CN103238565A - Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model - Google Patents
Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model Download PDFInfo
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Abstract
The invention relates to a transgenic drosophila model for screening EphB4 kinase activity inhibitors and a construction method of the transgenic drosophila model. Plasmids are injected into yw drosophila embryos, after eclosion female drosophilae are hybridized with yw wild type male drosophilae, eclosion male drosophilae are hybridized with yw wild type female drosophilae, obtained offspring red-eye male drosophilae are hybridized with GMR-Ga14 virgin drosophila to obtain I-generation drosophilae, and virgin drosophilae of the I-generation drosophilae are selected to hybridize with yw/Y; +Cyo male drosophilae, and descendant curly-wing rougheye male drosophilae are male drosophilae in the transgenic drosophila model; the obtained male drosophilae are hybridized with +/FM7;+Cyo female drosophilae, descendant bar-eye curly-wing rougheye female drosophilae are further hybridized with the male drosophilae in the model, and bar-eyeless curly-wing rougheye female drosophilae are female drosophilae in the transgenic drosophila model. The transgenic drosophila model can be used for screening targeted drugs more quickly and conveniently.
Description
Technical field
The present invention relates to animal model and construction method thereof, relate in particular to a kind of transgenic fly model and construction method thereof.
Background technology
The Eph acceptor has been formed the maximum family of receptor tyrosine kinase, plays a significant role in the graphic formation of embryo, neural target, vascular development and adult neovascularization with their film binding partner ephrin.Have more and more evidences show Eph receptor signal path promote the many tumours of human body generation, promote tumour to form indirectly as direct promotion tumor growth or by forming tumor vessel.Eph acceptor and part thereof are detected overexpression in tumours such as breast cancer, prostate cancer, colon cancer.
EphB4 particularly, it is maximum albumen of studying in the Eph family.Use siRNA or antisense oligonucleotides chain suppress the expression meeting of EphB4 and then suppress the invasion and attack of propagation, survival and the PC3 prostate gland cancer cell of cell.Use the ectodomain of soluble EphB4 can suppress tumor growth.Disturb the signal path of EphB4 in the endothelial cell can change tumor vascular form.These are observed we EphB4 of prompting and play a significant role in tumour and angiogenic growth thereof.EphB4(claims HTK again) and its part ephrinB2(HTKL), in the growth of blood vessel and lymphatic vessel network, play a significant role.The EphB4 specifically expressing is at the vein epithelial cell, and the commitment specifically expressing that ephrinB2 grows at blood vessel and lymphatic vessel network is at the artery epithelial cell.In the mouse, the obstacle that the defective of EphB4 gene will cause capillary to connect, and finally cause mouse in death embryonic period, embryonic phase.During the embryo was taken place, EphB4 was expressed in the position of haemocyte generation and vascular development.The unconventionality expression of EphB4 in breast tissue cause breast structure unordered, function of organization damage and tend to take place canceration.
Nai-Ying Yang etc. find that EphB4 regulates the migration of mouse melanin tumor cell.The melanoma cell that grade malignancy is high is expressed higher levels of EphB4, migration also than low fast of grade malignancy.When suppressing the forward signal path of EphB4, tumor cell migration is also slack-off.In contrast to this, crossing of active EphB4 expressed the transfer ability that has obviously strengthened cell.The effect of EphB4 on cell migration and cellular morphology needs its kinase activity, and when crossing the EphB4 that expresses no kinase activity in cell, the migration of cell is suppressed.Nai-Ying Yang mentions EphB4 in article why can to regulate cell migration be because participate in the transformation again of actin.Heroult M etc. has selected lineup's class tumor cell line, and determines to have the expression of EphB4.
Owing to be easy to raise and be convenient to the genetics operation, fruit bat is applied to the research of phenogenetics, signal transduction and cell biology by success as model organism.The conservative of high-caliber gene and signal, and the similitude of the cell physiological biochemical pathway between mammal and the fruit bat make the fruit bat model be convenient to study human gene.
Summary of the invention
The technical problem to be solved in the present invention is: the construction method that a kind of fruit bat model of the EphB4 of screening kinase activity inhibitor is provided.
For achieving the above object, the invention provides following concrete technical scheme:
Obtain sub-I for fruit bat by the hybridization of the virgin fly of male transgenic fly and female GMR-Gal4, choose its described sub-I for fruit bat virgin fly and yw/Y; The male fly hybridization of +/Cyo, the offspring selects the male fly that sticks up wing rough eye, is the male fruit bat in the fruit bat model; With the male fruit bat in the fruit bat model that obtains with +/FM7; +/Cyo hybridization, the offspring selects the bar eye, sticks up wing, the female fly of rough eye, the female fly that obtains again with the fruit bat model in male drosophila hybrid, the offspring selects no bar eye, sticks up wing, the female fly of rough eye, is the female fruit bat in the fruit bat model.
The genotype of described male transgenic fly is EphB4 Kinase domain/Y or EphB4 Kinase domain.
Described male transgenic fly is that 5 ' end is had people EphB4 Kinase domain from the yeast upstream activating sequence, is incorporated into to obtain in the ph chromosome genome.
The preparation method of described male transgenic fly is:
The first step is carried out the structure of carrier earlier, is about to PUAST and transform the PUAST-EGFP carrier as.
