CN106754923A - Crocea interferon d gene promoter sequences and its application - Google Patents
Crocea interferon d gene promoter sequences and its application Download PDFInfo
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- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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Abstract
Crocea interferon d gene promoter sequences and its application, are related to interferon IFNd gene promoters.Sequence, the activity assays of large yellow croaker IFNd gene promoters, the application of large yellow croaker IFNd gene promoters of large yellow croaker IFNd gene promoters are provided.Large yellow croaker IFNd gene promoters are cloned using the method for genomic walking, there is provided large yellow croaker IFNd gene promoter sequences；It is reported that genetic analysis experiment proves that large yellow croaker IFNd gene promoters can be by poly (I:C), LPS, PGN and bacterial genomes DNA induced activations.The checking of the clone and its strong promoter activity of large yellow croaker IFNd gene promoters, for study large yellow croaker IFNd genes expression regulation mechanism good experimental system is provided, application aspect be using the promoter construction of expression vector efficiently expressing exogenous gene or by the promoter be applied to genetically engineered fish structure create conditions.
Description
Technical field
The present invention relates to interferon IFNd gene promoters, more particularly, to crocea interferon IFNd gene promoter sequences
Row and its application.
Background technology
Interferon (Interferon, IFN) is that a class can induce vertebrate cells antiviral state, and antiviral anti-
Inductivity multigene family cell factor (the 1.Robertsen B.The interferon system played an important role in imperial
of teleost fish[J].Fish Shellfish Immunol,2006,20(2):172-191).Bony fish is used as low
Also possess the interferon system of comparatively perfect Deng vertebrate.It is the major classes of I type IFNs and II types IFNs two that fish IFNs is divided to, wherein
I types IFNs coordinates to be subject to more to pay close attention to (2.Zhang YB, Gui host anti-virus are immune in specific manner because of it
JF.Molecular regulation of interferon antiviral response in fish[J].Dev Comp
Immunol,2012,38(2):193-202.).Quantity and distribution according to cysteine in mature peptide, fish I types IFNs points is
Group I IFN (mature peptide contains 2 cysteines) and group II IFN (mature peptide contains 4 cysteines).Group I
IFN includes 3 subgroups of IFNa, IFNd and IFNe, and group II IFN include IFNb, IFNc and IFNf3 subgroup (3.Zou
J,Gorgoglione B,Taylor NG,et al.Salmonids have an extraordinary complex type
I IFN system:characterization of the IFN locus in rainbow trout oncorhynchus
mykiss reveals two novel IFN subgroups[J].J Immunol,2014,193(5):2273-2286).Base
The expression regulation of cause has turned into the focus in molecular biology research field, and promoter is the critical elements of gene expression regulation.Mirror
In importance of the fish I types IFNs in antiviral immunity and virus disease control, it will be logical to study its gene expression regulation mechanism
Overregulate the expression of the IFN thinking new to prevent and treat Virus disease of fish offer.To the flank Regulatory Sequence of fish I type IFNs genes 5 '
Analysis shows, there are multiple Binding site for transcription factor in fish I type IFNs gene promoters, such as IRF3, IRF7, NF- κ B and
ISRE etc., and with complicated expression and regulation mechanism.
The applicant passes through complete genome sequencing in early stage, it was found that 1 gene sequence of coding large yellow croaker I types IFN
Row (4.Ao JQ, Mu YN, Xiang LX, et al.Genome sequencing of the perciform fish
Larimichthys crocea provides insights into stress adaptation[J].Plos Genet,
2015,11(4):e1005118).Homologous comparison and evolutionary analysis show that I types IFN, for IFNd, belongs to fish group I
IFNs.It is that the expression for studying large yellow croaker IFNd genes is adjusted because promoter is the key factor of decision gene expression and its regulation and control
Control mechanism, 5 ' flanking promoter sequences of large yellow croaker IFNd genes has further been cloned from its genome, it is reported that genetic test
Experiment demonstrates the large yellow croaker IFNd gene promoters with stronger promoter activity.The large yellow croaker IFNd gene promoters
Clone and its checking of promoter activity, will provide well to study the expression regulation mechanism of large yellow croaker IFNd genes in theory
Experimental system, be using the strong promoter construction of expression vector efficiently expressing exogenous gene or by the promoter in application aspect
It is applied to genetically engineered fish structure and creates condition, with important theoretical and practical significance.
