CN102692506A - Use of CD133 in preparation of tumor marker and kit of CD133 - Google Patents
Use of CD133 in preparation of tumor marker and kit of CD133 Download PDFInfo
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Abstract
The invention relates to the fields of tumor molecular biology and tumor drug treatment. The invention provides a use of CD133 as a tumor cell invasion marker and an application with CD133 as a target for screening drugs that inhibit tumor cell invasion. According to the invention, technologies of plasmid transfection, rat glioma model establishment, vivo imaging, immunohistochemistry and Transwellare employed to verify the high expression of the CD133 in invasion foci around glioma, and it is verified that highly-expressed glioma cells are more susceptible to invasion in vivo and in vitro. Further research indicates thatthe CD133 makes glioma more susceptible to invasion by improving tumor cell migration capability and altering cytoskeletal. The CD133 can provide tumor treatment programme selection and antitumor drug screening with new ideas.
Description
Technical field
The present invention relates to molecular biology and tumour medicine field.Specifically, the present invention relates to the effect of a kind of tumor stem cell surface marker CD133 in glioma invasion and attack, and CD133 and downstream passages the drug screening of tumour with treat in potential application.
Background technology
The brain astroglioma is a kind of fatal and common ICT.In the routine operation treatment, under the prerequisite of chemicotherapy, patient's mean survival time is about 15 months.The reason that causes glioma to be difficult to cure is that glioma cell is prone to invasion and attack and sends out in the normal on every side brain tissue, causes excision not exclusively and then cause the recurrence of tumour.Yet at present the understanding to the mechanism of glioma invasion and attack and characteristic is still not enough, and therefore, the molecular mechanism of probing into the glioma invasion and attack is the keys of treating glioma.
Show in the existing research that CD133 is 5 transmembrane glycoproteins, is positioned at cell surface.CD133 all has expression in multiple entity tumor stem cells such as brain tumor stem cell and leukemic stem cells, colorectal cancer stem cell, prostate cancer stem cell, liver-cancer stem cell, can be used as the wide spectrum mark of related neoplasms stem cell.Find that in the research of tumour the negative cell of tumor cell ratio CD133 that CD133 is positive has stronger self and multiplication capacity, but also do not find its effect in the glioma invasion and attack at present.
Summary of the invention
The new purposes that the purpose of this invention is to provide CD133.The invention provides the purposes of CD133, and be the application that target sieving suppresses the medicine of tumor cell invasion with CD133, for the evaluation and the treatment of tumour provides reference frame as the sign of tumor cell invasion.
The invention provides the application of CD133, especially to glioma cell as the sign of tumor cell invasion.
During concrete the detection, can get sample to be tested earlier, analyze the expression that compares CD133 in sample to be tested and the normal control then.The CD133 expression is higher than normal control, and tumour cell is prone to attack.
Described sample to be tested can be a cell sample, tissue samples, perhaps the albumen extract of cell, tissue.Normal control can be normal person's a sample, is preferably to take from tissue or the cell sample that patient's cancer is other or tumor tissues is peripheral.
During detection, adopt associated antibodies etc. to detect CD133 content in the sample usually.Experimental result is represented with the mean value and the standard deviation of three independent experiments, is checked comparing difference with sided t.P＜0.01 thinks to have significant difference.All The data SPSS V13.0 softwares perhaps possess the software analysis of similar statistical function.During actual detected sample to be tested than the expression of CD133 in the normal control high 30% and more than positive result, show invasion and attack of tumour cell trend or migration.
CD133 of the present invention is the transmembrane protein that is positioned at cell surface, and its sequence and relevant information can be with reference to the genbank databases.For example use the sequence report of NP_006008.
The present invention use transfection the GL261 mouse glioma cell of luciferase plasmids set up mouse glioma model; Purpose is the distribution of glioma is positioned; And brain tissue slice carried out the immunohistochemical analysis of CD133, purpose is to probe into expression and the distribution of CD133 in the tumour.The result shows, CD133 is high expressed in the invasion and attack kitchen range around the glioma, apparently higher than the inside of normal surrounding tissue and tumour, the immunohistochemical analysis of human glioma histotomy also had identical result.Meanwhile; The present invention passes through GL261 mouse glioma cell transfection CD133-Flag plasmid; Mouse glioma model and the external transwell invasive ability test model of high expressed CD133 have been set up; And compare with transfection empty plasmid Flag group and control group; The effect that purpose is clear and definite CD133 in the glioma growth finds that thus the glioma cell of CD133 high expressed more is prone to attack, and in the nude mice model of the U87-GFP of other foundation and U87-CD133-GFP, has also observed identical phenomenon.Therefore the present invention is clear and definite, and CD133 makes glioma cell be prone to take place the new role of invasion and attack.
The present invention uses Flow cytometry cell cycle, the test of gelatin zymogram, cell adhesion test, external scratch test and cytoskeletal protein F-actin to dye to transfection CD133-Flag plasmid, transfection Flag empty plasmid and contrast three groups of cells and studies; Purpose is to probe into the reason that the CD133 high expressed causes that the glioma invasive ability strengthens; The result finds cell cycle, the mmp enzyme relevant with substrate degradation spectrum and the equal no significant difference of cell adhesion; And cell scratch test and immunofluorescence have confirmed that the external transfer ability of CD133 high expressed glioma also has enhancing; Cytoskeletal protein F-actin has the phenomenon that is the pencil arrangement; The transfer ability of clear and definite CD133 through improving tumour cell with change cytoskeleton and make glioma more be prone to attack, for the basis and the clinical research of being correlated with provides new thinking.
The present invention also provides CD133 to suppress the application in the tumor cell invasion medicine in preparation, and CD133 can be used as the medicine target that screening suppresses the tumor cell invasion medicine.The material that reduces the CD133 expression is the drug candidate that suppresses the tumor cell invasion medicine.Described inhibition tumor cell invasion medicine can be a medicine of subduing the tumor cell migration ability, also can be the medicine that suppresses the growth of tumour cell cytoskeleton.
Accordingly, the invention provides a kind of kit that suppresses the tumor cell invasion medicine that screens, this kit contains CD133.Preferably, this kit is to glioma cell.
Usually, contain the CD133 albumen reference substance of standard dose in the kit, operation manual, dilution or damping fluid etc.
The present invention has used the Westernblot technology that CD133 is expressed PI3K/AKT when changing, and FAK and ERK signal path are studied.The result finds that the level of pPDK1, pAkt in the CD133 high expressing cell obviously raises, and the level of pFAK and pERK is almost constant, has explained that CD133 can activate the PI3K/PDK/Akt path.Utilization suppressant Wortmannin can be in the glioma cell invasion and attack of vitro inhibition CD133 mediation, and this proof suppresses CD133 and the downstream effect thing can suppress the glioma cell invasion and attack.This is from another angle explanation, and CD133 of the present invention can be used as the medicine target that screening suppresses the medicine of glioma cell.
The present invention relates to effect and the related mechanism of tumor stem cell surface marker CD133 in the glioma invasion and attack.Experiment of the present invention shows that CD133 attacks to express in the kitchen range and increases around glioma, and CD133 crosses easier invasion and attack of glioma cell of expression; The present invention simultaneously finds that CD133 regulates the transfer ability of cytoskeletal protein F-actin and cell through interacting with downstream PI3K/Akt, and then makes glioma cell more be prone to attack.Although embodiments of the invention are model with the glioma cell,, the present invention also can be applied to some other tumour.Therefore the characteristic of CD133 and can new thinking be provided for the fundamental research of tumour and the research and development of antineoplastic with the molecular mechanism of downstream passages effect.