Second step, transform PUAST-EGFP as the PUAST-MYR-EGFP transgene carrier: at first design primer and prepare myristoylation modification recognition sequence (MYR, SEQ ID NO:1), the myristoylation of double digestion being modified recognition sequence is inserted in the PUAST-EGFP carrier that same enzyme cuts and namely obtains the PUAST-MYR-EGFP transgene carrier;
The 3rd step, PUAST-MYR-hEphB4-EGFP construction of recombinant plasmid: be SEQ ID NO:4 with the Kinase domain(of people hEphB4 gene) clone forms the PUAST-MYR-hEphB4-EGFP recombinant plasmid in PUAST-MYR-EGFP transgenic fly carrier;
In the 4th step, the gained microinjection screening of the 3rd step is obtained to be transgenic fly.The 3rd the step gained plasmid and plasmid 2-3 mix, by the microinjection instrument microinjection to from 25 ℃ of cultivations
Y wIn the drosophila embryos that the fruit bat strain is collected.Through after cultivating, the larva of survival chosen put in the fresh food.Through after cultivating, a pipe is put in the female fly that comes out sprouting wings and the male fly hybridization of yw wild type again, and a pipe is put in the male fly that comes out sprouting wings and the female fly hybridization of yw wild type.The filial generation fruit bat of resulting blood-shot eye illness namely is transgenic fly, and genotype is hEphB4 kinase domain.
Female fruit bat in the described fruit bat model and the eyes of male fruit bat all are coarse.
The kinase domain of the eyes overexpression people's of described fruit bat model EphB4, and have the phenotype of rough eye.
The fruit bat model of screening EphB4 kinase activity inhibitor involved in the present invention, the kinase domain of the fruit bat eyes overexpression people's of this model EphB4, and have the phenotype of rough eye.
The genotype of male fruit bat in the described fruit bat model is EphB4 Kinase domain/Y or EphB4 Kinase domain; The eyes of described fruit bat model are coarse; Described fruit bat model is to have cloned people's the kinase domain of EphB4 and 7 amino acid code areas of Src are fused to pUAST transgenic fly carrier, then microinjection screening gained.
The present invention also provides a kind of method of the EphB4 of screening kinase activity inhibitor, it is characterized in that, is with female fruit bat and male drosophila hybrid in the above-mentioned gained fruit bat model, and the sub-I that obtains is for fruit bat overexpression people EphB4 Kinase domain, and eyes are coarse; For the budding different phase of fruit bat, the medicine to be screened of feeding filters out and can make the fruit bat eyes become level and smooth medicine by coarse at this sub-I.Medicine to be screened mixes in the fruit bat medium.
Screening technique and the standard in each step are to see the form of sticking up wing and eyes.
The constructed characteristics of fruit bat model of the present invention are overexpression people EphB4 Kinase domain in the fruit bat eyes, thereby cause the eyes of fruit bat to become coarse.Two of characteristics are that this fruit bat model phenotype is easy to observe, and the medicine feed mode is easy, and drug screening work is quick relatively, can carry out a large amount of drug screening rapidly.
Generally be that medicine to be screened is mixed in the fruit bat medium.Concrete medicine feed mode is for to put into no medicine medium with the model fruit bat, and the embryo that it is produced has changed in the medicine medium then, observes the variation of adult fruit bat eyes, and the concentration series that can be divided into all kinds of series of medicine and medicine of the same race compares.
Screen medicine with the transgenic fly model, the incomparable advantage of other system is arranged.Utilize model of the present invention and this method, make quickly a large amount of compounds and Chinese herbal medicine screened to be achieved, can develop the medicine that suppresses the EphB4 kinase activity accordingly.
Transgenic fly model of the present invention is used for drug screening, add compound N VP-BHG712(receptor tyrosine kinase (RTK) in the feeding feed〉〉 Raf〉the Raf inhibitor, observing the fruit bat eye changes, ocular tumorization is inhibited as a result, eyes become smoothly by coarse, illustrate that the tumour of compound N VP-BHG712 has inhibitory action.
A is the wild type fruit bat in the accompanying drawing 3, and eye is sliding smooth; B is EphB4 Kinase domain transgenic fly, the eye tumourization; C adds compound N VP-BHG712(receptor tyrosine kinase (RTK) in the EphB4 Kinase domain transgenic fly feeding feed〉〉 Raf〉the Raf inhibitor) after, tumourization is inhibited, and the fruit bat eyes are become smoothly by coarse.Prove absolutely that this transgenic fly model can well be used for drug screening: the result is convenient to observe.
The model of gained of the present invention can be used for carrying out drug screening according to the key feature of the roughly step of Fig. 4 and technology, finally obtains medicine.
Description of drawings
Fig. 1 is PUAST carrier collection of illustrative plates;
Fig. 2 is the basic procedure that the microinjection screening obtains transgenic fly;
Fig. 3 is for fast screening the figure as a result of EphB4 kinase activity inhibitor with the fruit bat model.Electron-microscope scanning, condition: 20KV, X450,50um;
Fig. 4 is the roughly step of drug screening and the key feature of technology.
Embodiment
Be described in detail below in conjunction with the embodiment of the present invention of embodiment, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment carry out according to the described technology of the document in this area or condition or according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
It is synthetic that used primer is invitrogen company in following examples, and order-checking also is invitrogen company.
