length cDNA sequence of lefteye flounder CD39 described in SEQ ID No.1, it is applicable in using 5 software design of Primer glimmering in real time The specific primer of light RT-PCR detection, primer sequence are as follows:

CD39-q-F:5'-CCTTCCGACTCTACGGTAATGAC- 3'

CD39-q-R:5'-GGATCTGATATGGATACTGGACCT-3'

The PCR product estimated length of lefteye flounder CD39 be 127bp, agarose gel electrophoresis results display amplification after product length with Expected product length is consistent, and is single band, illustrates that designed primer has very strong specificity, suitable for glimmering in real time Light RT-PCR detection: agarose gel electrophoresis results are as shown in Figure 4.

Real-time fluorescence RT- is designed for according to the cds sequence (EU090804) of the lefteye flounder β-actin provided in NCBI simultaneously The primer of PCR internal reference, primer sequence are as follows:

β-actin-F:5'-AGGTTCCGTTGTCCCG -3'

β-actin-R:5'-TGGTTCCTCCAGATAGCAC -3'

The PCR product estimated length of β-actin is 150bp.Agarose gel electrophoresis results display amplification after product length with Expected product length is consistent, and is single band, illustrates that designed primer has very strong specificity, suitable for glimmering in real time It is expanded when light RT-PCR is detected as internal reference.Agarose gel electrophoresis results are as shown in Figure 5.

Embodiment 2:

External lipopolysaccharides (LPS) immunostimulation is detected to lefteye flounder head-kidney primary cell CD39 Gene Expression using the above method Experiment:

(1) healthy Paralichthys olivaceus (the long 45cm-50cm of body) 3 tails for not applying vaccine in the recent period, put to death after temporarily supporting in aquarium, dissect Separating head nephridial tissue is carried out originally culture after mechanical dispersion is at individual cells, is stimulated after overnight incubation using 20 μ g/ml LPS Cell, in different time sections lytic cell.

(2) Total RNAs extraction and purifying: with embodiment 2

(3) the first chain of cDNA synthesizes: using the GoScriptTM Reverse Transcription of Promega company System kit, reverse transcription primer use Random Primer or Oligo (dT) 15 Primer provided by kit, Using the total serum IgE of lefteye flounder head-kidney tissue or cell as templated synthesis the first chain of cDNA, reaction system is 20 μ L.

(4) real-time fluorescence RT-PCR: fluorescence RT-PCR uses the SYBR@Premix Ex TaqTM II of precious biotech firm Kit carries out.Using the first chain of cDNA synthesized in above-mentioned (2) as template, with the CD39-q-F and CD39-q- in design of primers R, β-actin-F and β-actin-R is special primer, carries out fluorescence RT-PCR amplified reaction, and each sample sets 3 parallel pipes, (fluorescence signal reaches circulation experienced when the threshold value of setting to the Ct value of 3 parallel pipes of gained in i.e. each reaction tube after amplification Number) it is averaged.

Fluorescence RT-PCR amplification reaction system is provided that

Fluorescence RT-PCR Amplification is provided that

95℃ 30 sec；

95 DEG C of 5 sec, 60 DEG C of 30 sec (40 circulations)；

55 DEG C -95 DEG C 30 sec(solubility curve).

(5) relative expression quantity of CD39 gene calculates in lefteye flounder head-kidney primary cell after LPS immunostimulation:

After the completion of fluorescence RT-PCR, according to after the stimulation of Ct value Computation immunity under lefteye flounder renal cell each period △ Ct, 2^-(△Ct)、2^-(△△Ct)Value, calculation are as follows:

△ Ct=Ct-Ct internal reference (β-actin)

Untreated cell when △ △ Ct=△ Ct-△ Ct(0)

With 2^-(△Ct)Numerical value indicates expression quantity of the CD39 gene relative to β-actin gene in lefteye flounder head-kidney；With 2^-(△△Ct)Numerical value Indicate expression quantity of the CD39 gene relative to untreated lefteye flounder renal cell CD39 gene in lefteye flounder head-kidney.