The content of the invention
An object of the present invention is the sequence for providing large yellow croaker IFNd gene promoters.
The second object of the present invention is to provide a kind of activity assays of large yellow croaker IFNd gene promoters.
The third object of the present invention is to provide the application of large yellow croaker IFNd gene promoters.
The sequence of the large yellow croaker IFNd gene promoters is：
The activity assays of the large yellow croaker IFNd gene promoters are as follows：
1) using large yellow croaker IFNd genes described in the luciferase reporter gene detecting system quantitative analysis of Promega companies
The active and artificial synthesized double-stranded RNA poly (I of promoter:C), e. coli lipopolysaccharide LPS, bacillus subtilis peptide gather
Sugared PGN and Aeromonas hydrophila genomic DNA stimulate the influence to its activity；
2) by large yellow croaker IFNd gene promoter areas fragment insertion Promega companies luciferase reporter gene carrier
In pGL3-Basic, firefly luciferase gene is set to be located under the control of the promoter, structure obtains recombinant vector pGL3-
IFNdP；EPC cells, 48h are transfected jointly with recombinant vector pGL3-IFNdP and Renilla luciferase reporter genophore pRL-TK
After collect transfectional cell；Firefly luciferase and sea pansy fluorescence are detected respectively using luciferase reporter gene detecting system
The enzymatic activity value of plain enzyme, the relative activity of luciferase in transfectional cell is drawn by the ratio for calculating the two enzymatic activity value；
3) with empty carrier pGL3-Basic and renilla luciferase control Reporter gene vector pRL-TK, that EPC is transfected jointly is thin
The relative luciferase activity of born of the same parents calculates the relative activity of the promoter as control, then with poly (I:C), lipopolysaccharides
LPS, peptide glycan PGN and bacterial genomes DNA stimulate the EPC cells that pGL3-IFNdP is transfected, by analyzing each treatment group fluorescence
The change of plain enzyme relative activity come determine large yellow croaker IFNd gene promoters activity whether stimulated thing induction influence.
The application of the large yellow croaker IFNd gene promoters is as follows：
1) identification of the clone and its promoter activity of the large yellow croaker IFNd gene promoters, is building large yellow croaker IFNd
Applied in the expression regulation mechanism experiment system of gene；
2) the large yellow croaker IFNd gene promoters can be thin in fish cell or mammal in structure carrier for expression of eukaryon
Applied in efficiently expressing exogenous gene in born of the same parents；
3) the large yellow croaker IFNd gene promoters are applied in genetically engineered fish is built.
Outstanding advantages of the invention and technique effect are as follows：
The present invention has cloned large yellow croaker IFNd gene promoters using the method for genomic walking, there is provided large yellow croaker IFNd
Gene promoter sequence；It is reported that genetic analysis experiment demonstrate the large yellow croaker IFNd gene promoters can be by poly (I:C)、
LPS, PGN and bacterial genomes DNA induced activations.Therefore, the clone of the large yellow croaker IFNd gene promoters and its strong promoter
The checking of activity, will provide good experimental system to study the expression regulation mechanism of large yellow croaker IFNd genes in theory,
Application aspect is to be applied to genetically engineered fish using the promoter construction of expression vector efficiently expressing exogenous gene or by the promoter
Structure creates condition, with important theoretical and practical significance.
Brief description of the drawings
Fig. 1 is the work using luciferase reporter gene detecting system quantitative analysis large yellow croaker IFNd gene promoters
Property.Abscissa pGL3 represents the relative luciferase activity (as control) of empty carrier pGL3-Basic transfection EPC cells；
PGL3-IFNdP is the relative luciferase activity that recombinant vector pGL3-IFNdP transfects EPC cells.As shown in figure 1, restructuring is carried
Relative luciferase activity in body pGL3-IFNdP transfection EPC cells is the 5.6 of empty carrier pGL3-Basic transfection EPC cells
Times, illustrate that large yellow croaker IFNd gene promoters can preferably start the transcription of luciferase reporter gene.Each experiment sets
Three repetitions, every time repeat set three it is parallel；Error target represents standard error of the mean.