Embodiment 1: fruit bat model and the structure thereof of screening EphB4 kinase activity inhibitor.
The 1:PUAST carrier is transformed (PUAST à PUAST-EGFP)
For can in fruit bat pupa time just can the testing goal gene whether changeing into the fruit bat body, need be with PUAST (Chinese plasmid vector strain cell pnca gene preservation center-Biovector Science Lab, Inc.pUAST, the carrier collection of illustrative plates is seen Fig. 1) carrier transforms, this has one section UAS (upstream activating sequence) sequence above carrier, the purpose of carrier transformation is that EGFP is linked in the PUAST carrier, and process is as follows:
Through XhoI and XbaI double digestion, it is standby that the recovery enzyme is cut afterproduct with the PUAST carrier; The PCR primer of design EGFP is as follows:
Xh-EGFP-F：G TAC TCG AGC TTT ATG GTG AGC AAG GGC GAG （SEQ ID NO:7）
X-EGFP-R：AAT CTA GAA CTT GTA CAG CTC GTC CAT G （SEQ ID NO:8）
Be masterplate PCR with above these 2 primers and pEGFP-N1 (Chinese plasmid vector strain cell pnca gene preservation center-Biovector Science Lab, Inc. pEGFP-N1), carry out the amplification of EGFP purpose fragment.
The PCR reaction system is as follows:
10×PCR buffer： 5 μL
Masterplate pEGFP-N1 1 μ L
Xh-EGFP-F 0.8 μL
X-EGFP-R 0.8 μL
dNTP(10mM)： 0.8 μL
Taq enzyme: 0.4 μ L
ddwater： 41.2 μL
Response procedures:
①95℃ 5min
57℃ 30s
Reclaim the PCR product, PUAST carrier after enzyme cut and the PCR product through reclaiming join in the 1.5mLEppendorff pipe in the ratio of 1:1,20-50ng carrier or fragment are used in a general coupled reaction, certainly our carrier and fragment amount that can add according to the connection effect adjustment of reality, but generally should not be greater than 150ng.Add 1 μ L, 10 * EXO III Buffer afterwards inside, mend to 10 μ L with sterile water, ice bath 5min behind the mixing adds 1 μ L EXO III (20U), mixing, ice bath 60min on ice.EXO III enzyme is exonuclease, and its enzyme in the time of 4 ℃ is cut speed and is approximately one hour 15bp, has been down to 4 ℃ so should guarantee the temperature of liquid in the pipe before adding EXO III enzyme.After enzyme is cut 1 hour, the EDTA(pH8.0 that adds 1 μ L0.5M) stops endonuclease reaction, whole system is placed 5min in 60 ℃, make the further sex change inactivation of enzyme, with being placed on ice bath 5min, guarantee carrier and insert stable bond between the fragment, afterwards all samples in the 1.5mLEppendorff pipe is joined competent cell DH
5Among the α, place on ice after three minutes and be coated with flat board, be placed on incubated overnight in the incubator afterwards.
Second day several monoclonal bacterium colony of picking from the flat board contains in the antibiotic LB culture fluid of AMP 37 ℃ of shaking table overnight incubation in 2 mL.Overnight culture is transferred to 1.5 mL Eppendorf pipe, centrifugal 30 seconds of 8,000 rpm.Abandon supernatant, add 250 μ L STET solution, shake resuspended bacterial sediment, afterwards with resuspended bacterium liquid on the Eppendorf pipe heater 100 ℃ the heating 2 minutes, centrifugal 5 minutes of 13,000 rpm choose the albumen precipitation of centrifuge tube bottom with the toothpick of sterilizing and to abandon.Add 250 μ L isopropyl alcohols (isopropanol), put upside down behind the mixing 13,000 rpm immediately centrifugal 2 minutes, abandon supernatant, the precipitation back-off dries the back and adds 50 μ L TE-RNase.The gained plasmid DNA can be used for restriction enzyme XhoI and XbaI enzyme cutting analysis.Enzyme is cut and is identified that correct plasmid is PUAST-EGFP.
The solution preparation:
STET solution: 0.1 mol/L NaCl, 10 mmol/L Tris-HCl(pH 8.0), 1 mmol/L EDTA(pH 8.0), 5% Triton X-100,0.22 μ m membrane filtration degerming.Facing with preceding adding final concentration is 1 mg/mL lysozyme (lysozyme).
TE-RNase: 10 mmol/L Tris-HCl（pH 8.0），1 mmol/L EDTA（pH 8.0），10 μg/mL RNase。
It is standby that PUAST-EGFP carrier after the extraction is put in-20 ℃ of refrigerators.
2: the structure of transgenic fly carrier PUAST-MYR-EGFP (PUAST-EGFP à PUAST-MYR-EGFP)
In order to make commentaries on classics can be positioned on the film advance the gene in the fruit bat; need transform the pUAST-EGFP carrier; used clone and microinjection carrier are pUAST-EGFP among the present invention; with 7 amino acid code areas of Src virus, just myristoylation is modified recognition sequence (MYR) interpolation in carrier.Myristoylation is modified recognition sequence: ATG GGG AGC AGC AAG AGC AAG(SEQ ID NO:1).In carrier is transformed, earlier carrier is cut with two restriction enzyme sites of EcoRI, BglII, these two restriction enzyme sites are PUAST carriers itself with, the carrier recovery after the incision is standby; Enzyme is cut system:
PUAST-EGFP 1ul (about 1ug)
EcoRI 1ul
BglII 1ul
10×buffer 5ul
ddw 42ul
Standby after the carrier recovery.