2^-(△Ct)Numerical value represents expression multiple of the CD39 gene relative to β-actin gene.2^-(△△Ct)Numerical value represents CD39 gene is relative to the expression multiple for not stimulating lefteye flounder cell CD39 gene.β-actin gene is house-keeping gene, in all classes It is all expressed in the cell of type, what the expression of house-keeping gene was only interacted by initiating sequence or promoter and RNA polymerase It influences, without the adjusting by other mechanism, by such environmental effects very little, expression is more constant.Therefore, house-keeping gene is normal It is used as measuring reference gene when target gene expression quantity.When 2 in this experiment^-(△Ct)Or 2^-(△△Ct)Numerical value is bigger, shows The expression quantity of CD39 gene is higher.

Fig. 6 is the expression variation that bacteria lipopolysaccharide LPS stimulates CD39 gene after lefteye flounder head-kidney primary cell.LPS stimulates 8h Afterwards, lefteye flounder CD39 gene expression amount raises rapidly, is 3.2 times for not stimulating cell.As a result prompt head-kidney primary cell by When pathogen associated molecular pattern molecule stimulates, CD39 gene relative expression quantity is shorter the time required to changing and difference is aobvious It writes, the innate immune responses of lefteye flounder may be taken part in.

Conclusion:

(1) bacteria lipopolysaccharide LPS stimulates lefteye flounder head-kidney primary cell that CD39 gene expression can be made significantly to raise.

(2) CD39 gene takes part in the innate immune responses of lefteye flounder.

Embodiment 3

Lefteye flounder live bacteria infection experiment is detected using the above method

(1) healthy Paralichthys olivaceus (the long 8-10cm of body) 50 tails of vaccine were not used in the recent period, were incubated in aquarium, every tail intraperitoneal injection 20 μ L(1x10 of viable bacteria⁶A tarda) continue to cultivate, lefteye flounder anatomical isolation cheek tissue is put to death in different time sections.

(2) Total RNAs extraction and purifying: with embodiment 2.

(3) the first chain of cDNA synthesizes: with application example 1.

(4) real-time fluorescence RT-PCR: with application example 1.

(5) relative expression quantity for infecting CD39 gene in the head-kidney tissue of lefteye flounder calculates: with application example 1

Fig. 7 is the relative expression quantity of CD39 gene in lefteye flounder cheek tissue after tarda is injected intraperitoneally.Moral is liked as seen from the figure CD39 gene expression amount is only 0.07 times of healthy Paralichthys olivaceus in lefteye flounder cheek tissue after Fahrenheit bacterium infection 4h, well below healthy Paralichthys olivaceus (i.e. the expression quantity of 0h) as a result prompts CD39 gene to take part in the lefteye flounder early immune response under bacterium infection, when CD39 gene When relative expression quantity reduces, lefteye flounder is likely to be at pathogen infection state, appoints although cannot observe that lefteye flounder has from naked eyes at this time What infection illness.Therefore the expression quantity of CD39 gene reflects the immune system activity of lefteye flounder to a certain extent, can pass through prison Survey lefteye flounder cheek tissue CD39 gene expression amount variation, tentatively judge lefteye flounder whether pathogenic infection, take prophylactic treatment to arrange ahead of time It applies, the development that avoids pushing comes to shove causes irremediable loss.

Embodiment 4

The cellular immunity factor IL-1 β gene that lefteye flounder head-kidney macrophage Extracellular ATP is induced using above method detection CD39 The adjusting of expression:

(1) according to the lefteye flounder IL-1 β (gene order BAM66989) provided on NCBI, designed for real-time fluorescence RT-PCR Primer.Design primer is as follows:

IL-1 β F:5'-CCTGTCGTTCTGGGCATCAA -3'

IL-1 β R:5'-CACCCCGCTGTCCTGCTT -3'

(2) lefteye flounder head-kidney macrophage preparation process is as follows:

1) healthy Paralichthys olivaceus (the long 45cm-50cm of body) 3 tails for not applying vaccine in the recent period, put to death after temporarily supporting in aquarium, dissect Separating head nephridial tissue carries out originally culture after mechanical dispersion is at individual cells.

