CN102174550A - Recombinant ubiquitin ligase PTB-U-box fusion gene and expression vector and application thereof - Google Patents
Recombinant ubiquitin ligase PTB-U-box fusion gene and expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant ubiquitin ligase PTB-U-box fusion gene and an expression vector and application thereof. The recombinant ubiquitin ligase PTB-U-box fusion gene is formed by connecting a PTB domain gene of IRS-1 and a U-box domain gene of carboxy terminus of Hsc70 Interacting protein (CHIP), and the specific nucleotide sequence is shown as SEQ ID No. 1. The constructed PTB-U-box fusion gene can be cloned to different expression vectors so as to enter tumor cells through different paths to play a role, and the expression vectors comprise eukaryotic expression vectors or adenovirus expression vectors. After the fusion gene transfects the tumor cells, the carcinogenic related protein IGF-1R of the host tumor cells can be regulated down, the cell proliferation and intrusion capacities of the tumor cells are remarkably reduced, and the fusion gene shows proliferation inhibition and intrusion inhibition on the tumor cells so as to achieve the anti-tumor effect.
Description
Technical field
The invention belongs to biological technical field, relate to gene recombination and expression thereof, particularly a kind of reorganization ubiquitin ligase PTB-U-box fusion gene and expression vector and purposes.
Background technology
1, the general introduction of CHIP
CHIP (Carboxy terminus of Hsc70 Interacting Protein) is a plasmosin class evolution conservative, that extensively distribute, is considered to a kind of chaperone (co-chaperone) altogether at first.Owing to contain a U-box structural domain in its molecule, therefore belong to a class E3 ubiquitin ligase in essence again.CHIP plays an important role in protein quality control, stable regulation process by promoting proteinic ubiquitinization, degraded.In breast cancer tissue, expression and the tumor grade of CHIP are negative correlation; Experimental evidence shows that also CHIP can suppress the propagation and the transfer of tumour.The effect of these research prompting enhancings CHIP might be as a kind of new oncotherapy strategy.
1) CHIP contains a TPR structural domain and a U-box structural domain
CHIP albumen is begun by N-terminal, is made of TPR structural domain (tetratricopeptide repeatdomain), Linker and U-box structural domain respectively.TPR structural domain mediation protein-protein interaction can and combine the proteic folding process of regulating heat shock protein substrate/client with the terminal identification mutually of heat shock protein(HSP) Hsc70/Hsp70, Hsp90C; The function of Linker is still indeterminate, and only understanding it at present is essential for mutually combining of TPR dependence; The U-box structural domain of C end has been given CHIP ubiquitin ligase activity, is responsible for raising ubiquitin binding enzyme E2 and the ubiquitin that will be combined on the E2 is transferred on TPR structural domain institute bonded mate molecule and the substrate protein thereof, promotes these molecules to degrade in proteasome.
2) the CHIP molecule is as a total molecule companion and a ubiquitin-like ligase enzyme, be intracellular protein folding-refolding machine and the uiquitin-protease enzyme system molecule hinge between these two kinds of paths of uniting, participate in the intracellular protein quality control
(protein quality control QC) continues to monitor the reparation or the degraded of the folding and misfolded protein of new polypeptide chain, to keep the stable state of cell to cell dependent protein matter quality control system.QC is made up of molecular chaperones (comprising Hsp70, Hsp40 etc.) and uiquitin-protease enzyme system system, and the former helps folding and refolding of new polypeptide chain, the latter's be responsible for degrading albumen of false folding or damage.
CHIP can mutually combine by its TPR structural domain and molecular chaperones Hsp70C end as a kind of total molecule companion, and adjusting molecular chaperones Hsp70 family member combines and release with substrate protein; In addition, CHIP with can stop the folding of Hsp70 after Hsp70 combines, and rely on its U-box structural domain and the active degraded that promotes substrate protein of ubiquitin ligase.By the way, CHIP participates in the quality control of intracellular protein directly.