Design the primer of PUAST-MYR-EGFP then, MYR sequence front is added part fragment on the carrier that comprises the EcoRI restriction enzyme site, the MYR back adds the carrier sequence (face forward primer F1 as follows) that comprises the BglII restriction enzyme site.The design of reverse primer (face reverse primer R1 as follows) only need design proper sequence and comprise the XbaI enzyme cutting site after the EGFP terminator, do enzyme with EcoRI and two restriction enzyme sites of XbaI and cut to identify whether last carrier transforms success, identify in conjunction with the stripe size (821bp) of amplified fragments simultaneously.The design primer:
Forward primer F1:ATT GG
G AAT TCA TGG GGA GCA GCA AGA GCA AGA TTC GTT AAC
AGA TCT(SEQ ID NO:2), wherein single underscore represents EcoR
IRestriction enzyme site; Double underline is the BglII restriction enzyme site, adds frame and partly is the MYR sequence.
Reverse primer R1:
TCTAGAGGATCTTTGTGAAGGA(SEQ ID NO:3), wherein single underscore is the XbaI enzyme cutting site.
Synthetic good primer (invitrogen company is synthetic) is diluted to 100 μ M with 1 * TE solution, gets 10 μ l and dilute good primer and 90 μ l ddw mixing in 1.5ml Eppendorf pipe, obtain the PCR product by pcr amplification reaction.
Reaction system:
10×PCR buffer： 5 μL
Masterplate pUAST-EGFP 1 μ L
Forward primer F1 0.8 μ L
Reverse primer R1 0.8 μ L
dNTP(10mM)： 0.8 μL
Taq enzyme: 0.4 μ L
ddwater： 41.2 μL
Response procedures:
①95℃ 5min
72℃ 10min
Preserve product for 4 ℃
Reclaim the PCR product, PUAST carrier after enzyme cut and the PCR product through reclaiming join in the 1.5mLEppendorff pipe in the ratio of 1:1,20-50ng carrier or fragment are used in a general coupled reaction, certainly our carrier and fragment amount that can add according to the connection effect adjustment of reality, but generally should not be greater than 150ng.Add 1 μ L, 10 * EXO III Buffer afterwards inside, mend to 10 μ L with sterile water, ice bath 5min behind the mixing adds 1 μ L EXO III (20U), mixing, ice bath 60min on ice.EXO III enzyme is exonuclease, and its enzyme in the time of 4 ℃ is cut speed and is approximately one hour 15bp, has been down to 4 ℃ so should guarantee the temperature of liquid in the pipe before adding EXO III enzyme.After enzyme is cut 1 hour, the EDTA(pH8.0 that adds 1 μ L0.5M) stops endonuclease reaction, whole system is placed 5min in 60 ℃, make the further sex change inactivation of enzyme, with being placed on ice bath 5min, guarantee carrier and insert stable bond between the fragment, afterwards all samples in the 1.5mLEppendorff pipe is joined competent cell DH
5Among the α, place on ice after three minutes and be coated with flat board, be placed on incubated overnight in the incubator afterwards.
Second day several monoclonal bacterium colony of picking from the flat board contains in the antibiotic LB culture fluid of AMP 37 ℃ of shaking table overnight incubation in 2 mL.Overnight culture is transferred to 1.5 mL Eppendorf pipe, centrifugal 30 seconds of 8,000 rpm.Abandon supernatant, add 250 μ L STET solution, shake resuspended bacterial sediment, afterwards with resuspended bacterium liquid on the Eppendorf pipe heater 100 ℃ the heating 2 minutes, centrifugal 5 minutes of 13,000 rpm choose the albumen precipitation of centrifuge tube bottom with the toothpick of sterilizing and to abandon.Add 250 μ L isopropyl alcohols (isopropanol), put upside down behind the mixing 13,000 rpm immediately centrifugal 2 minutes, abandon supernatant, the precipitation back-off dries the back and adds 50 μ L TE-RNase.The gained plasmid DNA can be used for restriction enzyme EcoRI and XbaI enzyme cutting analysis.Enzyme is cut and is identified that correct plasmid is the PUAST-MYR-EGFP transgene carrier.
The 3:PUAST-MYR-hEphB4-EGFP construction of recombinant plasmid
Kinase domain(http with people hEphB4 gene: //www.ncbi.nlm.nih.gov/nuccore/NM_004444.4, be SEQ ID NO:4) partial sequence clone in PUAST-MYR-EGFP transgenic fly carrier, form the PUAST-MYR-hEphB4-EGFP recombinant plasmid, this plasmid of microinjection screens and obtains transgenic fly then.
The first step: PUAST-MYR-EGFP transgenic fly carrier enzyme is cut
Select BglII and two restriction enzyme sites of XhoI on the PUAST-MYR-EGFP transgenic fly carrier, enzyme is cut system:
PUAST-MYR-EGFP 1ul (about 1ug)
XhoI 1ul
BglII 1ul
10×buffer 5ul
ddw 42ul
It is standby that carrier glue reclaims the back.