2) head-kidney is placed on the 200 mesh stainless steel mesh (about 80 μm of apertures) of sterilization treatment, sieve is placed on sterile glass On glass plate, 5 ml RPMI, 1640 culture medium is added, is lightly ground with the pestle vertical direction of sterilizing, wait organize homogenate to filter Under, aspirates tissue lapping liquid is simultaneously ground to tissue homogenate uniformly repeatedly again.

3) the Percoll solution of 4ml 51% is added in 15ml centrifuge tube, is slowly added to the renal cell of 3 times of dilution 4ml is finally slowly added to the Percoll solution of 4ml 34%.Trim centrifuge tube is centrifuged 30 min in 4 DEG C, 3000rpm.

4) centrifuge tube is taken out, horizontal rest, 51% Percoll solution is slowly sucked out with syringe and 34% Percoll is molten White cell suspension between liquid two-phase interface is transferred in a new 15ml centrifuge tube；By suspension in 4 DEG C, 3000rpm from 5 min of the heart abandons supernatant, 1640 culture medium of RPMI of 5ml serum-free is added, cell is suspended again rinsing.

5) 4 DEG C, 3000rpm centrifugation 5min, abandon supernatant, collect cell.It rinses for several times, until cell cleans up.It obtains at this time Cell, that is, lefteye flounder head-kidney the macrophage (head kidney macrophages, HKMs) obtained.

6) the HKMs cell of acquisition is suspended in 1640 culture medium of RPMI (10% FBS+1% p/s), uses blood counting chamber Estimate cell density, dilution is adjusted to every milliliter 10⁷A cell.

7) by HKMs cell suspension inoculation in 24 porocyte culture plates, every hole contains 5 × 10⁶A cell/500 μ l, in 21 DEG C Incubator is stayed overnight.

(3) 200 μM or 1000 μM of ATP stimulating expression of macrophage 2h are used after overnight incubation respectively, or uses extracellular core simultaneously Thuja acid enzyme inhibitor ARL 67156 handles cell, detects the variation of cell factor IL-1 beta gene expression level.Total RNAs extraction with Purifying: with embodiment 2.

(4) the first chain of cDNA synthesizes: with application example 1.

(5) real-time fluorescence RT-PCR: with application example 1.

(6) relative expression quantity of IL-1 β gene calculates in the head-kidney macrophage of lefteye flounder after stimulating:

After the completion of fluorescence RT-PCR, according to Ct value calculate lefteye flounder renal cell different disposal under △ Ct, 2^-(△Ct)、2^-(△△Ct) Value, calculation are as follows:

Internal reference (β-actin)

(untreated lefteye flounder head-kidney macrophage when 0)

WithNumerical value indicates expression quantity of the IL-1 β gene relative to β-actin gene in lefteye flounder head-kidney macrophage；

WithNumerical value indicates that IL-1 β gene is relative to untreated lefteye flounder head-kidney macrophage in lefteye flounder head-kidney macrophage The expression quantity of IL-1 β gene.

Numerical value represents expression multiple of the IL-1 β gene relative to β-actin gene.Numerical value represents Expression multiple of the IL-1 β gene relative to normal lefteye flounder head-kidney macrophage IL-1 β gene.β-actin gene is house-keeping gene, It is all expressed in all types of cells, the expression of house-keeping gene is only by initiating sequence or promoter and RNA polymerase phase The influence of interaction, without the adjusting by other mechanism, by such environmental effects very little, expression is more constant.Therefore, it manages Family's gene is often used as reference gene when measurement target gene expression quantity.In this experiment whenNumerical value is got over Greatly, show that the expression quantity of IL-1 β gene is higher.