3) CHIP of research prompting recently has the effect that suppresses tumor cell proliferation and transfer
CHIP is widely distributed, and especially its expression level is very high in the tissue that metabolism enlivens, as skeletal muscle, heart and brain.In cell, CHIP mainly is positioned in the kytoplasm, also has small part to be present in the nuclear.Nearest studies show that, in human breast carcinoma tissue, and the expression level of CHIP and malignancy of tumor degree and classification inverse correlation.Mechanism Study and cell experiment show that CHIP is by for example ubiquitinization, the degraded of HER2, ER-a, GR, SRC-3 etc. of the multiple carcinogenic protein of promotion, thus the propagation and the transfer of inhibition tumour.
2, the application of reorganization ubiquitin ligase
Ubiquitin ligase E3 is responsible for specific recognition and bound substrates in the ubiquitin process.A plurality of in recent years study group use recombinant technology and make up the reorganization ubiquitin ligase, are not some albumen of ubiquitin system natural substrate under the normal circumstances of successfully degrading.For example pass through to modify the reorganization F-box PROTEIN C FP of the substrate of ubiquitin ligase mixture Skp1-Cullin1-F-box (SCF), target degraded pRB and β-catenin in conjunction with the F-box of subunit formation.After recombinating from two RING of BRCA1 and BARD and proliferating cell nuclear antigen PCNA respectively, this recombinant molecule reduces the proteic level of p57 in the mode that proteasome relies on, and reduces its function.And the research of Li Xia etc. also shows, the reorganization ubiquitin ligase that utilizes Cb1 to make up can effectively be reduced the HER2 of high expression level in the breast cancer cell, thereby reaches the purpose that suppresses tumor growth.These studies confirm that the ubiquitin ligase that utilizes reorganization, target ground degraded some diseases associated molecule.But also do not utilize the active reorganization ubiquitin ligase that makes up of CHIP ubiquitin ligase to carry out the relevant report that gene therapy is studied at present.
Summary of the invention
The problem that the present invention solves is to provide a kind of reorganization ubiquitin ligase PTB-U-box fusion gene and expression vector and purposes, structure is based on the ubiquitin ligase active region gene fusion construct of CHIP, constructed fusion gene has the ubiquitinization and the degradation function of target, can be used for the preparation of anti-tumor drug or carries out gene therapy.
The present invention is achieved through the following technical solutions:
A kind of reorganization ubiquitin ligase PTB-U-box fusion gene is that the PTB domain gene with IRS-1 is connected with the U-box domain gene of CHIP.
The nucleotide sequence of described reorganization ubiquitin ligase PTB-U-box fusion gene is shown in SEQ.ID.NO.1.
Described reorganization ubiquitin ligase PTB-U-box fusion gene is cloned into carrier for expression of eukaryon pFLAG-CMV4 by EcoR I, EcoR V restriction enzyme site, and ubiquitin ligase carrier for expression of eukaryon pFLAG-CMV4-PTB-U-box obtains recombinating.
Described reorganization ubiquitin ligase PTB-U-box fusion gene cloning is gone among the destination carrier pAd/CMV/V5-DEST, obtains recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box.
Described reorganization ubiquitin ligase PTB-U-box fusion gene is applied to the preparation of anti-IGF-1R positive expression tumour medicine.
The application of described preparing anti-tumor medicine is that reorganization ubiquitin ligase PTB-U-box fusion gene cloning is gone into carrier for expression of eukaryon or adenovirus expression carrier.
Compared with prior art, the present invention has following beneficial technical effects:
The present invention can discern and in conjunction with the PTB domain gene of the IRS-1 of IGF-1R with have the U-box domain gene of the active CHIP of ubiquitin ligase, merge formation reorganization ubiquitin ligase PTB-U-box fusion gene by PCR, can the carcinogenic associated protein IGF-1R of target degraded after this fusion gene is expressed, wherein, the PTB structural domain is responsible for the combination of IGF-1R target, and U-box is responsible for the ubiquitin molecule is connected to substrate to promote substrate generation ubiquitinization and degraded.