Second step: the amplification of hEphB4 kinase domain gene
Proper sequence is selected in kinase domain (being SEQ ID NO:4) both sides at hEphB4, and considers the sequences Design primer of kinase domain., join in the LIC reaction system through after reclaiming with this purpose fragment (966bp) that primer PCR is come out, just can be connected with PUAST-MYR-EGFP transgenic fly carrier.
The PCR forward primer F2 of hEphB4:
ATTCGTGAAC
AGATCTGCCCTAATGAGGCTGTGAG(SEQ ID NO:5), wherein single underscore is the BglII restriction enzyme site.
The PCR reverse primer R2 of hEphB4:
CAGCCTCACTACTCAGCTGG
CTCGAGCTTTATGGTG (SEQ ID NO:6), wherein single underscore is the XhoI restriction enzyme site.
Synthetic good primer (invitrogen company is synthetic) is diluted to 100 μ M with 1 * TE solution, gets 10 μ l and dilute good primer and 90 μ l ddw mixing in 1.5ml Eppendorf pipe.
Reaction system:
10×PCR buffer： 5 μL
Masterplate (the DNA YW(Bloomington fruit bat center BL#1:Canton-S of fruit bat)) 1 μ L
PCR forward primer F2 0.8 μ L
PCR reverse primer R2 0.8 μ L
dNTP(10mM)： 0.8 μL
Taq enzyme: 0.4 μ L
ddwater： 41.2 μL
Response procedures:
①95℃ 5min
PCR is reacted purpose product glue to be reclaimed.
The 3rd step: the LIC reaction is undertaken by the catalysis of EXOIII DNA excision enzyme.
PUAST-MYR-EGFP transgenic fly carrier after enzyme cut and the above-mentioned second step PCR gained purpose fragment join in the 1.5mLEppendorff pipe in the ratio of 1:1,20-50ng carrier or fragment are used in a general coupled reaction, certainly our carrier and fragment amount that can add according to the connection effect adjustment of reality, but generally should not be greater than 150ng.Add 1 μ L, 10 * EXO III Buffer afterwards inside, mend to 10 μ L with sterile water, ice bath 5min behind the mixing adds 1 μ L EXO III (20U), mixing, ice bath 60min on ice.EXO III enzyme is exonuclease, and its enzyme in the time of 4 ℃ is cut speed and is approximately one hour 15bp, has been down to 4 ℃ so should guarantee the temperature of liquid in the pipe before adding EXO III enzyme.After enzyme is cut 1 hour, we add the EDTA(pH8.0 of 1 μ L0.5M) the termination endonuclease reaction, whole system is placed 5min in 60 ℃, make the further sex change inactivation of enzyme, with being placed on ice bath 5min, guarantee carrier and insert stable bond between the fragment, afterwards all samples in the 1.5mLEppendorff pipe is joined competent cell DH
5Among the α, place on ice after three minutes and be coated with flat board, be placed on incubated overnight in the incubator afterwards.
The 4th step: the extraction of PUAST-MYR-hEphB4-EGFP recombinant plasmid dna
Several monoclonal bacterium colonies of picking from the flat board contain in the antibiotic LB culture fluid of AMP 37 ℃ of shaking table overnight incubation in 2 mL.Overnight culture is transferred to 1.5 mL Eppendorf pipe, centrifugal 30 seconds of 8,000 rpm.Abandon supernatant, add 250 μ L STET solution, shake resuspended bacterial sediment, afterwards with resuspended bacterium liquid on the Eppendorf pipe heater 100 ℃ the heating 2 minutes, centrifugal 5 minutes of 13,000 rpm choose the albumen precipitation of centrifuge tube bottom with the toothpick of sterilizing and to abandon.Add 250 μ L isopropyl alcohols (isopropanol), put upside down behind the mixing 13,000 rpm immediately centrifugal 2 minutes, abandon supernatant, the precipitation back-off dries the back and adds 50 μ L TE-RNase.The gained plasmid DNA can be used for restriction analysis.It is standby to identify that correct PUAST-MYR-hEphB4-EGFP recombinant plasmid is put in-20 ℃ of refrigerators
The solution preparation:
STET solution: 0.1 mol/L NaCl, 10 mmol/L Tris-HCl(pH 8.0), 1 mmol/L EDTA(pH 8.0), 5% Triton X-100,0.22 μ m membrane filtration degerming.Facing with preceding adding final concentration is 1 mg/mL lysozyme (lysozyme).