Fig. 2 is in various dose poly (I:C) under incentive condition large yellow croaker IFNd gene promoters activity change.Horizontal seat
Mark is illustrated respectively in 0ng, 50ng, 100ng, 200ng poly (I:C) recombinant vector pGL3-IFNdP transfections EPC under incentive condition
The relative luciferase activity of cell.Ordinate is represented in transfection same dose poly (I:C under the conditions of), recombinant vector pGL3-
Relative luciferase activity transfects the luciferase of EPC cells relative to empty carrier pGL3-Basic in IFNdP transfection EPC cells
The increase multiple of relative activity (being defined as 1).As shown in Fig. 2 in poly (I:C) dosage is 0ng, 50ng, 100ng and 200ng
When, the relative luciferase activity in recombinant vector pGL3-IFNdP transfection EPC cells is empty carrier pGL3-Basic transfections EPC
4.7 times, 5.6 times, 11.8 times and 25.1 times of cell.Illustrate that the large yellow croaker IFNd gene promoters can be by poly (I:C) lure
Lead activation.Each experiment sets three repetitions, every time repeat set three it is parallel.Error target represents standard error of the mean.Using double
Tail T inspections fa calculates the significant difference for the treatment of group and control group, * p<0.05,**p<0.01.
Fig. 3 is the activity change of the large yellow croaker IFNd gene promoters under various concentrations LPS incentive conditions.Abscissa is distinguished
Represent the recombinant vector pGL3- under 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL LPS incentive conditions
IFNdP transfects the relative luciferase activity of EPC cells.Ordinate represented under same concentrations LPS incentive conditions, recombinant vector
Relative luciferase activity is glimmering relative to empty carrier pGL3-Basic transfection EPC cells in pGL3-IFNdP transfection EPC cells
The increase multiple of light element enzyme relative activity (being defined as 1).As shown in figure 3, being 0ng/mL, 100ng/mL, 500ng/ in LPS concentration
When mL, 1000ng/mL, 2000ng/mL, the relative luciferase activity in recombinant vector pGL3-IFNdP transfection EPC cells is
4.6 times, 5.8 times, 7.2 times, 7.4 times and 7.4 times of empty carrier pGL3-Basic transfection EPC cells.The large yellow croaker IFNd genes
Promoter can be by LPS induced activations.*p<0.05,**p<0.01.
Fig. 4 is the activity change of the large yellow croaker IFNd gene promoters under various concentrations PGN incentive conditions.Abscissa is distinguished
Represent the recombinant vector pGL3- under 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL LPS incentive conditions
IFNdP transfects the relative luciferase activity of EPC cells.Ordinate represented under same concentrations PGN incentive conditions, recombinant vector
Relative luciferase activity is glimmering relative to empty carrier pGL3-Basic transfection EPC cells in pGL3-IFNdP transfection EPC cells
The increase multiple of light element enzyme relative activity (being defined as 1).As shown in figure 4, being 0ng/mL, 100ng/mL, 500ng/ in PGN concentration
When mL, 1000ng/mL, 2000ng/mL, the relative luciferase activity in recombinant vector pGL3-IFNdP transfection EPC cells is
4.6 times, 6.1 times, 7.5 times, 6.6 times and 5.6 times of empty carrier pGL3-Basic transfection EPC cells.The large yellow croaker IFNd genes
Promoter can be by PGN induced activations.*p<0.05,**p<0.01.
Fig. 5 is the activity change of the large yellow croaker IFNd gene promoters under various dose bacterial genomes DNA incentive conditions.
Abscissa is illustrated respectively in recombinant vector pGL3- under 0ng, 10ng, 20ng, 50ng, 100ng bacterial genomes DNA incentive conditions
IFNdP transfects the relative luciferase activity of EPC cells.Ordinate is represented in transfection same dose bacterial genomes DNA conditions
Under, relative luciferase activity is transfected relative to empty carrier pGL3-Basic in recombinant vector pGL3-IFNdP transfection EPC cells
The increase multiple of the relative luciferase activity (being defined as 1) of EPC cells.As shown in figure 5, being in bacterial genomes DNA dosage
When 0ng, 10ng, 20ng, 50ng, 100ng, the relative luciferase activity in recombinant vector pGL3-IFNdP transfection EPC cells
It is 4.4 times, 4.5 times, 6.9 times, 10.2 times and 10.7 times of empty carrier pGL3-Basic transfection EPC cells.The large yellow croaker IFNd
Gene promoter can be by bacterial genomes DNA induced activations.*p<0.05,**p<0.01.