Constructed PTB-U-box fusion gene can be cloned into different expression vectors, plays a role thereby enter into tumour cell by different approach, and described expression vector comprises carrier for expression of eukaryon or adenovirus expression carrier.
The present invention is based on the PTB-U-box fusion gene, made up reorganization ubiquitin ligase carrier for expression of eukaryon pFLAG-CMV4-PTB-U-box, this carrier is after transfection tumor cell, can reduce the carcinogenic associated protein IGF-1R of host's tumour cell, the cell proliferation and the invasive ability of tumour cell significantly are lowered, the propagation that shows tumour cell suppresses and the invasion and attack inhibition, thereby reaches antineoplastic effect.
And based on the PTB-U-box fusion gene, the recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box of structure after generation comprises the virion of fusion gene, can be injected in the middle of the tumour entity after reaching certain titre.
Description of drawings
Fig. 1 is the electrophoresis detection of the PTB-U-box fusion gene that makes up figure as a result;
Fig. 2 is the double digestion qualification result figure of recombinant eukaryon expression vector pFLAG-CMV4-PTB-U-box;
Fig. 3 is the electrophoresis detection identified of the double digestion of entry vector pENTR-PTB-U-box figure as a result;
Fig. 4 detects the as a result figure of fusion gene PTB-U-box to the downward modulation of host's tumour cell IGF-1R albumen for immunoblotting;
Fig. 5 detects the cell proliferation graphic representation of the Hela cell of transfection different carriers for the MTT colorimetry;
Fig. 6 forms the Giemsa coloration result figure of ability for the Hela cell clone of transfection different carriers;
Fig. 7 is the column analytical results figure of the Hela cell clone rate of formation of transfection different carriers;
Fig. 8 is the cell invasion visual field observations figure of the Hela cell of transfection different carriers;
Fig. 9 counts column figure as a result for the cell invasion of the Hela cell of transfection different carriers.
Embodiment
The present invention utilizes the PTB structural domain (Phosphotyrosine Binding domain) of linkers IRS-1 (Insulin Receptor Substrate 1) can discern and bound insulin like growth factor acceptor (Insulin-like Growth Factor Receptor 1, IGF-1R) the ubiquitin ligase activity of characteristic and CHIP, structure can the carcinogenic associated protein IGF-1R of target degraded reorganization ubiquitin ligase PTB-U-box.Constructed fusion gene PTB-U-box clones the PTB domain gene of IRS-1 respectively and has the U-box domain gene of the active CHIP of ubiquitin ligase, adopts recombinant PCR to merge; Wherein, the PTB structural domain is responsible for the combination of IGF-1R target, and U-box is responsible for the ubiquitin molecule is connected to substrate to promote substrate generation ubiquitinization, degraded.Below in conjunction with the structure of fusion gene PTB-U-box, carrier for expression of eukaryon and recombinant adenovirus, the present invention is described in further detail to reach the degraded that promotes carcinogenic associated protein IGF-1R, and the explanation of the invention is not limited.
1, the structure of reorganization ubiquitin ligase PTB-U-box fusion gene
1.1PTB and the clone of U-box domain gene
According to the U-box gene order of the PTB gene order of IRS-1 among the Genbank, CHIP respectively design of amplification primers P is used to increase the PTB gene order in contrast, amplimer is used for the reorganization amplification of fusion gene to P1, P2, specific design is:
Primer is to P:
Upstream primer: ggcgaattca atggaggctg gggaggactt gag 33;
Downstream primer: ggcgatatcc tagcgagggc ggaactcatc ac 32;
Primer is to P1, P2:
Up：ggcgaattca?atggaggctg?gggaggactt?gag 33；
M1：gttcagccgg?cgagggcgga?actcatc 27；
M2：cgccctcgcc?ggctgaactt?cggggacg 28；
Down：ggcgatatcc?tagtagtcct?ccacccagcc?attc 34；
Wherein, introduce several reverse complemental Nucleotide of 5 ' end of U-box gene fragment among the downstream primer sequence M1 of amplification PTB, the upstream primer sequence M2 introducing PTB gene fragment 3 ' of amplification U-box-hold several Nucleotide is so that two truncates merge in subsequent recombination PCR reaction mutually.Simultaneously, in the upstream primer sequence Up of amplification PTB, introduce restriction enzyme site EcoR I, introduce restriction enzyme site EcoR V among the downstream primer sequence D own of amplification U-box.