Fig. 8 is the IL-1 β gene that CD39 inhibitor ARL 67156 handles that lefteye flounder head-kidney macrophage induces Extracellular ATP The opposite variation of expression, as seen from the figure, compared with the normal cell not stimulated, various concentration ATP stimulates lefteye flounder head-kidney macrophage Cell can lead to the up-regulation of IL-1 beta gene expression amount；When CD39 enzymatic activity is inhibited by its specific inhibitor ARL 67156, born of the same parents The IL-1 beta gene expression amount of outer ATP induction significantly increases.As a result it prompts, lefteye flounder Extracellular ATP hydrolase CD39 lures Extracellular ATP The pro-inflammatory cytokine IL-1 beta gene expression led has important negative regulation effect, and being one can be used for lefteye flounder disease prevention and cure Important potential target gene.

Conclusion:

(1) when lefteye flounder CD39 is inhibited by its inhibitor ARL 67156, the cell factor IL1-1 β of Extracellular ATP induction can be caused Gene expression is significantly raised.

Embodiment 5

The lefteye flounder head-kidney macrophage activity oxygen (ROS) that Extracellular ATP induces is generated using above method detection lefteye flounder CD39 Adjustment effect:

(1) lefteye flounder head-kidney macrophage preparation process is the same as embodiment 4.

(2) lefteye flounder head-kidney macrophage 2h is handled using CD39 inhibitor ARL 67156, to inhibit lefteye flounder CD39 enzyme activity Property, hereafter, head-kidney macrophage is stimulated with 1000 μM of ATP, is surveyed after ATP stimulates 5min, 10min, 20min, 30min Determine cell ROS and generates variation.

(3) specific step is as follows for ROS measurement:

1) before testing, all cells are fully transferred in EP pipe, 1000g is centrifuged 5min, abandons supernatant.

2) configured fresh culture is added in cell, gently piping and druming mixes, and every milliliter of culture medium of control group adds 50 μM of DCFH, it is 1000 μM that 50 μM of DCFH and ATP(ultimate densities are added in every milliliter of culture medium of experimental group), in incubator It is incubated for.

3) after handling the corresponding time, 1000g is centrifuged 5min, abandons supernatant.

4) three times, each 1000g is centrifuged 5min to cells rinsed with PBS.

5) cell is resuspended with PBS, under the conditions of excitation wavelength 500nm, measures the fluorescence intensity at launch wavelength 530nm.

As shown in figure 9,1000 μM of ATP stimulation lefteye flounder head-kidney macrophages can significant inducing cell generation ROS；Work as tooth Flounder CD39 enzymatic activity is inhibited by ARL 67156, after ATP stimulates 15min, 67156 experimental group ratio ATP stimulation group cell of ATP+ARL ROS yield it is significantly raised.Therefore, under ATP incentive condition, to inhibit CD39 enzymatic activity that can remarkably promote lefteye flounder head-kidney macrophage thin The ROS of born of the same parents' Extracellular ATP induction is generated.

Conclusion:

(1) ARL 67156 inhibits lefteye flounder CD39 enzymatic activity, the activity that lefteye flounder head-kidney macrophage Extracellular ATP can be caused to induce Oxygen generates significant increase.

(2) lefteye flounder Extracellular nucleotidase CD39 is to the gene expression of lefteye flounder pro-inflammatory cytokine, the conjunction of the congenital immunity factor It is the important potential target gene that can be used for lefteye flounder disease prevention and cure at important regulative.

SEQUENCE LISTING

<110>Tianjin Normal University

<120>a kind of cDNA full length sequence for encoding lefteye flounder Extracellular ATP hydrolase CD39 and its application