The present invention has confirmed that the expression of CD133 is relevant with the aggressive of glioma cell; And confirm the process of CD133, utilize the suppressant Wortmannin of PI3K can suppress the invasion and attack of glioma through having participated in the glioma invasion and attack with the effect of downstream PI3K/Akt signal path.Main experiment show of the present invention:
(1) CD133 attacks to express in the kitchen range around glioma and increases, and the glioma cell of CD133 high expressed is attacked with external more being prone in vivo;
(2) the CD133 high expressed causes that reason and cell cycle, extracellular matrix degradation and cell adhesion ability that the glioma invasion and attack strengthen strengthen no obvious relation, and strengthens relevant with skelemin F-actin structural change with the transfer ability of cell;
(3) PI3K/Akt path in CD133 downstream interacts and to cause that the glioma transfer ability strengthens with the F-actin structural change and then makes the invasive ability enhancing of glioma.
The present invention uses plasmid transfection, the modelling of mouse glioma, living imaging, SABC; Transwell technical identification CD133 is high expressed in the invasion and attack kitchen range around the glioma, and the glioma cell of CD133 high expressed more is prone to take place in the body and external invasion and attack.The detection of applying flow cytometry cell cycle, the test of gelatin zymogram, cell adhesion test, external scratch test and cytoskeleton staining technique find that CD133 makes glioma more be prone to attack through transfer ability and the change cytoskeleton that improves tumour cell.Use Westernblot technology and PI3K suppressant and find that CD133 and downstream PI3K/Akt path interact, suppress the glioma cell invasion and attack that this path can suppress the CD133 mediation.Therefore CD133 is not only the surface marker of glioma stem cell; Can also be as the sign of glioma invasive ability; CD133 and not only new thinking can be provided for the fundamental research of glioma with the new mechanism of the PI3K/Akt path effect in downstream can also provide reference for the research and the screening of clinical antineoplastic.
Description of drawings
Fig. 1 shows that representative people's glioblastoma sample CD133 immunohistochemical staining zone mainly is positioned at the invasion and attack zone of glioma.
Fig. 2 shows that CD133 can promote the interior invasion and attack of body of glioma.
A shows GL261, the HE dyeing of the plastidogenetic mouse glioma of GL261-Flag and GL261-CD133-Flag.
B shows the Flag immunohistochemical staining after the one-tenth knurl in the representative GL261-CD133-Flag brain.
Fig. 3-Fig. 5 shows that CD133 can promote the external invasion and attack and the migration of glioma cell.
Fig. 3, GL261-Mock under the light microscopic, the Transwell invasion and attack experimental analysis of GL261-Flag and GL261-CD133-Flag cell.
Fig. 4, GL261-Mock, the cut migration experimental analysis of GL261-Flag and GL261-CD133-Flag cell.
Fig. 5, GL261-Mock, GL261-Flag and GL261-CD133-Flag cell F-actin staining analysis.
Fig. 6-Figure 10 shows that CD133 is through activating the invasion and attack that the PI3K/Akt path promotes glioma.
Fig. 6 utilizes Western blot to analyze GL261-Mock, GL261-Flag and GL261-CD133-Flag cell Akt, PDK, the phosphorylation situation of ERK and FAK.
Fig. 7, PI3K suppressant Wortmannin suppresses the Akt activation that CD133 induces.
Fig. 8, the scratch experiment analysis of cell among the B.
Fig. 9, the quantitative test of cell Transwell invasion and attack experiment among the B.
Figure 10, cell F-actin staining analysis among the B.
Embodiment
Below in conjunction with accompanying drawing embodiment provided by the invention is elaborated.Experimental result is represented with the mean value and the standard deviation of three independent experiments, is checked comparing difference with sided t.P＜0.01 thinks to have significant difference.All The data SPSS V13.0 software analysis.
One, cellular incubation and transfection
This experiment utilizes people's glioma cell U87-MG and mouse glioma cell GL261.All cells all adopts the DMEM nutrient solution that contains 10% hyclone, 100 μ g/ml penicillin and 50 μ g/ml streptomysins, at the 5%CO2-95% air, cultivates under the condition of saturated humidity and 37 ℃.
Gene transfection is with reference to LipofectAMINE transfection reagent box operation manual.The cell that will be in exponential phase is with l * 10
5Cells/well is inoculated in the six porocyte culture plates, cultivates 16-24 h, during the about 60-80% of cell density, puts transfection reagent (A reagent: the 1 μ g plasmid+unparalleled anti-1640/DMEM nutrient culture media of 100 μ l serum-frees; B reagent: the unparalleled anti-1640/DMEM nutrient culture media of 5 μ l Lipofectamine+100 μ l serum-frees).With A reagent and the careful mixing of B reagent, room temperature leaves standstill 30-45 min, to form the DNA-liposome complex.Discard and train liquid in the culture hole, with twice of the unparalleled anti-RPMI1640/DMEM nutrient culture media washed cell of 2 ml serum-frees.In the DNA-liposome complex, add the unparalleled anti-RPMI1640/DMEM nutrient culture media of 800 μ l serum-frees, careful mixing, totally 1.0 ml add in the culture hole.37 ℃, 5%CO
2Cultivate 5 h, add the nutrient culture media that 1.0 ml contain 20% serum again, 37 ℃, 5%CO
2Cultivate 24 h, change fresh complete culture solution and continue to cultivate.With LV-GFP, LV-Luc, LV-Flag, LV-CD133-Flag (the CD133-Flag sequence is seen SEQ ID NO 1) and the corresponding cell of GST-CD133-CD expression plasmid transfection, obtain the stable transfection strain with liposome method after the screening.
Two, reagent
Active Src albumen is purchased the company limited in Millipore.Anti--CD133 antibody (k-18), anti--GAPDH antibody is purchased the Bioisystech Co., Ltd in Santa Cruz.Anti--p-ERK, anti--p-PDK1 (Ser241) antibody, anti--Akt antibody, anti--p-Akt (Ser473) antibody, anti--p-Akt (Thr308) antibody, anti--p-FAK (Tyr925) antibody, anti--mouse-HRP is two anti-, anti--and sheep-HRP are two anti-and anti--and rabbit-HRP two are anti-all to purchase the company limited in Cell Signaling.Anti--Flag antibody and PI3K suppressant Wortmannin purchase the company limited in Sigma.
Three, mouse/nude mice glioma modelling
Steady phase inversion answers plasmid after 72 hours, and 10
5Individual GL261 or U87-MG injection cell go into around age male C57BL/6 mouse or all around age male nude mouse RCN.The inserting needle position is the right side brain, moves 0.5mm behind the Breggma point, then about sidesway 2.2mm, downwards about 3mm.Detect the ICT growing state with toy living imaging system after 4～6 weeks.Anesthesia and with 4% paraformaldehyde perfusion mouse, get brain, 4% paraformaldehyde is fixed, FFPE, the section.