Then, be template with the cDNA of Hela cell, respectively with primer to P, P1 (Up is that amplimer is right M1), pcr amplification PTB domain gene, the pcr amplification program is: 94 ℃ of 5min, 94 ℃ of 30s, 57 ℃ of 45s, 72 ℃ of 30s, 35cycle;
CDNA with the Hela cell is a template, and (M2 is that amplimer is right Down), and pcr amplification U-box domain gene, pcr amplification program are 94 ℃ of 5min, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, 35cycle to P2 with primer.
Collect pcr amplification product, obtain PTB domain gene and U-box domain gene behind the purifying.
1.2PTB-U-box the structure of fusion gene
Utilize recombinant PCR, the PTB domain gene of amplification and the U-box domain gene of amplification carried out gene fusion:
Is template with primer to the U-box domain gene equal amount of mixture (mixing at 1: 1) that P2 increases to the PTB domain gene that P1 increases with primer, Down is right as primer with PTB upstream primer Up, U-box downstream primer, carry out recombinant PCR, PTB is merged mutually with U-box, the pcr amplification program is: 94 ℃ of 5min, 94 ℃ of 30s, 61 ℃ of 45s, 72 ℃ of 1min, 35cycle.Collect the product of recombinant PCR amplification, obtain the PTB-U-box fusion gene behind the purifying.
The result that PTB domain gene and PTB-U-box fusion gene are carried out detected through gel electrophoresis as shown in Figure 1, wherein swimming lane M is Marker, the clip size of PTB domain gene is 372bp, and the clip size of PTB-U-box (PTB-U) fusion gene is 906bp, is consistent with desired design.
2, the structure of the recombinant eukaryon expression vector of PTB-U-box fusion gene, recombinant adenoviral vector
2.1 the structure of recombinant eukaryon expression vector pFLAG-CMV4-PTB-U-box
With restriction enzyme EcoR I, EcoR V PTB-U-box fusion gene and carrier for expression of eukaryon pFLAG-CMV4 are carried out double digestion respectively respectively, reclaim after the endonuclease bamhi, the PTB-U-box fusion gene after with the T4 ligase enzyme enzyme being cut is connected with the pFLAG-CMV4 carrier.
After ligation is finished, connect product and transform DH5 α competent cell, after the overnight incubation, the single clone of picking shakes bacterium and cultivates, and extracts plasmid and carries out EcoR I, the evaluation of EcoR V double digestion, carries out sequence verification at last; With constructed successful recombinant molecule called after pFLAG-CMV4-PTB-U-box, this is reorganization ubiquitin ligase PTB-U-box carrier for expression of eukaryon.
The electrophoresis result that double digestion is identified as shown in Figure 2, wherein swimming lane M is Marker, swimming lane CMV4-PTB inserts carrier with the PTB domain gene to cut the detection contrast as enzyme, swimming lane CMV4-PTB-U is that the enzyme of the carrier for expression of eukaryon of fusion gene is cut detected result, two fragments of all digested one-tenth size of two carriers as can be seen, and small segment PTB-U fusion gene wherein is greater than in contrast PTB domain gene.
The contrast sequencing result, the nucleotide sequence of PTB-U-box fusion gene is shown in SEQ.ID.NO.1.