Specific embodiment
Following examples will the invention will be further described with reference to accompanying drawing.
1. the clone of large yellow croaker IFNd gene promoters
1.1 build 4 kinds of limitations respectively using the GenomeWalker Universal Kit kits of Clontech companies
Property restriction endonuclease large yellow croaker genome single endonuclease digestion library, 4 kinds of DNA endonucleases used are Dra I, EcoR V, Pv μ II and
StμI.Concrete operations are：
1.1.1 genome purity analysis.
1. 1 μ L large yellow croakers genomic DNAs electrophoresis on 0.6% Ago-Gel is taken.
2. endonuclease Dra I trial cuts are used.System is：
1.1.2 genome is digested.
1. centrifuge tube DL1, DL2, DL3, DL4 and a positive control of 5 1.5mL of mark.
2. each endonuclease digestion system component includes：
3. 5~10s of low speed vortex and brief centrifugation, 37 DEG C of digested overnights.
1.1.3 Genomic DNA Purification.
1. isometric phenol is added in above-mentioned each reaction tube.
2. 5~10s of low speed vortex and brief centrifugation.
3. upper strata aqueous phase is transferred in another 1.5mL centrifuge tube, and adds isometric chloroform.
4. 5~10s of low speed vortex and brief centrifugation, in transfer upper strata aqueous phase to a new centrifuge tube, and are added to 2 to water
95% ethanol of times volume precooling, the sodium acetate (pH4.5) of 1/10 volume and the glucosides of 20 μ g.
5. 5~10s of low speed vortex and under the conditions of 4 DEG C with 14000r/min be centrifuged 15min.
6. abandon supernatant and washed with 80% ethanol of 100 μ L precoolings, be then centrifuged with 14000r/min under the conditions of 4 DEG C
10min。
7. abandon supernatant and dissolved with 20 μ L TE buffer solutions (pH7.5) after drying in atmosphere.
1.1.4 joint Adaptor is connected on the genomic fragment digested respectively through above-mentioned 4 kinds of restriction enzymes.
1. linked system：
2. condition of contact：16 DEG C overnight connect.
3. condition of contact is terminated：70℃ 5min.
4. often pipe adds 75 μ L TE buffer solutions (pH7.5), that is, obtain 4 kinds of large yellow croaker genome lists of restriction enzyme
Digestion library, for the clone of promoter.
1.2 amplify large yellow croaker IFNd gene promoter sequences using two-wheeled touchdown PCR method, and specific steps are pressed
GenomeWalker Universal Kit kit specifications are carried out.Comprise the following steps that：
1.2.1 first round touchdown PCR reaction system：
PCR programs：
1.2.2 second touchdown PCR is taken turns:Respectively with first round PCR primer as template, using nido adapter-primer AP2 and base
Because special primer GSP2 is carried out, other compositions and reaction system are identical with the first round.
PCR programs：
The PCR primer that 1.3 amplifications are obtained is connected to TaKaRa companies pMD18T-Simple vector carriers carries out sequence
Determine and assemble, so as to obtain the large yellow croaker IFNd gene promoter sequences.
2. the activity analysis of large yellow croaker IFNd gene promoters
The structure of the restructuring luciferase reporter gene carrier pGL3-IFNdP of 2.1 promoter gene fragments of IFNd containing large yellow croaker
Build
By large yellow croaker IFNd promoter gene fragments insertion Promega companies luciferase reporter gene carriers pGL3-
In Basic, the expression of firefly luciferase (Luciferase) reporter gene is set to be controlled by IFNd promoters, what structure was obtained
Recombinant vector is named as pGL3-IFNdP.
Specific steps：
1. synthesis carries the forward primer of Xho I restriction enzyme sites：5'-CCGCTCGAGTTCATGCTCAGGTAA-3', carries
The reverse primer in Hind III digestions site：5'-CCCAAGCTTAACTGCAATGCTGAAT-3'.