Four, histopathology and SABC
The glioma sample is fixed in 4% paraformaldehyde, and paraffin encapsulates, section, tissues observed pathomorphism under the light microscopic of HE dyeing back.The dewaxing back is carried out SABC with anti--CD133 with anti--Flag antibody and is detected.Photo obtains with Motic Image Advanced 3.2 picture analyzing systems.
Five, Transwell cell invasion test
On Transwell chamber, 24 hole polycarbonate membrane (membrane aperture 8 mm), be coated with the matrigel 40 μ l of 1mg/ml, hatch 5 h for 37 ℃, make it on miillpore filter, to be reassembled as basement membrane structure.Chamber residual liquid in the sucking-off adds the DMEM aquation basilar memebrane that 70 μ l do not contain serum afterwards, hatches 30 min for 37 ℃.The A2780/s that takes the logarithm growth period, A2780/cis, Mortalin/HSPA9/Grp75 SiRNA cell are l * 10 with the DMEM nutrient solution adjustment cell number of increase serum not
5Individual/ml.It is upward indoor to get 200 μ l cell suspension inoculation Transwell, and following chamber adds the DMEM nutrient solution 600 μ l of 10%FBS, 37 ℃ of 5%CO
2Cultivate 24 h in the incubator.Discard nutrient solution in the hole behind 24 h, PBS washes 2 times, and methyl alcohol is fixed 15 min, 0.1% violet staining, 15 min, d
2H
2O
2Wash 3 times, confluent monolayer cells on the filter membrane erased with cotton swab gently, under 400 times of light microscopics selective membrane up and down in 5 different visuals field wear the theca cell number, average.
Six, scratch test
Get and grow to 80% cell that merges, cell is resuspended in the serum-free medium 24 hours.Behind the trypsinization with l * 10
6Individual cells/well is inoculated in 6 porocyte culture plates, cultivates 24 h cells and merges basically.Vertically at the bottom of culture plate, draw parallel, vertical 4 " cuts " with aseptic l ml rifle head, the cell under drawing with the flush away quilt with training liquid flushing several times.Put at fixed time and under inverted phase contrast microscope, count, establish two multiple holes, 3 independent experiments for every group apart from the cell number in cut 0.2 mm.
Seven, cell adhesion test
Cell is resuspended in the serum-free medium 24 hours.24 orifice plates encapsulate matrigel (Matrigel), and every hole adds and contains 2.5*10
50.5 ml cell suspension of individual cell, 37 degree are hatched 1h.PBS washes (flush away is attached cell not) 4 times.0.5% crystal violet (20% methanol solution) dyeing 10min.DdH
2O washs 10min.Every hole adds 10% acetic acid 0.25ml, and lysate is colorimetric in the 590nm place.
Eight, gelatin zymogram test
Prepare 10% gel.During the preparation separation gel, add the 10mg/ml gelatin WS, 65 ℃ of water-baths make that the gelatin final concentration is 1mg/ml.Protein sample preparation: 1) supernatant: cell non-serum is cultivated 24h.Collect supernatant on ice, centrifugal removal cell of 3000rpm * 10min and remains concentrate 20 times, and the back is in-4 ℃ of preservations.2) cell pyrolysis liquid: cell lysis (lysate: contain 1 * TBS solution of 1% Triton X-100,30l/60mm dish), cocktail 1:200 places and shakes 30 min on ice, and the back is in-70 ℃ of preservations.12000rpm * 10min is centrifugal in the dissolving back, quantitatively draws supernatant.Add equal-volume Tris-Glycine SDS Sample Buffer (2 *) dilution, room temperature is placed 10 min, adds bromophenol blue 0.5l.Electrophoresis to bromophenol blue is run out of glue.1 * Developing Buffer100ml, room temperature is shaken balanced gel 30min slowly, 37 ℃ of equilibrate overnight.0.5% Coomassie brilliant blue R-250 (methyl alcohol: glacial acetic acid: the dyeing of water=30:10:60) 30min, destainer (methyl alcohol: glacial acetic acid: decolouring in the water=50:10:40), take pictures.
Nine, Western blotting
The cell in growth period of taking the logarithm is removed nutrient culture media, uses 0.25% trypsinization, and 0.01M PBS blows and beats cell, is collected into 10ml EP pipe, and is centrifugal, twice of PBS washed cell.Add an amount of SDS protein lysate and protease inhibitors cocktail according to cell concentration.Behind the SDS-PAGE electrophoresis, wet turning egg(s) is in vain to pvdf membrane, and 5%BSA (TBS/0.1% Tween 20 dissolvings) sealing 2 hours afterwards with anti-at room temperature hatched 2 hours or 4 ℃ of incubated overnight, is given a baby a bath on the third day after its birth time with TBS/0.1% Tween 20, at every turn 10min.Incubated at room two is anti-2 hours again, and TBS/0.1% Tween 20 gives a baby a bath on the third day after its birth time, each 10min.Detect with the ECL chemiluminescence detection kit.
Embodiment 1
Make up the former bit model of C57 mouse of GL261, toy living imaging detection of dynamic tumor growth with the GL261 mouse glioma cell of luciferase mark.Utilize mouse glioma model and people's glioblastoma sample; Detect expression characteristic and the difference of CD133 (the CD133-Flag sequence is seen SEQ ID NO 1, and its luciferase sequence is seen SEQ ID NO 2) in glioma inside, glioma and normal tissues boundary and normal tissues through SABC.The result shows that glioma is to the obviously invasion and attack of normal cerebral tissue zone.The CD133 immunohistochemical staining of brain sheet shows, tumor invasion zone C D133 high expressed, and inside tumor and the CD133 of normal cerebral tissue expression are less.Same result has also obtained checking in people's glioma sample.To the CD133 immunohistochemical staining prompting that 18 examples have the glioma sample of tumour and normal tissues boundary to carry out, the CD133 expression of junctional area is far above inside tumor and normal cerebral tissue.
Therefore, CD133 can also be as the sign of glioma cell invasion and attack.
Embodiment 2
Make up the plasmid of expressing CD133, in above-mentioned two kinds of glioma cells, crossed expression CD133.Utilize the C57 mouse model of GL261 and the nude mice model of U87, observation CD133 crosses border, daughter knurl number, corpus callosum invasion and attack and the ventricles of the brain of expressing the back tumour and forming and sends out situation.The result shows that the borderline tumor that Mock and Flag group form is smooth, and the tumour of CD133 group presents high aggressive, shows as to normal cerebral tissue's invasion and attack obviously, and a large amount of cerebrospinal fluid is sent out and planted.These are sent out kitchen range and are CD133+, point out their CD133 cells that all derives from external source.Observed phenomenon same as described above in the nude mice model of U87-GFP and U87-CD133-GFP, promptly the aggressive of CD133 group tumour obviously is better than the Flag group, shows as the CD133 group and forms a large amount of daughter knurls, and quantity is 5 times of Flag group daughter knurl approximately.
This explanation, CD133 can be used in the aggressive that promotes tumour.CD133 is not only the surface marker of glioma stem cell, can also be as the sign of glioma invasive ability.
Embodiment 3
Utilized the GL261 and the U87 glioma cell of expressing CD133, further analyzed the effect of CD133 in the glioma invasive procedure through Transwell model, Flow cytometry cell cycle, the test of gelatin zymogram, cell adhesion test, external scratch test and cytoskeletal protein F-actin dyeing.