1.3 the structure of recombinant adenoviral vector pDEST-PTB-U-box
The structure of recombinant adenovirus uses the ViraPower of Invitrogen company
TMAdenovirus system, this system comprises entry vector pENTR
TM, destination carrier pAd/CMV/V5-DEST, LR clonase, Proteinase K etc.At first Kuo Zeng PTB domain gene and PTB-U-box fusion gene utilize restriction enzyme EcoR I, EcoR V and T4 ligase enzyme, respectively this two bar segment are inserted into entry vector pENTR
TMAmong the 3C.Through enzyme cut identify and sequence verification after, carry out LR and react, in PH is 8.0 TE buffer, under the effect of LR recombinase, 25 ℃ of reaction 1h with purpose fragment PTB or PTB-U-box fusion gene, transfer to the destination carrier pAd/CMV/V5-DEST from entry vector.Add Proteinase K then, 10 minutes termination reactions of 37 ℃ of effects, get recombinant products 1 μ L and transform the Top10 competent cell, the picking mono-clonal, shaking bacterium cultivates and screens with the penbritin LB nutrient solution negativity that contains 30 μ g/ml paraxin, the paraxin sensitive strain is extracted plasmid, and double digestion is identified, last sequence verification; The correct fusion gene that checks order is recombined into the carrier called after recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box of destination carrier.
The electrophoresis detection result that the double digestion of entry vector pENTR-PTB-U-box is identified as shown in Figure 3, wherein Fig. 3 a is a pENTR-PTB carrier in contrast, Fig. 3 b is the double digestion qualification result of pENTR-PTB-U-box entry vector, two fragments of all digested one-tenth size of two carriers as can be seen, and small segment PTB-U fusion gene wherein is greater than in contrast PTB domain gene.
After recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box structure is finished, with its infected person embryonic kidney embryo HEK-293 cell, generation has communicable virion, measure the titre of recombinant virus particle with spot hybridization, after obtaining enough titres, can carry out mice with tumor knurl body local injection, be somebody's turn to do the function of tumor inhibition effect of reorganization ubiquitin ligase in the animal level detection.
3, carrier for expression of eukaryon transfection IGF-1R positive tumor cell is Hela, promotes the degraded of IGF-1R, and suppresses cell proliferation and invasion and attack
The Hela tumour cell of IGF-1R being expressed the male logarithmic phase is with every hole 2 * 10
5The density of cell is inoculated in 6 well culture plates, and nutrient solution is the DMEM that contains 10% calf serum, cultivates in CO2gas incubator (37 ℃).Next day is when growing to 80% when cell, according to Lipofectamine
TM2000 specification sheetss carry out liposome transfection, with carrier for expression of eukaryon pFLAG-CMV4-PTB-U-box transfection Hela tumour cell.
3.1 immunoblotting detects fusion rotein PTB-U-box to the proteic downward modulation of host's tumour cell IGF-1R
Collect the reorganization Hela tumour cell of cultivating, add cell pyrolysis liquid, lysing cell 30min on ice, the collecting cell extract, utilize immunoblotting to detect the variation of the proteic content of IGF-1R in the tumour cell of transfection fusion gene PTB-U-box, with transfection the Hela tumour cell of pFLAG-CMV4 empty plasmid, transfection pFLAG-CMV4-PTB carrier as the contrast of the Hela tumour cell of transfection fusion gene.
When carrying out the immunoblotting detection, anti-FLAG antibody (M2) is available from sigma company, and anti-IGF-1R α/β antibody is available from Santa Cruz Biotechnology company, and fluorescently-labeled sheep anti-mouse igg and goat anti-rabbit igg are available from Odyssey company.
The immunoblotting detected result after transfection pFLAG-CMV4-PTB carrier, the pFLAG-CMV4-PTB-U-box carrier, shows that with anti-FLAG tag antibody detected result the purpose fragment PTB of transfection or PTB-U-box all correspondingly obtain expressing as shown in Figure 4.
