CN108893491A - A method of screening CREB signal path agonist or inhibitor - Google Patents
A method of screening CREB signal path agonist or inhibitor Download PDFInfo
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Abstract
A kind of method of screening CREB signal path agonist or inhibitor the invention discloses CREB signal path luciferase reporter gene carrier and based on the carrier.The present invention is by the way that HTLV-1 virus LTR regional sequence segment to be cloned into luciferase reporter gene carrier, a kind of CREB signal path luciferase reporter gene carrier is made, it can be used for detecting CREB signal path activity, can be used for the agonist or inhibitor of screening CREB signal path.The fucoidin of Targeted-control CREB signal path is screened simultaneously, and fucoidin negative regulation inhibits CREB Pathway Activation.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of screening CREB signal path agonist or inhibitor
Method.
Background technique
CREB (cAMP response element-binding protein, cyclic adenosine monophosphate response element combination egg
It is white) be a kind of Intracellular transcription factor, by be integrated to cyclic adenosine monophosphate response element (cAMP response element,
CRE enhance the expression of downstream gene on).CREB signal path regulates and controls numerous vital movements, including tissue development, memory shape
At, circadian rhythm etc..The abnormal generation that will lead to the diseases such as cancer, alzheimer's disease of the activity of CREB signal path, and
It may finally lead to death.Therefore, sensitive to reliably detect CREB signal path activity and artificial adjustment CREB signal path
Activity all has huge value for scientific research and clinical application.
HTLV-1 (Human T-lymphotropic virus 1, I type of human T-leukemia virus) is a kind of c-type
RNA retrovirus causes global ten thousand people of 1500-2000 to infect, to cause the diseases such as adult T cell leukemia (ATL).
There are three CRE in region by the LTR (long terminal repeat, long end repeat) of HTLV-1 genome, can be with host
CREB in conjunction with and originate transcription to realizing self-replacation.Meanwhile HTLV-1 genome encoding Tax albumen can activate host
CREB signal path and NF- κ B signal access are to promote viral own survival and breeding.Therefore, the region HTLV-1LTR is
One sensitive CREB signal path responds original part, and Tax albumen is an efficient CREB signal path activator.
Polysaccharide is a kind of macromolecule biological activity substance being made of carbohydrate.In addition to directly providing energy, very much
Polysaccharide has significant physiological function because of its unique property.Existing research surface, polysaccharide is in strengthen immunity, anti-swollen
Tumor, anti-oxidant, antiviral, reducing blood lipid aspect all have ideal effect.Algal polysaccharides are the distinctive carbon aquations of a kind of algae
Object is closed, usually there are some special physiological functions due to its distinguished structure.It is first to occupy the whole world for China's seaweed abundance
Position, therefore also have algal polysaccharides resource abundant.Make full use of algal polysaccharides that will generate huge economy and society
Value, the novel physiological function of excavating algal polysaccharides have potential scientific meaning and application value.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provides a kind of screening CREB signal path agonist or inhibition
The method of agent obtains a HTLV-1 virus LTR regional sequence segment by screening and cloning, can rise in conjunction with host CREB
Transcription begin to realize self-replacation, which is built into a luciferase reporter gene carrier system
System, can be used for screening the agonist or inhibitor of CREB signal path.
A kind of CREB signal path luciferase reporter gene carrier, including firefly luciferase reporter gene and
HTLV-1 virus LTR regional sequence segment, the HTLV-1 virus LTR regional sequence segment are located at firefly luciferase report
Upstream region of gene and firefly luciferase reporter gene expression can be started after CREB protein activation.
The sequence of the HTLV-1 virus LTR regional sequence segment is as shown in SEQ ID No.1.The sequence is sick from HTLV-1
It clones and obtains in virus gene group, include three CRE, transcription can be originated in conjunction with host CREB to realize self-replacation.
The CREB signal path luciferase reporter gene carrier, sequence is as shown in SEQ ID No.2.The sequence
Skeleton including pGL4.20 [Luc2/Puro] plasmid (Promega company), contains restriction enzyme BglII and HindIII
Above-mentioned HTLV-1 virus LTR regional sequence segment is connected to pGL4.20 [Luc2/ by digestion method by restriction enzyme site
Puro] plasmid BglII and HindIII between, obtain carrier be named as pGL4.20-HTLV-1-LTR-2.
The present invention also provides a kind of methods of screening CREB signal path agonist or inhibitor, include the following steps：
(1) the CREB signal path luciferase reporter gene carrier and internal reference carrier, positive control vector is same
When transfection cell after cultivate, the internal reference carrier includes the Renilla luciferase reporter gene of sustainable stable expression, the sun
Property control vector include that expression product is capable of the tax gene of activating ELK 1 B signal access；
(2) continue to cultivate after substance to be screened is added into cell culture medium；
(3) uciferase activity for detecting each groups of cells, if the groups of cells uciferase activity ratio of substance to be screened is added
Not plus the groups of cells of substance to be screened is strong, then the substance to be screened is CREB signal path agonist；If substance to be screened is added
Groups of cells uciferase activity than not plus substance to be screened groups of cells it is weak, then the substance to be screened be CREB signal path press down
Preparation.