Transwell invasion and attack experimental result shows CD133 group invasion and attack cell number obviously more than Mock group and Flag group, and CD133 is in the external invasion and attack that can obviously promote the GL261 glioma cell in prompting.The cell cycle distribution of three groups of cells does not have significant difference, and this has just got rid of the influence of cell cycle to Transwell invasion and attack experimental result.Mmp enzyme spectrum experimental result points out the activity of three groups of cell MMP-2 and MMP-9 not have difference, explains that CD133 is to the not obviously influence of glioma cell matrix degradation ability.The cell adhesion experimental result has explained that CD133 sticks ability not obviously influence equally to glioma cell.At last, the transfer ability of in scratch experiment, observing CD133 group cell is apparently higher than all the other two groups.Coloration result to F-actin has confirmed this phenomenon.The actin disperse of Flag cell is distributed in the endochylema, and the actin of CD133 cell assembles bunchy, forms projection, is distributed in cell edges more.Explain that CD133 can regulate the tissue of actin cytoskeleton.
Above-mentioned experiment has confirmed that further CD133 can promote the invasion and attack of glioma cell.And CD133 is through strengthening the cell migration ability and regulating actin cytoskeleton promotion invasion by tumor cells.
Embodiment 4
Detect CD133 through Western blot and express change, comprise PI3K/AKT, the effect of FAK and ERK signal path glioma invasion and attack adjusting path.Utilize the suppressant Wortmannin of PI3K to detect the biological function of PI3K/AKT signal path in the invasion and attack of CD133 regulation and control glioma cell, wherein detection method comprises Transwell model, external scratch test and cytoskeletal protein F-actin dyeing.
The result shows that than Mock group and Flag group, the level of pPDK1, pAkt obviously raises in the CD133 group cell, and the level of pFAK and pERK is almost constant, has explained that CD133 can activate the PI3K/PDK/Akt path.Western blot result shows that Wortmannin can obviously suppress the phosphorylation of Akt.Scratch experiment and Transwell invasion and attack experimental result show that Wortmannin can obviously suppress the transfer ability of glioma cell.Dyeing to F-actin has confirmed that Wortmannin can significantly suppress the tissue of actin cytoskeleton.
Therefore, CD133 and with the PI3K/Akt path in downstream, not only new thinking can be provided for the fundamental research of glioma, can also reference be provided for the research and the screening of clinical antineoplastic.For example, Wortmannin can obviously suppress the phosphorylation of Akt, just can obviously suppress the invasion and attack of glioma cell.CD133 and Akt can be used to screen the medicine that suppresses tumor cell invasion, especially suppress the medicine of glioma cell cell invasion.
SEQUENCE?LISTING
< 110>Fudan University
< 120>purposes and the kit thereof of CD133 in the preparation tumor markers
<130> 1032
<160> 2
<170> PatentIn?version?3.1
<210> 1
<211> 9292
<212> DNA
<213> Artificial
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tatgctgtgt?cctggggctg?ctgtttatta?ttctgatgcc?tctggtgggg?tatttctttt 2880
gtatgtgtcg?ttgctgtaac?aaatgtggtg?gagaaatgca?ccagcgacag?aaggaaaatg 2940
ggcccttcct?gaggaaatgc?tttgcaatct?ccctgttggt?gatttgtata?ataataagca 3000
ttggcatctt?ctatggtttt?gtggcaaatc?accaggtaag?aacccggatc?aaaaggagtc 3060
ggaaactggc?agatagcaat?ttcaaggact?tgcgaactct?cttgaatgaa?actccagagc 3120
aaatcaaata?tatattggcc?cagtacaaca?ctaccaagga?caaggcgttc?acagatctga 3180
acagtatcaa?ttcagtgcta?ggaggcggaa?ttcttgaccg?actgagaccc?aacatcatcc 3240
ctgttcttga?tgagattaag?tccatggcaa?cagcgatcaa?ggagaccaaa?gaggcgttgg 3300
agaacatgaa?cagcaccttg?aagagcttgc?accaacaaag?tacacagctt?agcagcagtc 3360
tgaccagcgt?gaaaactagc?ctgcggtcat?ctctcaatga?ccctctgtgc?ttggtgcatc 3420
catcaagtga?aacctgcaac?agcatcagat?tgtctctaag?ccagctgaat?agcaaccctg 3480
aactgaggca?gcttccaccc?gtggatgcag?aacttgacaa?cgttaataac?gttcttagga 3540
cagatttgga?tggcctggtc?caacagggct?atcaatccct?taatgatata?cctgacagag 3600
tacaacgcca?aaccacgact?gtcgtagcag?gtatcaaaag?ggtcttgaat?tccattggtt 3660
cagatatcga?caatgtaact?cagcgtcttc?ctattcagga?tatactctca?gcattctctg 3720
tttatgttaa?taacactgaa?agttacatcc?acagaaattt?acctacattg?gaagagtatg 3780
attcatactg?gtggctgggt?ggcctggtca?tctgctctct?gctgaccctc?atcgtgattt 3840
tttactacct?gggcttactg?tgtggcgtgt?gcggctatga?caggcatgcc?accccgacca 3900
cccgaggctg?tgtctccaac?accggaggcg?tcttcctcat?ggttggagtt?ggattaagtt 3960
tcctcttttg?ctggatattg?atgatcattg?tggttcttac?ctttgtcttt?ggtgcaaatg 4020
tggaaaaact?gatctgtgaa?ccttacacga?gcaaggaatt?attccgggtt?ttggatacac 4080