TE-RNase: 10 mmol/L Tris-HCl（pH 8.0），1 mmol/L EDTA（pH 8.0），10 μg/mL RNase。
4, the screening of transgenic fly model
1) obtain the PUAST-MYR-hEphB4-EGFP recombinant plasmid of above-mentioned steps after, through order-checking (invitrogen company), cloned sequence is entirely true, extracts concentration again greater than the material of 1 μ g/ul from this plasmid, dissolves with ddw.Plasmid PUAST-MYR-hEphB4-EGFP and plasmid 2-3(Tissue Specificity of Drosophila P Element Transposition Is Regulated at the Level of mRNA Splicing Cell, Vol. 44,7-19, January 17,1986,) mix, final concentration is respectively 200 ng/ μ l and 50 ng/ μ l.Be put in 4 ℃ of preservations afterwards, be preferably in the week and injected.Before the injection, mixed liquor is centrifugal again, and in the process of microinjection, mixed liquor need be stored in 4 ℃.By the microinjection instrument microinjection to from 25 ℃ of cultivations
Y wIn the drosophila embryos that fruit bat (Bloomington fruit bat center BL#1:Canton-S) strain is collected.See Fig. 2, the cover glass that fills drosophila embryos is placed in the culture dish, put a filter paper that soaks in the culture dish, again culture dish is placed in the 18 degree insulating boxs.After 24 hours, cover glass is taken out, the larva of survival is chosen put in the fresh food.The pipe that fills food is placed in the 25 degree insulating boxs, after about 10 days, a pipe is put in the female fly that comes out sprouting wings and a male fly of yw wild type (Bloomington fruit bat center BL#1:Canton-S) hybridization, a pipe is put in the male fly that comes out sprouting wings and a female fly of yw wild type (Bloomington fruit bat center BL#1:Canton-S) hybridization.After about 10 days, after the filial generation fruit bat comes out, at the microscopically microscopy, find the blood-shot eye illness fruit bat, be transgenic fly, genotype is hEphB4 kinase domain;
2) virgin fly (from the Bloomington fruit bat center BL#9146) hybridization of choosing the transgenic fly of male hEphB4 kinase domain of gained and female GMR-Gal4 obtains sub-I (genotype is hEphB4 kinase domain for fruit bat; GMR-Gal4).Obtain the method for the male fruit bat in the transgenic fly model: choose above-mentioned sub-I for fruit bat the virgin fly with
Y w/ Y; Male fly (the BL#1495:y of +/Cyo
[1]w
[1]BL#4558:FM7a, l (1) TW24[1]/oc[1] ptg[3] l (1) TW1[cs]; CyO/l (2) DTS91[1], select after the hybridization
Y w/ Y; The male fly of +/Cyo) hybridization, the offspring selects the male fly that sticks up wing rough eye.Obtain the method for the female fruit bat in the fruit bat model: with the male fruit bat in the fruit bat model that obtains with +/FM7; Female fruit bat (the BL#1495:y of +/Cyo
[1]w
[1]BL#4558:FM7a, l (1) TW24[1]/oc[1] ptg[3] l (1) TW1[cs]; CyO/l (2) DTS91[1], select after the hybridization +/FM7; The female fruit bat of +/Cyo) hybridization, the offspring selects the bar eye, sticks up wing, the female fly of rough eye, the female fly that obtains again with the fruit bat model in male drosophila hybrid, the offspring selects no bar eye, sticks up wing, the female fly of rough eye, i.e. female fruit bat in the transgenic fly model.The genotype of male and female fruit bat in the resulting transgenic models is hEphB4 Kinase domain/Y or hEphB4Kinase domain; GMR-Gal4/Cyo.
Embodiment 2, the transgenic fly model is used for drug screening
The concrete steps that the fruit bat model is used for drug screening are: the hEphB4 transgenic fly model that top embodiment 1 is obtained is the fruit bat feeding feed interpolation compound N VP-BHG712(receptor tyrosine kinase (RTK) that the eye tumourization takes place eye〉〉 Raf〉the Raf inhibitor, U.S. Selleck company, network address: http://www.selleck.cn/), observing the fruit bat eye changes, ocular tumorization is inhibited as a result, eyes become smoothly by coarse, illustrate that the tumour of compound N VP-BHG712 has inhibitory action.
Fig. 3 is for fast screening the figure as a result of hEphB4 kinase activity inhibitor with the fruit bat model.A is wild type fruit bat (from Bloomington fruit bat center BL#1:Canton-S) in the drawings, and eye is sliding smooth; B is hEphB4 Kinase domain; The GMR-Gal4/Cyo transgenic fly, the eye tumourization; C is hEphB4 Kinase domain; Add compound N VP-BHG712(receptor tyrosine kinase (RTK) in the GMR-Gal4/Cyo transgenic fly feeding feed〉〉 Raf〉the Raf inhibitor, U.S. Selleck company, network address: http://www.selleck.cn/), tumourization is inhibited, and the fruit bat eyes are become smoothly by coarse.Illustrate that this fruit bat model can well be used for drug screening: the result is convenient to observe.