2. enter performing PCR with Takara companies PrimeSTAR HS DNA Polymerase polymerase systems to expand.
PCR reaction systems are：
PCR programs are as follows：
3. PCR primer recovery is carried out with Omega companies glue reclaim kit.
4. PCR primer and carrier pGL3-Basic after reclaiming carry out Xho I/Hind III double digestions respectively.Digestion body
System：
Digestion condition：37 DEG C of reactions are overnight.
5. it is separately recovered above-mentioned PCR primer and carrier through Xho I/Hind III double digestions with glue reclaim kit
PGL3-Basic, and connected through the PCR primer and carrier pGL3-Basic of double digestion, connector with the ligase of Takara company's Ts 4
System：
Reaction condition：16 DEG C of reactions are overnight.
6. above-mentioned connection product conversion E. coli DH5 α competent cells, positive gram is screened through bacterium colony PCR
It is grand, plasmid is extracted with mini-scale plasmid kit, and confirm the correctness that promoter fragment is inserted through sequencing, so as to obtain containing rheum officinale
The recombinant vector pGL3-IFNdP of fish IFNd promoter gene fragments.
2.2 Basal activities that large yellow croaker IFNd gene promoters are analyzed using luciferase reporter gene detecting system
1. the preferable EPC cells of state are seeded to (1 × 10 in 96 orifice plates5/ hole), L15 culture mediums are added, it is transferred to constant temperature
Incubated overnight in 28 DEG C of incubator, makes its adherent and recovers to the state of exponential phase.
L15 culture medium prescriptions：L15 basal mediums, 10%Gibco Australia hyclone.
2. 2h changes cell culture medium before transfecting.During transfection, 30 μ L L15 culture mediums, 0.6 μ g are added in 1.5mL Ep pipes
Recombinant plasmid pGL3-IFNdP, 6ng renilla luciferase internal reference Reporter gene vector pRL-TK and 12 μ LHD turns
Transfection reagent (Promega) simultaneously blows and beats incubation at room temperature 15min cotransfection EPC cells (three parallel amounts) after mixing.Meanwhile, use
The EPC cells of 0.6 μ g empty carrier pGL3-Basic and 6ng pRL-TK cotransfections are used as control.
Transfectional cell is collected after 48h, firefly luciferase is read respectively with luciferase reporter gene detecting system
With the enzymatic activity value of renilla luciferase, the phase of luciferase in transfectional cell is drawn by the ratio for calculating the two enzymatic activity value
To activity.Meanwhile, the EPC cells using the common transfection of empty carrier pGL3-Basic and pRL-TK calculate the startup as control
The relative activity of son.The method of luciferase enzyme activity determination is with reference to the double luciferase report gene detecting systems of Promega companies
Specification is carried out, and is concretely comprised the following steps：
3. after transfection EPC cells 48h, upper strata culture medium is sucked, with PBS washed cell 2 times；
4. 100 μ L PLB lysates (Passive Lysis Buffer, kit is provided) are added to split at room temperature per hole
Solution cell 15min, period is light and slow to rock culture plate, makes its cracking complete, and cell lysate supernatant is collected by centrifugation；
5. 100 μ L luciferases test agent II LARII and the 20 above-mentioned cell lysate supernatants of μ L are mixed in detection pipe
Close, the activity of firefly luciferase is measured with chemiluminescence detector Luminometer TD 20/20 immediately；
6. to 100 μ L Stop＆Glo reagents are added in detection pipe, by above-mentioned reaction quenching, while starting sea pansy fluorescein
Enzyme reaction, measurement renilla luciferase activity；
7. the enzymatic activity value of firefly luciferase and renilla luciferase is read respectively, calculates the ratio of the two enzymatic activity value
It is worth the relative activity of luciferase in transfectional cell.Meanwhile, transfected jointly with empty carrier pGL3-Basic and pRL-TK
EPC cells calculate the relative activity (Fig. 1) of the promoter as control.