The internal reference carrier is pRL-CMV, and sequence is as shown in SEQ ID No.3.Internal reference carrier pRL-CMV is purchased from
Promega company includes Renilla luciferase reporter gene, can sustainedly and stably give expression to renilla luciferase, be used for
Error is eliminated as internal reference.
The positive control vector is pcDNA3.1-Tax, and sequence is as shown in SEQ ID No.4.The positive control vector is
Tax gene insertion pcDNA3.1 plasmid (pcDNA3.1 plasmid is purchased from Promega company) in HTLV-1 genome is obtained,
The Tax albumen of tax gene coding in HTLV-1 genome can activate host CREB signal path to promote virus itself
Survival and breeding, since host cell itself CREB signal path activation level is lower, so the CREB signal path is double
After in luciferase reporter gene vector introduction host cell, the expression of firefly luciferase reporter gene has very much
Limit, it is not easy to detect, by importing tax gene expression Tax albumen again, host's CREB signal path can be activated, to make firefly
The expression of fireworm luciferase reporter gene is greatly improved, and convenient for detection, reduces detection error.
Application the present invention also provides fucoidin as CREB signal pathway inhibitor.The use of the fucoidin
Concentration is 1 μ g/mL -1mg/mL.The present invention screens a variety of polysaccharide using the above method, and finally screening obtains fucoidin to CREB
Signal path is inhibited, can be used as the inhibitor of CREB signal path.
The present invention by the way that HTLV-1 virus LTR regional sequence segment is cloned into luciferase reporter gene carrier,
A kind of CREB signal path luciferase reporter gene carrier is made, can be used for detecting CREB signal path activity, it can also
With the agonist or inhibitor for screening CREB signal path.The rock algae of Targeted-control CREB signal path is screened simultaneously
Polysaccharide, fucoidin negative regulation inhibit CREB Pathway Activation.
Detailed description of the invention
Fig. 1 is HTLV-1LTR region segments PCR result electrophoresis detection figure, wherein swimming lane 1 indicates DNA Marker
DL2000 (TaKaRa), swimming lane 2 indicate that PCR product, arrow meaning indicate the area LTR of target fragment HTLV-1 genome.
Fig. 2 is the structural schematic diagram of pGL4.20 carrier.
Fig. 3 is carrier pGL4.20-HTLV-1-LTR-2 bacterium colony PCR result electrophoresis detection figure, wherein swimming lane 1 indicates DNA
Marker DL2000 (TaKaRa), swimming lane 2-7 indicate that the bacterium colony PCR qualification result of single colonie, arrow indicate correct target patch
The area LTR of section HTLV-1 genome (2-5 is correct, 6-7 mistake).
Fig. 4 is the selection result figure of the fucoidin as CREB signal pathway inhibitor.
Fig. 5 is that fucoidin inhibits CREB signal path specificity verification result figure.
Specific embodiment
Embodiment 1
According to the genome sequence (ID of the HTLV-1 in GenBank：9632565) area Zhong LTR uses Primer
Premier5.0 software design PCR primer, amplimer sequence are as follows：
Upstream primer FP1：5'-GCCAGATCTTGACAATGACCATGAGCCC-3'；
Downstream primer RP1：5'-GGCAAGCTTCTCCTGAACTGTCTCCACGC-3'.
Above-mentioned primer is synthesized by Shanghai Sheng Gong Biotechnology Co., Ltd.Target patch segment DNA size is 353bp.
Firstly, carrying out PCR amplification, (Tiangeng biological cell genome extraction kit is used using C8166 cellular genome
(DP304) extract) it is used as PCR amplification template, reaction system is as follows, and system total volume is 50 μ L：
Response procedures are：98 DEG C of 2min initial denaturations；98 DEG C of denaturation 10s, 60 DEG C of annealing 10s, 72 DEG C of extension 30s in recycling,
30 circulations；Continue to extend 3min for 72 DEG C after circulation；Last 4 DEG C of preservations.
PCR, which has reacted, takes 5 μ L PCR products to carry out 2% agarose electrophoretic analysis, as a result as shown in Figure 1,1 table of swimming lane in figure
Show that DNA Marker DL2000 (TaKaRa), swimming lane 2 indicate PCR product, arrow indicates target fragment HTLV-1 genome
The area LTR.
Then, the correct PCR product of verifying is purified, with two kinds of restriction endonuclease BglII and HindIII to above-mentioned PCR
Product carries out double digestion, and endonuclease reaction system is as follows, and system total volume is 50 μ L：
Response procedures are：In 37 DEG C of digestion 1h, recovery purifying is then carried out.
Embodiment 2
The structural schematic diagram of pGL4.20 [Luc2/Puro] carrier (Promega company) as shown in Fig. 2, carrier containing restricted
Property restriction endonuclease BglII and HindIII restriction enzyme site.Above-mentioned PCR product is carried out with two kinds of restriction endonuclease BglII and HindIII double
Digestion, endonuclease reaction system is as follows, and system total volume is 50 μ L：
Response procedures are：In 37 DEG C of digestion 1h, recovery purifying is then carried out.