Sequence table
SEQUENCE LISTING
＜110〉the Xiamen University ocean is three
＜120〉a kind of transgenic fly model and construction method thereof that screens EphB4 kinase activity inhibitor
<130> P6700
<160> 8
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tctagaggat ctttgtgaag ga 22
<210> 4
<211> 4369
<212> DNA
<213> Homo sapiens
<400> 4
ttccagcgca gctcagcccc tgcccggccc ggcccgcccg gctccgcgcc gcagtctccc 60
tccctcccgc tccgtccccg ctcgggctcc caccatcccc gcccgcgagg agagcactcg 120
gcccggcggc gcgagcagag ccactccagg gaggggggga gaccgcgagc ggccggctca 180
gcccccgcca cccggggcgg gaccccgagg ccccggaggg accccaactc cagccacgtc 240
ttgctgcgcg cccgcccggc gcggccactg ccagcacgct ccgggcccgc cgcccgcgcg 300
cgcggcacag acgcggggcc acacttggcg ccgccgcccg gtgccccgca cgctcgcatg 360
ggcccgcgct gagggccccg acgaggagtc ccgcgcggag tatcggcgtc cacccgccca 420
gggagagtca gacctggggg ggcgagggcc ccccaaactc agttcggatc ctacccgagt 480
gaggcggcgc catggagctc cgggtgctgc tctgctgggc ttcgttggcc gcagctttgg 540
aagagaccct gctgaacaca aaattggaaa ctgctgatct gaagtgggtg acattccctc 600
aggtggacgg gcagtgggag gaactgagcg gcctggatga ggaacagcac agcgtgcgca 660
cctacgaagt gtgtgacgtg cagcgtgccc cgggccaggc ccactggctt cgcacaggtt 720
gggtcccacg gcggggcgcc gtccacgtgt acgccacgct gcgcttcacc atgctcgagt 780
gcctgtccct gcctcgggct gggcgctcct gcaaggagac cttcaccgtc ttctactatg 840
agagcgatgc ggacacggcc acggccctca cgccagcctg gatggagaac ccctacatca 900
aggtggacac ggtggccgcg gagcatctca cccggaagcg ccctggggcc gaggccaccg 960
ggaaggtgaa tgtcaagacg ctgcgtctgg gaccgctcag caaggctggc ttctacctgg 1020
ccttccagga ccagggtgcc tgcatggccc tgctatccct gcacctcttc tacaaaaagt 1080
gcgcccagct gactgtgaac ctgactcgat tcccggagac tgtgcctcgg gagctggttg 1140
tgcccgtggc cggtagctgc gtggtggatg ccgtccccgc ccctggcccc agccccagcc 1200
tctactgccg tgaggatggc cagtgggccg aacagccggt cacgggctgc agctgtgctc 1260
cggggttcga ggcagctgag gggaacacca agtgccgagc ctgtgcccag ggcaccttca 1320
agcccctgtc aggagaaggg tcctgccagc catgcccagc caatagccac tctaacacca 1380
ttggatcagc cgtctgccag tgccgcgtcg ggtacttccg ggcacgcaca gacccccggg 1440
gtgcaccctg caccacccct ccttcggctc cgcggagcgt ggtttcccgc ctgaacggct 1500
cctccctgca cctggaatgg agtgcccccc tggagtctgg tggccgagag gacctcacct 1560
acgccctccg ctgccgggag tgccgacccg gaggctcctg tgcgccctgc gggggagacc 1620
tgacttttga ccccggcccc cgggacctgg tggagccctg ggtggtggtt cgagggctac 1680
gtcctgactt cacctatacc tttgaggtca ctgcattgaa cggggtatcc tccttagcca 1740
cggggcccgt cccatttgag cctgtcaatg tcaccactga ccgagaggta cctcctgcag 1800
tgtctgacat ccgggtgacg cggtcctcac ccagcagctt gagcctggcc tgggctgttc 1860
cccgggcacc cagtggggct gtgctggact acgaggtcaa ataccatgag aagggcgccg 1920
agggtcccag cagcgtgcgg ttcctgaaga cgtcagaaaa ccgggcagag ctgcgggggc 1980
tgaagcgggg agccagctac ctggtgcagg tacgggcgcg ctctgaggcc ggctacgggc 2040
ccttcggcca ggaacatcac agccagaccc aactggatga gagcgagggc tggcgggagc 2100
agctggccct gattgcgggc acggcagtcg tgggtgtggt cctggtcctg gtggtcattg 2160
tggtcgcagt tctctgcctc aggaagcaga gcaatgggag agaagcagaa tattcggaca 2220
aacacggaca gtatctcatc ggacatggta ctaaggtcta catcgacccc ttcacttatg 2280
aagaccctaa tgaggctgtg agggaatttg caaaagagat cgatgtctcc tacgtcaaga 2340
ttgaagaggt gattggtgca ggtgagtttg gcgaggtgtg ccgggggcgg ctcaaggccc 2400
cagggaagaa ggagagctgt gtggcaatca agaccctgaa gggtggctac acggagcggc 2460
agcggcgtga gtttctgagc gaggcctcca tcatgggcca gttcgagcac cccaatatca 2520
tccgcctgga gggcgtggtc accaacagca tgcccgtcat gattctcaca gagttcatgg 2580
agaacggcgc cctggactcc ttcctgcggc taaacgacgg acagttcaca gtcatccagc 2640
tcgtgggcat gctgcggggc atcgcctcgg gcatgcggta ccttgccgag atgagctacg 2700
tccaccgaga cctggctgct cgcaacatcc tagtcaacag caacctcgtc tgcaaagtgt 2760
ctgactttgg cctttcccga ttcctggagg agaactcttc cgatcccacc tacacgagct 2820
ccctgggagg aaagattccc atccgatgga ctgccccgga ggccattgcc ttccggaagt 2880
tcacttccgc cagtgatgcc tggagttacg ggattgtgat gtgggaggtg atgtcatttg 2940
gggagaggcc gtactgggac atgagcaatc aggacgtgat caatgccatt gaacaggact 3000
accggctgcc cccgccccca gactgtccca cctccctcca ccagctcatg ctggactgtt 3060
ggcagaaaga ccggaatgcc cggccccgct tcccccaggt ggtcagcgcc ctggacaaga 3120
tgatccggaa ccccgccagc ctcaaaatcg tggcccggga gaatggcggg gcctcacacc 3180
ctctcctgga ccagcggcag cctcactact cagcttttgg ctctgtgggc gagtggcttc 3240
gggccatcaa aatgggaaga tacgaagaaa gtttcgcagc cgctggcttt ggctccttcg 3300
agctggtcag ccagatctct gctgaggacc tgctccgaat cggagtcact ctggcgggac 3360
accagaagaa aatcttggcc agtgtccagc acatgaagtc ccaggccaag ccgggaaccc 3420