2.3 immunostimulations are tested
Two schemes are implemented in immunostimulation experiment altogether：1) by above-mentioned recombinant vector pGL3-IFNdP, pRL-TK from different doses
Poly (the I of amount:C) or bacterial genomes DNA cotransfection EPC cells, transfectional cell is collected after 48h.2) above-mentioned recombinant vector
After pGL3-IFNdP, pRL-TK transfection EPC cells 6h, LPS and PGN to different final concentrations are separately added into cell culture medium,
Transfectional cell is collected after 36h.
The activity change of large yellow croaker IFNd gene promoters is referring to Fig. 3 under various concentrations LPS incentive conditions.It is dense in LPS
Spend during for 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, recombinant vector pGL3-IFNdP transfections EPC
Relative luciferase activity in cell is 4.6 times of empty carrier pGL3-Basic transfection EPC cells, 5.8 times, 7.2 times, 7.4
Times and 7.4 times.The large yellow croaker IFNd gene promoters can be by LPS induced activations.*p<0.05,**p<0.01.
The activity change of large yellow croaker IFNd gene promoters is referring to Fig. 4 under various concentrations PGN incentive conditions.It is dense in PGN
Spend during for 0ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, recombinant vector pGL3-IFNdP transfections EPC
Relative luciferase activity in cell is 4.6 times of empty carrier pGL3-Basic transfection EPC cells, 6.1 times, 7.5 times, 6.6
Times and 5.6 times.The large yellow croaker IFNd gene promoters can be by PGN induced activations.*p<0.05,**p<0.01.
Calculate the medicine irritation bar in various dose or concentration respectively with above-mentioned luciferase reporter gene detecting system
Relative luciferase activity in transfectional cell under part, and compared with relative luciferase activity in unprocessed transfectional cell.
From Fig. 2 and Fig. 5 it can be found that the poly (I of various dose:) and the stimulation of bacterial genomes DNA can significantly increase C
The relative luciferase activity of strong transfectional cell, illustrates that large yellow croaker IFNd gene promoters can be by poly (I:C)、LPS、PGN
And bacterial genomes DNA induced activations.
Sequence table
<110>State Oceanic Administration Bureau The Third Oceanography Institute
<120>Crocea interferon d gene promoter sequences and its application
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1005
<212> DNA
<213>Large yellow croaker< Larimichthys crocea>
<400> 1
caaagttccc acgaagtcct atcttatcta aagacacgtg tctgtcaaca ctgcagcact 1
tcagtgactc ccagccctca tatgacacag tgctgatttg tttcaggtgt tctgatgaag 61
agctggagga gaatcccctc ctgcacagac agactgcaga cactgcagca gcagcagcag 121
cttcatgctc tcaggtaaga caacgtgaaa tgtcacgcag ctctcatcaa tgaacgtcag 181
gccacagtga cggctcactt tcattttctc tgcagggaaa cttccgcttt atggctcctc 241
ttcctcattc atcaccaatc aacacaagtc atgatgcaag tcaattaaac acaaacagac 301
gtgatgacga cattaaaaca tacgtaatca tcataaatat gaaatactgt aaactgatta 361
aattcaacag ataaactaaa gtttaaatca ttatttatga aggtgatttt aattcaagga 421
tcatttacag tcattttcta tttgatctga aatattccga taataatcca taaattattg 481
aaataagtta ctcaaataac agtgagctgt gtttatcttt atgtttttaa ttttctatgt 541
tttttaaatt atgttttaac tttgctgtta ttgttgttca tgcttttatt ttgtgacatt 601
tcatattccc aggaacatta attcaaatcc gaaatgtgtg acctgggttt gagccgcgtt 661
agccacagtc cacgtcgcga ttcccacagg tgaagtttct gtgtttgttt caaatgatgt 721
tttaactttg ttgttcatgt ttttattttg tgacatttca tattcccagg aataattcaa 781
atccgaagtg tgtgggctgg tagattccca caggtggatg tgaggaaaat gaaataggtg 841
cgtcctgcta cagtataaat gagccgctgc aggtgagttt gaacacaaca cctggacaca 901
cccgactttt gccactcaaa gacactttgt ctgtttgtaa agatg 961
Claims (5)
1. large yellow croaker IFNd gene promoters, it is characterised in that its sequence is：
2. activity assays of large yellow croaker IFNd gene promoters as claimed in claim 1, it is characterised in that including following step
Suddenly：
1) using large yellow croaker IFNd gene promoters described in the luciferase reporter gene detecting system quantitative analysis of Promega companies