IGF-1R α, β immunoblotting detected result show: with transfection the control cells of pFLAG-CMV4 empty plasmid and pFLAG-CMV4-PTB (PTB) compare, transfection in the Hela cell of pFLAG-CMV4-PTB-U-box its IGF-1R α, β band color all obviously shoal, also be that its IGF-1R α, β content reduce accordingly, and its immunoblotting of Anti-tubulin in contrast detects demonstration unanimity, no considerable change; This explanation is under identical transfection conditions, and the expression of fusion gene PTB-U-box has produced the target degraded to IGF-1R albumen in the Hela tumour cell, makes its IGF-1R albumen reduce.
3.2MTT colorimetric experiment and clone's formation experiment detection fusion gene PTB-U-box express the influence to host's tumor cell proliferation
The Hela cell strain of difference transfection pFLAG-CMV4 empty carrier (CMV4), pFLAG-CMV4-PTB (PTB) and pFLAG-CMV4-PTB-U-box (PTB-U), density with 2000 cells/well after 24 hours is inoculated in 96 orifice plates, carry out the detection of MTT colorimetry back 1～5 day every day in inoculating, the cell proliferation curve is drawn in the relatively cell proliferation of three kinds of transfectional cells.The result as shown in Figure 5, wherein X-coordinate is the fate after the transfection, ordinate zou is the absorbance of reaction viable count, as can be seen, from the 2nd day, the propagation situation basically identical of the tumour cell of transfection empty carrier and PTB, and the propagation of the tumour cell of transfection PTB-U-box fusion gene obviously lags behind the above two, this show fusion gene expression inhibiting the propagation of tumour cell.
The Hela cell strain of difference transfection pFLAG-CMV4 empty carrier (CMV4), pFLAG-CMV4-PTB (PTB) and pFLAG-CMV4-PTB-U-box (PTB-U), be inoculated in the 60mm culture dish with 200 cell count after 24 hours, add G418 simultaneously to final concentration 600 μ g/ml, cultivated 14 days, pair cell carries out the dyeing of Giemsa dye liquor, takes a picture and calculates cloning efficiency, and the plate clone that detects transfectional cell forms ability.Photo after the dyeing as shown in Figure 6, the column analytical results of cloning efficiency as shown in Figure 7, in conjunction with Fig. 6, Fig. 7, can obviously find out, the formed number of cell clones of Hela cell of transfection empty carrier and PTB does not have significant difference, and the formed number of cell clones of Hela cell of transfection PTB-U is compared obvious minimizing with the above two, show that tumour cell transfection fusion gene is after expressing, the clonality that obviously suppresses the Hela cell further illustrates fusion gene and expresses the back to tumor cell proliferation inhibition.
3.3Transwell experiment detects the influence of fusion gene PTB-U-box expression to host's tumor cell invasion
The Hela cell strain of transfection pFLAG-CMV4 empty carrier (CMV4), pFLAG-CMV4-PTB (PTB) and pFLAG-CMV4-PTB-U-box (PTB-U) carries out transwell invasion and attack experiment respectively, and cell dilution is become 10
4Individual/ml, each transwell cell (spreading matrigel 80 μ l in advance) inoculation 200 μ l, fully wipe matrigel in the cell with cotton swab behind the 24h, carry out cell dyeing with the Giemsa dye liquor, (200 *) are observed and are taken a picture under the inverted microscope, and carry out cell counting, and count 10 visuals field at random, calculate average invasion and attack cell count.
The observations of inverted microscope as shown in Figure 8, figure is as shown in Figure 9 as a result for the cell counting column, in conjunction with Fig. 8, Fig. 9, can obviously find out after the transwell cell is inoculated the cell 24h of same quantity, the cell quantity that invasion and attack take place the Hela cell of transfection empty carrier and PTB does not have significant difference, and the Hela cell invasion number of transfection fusion gene is compared obvious minimizing with the above two, show that tumour cell transfection fusion gene is after expressing, obviously suppress the invasive ability of Hela cell, illustrate that the expression of fusion gene PTB-U-box can suppress the invasion by tumor cells ability.