Embodiment 3
Target patch segment DNA and carrier that embodiment 1 and the processing of 2 double digestion of embodiment are prepared are attached reaction,
Coupled reaction system is as follows, and system total volume is 10 μ L：
Response procedures are：In 16 DEG C of connection 1h.
Connection product is transformed into competent cell, row bacterium colony PCR identification is dropped into positive colony bacterium, as a result such as Fig. 3 institute
Showing, swimming lane 1 indicates DNA Marker DL2000 (TaKaRa) in figure, and swimming lane 2-7 indicates the bacterium colony PCR qualification result of single colonie,
Arrow indicates the area LTR of correct target fragment HTLV-1 genome (2-5 is correct, 6-7 mistake)；Further, electrophoresis is examined
It is correct to survey correct bacterium colony progress sequence verification, is named as pGL4.20-HTLV-1-LTR-2.
Embodiment 4
CREB signal path agonist or inhibitor screening.
Screening technique is as follows：
Cell (293T cell) culture is to carry out under normal conditions, 37 DEG C of temperature, CO2Concentration 5%, cell type are
293T cell.Logarithmic growth phase is grown to cell, is counted with cell counting board, is inoculated in 96 orifice plates by required cell concentration, is trained
Support the transfection for carrying out cell afterwards for 24 hours.
25 μ L OPTI-MEM culture mediums are taken to dilute 5ng pGL4.20-HTLV-1-LTR-2,1ng pRL-CMV (is purchased from
Promega, sequence is as shown in SEQ ID No.3) and 100ng pcDNA3.1 (being purchased from Promega) empty carrier, 25 μ L OPTI-
MEM culture medium dilutes 0.25 μ L Lipofectamine, 2000 reagent, and the mixing of 5min latter three, totally 50 μ L, gently shakes up, quiet
Set 20min.Culture cell per well first siphons away 50 μ L culture mediums, then adds above-mentioned 50 μ L mixed liquor, final every hole total volume
100μL.Every group sets 3 multiple holes.Add 100 μ L fresh cultures and prescribed concentration to be screened (1 μ g/mL -1mg/ after 6h again
ML) natural products.
Each experimental group sets 3 parallel holes, and every group of experiment is repeated 3 times.Transfection cell is grouped as follows：
1. negative control group：The natural products being added is replaced with equivalent PBS；
2. positive controls：The natural products being added is replaced, with equivalent PBS with pcDNA3.1-Tax (sequence such as SEQ ID
Shown in No.4) positive control vector replace pcDNA3.1 empty carrier；
3. experimental group：On the basis of 2., prescribed concentration (1 μ g/mL -1mg/mL) natural products is added.
After each transfection group culture for 24 hours, vitellophag detects the uciferase activity of each group.
Luciferase assays use Dual-Luciferase detection kit (Invitrogen company), by specification behaviour
Make：The luciferase substrate of kit and buffer are mixed into luciferase reagent packing, -80 DEG C of storages first.stop&
Glo buffer with stop&Glo substrate is mixed into stop&Glo reagent after restoring to room temperature, ready-to-use.After cell transfecting 48h
Culture medium is sucked out, fresh culture is added with 35 holes μ L/, is firstly added equivalent luciferase reagent, vibrates 10min, measures glimmering
Light value adds 30 μ L stop&Glo reagents, vibrates 10min, measures fluorescent value with fluorescence illumination photometer.
All data are indicated with mean+SD.Statistical analysis uses 17.0 software of SPSS, has between two groups significant
Difference is expressed as P<0.05, there is extremely significant difference to be expressed as P between two groups<0.01.
As a result as shown in figure 4, we have observed that, comparing negative control group, positive control from luciferase assays result
Group CREB signal path has about 80 times of activation, and (be purchased from Shaanxi source Thailand biotechnology has low concentration (1 μ g/mL) fucoidin
Limit company, average relative molecular mass 189kDa, purity 99%) activation is suppressed to 62 times or so (P<0.05), high concentration
Activation is suppressed to 50 times or so (P by (1mg/mL) fucoidin<0.01), statistically there is significant difference.Illustrate rock algae
Polysaccharide can be used as CREB signal pathway inhibitor.
Embodiment 5
Fucoidin inhibits CREB signal path specificity verification.
Screening technique is as follows：
Cell (293T cell) culture is to carry out under normal conditions, 37 DEG C of temperature, CO2Concentration 5%, cell type are
293T cell.Logarithmic growth phase is grown to cell, is counted with cell counting board, is inoculated in 96 orifice plates by required cell concentration, is trained
Support the transfection for carrying out cell afterwards for 24 hours.
Take 25 μ L OPTI-MEM culture mediums dilution 5ng Reporter gene vector (pGL4.20-HTLV-1-LTR-2 or
PGL4.20-NF- κ B (sequence is as shown in SEQ ID No.7)), 1ng pRL-CMV and 100ng pcDNA3.1-Tax, 25 μ L
OPTI-MEM culture medium dilutes 0.25 μ L Lipofectamine, 2000 reagent, and the mixing of 5min latter three, totally 50 μ L, gently shakes
It is even, stand 20min.Culture cell per well first siphons away 50 μ L culture mediums, then adds above-mentioned 50 μ L mixed liquor, and final every hole is total
100 μ L of volume.Every group sets 3 multiple holes.Add 100 μ L fresh cultures and 1mg/mL fucoidin after 6h again.