cctacttact?aaatgaagac?tgggaatact?atctctctgg?gaagctattt?aataaatcaa 4140
aaatgaagct?cacttttgaa?caagtttaca?gtgactgcaa?aaaaaataga?ggcacttacg 4200
gcactcttca?cctgcagaac?agcttcaata?tcagtgaaca?tctcaacatt?aatgagcata 4260
ctggaagcat?aagcagtgaa?ttggaaagtc?tgaaggtaaa?tcttaatatc?tttctgttgg 4320
gtgcagcagg?aagaaaaaac?cttcaggatt?ttgctgcttg?tggaatagac?agaatgaatt 4380
atgacagcta?cttggctcag?actggtaaat?cccccgcagg?agtgaatctt?ttatcatttg 4440
catatgatct?agaagcaaaa?gcaaacagtt?tgcccccagg?aaatttgagg?aactccctga 4500
aaagagatgc?acaaactatt?aaaacaattc?accagcaacg?agtccttcct?atagaacaat 4560
cactgagcac?tctataccaa?agcgtcaaga?tacttcaacg?cacagggaat?ggattgttgg 4620
agagagtaac?taggattcta?gcttctctgg?attttgctca?gaacttcatc?acaaacaata 4680
cttcctctgt?tattattgag?gaaactaaga?agtatgggag?aacaataata?ggatattttg 4740
aacattatct?gcagtggatc?gagttctcta?tcagtgagaa?agtggcatcg?tgcaaacctg 4800
tggccaccgc?tctagatact?gctgttgatg?tctttctgtg?tagctacatt?atcgacccct 4860
tgaatttgtt?ttggtttggc?ataggaaaag?ctactgtatt?tttacttccg?gctctaattt 4920
ttgcggtaaa?actggctaag?tactatcgtc?gaatggattc?ggaggacgtg?tacgatgatg 4980
ttgaaactat?acccatgaaa?aatatggaaa?atggtaataa?tggttatcat?aaagatcatg 5040
tatatggtat?tcacaatcct?gttatgacaa?gcccatcaca?acatatggac?tacaaagacg 5100
atgacgacaa?gtgataaacc?ggtcgccacc?agcggccgcg?tcgacaatca?acctctggat 5160
tacaaaattt?gtgaaagatt?gactggtatt?cttaactatg?ttgctccttt?tacgctatgt 5220
ggatacgctg?ctttaatgcc?tttgtatcat?gctattgctt?cccgtatggc?tttcattttc 5280
tcctccttgt?ataaatcctg?gttgctgtct?ctttatgagg?agttgtggcc?cgttgtcagg 5340
caacgtggcg?tggtgtgcac?tgtgtttgct?gacgcaaccc?ccactggttg?gggcattgcc 5400
accacctgtc?agctcctttc?cgggactttc?gctttccccc?tccctattgc?cacggcggaa 5460
ctcatcgccg?cctgccttgc?ccgctgctgg?acaggggctc?ggctgttggg?cactgacaat 5520
tccgtggtgt?tgtcggggaa?gctgacgtcc?tttccatggc?tgctcgcctg?tgttgccacc 5580
tggattctgc?gcgggacgtc?cttctgctac?gtcccttcgg?ccctcaatcc?agcggacctt 5640
ccttcccgcg?gcctgctgcc?ggctctgcgg?cctcttccgc?gtcttcgcct?tcgccctcag 5700
acgagtcgga?tctccctttg?ggccgcctcc?ccgcctggaa?ttcgagctcg?gtacctttaa 5760
gaccaatgac?ttacaaggca?gctgtagatc?ttagccactt?tttaaaagaa?aaggggggac 5820
tggaagggct?aattcactcc?caacgaagac?aagatctgct?ttttgcttgt?actgggtctc 5880
tctggttaga?ccagatctga?gcctgggagc?tctctggcta?actagggaac?ccactgctta 5940
agcctcaata?aagcttgcct?tgagtgcttc?aagtagtgtg?tgcccgtctg?ttgtgtgact 6000
ctggtaacta?gagatccctc?agaccctttt?agtcagtgtg?gaaaatctct?agcagtagta 6060
gttcatgtca?tcttattatt?cagtatttat?aacttgcaaa?gaaatgaata?tcagagagtg 6120
agaggaactt?gtttattgca?gcttataatg?gttacaaata?aagcaatagc?atcacaaatt 6180
tcacaaataa?agcatttttt?tcactgcatt?ctagttgtgg?tttgtccaaa?ctcatcaatg 6240
tatcttatca?tgtctggctc?tagctatccc?gcccctaact?ccgcccagtt?ccgcccattc 6300
tccgccccat?ggctgactaa?ttttttttat?ttatgcagag?gccgaggccg?cctcggcctc 6360
tgagctattc?cagaagtagt?gaggaggctt?ttttggaggc?ctaggctttt?gcgtcgagac 6420
gtacccaatt?cgccctatag?tgagtcgtat?tacgcgcgct?cactggccgt?cgttttacaa 6480
cgtcgtgact?gggaaaaccc?tggcgttacc?caacttaatc?gccttgcagc?acatccccct 6540
ttcgccagct?ggcgtaatag?cgaagaggcc?cgcaccgatc?gcccttccca?acagttgcgc 6600
agcctgaatg?gcgaatggcg?cgacgcgccc?tgtagcggcg?cattaagcgc?ggcgggtgtg 6660
gtggttacgc?gcagcgtgac?cgctacactt?gccagcgccc?tagcgcccgc?tcctttcgct 6720
ttcttccctt?cctttctcgc?cacgttcgcc?ggctttcccc?gtcaagctct?aaatcggggg 6780
ctccctttag?ggttccgatt?tagtgcttta?cggcacctcg?accccaaaaa?acttgattag 6840
ggtgatggtt?cacgtagtgg?gccatcgccc?tgatagacgg?tttttcgccc?tttgacgttg 6900
gagtccacgt?tctttaatag?tggactcttg?ttccaaactg?gaacaacact?caaccctatc 6960
tcggtctatt?cttttgattt?ataagggatt?ttgccgattt?cggcctattg?gttaaaaaat 7020
gagctgattt?aacaaaaatt?taacgcgaat?tttaacaaaa?tattaacgtt?tacaatttcc 7080
caggtggcac?ttttcgggga?aatgtgcgcg?gaacccctat?ttgtttattt?ttctaaatac 7140
attcaaatat?gtatccgctc?atgagacaat?aaccctgata?aatgcttcaa?taatattgaa 7200
aaaggaagag?tatgagtatt?caacatttcc?gtgtcgccct?tattcccttt?tttgcggcat 7260
tttgccttcc?tgtttttgct?cacccagaaa?cgctggtgaa?agtaaaagat?gctgaagatc 7320
agttgggtgc?acgagtgggt?tacatcgaac?tggatctcaa?cagcggtaag?atccttgaga 7380
gttttcgccc?cgaagaacgt?tttccaatga?tgagcacttt?taaagttctg?ctatgtggcg 7440
cggtattatc?ccgtattgac?gccgggcaag?agcaactcgg?tcgccgcata?cactattctc 7500
agaatgactt?ggttgagtac?tcaccagtca?cagaaaagca?tcttacggat?ggcatgacag 7560
taagagaatt?atgcagtgct?gccataacca?tgagtgataa?cactgcggcc?aacttacttc 7620
tgacaacgat?cggaggaccg?aaggagctaa?ccgctttttt?gcacaacatg?ggggatcatg 7680
taactcgcct?tgatcgttgg?gaaccggagc?tgaatgaagc?cataccaaac?gacgagcgtg 7740
acaccacgat?gcctgtagca?atggcaacaa?cgttgcgcaa?actattaact?ggcgaactac 7800
ttactctagc?ttcccggcaa?caattaatag?actggatgga?ggcggataaa?gttgcaggac 7860
cacttctgcg?ctcggccctt?ccggctggct?ggtttattgc?tgataaatct?ggagccggtg 7920
agcgtgggtc?tcgcggtatc?attgcagcac?tggggccaga?tggtaagccc?tcccgtatcg 7980
tagttatcta?cacgacgggg?agtcaggcaa?ctatggatga?acgaaataga?cagatcgctg 8040
agataggtgc?ctcactgatt?aagcattggt?aactgtcaga?ccaagtttac?tcatatatac 8100