cgggtgggac aggaggaccg gccccgcagt actgacctgc aggaactccc caccccaggg 3480
acaccgcctc cccattttcc ggggcagagt ggggactcac agaggccccc agccctgtgc 3540
cccgctggat tgcactttga gcccgtgggg tgaggagttg gcaatttgga gagacaggat 3600
ttgggggttc tgccataata ggaggggaaa atcacccccc agccacctcg gggaactcca 3660
gaccaagggt gagggcgcct ttccctcagg actgggtgtg accagaggaa aaggaagtgc 3720
ccaacatctc ccagcctccc caggtgcccc cctcaccttg atgggtgcgt tcccgcagac 3780
caaagagagt gtgactccct tgccagctcc agagtggggg ggctgtccca gggggcaaga 3840
aggggtgtca gggcccagtg acaaaatcat tggggtttgt agtcccaact tgctgctgtc 3900
accaccaaac tcaatcattt ttttcccttg taaatgcccc tcccccagct gctgccttca 3960
tattgaaggt ttttgagttt tgtttttggt cttaattttt ctccccgttc cctttttgtt 4020
tcttcgtttt gtttttctac cgtccttgtc ataactttgt gttggaggga acctgtttca 4080
ctatggcctc ctttgcccaa gttgaaacag gggcccatca tcatgtctgt ttccagaaca 4140
gtgccttggt catcccacat ccccggaccc cgcctgggac ccccaagctg tgtcctatga 4200
aggggtgtgg ggtgaggtag tgaaaagggc ggtagttggt ggtggaaccc agaaacggac 4260
gccggtgctt ggaggggttc ttaaattata tttaaaaaag taactttttg tataaataaa 4320
agaaaatggg acgtgtccca gctccagggg taaaaaaaaa aaaaaaaaa 4369
<210> 5
<211> 35
<212> DNA
＜213〉artificial synthetic
<400> 5
attcgtgaac agatctgccc taatgaggct gtgag 35
<210> 6
<211> 36
<212> DNA
＜213〉artificial synthetic
<400> 6
cagcctcact actcagctgg ctcgagcttt atggtg 36
<210> 7
<211> 31
<212> DNA
＜213〉artificial synthetic
<400> 7
gtactcgagc tttatggtga gcaagggcga g 31
<210> 8
<211> 28
<212> DNA
＜213〉artificial synthetic
<400> 8
aatctagaac ttgtacagct cgtccatg 28
Claims (9)
1. fruit bat model that screens EphB4 kinase activity inhibitor is characterized in that: the kinase domain of the fruit bat eyes overexpression people's of this model EphB4, and have the phenotype of rough eye.
2. the described fruit bat model of claim 1 is characterized in that, the genotype of the male fruit bat in this fruit bat model is EphB4 Kinase domain/Y or EphB4 Kinase domain; The eyes of described fruit bat model are coarse; Described fruit bat model is to have cloned people's the kinase domain of EphB4 and 7 amino acid code areas of Src are fused to pUAST transgenic fly carrier, then microinjection screening gained.
3. a construction method that screens the fruit bat model of EphB4 kinase activity inhibitor is characterized in that, is to obtain sub-I for fruit bat by the hybridization of the virgin fly of male transgenic fly and female GMR-Gal4, choose its described sub-I for fruit bat virgin fly and yw/Y; The male fly hybridization of +/Cyo, the offspring selects the male fly that sticks up wing rough eye, is the male fruit bat in the fruit bat model; With the male fruit bat in the fruit bat model that obtains with +/FM7; +/Cyo hybridization, the offspring selects the bar eye, sticks up wing, the female fly of rough eye, the female fly that obtains again with the fruit bat model in male drosophila hybrid, the offspring selects no bar eye, sticks up wing, the female fly of rough eye, is the female fruit bat in the fruit bat model.
4. the construction method of claim 3, it is characterized in that: the genotype of described male transgenic fly is EphB4 Kinase domain/Y or EphB4 Kinase domain.
5. the construction method of claim 3 is characterized in that: described male transgenic fly is that 5 ' end is had people EphB4 Kinase domain from the yeast upstream activating sequence, is incorporated into to obtain in the ph chromosome genome.
6. the construction method of claim 3, it is characterized in that: the female fruit bat in the described fruit bat model and the eyes of male fruit bat all are coarse.
7. the construction method of claim 3 is characterized in that: the kinase domain of the eyes overexpression people's of this fruit bat model EphB4, and have the phenotype of rough eye.
8. method of screening EphB4 kinase activity inhibitor, it is characterized in that, be with female fruit bat and male drosophila hybrid in the arbitrary described fruit bat model of claim 3-7, the sub-I that obtains is for fruit bat overexpression people EphB4 Kinase domain, and eyes are coarse; For the budding different phase of fruit bat, the medicine to be screened of feeding filters out and can make the fruit bat eyes become level and smooth medicine by coarse at this sub-I.
9. the described method of claim 8, it is characterized in that: medicine to be screened mixes in the fruit bat medium.
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