The active and artificial synthesized double-stranded RNA poly (I of son:C), e. coli lipopolysaccharide LPS, bacillus subtilis peptide glycan PGN
Stimulate the influence to its activity with Aeromonas hydrophila genomic DNA；
2) by large yellow croaker IFNd gene promoter areas fragment insertion Promega companies luciferase reporter gene carrier
In pGL3-Basic, firefly luciferase gene is set to be located under the control of the promoter, structure obtains recombinant vector pGL3-
IFNdP；EPC cells, 48h are transfected jointly with recombinant vector pGL3-IFNdP and Renilla luciferase reporter genophore pRL-TK
After collect transfectional cell；Firefly luciferase and sea pansy fluorescence are detected respectively using luciferase reporter gene detecting system
The enzymatic activity value of plain enzyme, the relative activity of luciferase in transfectional cell is drawn by the ratio for calculating the two enzymatic activity value；
3) EPC cells are transfected with empty carrier pGL3-Basic and renilla luciferase control Reporter gene vector pRL-TK jointly
Relative luciferase activity calculates the relative activity of the promoter as control, then with poly (I:C), lipopolysaccharides LPS, peptide
Glycan PGN and bacterial genomes DNA stimulates the EPC cells that pGL3-IFNdP is transfected, by analyzing each treatment group luciferase phase
To activity change come determine large yellow croaker IFNd gene promoters activity whether stimulated thing induction influence.
3. large yellow croaker IFNd gene promoters as claimed in claim 1, it is characterised in that the mirror of its clone and its promoter activity
It is fixed, applied in the expression regulation mechanism experiment system for building large yellow croaker IFNd genes.
4. large yellow croaker IFNd gene promoters as claimed in claim 1 are dynamic in fish cell or lactation in structure carrier for expression of eukaryon
Applied in efficiently expressing exogenous gene in thing cell.
5. large yellow croaker IFNd gene promoters as claimed in claim 1 are applied in genetically engineered fish is built.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326025A (en) * | 2017-06-16 | 2017-11-07 | 集美大学 | A kind of crocea interferon IFN1 promoters, nucleic acid construct, cell and its production and use |
| CN113088522A (en) * | 2021-04-29 | 2021-07-09 | 集美大学 | Japanese eel transcription factor c-Rel gene promoter and application thereof |
| CN113201541A (en) * | 2021-04-25 | 2021-08-03 | 集美大学 | Japanese eel transcription factor AP-1 gene promoter and application thereof |
| CN113717973A (en) * | 2021-08-17 | 2021-11-30 | 集美大学 | Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof |
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2016
- 2016-12-15 CN CN201611159343.8A patent/CN106754923B/en active Active
Non-Patent Citations (2)
| Title |
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| DING Y等: "GenBank 登录号：KU144880.1", 《NCBI》 * |
| YANG DING 等: "Identification of Two Subgroups of TypeⅠIFNs in Perciforme Fish Large Yellow Croaker Larimichthys crocea Provides Novel Insights into Function and Regulation of Fish TypeⅠIFNs", 《FRONTIERS IN IMMUNOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326025A (en) * | 2017-06-16 | 2017-11-07 | 集美大学 | A kind of crocea interferon IFN1 promoters, nucleic acid construct, cell and its production and use |
| CN113201541A (en) * | 2021-04-25 | 2021-08-03 | 集美大学 | Japanese eel transcription factor AP-1 gene promoter and application thereof |
| CN113201541B (en) * | 2021-04-25 | 2023-08-15 | 集美大学 | Japanese eel transcription factor AP-1 gene promoter and application thereof |
| CN113088522A (en) * | 2021-04-29 | 2021-07-09 | 集美大学 | Japanese eel transcription factor c-Rel gene promoter and application thereof |
| CN113088522B (en) * | 2021-04-29 | 2023-07-14 | 集美大学 | Japanese eel transcription factor c-Rel gene promoter and application thereof |
| CN113717973A (en) * | 2021-08-17 | 2021-11-30 | 集美大学 | Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof |
| CN113717973B (en) * | 2021-08-17 | 2023-10-24 | 集美大学 | Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and application thereof |
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