tttagattga?tttaaaactt?catttttaat?ttaaaaggat?ctaggtgaag?atcctttttg 8160
ataatctcat?gaccaaaatc?ccttaacgtg?agttttcgtt?ccactgagcg?tcagaccccg 8220
tagaaaagat?caaaggatct?tcttgagatc?ctttttttct?gcgcgtaatc?tgctgcttgc 8280
aaacaaaaaa?accaccgcta?ccagcggtgg?tttgtttgcc?ggatcaagag?ctaccaactc 8340
tttttccgaa?ggtaactggc?ttcagcagag?cgcagatacc?aaatactgtc?cttctagtgt 8400
agccgtagtt?aggccaccac?ttcaagaact?ctgtagcacc?gcctacatac?ctcgctctgc 8460
taatcctgtt?accagtggct?gctgccagtg?gcgataagtc?gtgtcttacc?gggttggact 8520
caagacgata?gttaccggat?aaggcgcagc?ggtcgggctg?aacggggggt?tcgtgcacac 8580
agcccagctt?ggagcgaacg?acctacaccg?aactgagata?cctacagcgt?gagctatgag 8640
aaagcgccac?gcttcccgaa?gggagaaagg?cggacaggta?tccggtaagc?ggcagggtcg 8700
gaacaggaga?gcgcacgagg?gagcttccag?ggggaaacgc?ctggtatctt?tatagtcctg 8760
tcgggtttcg?ccacctctga?cttgagcgtc?gatttttgtg?atgctcgtca?ggggggcgga 8820
gcctatggaa?aaacgccagc?aacgcggcct?ttttacggtt?cctggccttt?tgctggcctt 8880
ttgctcacat?gttctttcct?gcgttatccc?ctgattctgt?ggataaccgt?attaccgcct 8940
ttgagtgagc?tgataccgct?cgccgcagcc?gaacgaccga?gcgcagcgag?tcagtgagcg 9000
aggaagcgga?agagcgccca?atacgcaaac?cgcctctccc?cgcgcgttgg?ccgattcatt 9060
aatgcagctg?gcacgacagg?tttcccgact?ggaaagcggg?cagtgagcgc?aacgcaatta 9120
atgtgagtta?gctcactcat?taggcacccc?aggctttaca?ctttatgctt?ccggctcgta 9180
tgttgtgtgg?aattgtgagc?ggataacaat?ttcacacagg?aaacagctat?gaccatgatt 9240
acgccaagcg?cgcaattaac?cctcactaaa?gggaacaaaa?gctggagctg?ca 9292
<210> 2<211> 8304<212> DNA<213> Artificial<400> 2
agcttaatgt?agtcttatgc?aatactcttg?tagtcttgca?acatggtaac?gatgagttag 60
caacatgcct?tacaaggaga?gaaaaagcac?cgtgcatgcc?gattggtgga?agtaaggtgg 120
tacgatcgtg?ccttattagg?aaggcaacag?acgggtctga?catggattgg?acgaaccact 180
gaattgccgc?attgcagaga?tattgtattt?aagtgcctag?ctcgatacaa?taaacgggtc 240
tctctggtta?gaccagatct?gagcctggga?gctctctggc?taactaggga?acccactgct 300
taagcctcaa?taaagcttgc?cttgagtgct?tcaagtagtg?tgtgcccgtc?tgttgtgtga 360
ctctggtaac?tagagatccc?tcagaccctt?ttagtcagtg?tggaaaatct?ctagcagtgg 420
cgcccgaaca?gggacctgaa?agcgaaaggg?aaaccagagc?tctctcgacg?caggactcgg 480
cttgctgaag?cgcgcacggc?aagaggcgag?gggcggcgac?tggtgagtac?gccaaaaatt 540
ttgactagcg?gaggctagaa?ggagagagat?gggtgcgaga?gcgtcagtat?taagcggggg 600
agaattagat?cgcgatggga?aaaaattcgg?ttaaggccag?ggggaaagaa?aaaatataaa 660
ttaaaacata?tagtatgggc?aagcagggag?ctagaacgat?tcgcagttaa?tcctggcctg 720
ttagaaacat?cagaaggctg?tagacaaata?ctgggacagc?tacaaccatc?ccttcagaca 780
ggatcagaag?aacttagatc?attatataat?acagtagcaa?ccctctattg?tgtgcatcaa 840
aggatagaga?taaaagacac?caaggaagct?ttagacaaga?tagaggaaga?gcaaaacaaa 900
agtaagacca?ccgcacagca?agcggccgct?gatcttcaga?cctggaggag?gagatatgag 960
ggacaattgg?agaagtgaat?tatataaata?taaagtagta?aaaattgaac?cattaggagt 1020
agcacccacc?aaggcaaaga?gaagagtggt?gcagagagaa?aaaagagcag?tgggaatagg 1080
agctttgttc?cttgggttct?tgggagcagc?aggaagcact?atgggcgcag?cctcaatgac 1140
gctgacggta?caggccagac?aattattgtc?tggtatagtg?cagcagcaga?acaatttgct 1200
gagggctatt?gaggcgcaac?agcatctgtt?gcaactcaca?gtctggggca?tcaagcagct 1260
ccaggcaaga?atcctggctg?tggaaagata?cctaaaggat?caacagctcc?tggggatttg 1320
gggttgctct?ggaaaactca?tttgcaccac?tgctgtgcct?tggaatgcta?gttggagtaa 1380
taaatctctg?gaacagattt?ggaatcacac?gacctggatg?gagtgggaca?gagaaattaa 1440
caattacaca?agcttaatac?actccttaat?tgaagaatcg?caaaaccagc?aagaaaagaa 1500
tgaacaagaa?ttattggaat?tagataaatg?ggcaagtttg?tggaattggt?ttaacataac 1560
aaattggctg?tggtatataa?aattattcat?aatgatagta?ggaggcttgg?taggtttaag 1620
aatagttttt?gctgtacttt?ctatagtgaa?tagagttagg?cagggatatt?caccattatc 1680
gtttcagacc?cacctcccaa?ccccgagggg?acccgacagg?cccgaaggaa?tagaagaaga 1740
aggtggagag?agagacagag?acagatccat?tcgattagtg?aacggatctc?gacggtatcg 1800
gttaactttt?aaaagaaaag?gggggattgg?ggggtacagt?gcaggggaaa?gaatagtaga 1860
cataatagca?acagacatac?aaactaaaga?attacaaaaa?caaattacaa?aaattcaaaa 1920
ttttatcgat?cacgagacta?gcctcgagaa?gcttgatatc?gaattcccac?ggggttgggg 1980
ttgcgccttt?tccaaggcag?ccctgggttt?gcgcagggac?gcggctgctc?tgggcgtggt 2040
tccgggaaac?gcagcggcgc?cgaccctggg?tctcgcacat?tcttcacgtc?cgttcgcagc 2100
gtcacccgga?tcttcgccgc?tacccttgtg?ggccccccgg?cgacgcttcc?tgctccgccc 2160
ctaagtcggg?aaggttcctt?gcggttcgcg?gcgtgccgga?cgtgacaaac?ggaagccgca 2220
cgtctcacta?gtaccctcgc?agacggacag?cgccagggag?caatggcagc?gcgccgaccg 2280
cgatgggctg?tggccaatag?cggctgctca?gcggggcgcg?ccgagagcag?cggccgggaa 2340
ggggcggtgc?gggaggcggg?gtgtggggcg?gtagtgtggg?ccctgttcct?gcccgcgcgg 2400
tgttccgcat?tctgcaagcc?tccggagcgc?acgtcggcag?tcggctccct?cgttgaccga 2460
atcaccgacc?tctctcccca?gggggatcca?tggaagacgc?caaaaacata?aagaaaggcc 2520
cggcgccatt?ctatccgctg?gaagatggaa?ccgctggaga?gcaactgcat?aaggctatga 2580
agagatacgc?cctggttcct?ggaacaattg?cttttacaga?tgcacatatc?gaggtggaca 2640
tcacttacgc?tgagtacttc?gaaatgtccg?ttcggttggc?agaagctatg?aaacgatatg 2700
ggctgaatac?aaatcacaga?atcgtcgtat?gcagtgaaaa?ctctcttcaa?ttctttatgc 2760
cggtgttggg?cgcgttattt?atcggagttg?cagttgcgcc?cgcgaacgac?atttataatg 2820