Each experimental group sets 3 parallel holes, and every group of experiment is repeated 3 times.Transfection cell is grouped as follows：
1. Reporter gene vector is pGL4.20-HTLV-1-LTR-2, the fucoidin being added is replaced with equivalent PBS；
2. 1mg/mL fucoidin is added on the basis of 1.；
3. Reporter gene vector is changed to pGL4.20-NF- κ B on the basis of 1.)；
4. 1mg/mL fucoidin is added on the basis of 3..
After each transfection group culture for 24 hours, vitellophag detects the uciferase activity of each group.
Luciferase assays use Dual-Luciferase detection kit (Invitrogen company), by specification behaviour
Make：The luciferase substrate of kit and buffer are mixed into luciferase reagent packing, -80 DEG C of storages first.stop&
Glo buffer with stop&Glo substrate is mixed into stop&Glo reagent after restoring to room temperature, ready-to-use.After cell transfecting 48h
Culture medium is sucked out, fresh culture is added with 35 holes μ L/, is firstly added equivalent luciferase reagent, vibrates 10min, measures glimmering
Light value adds 30 μ L stop&Glo reagents, vibrates 10min, measures fluorescent value with fluorescence illumination photometer.
All data are indicated with mean+SD.Statistical analysis uses 17.0 software of SPSS, has between two groups significant
Difference is expressed as P<0.05, there is extremely significant difference to be expressed as P between two groups<0.01.
Tax albumen is in addition to can be with activating ELK 1 B signal access, moreover it is possible to activate NF- κ B signal access.As a result as shown in figure 5,
1mg/mL fucoidin can significantly inhibit the activation of CREB signal path, but not have active effects to NF- κ B signal pathway activity,
Illustrate that fucoidin is direct inhibition CREB signal path rather than is pressed down indirectly by inhibiting tax gene or Tax protein active
CREB signal path processed.
Sequence table
<110>Zhejiang Prov Industrial And Commercial University
<120>A method of screening CREB signal path agonist or inhibitor
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 352
<212> DNA
<213>I type of human T-leukemia virus (Human T-lymphotropic virus 1)
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gacaatgacc atgagcccca aatatccccc gggggcttag agcctcctag tgaaaaacat 60
ttccgagaaa cagaagtctg aaaaggtcag ggcccagact aaggctctga cgtctccccc 120
cggaggacag ctcagcaccg gctcgggcta ggccctgacg tgtccccctg aagacaaatc 180
ataagctcag acctccggga agccaccggg aaccacctat ttcctcccta tgtttgtcaa 240
gccgtcctca ggcgttgacg acaacccctc acctcaaaaa aaacttttca tggcacgcat 300
atggctcaat aaactaacag gagtctataa aagcgtggag acagttcagg ag 352
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<213>Artificial sequence (Artificial)
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ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tgacaatgac 60
catgagcccc aaatatcccc cgggggctta gagcctccta gtgaaaaaca tttccgagaa 120
acagaagtct gaaaaggtca gggcccagac taaggctctg acgtctcccc ccggaggaca 180
gctcagcacc ggctcgggct aggccctgac gtgtccccct gaagacaaat cataagctca 240
gacctccggg aagccaccgg gaaccaccta tttcctccct atgtttgtca agccgtcctc 300
aggcgttgac gacaacccct cacctcaaaa aaaacttttc atggcacgca tatggctcaa 360
taaactaaca ggagtctata aaagcgtgga gacagttcag gagaagcttg gcaatccggt 420
actgttggta aagccaccat ggaagatgcc aaaaacatta agaagggccc agcgccattc 480
tacccactcg aagacgggac cgccggcgag cagctgcaca aagccatgaa gcgctacgcc 540
ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat tacctacgcc 600
gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg gctgaataca 660
aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc cgtgttgggt 720
gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga gcgcgagctg 780
ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa agggctgcaa 840
aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat catggatagc 900
aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca tttgccaccc 960
ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac catcgccctg 1020
atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc gcaccgcacc 1080
gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat catccccgac 1140
accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac cacgctgggc 1200
tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga gctattcttg 1260
cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact atttagcttc 1320
ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga gatcgccagc 1380
ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt ccacctacca 1440
ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat cacccccgaa 1500
ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc taaggtggtg 1560
gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg cgtccgtggc 1620
cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct catcgacaag 1680
gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca cttcttcatc 1740
gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc agccgaactg 1800
gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg cctgcccgac 1860
gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa aaccatgacc 1920
gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa gctgcgcggt 1980
ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga cgcccgcaag 2040
atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgta ataattctag 2100
agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa 2160
accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220
ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280
atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2340
tgtggtaaaa tcgataagga tccgtttgcg tattgggcgc tcttccgctg atctgcgcag 2400
caccatggcc tgaaataacc tctgaaagag gaacttggtt agctaccttc tgaggcggaa 2460
agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag 2520
gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag 2580
gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc 2640
cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 2700
atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat 2760
tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcgattctt 2820
ctgacactag cgccaccatg accgagtaca agcctaccgt gcgcctggcc actcgcgatg 2880
atgtgccccg cgccgtccgc actctggccg ccgctttcgc cgactacccc gctacccggc 2940
acaccgtgga ccccgaccgg cacatcgagc gtgtgacaga gttgcaggag ctgttcctga 3000
cccgcgtcgg gctggacatc ggcaaggtgt gggtagccga cgacggcgcg gccgtggccg 3060
tgtggactac ccccgagagc gttgaggccg gcgccgtgtt cgccgagatc ggcccccgaa 3120
tggccgagct gagcggcagc cgcctggccg cccagcagca aatggagggc ctgcttgccc 3180
cccatcgtcc caaggagcct gcctggtttc tggccactgt aggagtgagc cccgaccacc 3240
agggcaaggg cttgggcagc gccgtcgtgt tgcccggcgt agaggccgcc gaacgcgccg 3300
gtgtgcccgc ctttctcgaa acaagcgcac caagaaacct tccattctac gagcgcctgg 3360
gcttcaccgt gaccgccgat gtcgaggtgc ccgagggacc taggacctgg tgtatgacac 3420
gaaaacctgg cgcctaatga tctagaaccg gtcatggccg caataaaata tctttatttt 3480
cattacatct gtgtgttggt tttttgtgtg ttcgaactag atgctgtcga ccgatgccct 3540
tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg 3600
cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc 3660
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3720
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3780
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3840
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3900
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3960
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 4020
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 4080
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 4140
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 4200
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 4260
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 4320
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 4380
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4440
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 4500
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 4560
atctaaagta tatatgagta aacttggtct gacagcggcc gcaaatgcta aaccactgca 4620
gtggttacca gtgcttgatc agtgaggcac cgatctcagc gatctgccta tttcgttcgt 4680
ccatagtggc ctgactcccc gtcgtgtaga tcactacgat tcgtgagggc ttaccatcag 4740
gccccagcgc agcaatgatg ccgcgagagc cgcgttcacc ggcccccgat ttgtcagcaa 4800
tgaaccagcc agcagggagg gccgagcgaa gaagtggtcc tgctactttg tccgcctcca 4860
tccagtctat gagctgctgt cgtgatgcta gagtaagaag ttcgccagtg agtagtttcc 4920
gaagagttgt ggccattgct actggcatcg tggtatcacg ctcgtcgttc ggtatggctt 4980
cgttcaactc tggttcccag cggtcaagcc gggtcacatg atcacccata ttatgaagaa 5040
atgcagtcag ctccttaggg cctccgatcg ttgtcagaag taagttggcc gcggtgttgt 5100
cgctcatggt aatggcagca ctacacaatt ctcttaccgt catgccatcc gtaagatgct 5160
tttccgtgac cggcgagtac tcaaccaagt cgttttgtga gtagtgtata cggcgaccaa 5220
gctgctcttg cccggcgtct atacgggaca acaccgcgcc acatagcagt actttgaaag 5280
tgctcatcat cgggaatcgt tcttcggggc ggaaagactc aaggatcttg ccgctattga 5340
gatccagttc gatatagccc actcttgcac ccagttgatc ttcagcatct tttactttca 5400
ccagcgtttc ggggtgtgca aaaacaggca agcaaaatgc cgcaaagaag ggaatgagtg 5460
cgacacgaaa atgttggatg ctcatactcg tcctttttca atattattga agcatttatc 5520
agggttacta gtacgtctct caaggataag taagtaatat taaggtacgg gaggtattgg 5580
acaggccgca ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa 5640
tcgatagtac taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa 5700
taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc tct 5743
<210> 3
<211> 4079
<212> DNA
<213>Artificial sequence (Artificial)
<400> 3
agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60
tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120
atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180
tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240
tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300
tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360
aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420
caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480
tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540
gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600
tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660
taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag 720
cagagctcgt ttagtgaacc gtcagatcac tagaagcttt attgcggtag tttatcacag 780
ttaaattgct aacgcagtca gtgcttctga cacaacagtc tcgaacttaa gctgcagaag 840
ttggtcgtga ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca 900
atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg 960
tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt tcaattacag 1020
ctcttaaggc tagagtactt aatacgactc actataggct agccaccatg acttcgaaag 1080
tttatgatcc agaacaaagg aaacggatga taactggtcc gcagtggtgg gccagatgta 1140
aacaaatgaa tgttcttgat tcatttatta attattatga ttcagaaaaa catgcagaaa 1200
atgctgttat ttttttacat ggtaacgcgg cctcttctta tttatggcga catgttgtgc 1260
cacatattga gccagtagcg cggtgtatta taccagacct tattggtatg ggcaaatcag 1320
gcaaatctgg taatggttct tataggttac ttgatcatta caaatatctt actgcatggt 1380
ttgaacttct taatttacca aagaagatca tttttgtcgg ccatgattgg ggtgcttgtt 1440
tggcatttca ttatagctat gagcatcaag ataagatcaa agcaatagtt cacgctgaaa 1500
gtgtagtaga tgtgattgaa tcatgggatg aatggcctga tattgaagaa gatattgcgt 1560
tgatcaaatc tgaagaagga gaaaaaatgg ttttggagaa taacttcttc gtggaaacca 1620
tgttgccatc aaaaatcatg agaaagttag aaccagaaga atttgcagca tatcttgaac 1680
cattcaaaga gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt gaaatcccgt 1740
tagtaaaagg tggtaaacct gacgttgtac aaattgttag gaattataat gcttatctac 1800
gtgcaagtga tgatttacca aaaatgttta ttgaatcgga cccaggattc ttttccaatg 1860
ctattgttga aggtgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc 1920
atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc 1980
gagttctcaa aaatgaacaa taattctaga gcggccgctt cgagcagaca tgataagata 2040
cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 2100
aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 2160
caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 2220
caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccaggtggca cttttcgggg 2280
aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 2340
catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 2400
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 2460
tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 2520
ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 2580
ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 2640
cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 2700
ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 2760
tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 2820
gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 2880
ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 2940
aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 3000
acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 3060
tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 3120
cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 3180
gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 3240
taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3300
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3360
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3420
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3480
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3540
cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 3600
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 3660
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 3720
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 3780
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 3840
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 3900
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 3960
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4020
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tggctcgac 4079
<210> 4
<211> 6429
<212> DNA
<213>Artificial sequence (Artificial)
<400> 4
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttatgg cccacttccc agggtttgga cagagtcttc ttttcggata 960
cccagtctac gtgtttggag actgtgtaca aggcgactgg tgccccatct ctgggggact 1020
atgttcggcc cgcctacatc gtcacgccct actggccacc tgtccagagc atcagatcac 1080
ctgggacccc atcgatggac gcgttatcgg ctcagctcta cagttcctta tccctcgact 1140
cccctccttc cccacccaga gaacctctaa gacccttaag gtccttaccc cgccaatcac 1200
tcatacaacc cccaacattc caccctcctt cctccaggcc atgcgcaaat actccccctt 1260
ccgaaatgga tacatggaac ccacccttgg gcagcacctc ccaaccctgt cttttccaga 1320
ccccggactc cggccccaaa acctgtacac cctctgggga ggctccgttg tctgcatgta 1380
cctctaccag ctttcccccc ccatcacctg gcccctcctg ccccatgtga ttttttgcca 1440
ccccggccag ctcggggcct tcctcaccaa tgttccctac aaacgaatag aaaaactcct 1500
ctataaaatt tcccttacca caggggccct aataattcta cccgaggact gtttgcccac 1560
cacccttttc cagcctgcta gggcacccgt cacgctgaca gcctggcaaa acggcctcct 1620
tccgttccac tcaaccctca ccactccagg ccttatttgg acatttaccg atggcacgcc 1680
tatgatttcc gggccctgcc ctaaagatgg ccagccatct ttagtactac agtcctcctc 1740
ctttatattt cacaaatttc aaaccaaggc ctaccacccc tcatttctac tctcacacgg 1800
cctcatacag tactcttcct ttcataattt gcatctccta tttgaagaat acaccaacat 1860
ccccatttct ctacttttta acgaaaaaga ggcagatgac aatgaccatg agccccaaat 1920
atcccccggg ggcttagagc ctctcagtga aaaacatttc cgtgaaacag aagtctgagc 1980
ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 2040
gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 2100
ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 2160
attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 2220
gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg 2280
gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2340
ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2400
tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2460
ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2520
atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2580
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2640
tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2700
tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg 2760
aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 2820
aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 2880
caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 2940
cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 3000
ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 3060
cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg 3120
atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3180
ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 3240
cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 3300
tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt 3360
tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 3420
cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 3480
catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 3540
ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 3600
ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 3660
ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 3720
tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 3780
ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 3840
atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 3900
cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac 3960
caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 4020
ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 4080
atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa 4140
agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 4200
ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 4260
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 4320
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 4380
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 4440
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 4500
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 4560
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 4620
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 4680
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 4740
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 4800
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 4860