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1668

<212> DNA

<213>artificial sequence

<400> 1

tgagagagtg aaagaggggg agacagaaag agagagagcg agagagaggg ggggcaaaga 60

atagaaagag cattgtgttg ctgctgagaa agaacccatg tccgcacaaa gagagatgaa 120

agagaagaac ccctggcaca cgccagtgac catcatcttc actgtgattg gtgtcatagc 180

gattgttgcc ttggtgacgg tagcagtttt gcagaacagg cctctccccc aaaagtacaa 240

gtatggaatc gtgctggacg ctggatcctc ccacacagct ctgtatatct acgagtggcc 300

ggcagagaaa gataacaaca ctggcagagt tgaacagacg cattcctgca aagtcaaagg 360

cccaggtatt tccagctacg cgtctgttcc taggaaagcc ggggagtctc tgagtgagtg 420

catgcaggag gccaggcagc gagtccctga aaagagacac agcgagaccc ctctttacct 480

gggtgctact gcggggatga gattacttaa tttagagaac agcttggcgt cagacaaggt 540

ctttcaggct gtggaggaag cgctgcaaaa gttccccttt tcctttcagg gagcgagaat 600

cctcagcggc caggaagagg gtgccttcgg atgggttacg gtcaactact tggatgatcg 660

cctcaaacag agcttggaaa ccagaggcgc cctcgacctt ggtggggcct ccactcaaat 720

tagctttgtg tcagacgatt ttgacggctc cgagtccccc gacaacggcg tcgccttccg 780

actctacggt aatgactaca acctgtacac tcacagcttc ctgtgttacg ggaaagacca 840

agcattacgg atgacgctgg caaaacagac tcagtcaggt ccagtatcca tatcagatcc 900

ctgtttcaac cctggttaca tcgagacaaa gaactactcc atcgtctatg acagcccctg 960

cgtgtccaac atgaaacccc agggagctcc tgccaccttc actcacacag ggaaaggaaa 1020

cttctctcag tgccaagaag tcgtcaaacg caacttcaac ttcaaacagt gcaaatacag 1080

ccagtgctct ttcaatgggg tcttccagcc acggttgcag gggccatttg gggccttctc 1140

tgcatactac tttgtgatga acttcctcaa tctggcagac acatccatcc ctcttgaagc 1200

tgtcactgag aagctatcac gctactgtgc taccccttgg aaccagataa agcagcagca 1260

tccaggagtg aaactaaaat atctggccga gtattgtttc tctggcacgt acatcatcac 1320

cctgctgaca gaaggataca acttcacatc acagaactac cccaacataa aattcatcaa 1380

gaagataaaa ggcagtgacg caggctggac gctgggctac atgttgaacc tgaccaacat 1440

gattccagcc gaggcccctg actccccgcc tctgccccac gctggctacg tctctattgt 1500

cactgtcatc gcaatactgc tcttcgtcct cttcatcctc agcctgcgtc ccctctggcc 1560

ccggtgctcc aaacaaccac agatcatata aaatatacgc acacacacaa acatatatga 1620

tgtatgtttg agtaaagttg gtgaagggaa aaaaaaaaaa aaaaaaaa 1668

<210> 2

<211> 497

<212> PRT

<213>lefteye flounder CD39 amino acid sequence

<400> 2

Met Ser Ala Gln Arg Glu Met Lys Glu Lys Asn Pro Trp His Thr Pro

1 5 10 15

Val Thr Ile Ile Phe Thr Val Ile Gly Val Ile Ala Ile Val Ala Leu

20 25 30

Val Thr Val Ala Val Leu Gln Asn Arg Pro Leu Pro Gln Lys Tyr Lys

35 40 45

Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ala Leu Tyr Ile

50 55 60

Tyr Glu Trp Pro Ala Glu Lys Asp Asn Asn Thr Gly Arg Val Glu Gln

65 70 75 80

Thr His Ser Cys Lys Val Lys Gly Pro Gly Ile Ser Ser Tyr Ala Ser

85 90 95

Val Pro Arg Lys Ala Gly Glu Ser Leu Ser Glu Cys Met Gln Glu Ala

100 105 110

Arg Gln Arg Val Pro Glu Lys Arg His Ser Glu Thr Pro Leu Tyr Leu

115 120 125

Gly Ala Thr Ala Gly Met Arg Leu Leu Asn Leu Glu Asn Ser Leu Ala

130 135 140

Ser Asp Lys Val Phe Gln Ala Val Glu Glu Ala Leu Gln Lys Phe Pro

145 150 155 160