aacgtgaatt?gctcaacagt?atgggcattt?cgcagcctac?cgtggtgttc?gtttccaaaa 2880
aggggttgca?aaaaattttg?aacgtgcaaa?aaaagctccc?aatcatccaa?aaaattatta 2940
tcatggattc?taaaacggat?taccagggat?ttcagtcgat?gtacacgttc?gtcacatctc 3000
atctacctcc?cggttttaat?gaatacgatt?ttgtgccaga?gtccttcgat?agggacaaga 3060
caattgcact?gatcatgaac?tcctctggat?ctactggtct?gcctaaaggt?gtcgctctgc 3120
ctcatagaac?tgcctgcgtg?agattctcgc?atgccagaga?tcctattttt?ggcaatcaaa 3180
tcattccgga?tactgcgatt?ttaagtgttg?ttccattcca?tcacggtttt?ggaatgttta 3240
ctacactcgg?atatttgata?tgtggatttc?gagtcgtctt?aatgtataga?tttgaagaag 3300
agctgtttct?gaggagcctt?caggattaca?agattcaaag?tgcgctgctg?gtgccaaccc 3360
tattctcctt?cttcgccaaa?agcactctga?ttgacaaata?cgatttatct?aatttacacg 3420
aaattgcttc?tggtggcgct?cccctctcta?aggaagtcgg?ggaagcggtt?gccaagaggt 3480
tccatctgcc?aggtatcagg?caaggatatg?ggctcactga?gactacatca?gctattctga 3540
ttacacccga?gggggatgat?aaaccgggcg?cggtcggtaa?agttgttcca?ttttttgaag 3600
cgaaggttgt?ggatctggat?accgggaaaa?cgctgggcgt?taatcaaaga?ggcgaactgt 3660
gtgtgagagg?tcctatgatt?atgtccggtt?atgtaaacaa?tccggaagcg?accaacgcct 3720
tgattgacaa?ggatggatgg?ctacattctg?gagacatagc?ttactgggac?gaagacgaac 3780
acttcttcat?cgttgaccgc?ctgaagtctc?tgattaagta?caaaggctat?caggtggctc 3840
ccgctgaatt?ggaatccatc?ttgctccaac?accccaacat?cttcgacgca?ggtgtcgcag 3900
gtcttcccga?cgatgacgcc?ggtgaacttc?ccgccgccgt?tgttgttttg?gagcacggaa 3960
agacgatgac?ggaaaaagag?atcgtggatt?acgtcgccag?tcaagtaaca?accgcgaaaa 4020
agttgcgcgg?aggagttgtg?tttgtggacg?aagtaccgaa?aggtcttacc?ggaaaactcg 4080
acgcaagaaa?aatcagagag?atcctcataa?aggccaagaa?gggcggaaag?atcgccgtgt 4140
aaagcggccg?cgtcgacaat?caacctctgg?attacaaaat?ttgtgaaaga?ttgactggta 4200
ttcttaacta?tgttgctcct?tttacgctat?gtggatacgc?tgctttaatg?cctttgtatc 4260
atgctattgc?ttcccgtatg?gctttcattt?tctcctcctt?gtataaatcc?tggttgctgt 4320
ctctttatga?ggagttgtgg?cccgttgtca?ggcaacgtgg?cgtggtgtgc?actgtgtttg 4380
ctgacgcaac?ccccactggt?tggggcattg?ccaccacctg?tcagctcctt?tccgggactt 4440
tcgctttccc?cctccctatt?gccacggcgg?aactcatcgc?cgcctgcctt?gcccgctgct 4500
ggacaggggc?tcggctgttg?ggcactgaca?attccgtggt?gttgtcgggg?aagctgacgt 4560
cctttccatg?gctgctcgcc?tgtgttgcca?cctggattct?gcgcgggacg?tccttctgct 4620
acgtcccttc?ggccctcaat?ccagcggacc?ttccttcccg?cggcctgctg?ccggctctgc 4680
ggcctcttcc?gcgtcttcgc?cttcgccctc?agacgagtcg?gatctccctt?tgggccgcct 4740
ccccgcctgg?aattcgagct?cggtaccttt?aagaccaatg?acttacaagg?cagctgtaga 4800
tcttagccac?tttttaaaag?aaaagggggg?actggaaggg?ctaattcact?cccaacgaag 4860
acaagatctg?ctttttgctt?gtactgggtc?tctctggtta?gaccagatct?gagcctggga 4920
gctctctggc?taactaggga?acccactgct?taagcctcaa?taaagcttgc?cttgagtgct 4980
tcaagtagtg?tgtgcccgtc?tgttgtgtga?ctctggtaac?tagagatccc?tcagaccctt 5040
ttagtcagtg?tggaaaatct?ctagcagtag?tagttcatgt?catcttatta?ttcagtattt 5100
ataacttgca?aagaaatgaa?tatcagagag?tgagaggaac?ttgtttattg?cagcttataa 5160
tggttacaaa?taaagcaata?gcatcacaaa?tttcacaaat?aaagcatttt?tttcactgca 5220
ttctagttgt?ggtttgtcca?aactcatcaa?tgtatcttat?catgtctggc?tctagctatc 5280
ccgcccctaa?ctccgcccag?ttccgcccat?tctccgcccc?atggctgact?aatttttttt 5340
atttatgcag?aggccgaggc?cgcctcggcc?tctgagctat?tccagaagta?gtgaggaggc 5400
ttttttggag?gcctaggctt?ttgcgtcgag?acgtacccaa?ttcgccctat?agtgagtcgt 5460
attacgcgcg?ctcactggcc?gtcgttttac?aacgtcgtga?ctgggaaaac?cctggcgtta 5520
cccaacttaa?tcgccttgca?gcacatcccc?ctttcgccag?ctggcgtaat?agcgaagagg 5580
cccgcaccga?tcgcccttcc?caacagttgc?gcagcctgaa?tggcgaatgg?cgcgacgcgc 5640
cctgtagcgg?cgcattaagc?gcggcgggtg?tggtggttac?gcgcagcgtg?accgctacac 5700
ttgccagcgc?cctagcgccc?gctcctttcg?ctttcttccc?ttcctttctc?gccacgttcg 5760
ccggctttcc?ccgtcaagct?ctaaatcggg?ggctcccttt?agggttccga?tttagtgctt 5820
tacggcacct?cgaccccaaa?aaacttgatt?agggtgatgg?ttcacgtagt?gggccatcgc 5880
cctgatagac?ggtttttcgc?cctttgacgt?tggagtccac?gttctttaat?agtggactct 5940
tgttccaaac?tggaacaaca?ctcaacccta?tctcggtcta?ttcttttgat?ttataaggga 6000
ttttgccgat?ttcggcctat?tggttaaaaa?atgagctgat?ttaacaaaaa?tttaacgcga 6060
attttaacaa?aatattaacg?tttacaattt?cccaggtggc?acttttcggg?gaaatgtgcg 6120
cggaacccct?atttgtttat?ttttctaaat?acattcaaat?atgtatccgc?tcatgagaca 6180
ataaccctga?taaatgcttc?aataatattg?aaaaaggaag?agtatgagta?ttcaacattt 6240
ccgtgtcgcc?cttattccct?tttttgcggc?attttgcctt?cctgtttttg?ctcacccaga 6300
aacgctggtg?aaagtaaaag?atgctgaaga?tcagttgggt?gcacgagtgg?gttacatcga 6360
actggatctc?aacagcggta?agatccttga?gagttttcgc?cccgaagaac?gttttccaat 6420
gatgagcact?tttaaagttc?tgctatgtgg?cgcggtatta?tcccgtattg?acgccgggca 6480
agagcaactc?ggtcgccgca?tacactattc?tcagaatgac?ttggttgagt?actcaccagt 6540
cacagaaaag?catcttacgg?atggcatgac?agtaagagaa?ttatgcagtg?ctgccataac 6600
catgagtgat?aacactgcgg?ccaacttact?tctgacaacg?atcggaggac?cgaaggagct 6660
aaccgctttt?ttgcacaaca?tgggggatca?tgtaactcgc?cttgatcgtt?gggaaccgga 6720
gctgaatgaa?gccataccaa?acgacgagcg?tgacaccacg?atgcctgtag?caatggcaac 6780
aacgttgcgc?aaactattaa?ctggcgaact?acttactcta?gcttcccggc?aacaattaat 6840