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 4920
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 4980
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 5040
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 5100
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 5160
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt 5220
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5280
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5340
ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5400
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5460
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 5520
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 5580
cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 5640
agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 5700
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5760
gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5820
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5880
gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5940
cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6000
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 6060
accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 6120
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 6180
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 6240
caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6300
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6360
gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 6420
cctgacgtc 6429
<210> 5
<211> 28
<212> DNA
<213>Artificial sequence (Artificial)
<400> 5
gccagatctt gacaatgacc atgagccc 28
<210> 6
<211> 29
<212> DNA
<213>Artificial sequence (Artificial)
<400> 6
ggcaagcttc tcctgaactg tctccacgc 29
<210> 7
<211> 5678
<212> DNA
<213>Artificial sequence (Artificial)
<400> 7
ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tagcttgggc 60
tgcaggtcga ggctggggat tcccatcctc gaggctgggg attccccatc tcgaggctgg 120
ggattcccca tctcgaggct ggggattccc catctcgacc gtccatccat tcacagcgct 180
tctataaagg cgccagctga ggcgcctact actccaaccg cgactgcagc gagcaactga 240
gaagactgga tagagccggc ggttccgcga acgagcagtg accgcgctcc cacccagctc 300
tgctctgcag ctcccaccag tgtctctcta gaggatctaa gcttggcaat ccggtactgt 360
tggtaaagcc accatggaag atgccaaaaa cattaagaag ggcccagcgc cattctaccc 420
actcgaagac gggaccgccg gcgagcagct gcacaaagcc atgaagcgct acgccctggt 480
gcccggcacc atcgccttta ccgacgcaca tatcgaggtg gacattacct acgccgagta 540
cttcgagatg agcgttcggc tggcagaagc tatgaagcgc tatgggctga atacaaacca 600
tcggatcgtg gtgtgcagcg agaatagctt gcagttcttc atgcccgtgt tgggtgccct 660
gttcatcggt gtggctgtgg ccccagctaa cgacatctac aacgagcgcg agctgctgaa 720
cagcatgggc atcagccagc ccaccgtcgt attcgtgagc aagaaagggc tgcaaaagat 780
cctcaacgtg caaaagaagc taccgatcat acaaaagatc atcatcatgg atagcaagac 840
cgactaccag ggcttccaaa gcatgtacac cttcgtgact tcccatttgc cacccggctt 900
caacgagtac gacttcgtgc ccgagagctt cgaccgggac aaaaccatcg ccctgatcat 960
gaacagtagt ggcagtaccg gattgcccaa gggcgtagcc ctaccgcacc gcaccgcttg 1020
tgtccgattc agtcatgccc gcgaccccat cttcggcaac cagatcatcc ccgacaccgc 1080
tatcctcagc gtggtgccat ttcaccacgg cttcggcatg ttcaccacgc tgggctactt 1140
gatctgcggc tttcgggtcg tgctcatgta ccgcttcgag gaggagctat tcttgcgcag 1200
cttgcaagac tataagattc aatctgccct gctggtgccc acactattta gcttcttcgc 1260
taagagcact ctcatcgaca agtacgacct aagcaacttg cacgagatcg ccagcggcgg 1320
ggcgccgctc agcaaggagg taggtgaggc cgtggccaaa cgcttccacc taccaggcat 1380
ccgccagggc tacggcctga cagaaacaac cagcgccatt ctgatcaccc ccgaagggga 1440
cgacaagcct ggcgcagtag gcaaggtggt gcccttcttc gaggctaagg tggtggactt 1500
ggacaccggt aagacactgg gtgtgaacca gcgcggcgag ctgtgcgtcc gtggccccat 1560
gatcatgagc ggctacgtta acaaccccga ggctacaaac gctctcatcg acaaggacgg 1620
ctggctgcac agcggcgaca tcgcctactg ggacgaggac gagcacttct tcatcgtgga 1680
ccggctgaag agcctgatca aatacaaggg ctaccaggta gccccagccg aactggagag 1740
catcctgctg caacacccca acatcttcga cgccggggtc gccggcctgc ccgacgacga 1800
tgccggcgag ctgcccgccg cagtcgtcgt gctggaacac ggtaaaacca tgaccgagaa 1860
ggagatcgtg gactatgtgg ccagccaggt tacaaccgcc aagaagctgc gcggtggtgt 1920
tgtgttcgtg gacgaggtgc ctaaaggact gaccggcaag ttggacgccc gcaagatccg 1980
cgagattctc attaaggcca agaagggcgg caagatcgcc gtgtaataat tctagagtcg 2040
gggcggccgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac 2100
aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 2160
tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 2220
tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 2280
taaaatcgat aaggatccgt ttgcgtattg ggcgctcttc cgctgatctg cgcagcacca 2340
tggcctgaaa taacctctga aagaggaact tggttagcta ccttctgagg cggaaagaac 2400
cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 2460
agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 2520
ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 2580
ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 2640
tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag 2700
aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcga ttcttctgac 2760
actagcgcca ccatgaccga gtacaagcct accgtgcgcc tggccactcg cgatgatgtg 2820
ccccgcgccg tccgcactct ggccgccgct ttcgccgact accccgctac ccggcacacc 2880
gtggaccccg accggcacat cgagcgtgtg acagagttgc aggagctgtt cctgacccgc 2940
gtcgggctgg acatcggcaa ggtgtgggta gccgacgacg gcgcggccgt ggccgtgtgg 3000
actacccccg agagcgttga ggccggcgcc gtgttcgccg agatcggccc ccgaatggcc 3060
gagctgagcg gcagccgcct ggccgcccag cagcaaatgg agggcctgct tgccccccat 3120
cgtcccaagg agcctgcctg gtttctggcc actgtaggag tgagccccga ccaccagggc 3180
aagggcttgg gcagcgccgt cgtgttgccc ggcgtagagg ccgccgaacg cgccggtgtg 3240
cccgcctttc tcgaaacaag cgcaccaaga aaccttccat tctacgagcg cctgggcttc 3300
accgtgaccg ccgatgtcga ggtgcccgag ggacctagga cctggtgtat gacacgaaaa 3360
cctggcgcct aatgatctag aaccggtcat ggccgcaata aaatatcttt attttcatta 3420
catctgtgtg ttggtttttt gtgtgttcga actagatgct gtcgaccgat gcccttgaga 3480
gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 3540
atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc 3600