Through above-mentioned checking surface: the eukaryon expression plasmid pFLAG-CMV4-PTB-U-box of reorganization ubiquitin ligase PTB-U-box, after transfection IGF-1R positive tumor cell is Hela, by promoting the IGF-1R downward modulation, can effectively suppress the propagation and the invasion and attack of IGF-1R positive cell, have the effect and the purposes of anti-IGF-1R positive tumor.
Claims (6)
1. a reorganization ubiquitin ligase PTB-U-box fusion gene is characterized in that, is that the PTB domain gene with IRS-1 is connected with the U-box domain gene of CHIP.
2. reorganization ubiquitin ligase PTB-U-box fusion gene as claimed in claim 1 is characterized in that the nucleotide sequence of described reorganization ubiquitin ligase PTB-U-box fusion gene is shown in SEQ.ID.NO.1.
3. reorganization ubiquitin ligase PTB-U-box fusion gene as claimed in claim 1 or 2, it is characterized in that, described reorganization ubiquitin ligase PTB-U-box fusion gene is cloned into carrier for expression of eukaryon pFLAG-CMV4 by EcoR I, EcoR V restriction enzyme site, and ubiquitin ligase carrier for expression of eukaryon pFLAG-CMV4-PTB-U-box obtains recombinating.
4. reorganization ubiquitin ligase PTB-U-box fusion gene as claimed in claim 1 or 2, it is characterized in that, described reorganization ubiquitin ligase PTB-U-box fusion gene cloning is gone among the destination carrier pAd/CMV/V5-DEST, obtains recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box.
5. claim 1 or 2 described reorganization ubiquitin ligase PTB-U-box fusion genes are applied to the preparation of anti-IGF-1R positive expression tumour medicine.
6. the application of preparing anti-tumor medicine as claimed in claim 5 is characterized in that, the ubiquitin ligase PTB-U-box fusion gene cloning of will recombinating is gone into carrier for expression of eukaryon or adenovirus expression carrier.
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| CN102517312A (en) * | 2011-11-29 | 2012-06-27 | 中国人民解放军第四军医大学 | Recombination ubiquitin ligase SH2-U-box fusion gene as well as expression vectors and applications thereof |
| CN103353527A (en) * | 2013-07-01 | 2013-10-16 | 浙江大学 | PNC detection kit for detecting tumor invasion and application thereof |
| CN104673762A (en) * | 2015-01-19 | 2015-06-03 | 江苏大学 | Specific antibody for ubiquitin ligase Nedd4-1 and application of specific antibody |
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| CN1314811C (en) * | 2005-07-25 | 2007-05-09 | 中国人民解放军第四军医大学 | Recombinant chimeric eucaryotic expression vector Cb1-Grb7(SH2)-RING and use of anti-tumour gene drug |
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| CN1314811C (en) * | 2005-07-25 | 2007-05-09 | 中国人民解放军第四军医大学 | Recombinant chimeric eucaryotic expression vector Cb1-Grb7(SH2)-RING and use of anti-tumour gene drug |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517312A (en) * | 2011-11-29 | 2012-06-27 | 中国人民解放军第四军医大学 | Recombination ubiquitin ligase SH2-U-box fusion gene as well as expression vectors and applications thereof |
| CN103353527A (en) * | 2013-07-01 | 2013-10-16 | 浙江大学 | PNC detection kit for detecting tumor invasion and application thereof |
| CN104673762A (en) * | 2015-01-19 | 2015-06-03 | 江苏大学 | Specific antibody for ubiquitin ligase Nedd4-1 and application of specific antibody |
| CN104673762B (en) * | 2015-01-19 | 2017-10-20 | 江苏大学 | Anti- ubiquitin ligase Nedd4 1 specific antibody and its application |
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