Phe Ser Phe Gln Gly Ala Arg Ile Leu Ser Gly Gln Glu Glu Gly Ala

165 170 175

Phe Gly Trp Val Thr Val Asn Tyr Leu Asp Asp Arg Leu Lys Gln Ser

180 185 190

Leu Glu Thr Arg Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile

195 200 205

Ser Phe Val Ser Asp Asp Phe Asp Gly Ser Glu Ser Pro Asp Asn Gly

210 215 220

Val Ala Phe Arg Leu Tyr Gly Asn Asp Tyr Asn Leu Tyr Thr His Ser

225 230 235 240

Phe Leu Cys Tyr Gly Lys Asp Gln Ala Leu Arg Met Thr Leu Ala Lys

245 250 255

Gln Thr Gln Ser Gly Pro Val Ser Ile Ser Asp Pro Cys Phe Asn Pro

260 265 270

Gly Tyr Ile Glu Thr Lys Asn Tyr Ser Ile Val Tyr Asp Ser Pro Cys

275 280 285

Val Ser Asn Met Lys Pro Gln Gly Ala Pro Ala Thr Phe Thr His Thr

290 295 300

Gly Lys Gly Asn Phe Ser Gln Cys Gln Glu Val Val Lys Arg Asn Phe

305 310 315 320

Asn Phe Lys Gln Cys Lys Tyr Ser Gln Cys Ser Phe Asn Gly Val Phe

325 330 335

Gln Pro Arg Leu Gln Gly Pro Phe Gly Ala Phe Ser Ala Tyr Tyr Phe

340 345 350

Val Met Asn Phe Leu Asn Leu Ala Asp Thr Ser Ile Pro Leu Glu Ala

355 360 365

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

370 375 380

Lys Gln Gln His Pro Gly Val Lys Leu Lys Tyr Leu Ala Glu Tyr Cys

385 390 395 400

Phe Ser Gly Thr Tyr Ile Ile Thr Leu Leu Thr Glu Gly Tyr Asn Phe

405 410 415

Thr Ser Gln Asn Tyr Pro Asn Ile Lys Phe Ile Lys Lys Ile Lys Gly

420 425 430

Ser Asp Ala Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met

435 440 445

Ile Pro Ala Glu Ala Pro Asp Ser Pro Pro Leu Pro His Ala Gly Tyr

450 455 460

Val Ser Ile Val Thr Val Ile Ala Ile Leu Leu Phe Val Leu Phe Ile

465 470 475 480

Leu Ser Leu Arg Pro Leu Trp Pro Arg Cys Ser Lys Gln Pro Gln Ile

485 490 495

Ile

<210> 3

<211> 23

<212> DNA

<213>artificial sequence

<400> 3

ccttccgact ctacggtaat gac 23

<210> 4

<211> 24

<212> DNA

<213>artificial sequence

<400> 4

ggatctgata tggatactgg acct 24

Claims

1. lefteye flounder Extracellular ATP hydrolase CD39 gene cDNA sequence, gene cDNA overall length 1685bp, the opening containing 1494bp Reading frame has nucleotide sequence shown in SEQ ID NO.1；It encodes 497 amino acid, has shown in SEQ ID NO.2 Amino acid sequence.

2. lefteye flounder Extracellular ATP hydrolase CD39 gene order described in claim 1 is used as lefteye flounder disease prevention and cure target base in preparation Because of the application of aspect.

3. lefteye flounder Extracellular ATP hydrolase CD39 gene order described in claim 1 is used as lefteye flounder disease real-time fluorescence RT- in preparation The application of PCR context of detection.

4. lefteye flounder Extracellular ATP hydrolase CD39 gene order described in claim 1 is in terms of preparation is as lefteye flounder disease prevention and cure Using.

5. the specific primer for detecting lefteye flounder Extracellular ATP hydrolase CD39 gene cDNA sequence, it is characterised in that:

Forward primer: 5 '-CCTTCCGACTCTACGGTAATGAC-3 ' SEQ ID NO.3

Reverse primer: 5 '-GGATCTGATATGGATACTGGACCT-3 ' SEQ ID NO.4.