agactggatg?gaggcggata?aagttgcagg?accacttctg?cgctcggccc?ttccggctgg 6900
ctggtttatt?gctgataaat?ctggagccgg?tgagcgtggg?tctcgcggta?tcattgcagc 6960
actggggcca?gatggtaagc?cctcccgtat?cgtagttatc?tacacgacgg?ggagtcaggc 7020
aactatggat?gaacgaaata?gacagatcgc?tgagataggt?gcctcactga?ttaagcattg 7080
gtaactgtca?gaccaagttt?actcatatat?actttagatt?gatttaaaac?ttcattttta 7140
atttaaaagg?atctaggtga?agatcctttt?tgataatctc?atgaccaaaa?tcccttaacg 7200
tgagttttcg?ttccactgag?cgtcagaccc?cgtagaaaag?atcaaaggat?cttcttgaga 7260
tccttttttt?ctgcgcgtaa?tctgctgctt?gcaaacaaaa?aaaccaccgc?taccagcggt 7320
ggtttgtttg?ccggatcaag?agctaccaac?tctttttccg?aaggtaactg?gcttcagcag 7380
agcgcagata?ccaaatactg?tccttctagt?gtagccgtag?ttaggccacc?acttcaagaa 7440
ctctgtagca?ccgcctacat?acctcgctct?gctaatcctg?ttaccagtgg?ctgctgccag 7500
tggcgataag?tcgtgtctta?ccgggttgga?ctcaagacga?tagttaccgg?ataaggcgca 7560
gcggtcgggc?tgaacggggg?gttcgtgcac?acagcccagc?ttggagcgaa?cgacctacac 7620
cgaactgaga?tacctacagc?gtgagctatg?agaaagcgcc?acgcttcccg?aagggagaaa 7680
ggcggacagg?tatccggtaa?gcggcagggt?cggaacagga?gagcgcacga?gggagcttcc 7740
agggggaaac?gcctggtatc?tttatagtcc?tgtcgggttt?cgccacctct?gacttgagcg 7800
tcgatttttg?tgatgctcgt?caggggggcg?gagcctatgg?aaaaacgcca?gcaacgcggc 7860
ctttttacgg?ttcctggcct?tttgctggcc?ttttgctcac?atgttctttc?ctgcgttatc 7920
ccctgattct?gtggataacc?gtattaccgc?ctttgagtga?gctgataccg?ctcgccgcag 7980
ccgaacgacc?gagcgcagcg?agtcagtgag?cgaggaagcg?gaagagcgcc?caatacgcaa 8040
accgcctctc?cccgcgcgtt?ggccgattca?ttaatgcagc?tggcacgaca?ggtttcccga 8100
ctggaaagcg?ggcagtgagc?gcaacgcaat?taatgtgagt?tagctcactc?attaggcacc 8160
ccaggcttta?cactttatgc?ttccggctcg?tatgttgtgt?ggaattgtga?gcggataaca 8220
atttcacaca?ggaaacagct?atgaccatga?ttacgccaag?cgcgcaatta?accctcacta 8280
aagggaacaa?aagctggagc?tgca 8304
Claims (9)
1.CD133 the purposes in the preparation tumor markers, wherein, described CD133 is the sign of tumor cell invasion.
2. application as claimed in claim 1 is characterized in that described tumour cell is a glioma cell.
3. application as claimed in claim 1 is characterized in that, gets sample to be tested, detects the expression of CD133; The CD133 expression of sample to be tested is higher than the normal control sample, and tumour cell is prone to attack.
4. application as claimed in claim 3 is characterized in that described sample to be tested is a cell sample, tissue samples, perhaps the albumen extract of cell, tissue.
5.CD133 suppress the application in the tumor cell invasion medicine in preparation, wherein, CD133 is the medicine target that screening suppresses the tumor cell invasion medicine.
6. application as claimed in claim 5 is characterized in that, described inhibition tumor cell invasion medicine is to reduce the CD133 expression.
7. application as claimed in claim 5 is characterized in that, described inhibition tumor cell invasion medicine is a medicine of subduing the tumor cell migration ability.
8. one kind is screened the kit that suppresses the tumor cell invasion medicine, it is characterized in that this kit contains CD133.
9. kit as claimed in claim 8 is characterized in that described tumour cell is a glioma cell.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011100677634A CN102692506A (en) | 2011-03-21 | 2011-03-21 | Use of CD133 in preparation of tumor marker and kit of CD133 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100677634A CN102692506A (en) | 2011-03-21 | 2011-03-21 | Use of CD133 in preparation of tumor marker and kit of CD133 |
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| CN102692506A true CN102692506A (en) | 2012-09-26 |
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| CN2011100677634A Pending CN102692506A (en) | 2011-03-21 | 2011-03-21 | Use of CD133 in preparation of tumor marker and kit of CD133 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103424553A (en) * | 2013-07-18 | 2013-12-04 | 上海交通大学医学院附属瑞金医院 | HbA1c level detection reagent used as NAFLD detection reagent and used for screening medicine for treating NAFLD |
| CN107884583A (en) * | 2016-09-30 | 2018-04-06 | 复旦大学 | Applications of the CD133 of cytoplasm positioning as prognosis in hcc mark |
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| WO2008039874A2 (en) * | 2006-09-26 | 2008-04-03 | Cedars-Sinai Medical Center | Cancer stem cell antigen vaccines and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103424553A (en) * | 2013-07-18 | 2013-12-04 | 上海交通大学医学院附属瑞金医院 | HbA1c level detection reagent used as NAFLD detection reagent and used for screening medicine for treating NAFLD |
| CN107884583A (en) * | 2016-09-30 | 2018-04-06 | 复旦大学 | Applications of the CD133 of cytoplasm positioning as prognosis in hcc mark |
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Application publication date: 20120926 |