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 3660
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 3720
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 3780
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 3840
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 3900
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 3960
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4020
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4080
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4140
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4200
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4260
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 4320
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4380
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 4440
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 4500
aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca ctgcagtggt 4560
taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg ttcgtccata 4620
gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc atcaggcccc 4680
agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc agcaatgaac 4740
cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc ctccatccag 4800
tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag tttccgaaga 4860
gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat ggcttcgttc 4920
aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg aagaaatgca 4980
gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt gttgtcgctc 5040
atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag atgcttttcc 5100
gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg accaagctgc 5160
tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt gaaagtgctc 5220
atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct attgagatcc 5280
agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac tttcaccagc 5340
gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat gagtgcgaca 5400
cgaaaatgtt ggatgctcat actcgtcctt tttcaatatt attgaagcat ttatcagggt 5460
tactagtacg tctctcaagg ataagtaagt aatattaagg tacgggaggt attggacagg 5520
ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat 5580
agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc 5640
tgtccccagt gcaagtgcag gtgccagaac atttctct 5678
Claims (8)
1. a kind of CREB signal path luciferase reporter gene carrier, which is characterized in that including firefly luciferase report
It accuses gene and HTLV-1 virus LTR regional sequence segment, the HTLV-1 virus LTR regional sequence segment is located at firefly fluorescence
Plain enzyme reporter gene upstream and firefly luciferase reporter gene expression can be started after CREB protein activation.
2. CREB signal path luciferase reporter gene carrier as described in claim 1, which is characterized in that described
The sequence of HTLV-1 virus LTR regional sequence segment is as shown in SEQ ID No.1.
3. CREB signal path luciferase reporter gene carrier as described in claim 1, which is characterized in that sequence is such as
Shown in SEQ ID No.2.
4. a kind of method of screening CREB signal path agonist or inhibitor, which is characterized in that include the following steps：
(1) will as described in claims 1 to 3 is any CREB signal path luciferase reporter gene carrier and internal reference carrier,
Positive control vector is cultivated after transfecting cell simultaneously, and the internal reference carrier includes the renilla luciferase report of sustainable stable expression
Gene is accused, the positive control vector includes the tax gene that expression product is capable of activating ELK 1 B signal access；
(2) continue to cultivate after substance to be screened is added into cell culture medium；
(3) uciferase activity of each groups of cells is detected, if the groups of cells uciferase activity of substance to be screened is added than not adding
The groups of cells of substance to be screened is strong, then the substance to be screened is CREB signal path agonist；If the thin of substance to be screened is added
Born of the same parents organize uciferase activity than not adding the groups of cells of substance to be screened weak, then the substance to be screened is the inhibition of CREB signal path
Agent.
5. method as claimed in claim 4, which is characterized in that the internal reference carrier is pRL-CMV, sequence such as SEQ ID
Shown in No.3.
6. method as claimed in claim 4, which is characterized in that the positive control vector is pcDNA3.1-Tax, and sequence is such as
Shown in SEQ ID No.4.
7. application of the fucoidin as CREB signal pathway inhibitor.
8. the use as claimed in claim 7, which is characterized in that the use concentration of the fucoidin is 1 μ g/mL -1mg/mL.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107893085A (en) * | 2017-12-19 | 2018-04-10 | 浙江大学 | A kind of luciferase reporter gene plasmid and its structure available for lonely nuclear receptor activities regulator screening is with applying |
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2018
- 2018-06-12 CN CN201810599982.9A patent/CN108893491A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107893085A (en) * | 2017-12-19 | 2018-04-10 | 浙江大学 | A kind of luciferase reporter gene plasmid and its structure available for lonely nuclear receptor activities regulator screening is with applying |
Non-Patent Citations (4)
| Title |
|---|
| ABDIZADEH, MF等: "Examination of a Reporter Vector for HTLV-1 Infectivity Using MT2, a HTLV-1 Producer Cell Line", 《JUNDISHAPUR JOURNAL OF MICROBIOLOGY》 * |
| O"MAHONY, AM等: "Human T-cell lymphotropic virus type 1 tax induction of biologically active NF-kappa B requires I kappa B kinase-1-mediated phosphorylation of RelA/p65", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 王卓娅等: "雄激素受体激动剂与拮抗剂高通量筛选细胞模型的构建", 《基因组学与应用生物学》 * |
| 黄倩等: "人参多糖通过cAMP/PKA/CREB信号通路抗糖尿病肾病肾纤维化作用机制研究", 《中国药理学通报》 * |
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Application publication date: 20181127 |