CN100347307C - Recombination chimeric molecule Cb1-Grb2(SH2)-RING eukaryotic expression plasmid and antitumor genetic medicine use - Google Patents
Recombination chimeric molecule Cb1-Grb2(SH2)-RING eukaryotic expression plasmid and antitumor genetic medicine use Download PDFInfo
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Abstract
The present invention discloses a construction method of a recombinant inlaid Cb1 ubiquitin ligase Cb1-Grb2(SH2)-RING eukaryotic expression carrier, and the constructed expression plasmid pcDNA3.1(+)-Cb1-Grb7(SH2)-RING can be used as a gene medicine for resisting HER2 positive tumor. The experiment proves that the ubiquitining and down regulating of a recombinant inlaid molecule Cb1-Grb2(SH2)-RING eukaryotic expression plasmid are promoted by being combined with HER2 proteins after the eukaryotic expression plasmid transfects an HER2 positive breast cancer cell line SK-BR-3, the proliferation of cells relevant to overactivation of HER2 can be effectively inhibited, so the eukaryotic expression plasmid has the purpose of resisting HER2 positive tumor.
Description
Technical field
The invention belongs to field of tumor gene therapy, relate to reorganization chimeric Cbl ubiquitin ligase Cbl-Grb2 (SH2)-RING Construction of eukaryotic and be used for the purposes of the genomic medicine of anti-HER2 positive tumor.
Background technology
Cbl (Casitas B-lineage lymphoma) is the plasmosin that a class is evolved and upward guarded, extensively distribute, form by 906 amino acid, include a plurality of different structural domains, can mediate with different cells in the molecule combination, therefore in the cell activation process, can be used as linkers or support molecule and participate in signal transduction; The function that intramolecular RING structural domain of while is given the Cbl ubiquitin ligase promotes its bonded target molecule generation ubiquitinization, downward modulation, thereby participates in the negative regulation mechanism of intracellular signal transduction.Cbl sudden change or dysfunction can cause cell to take place to transform or immune dysfunction.
1.Cbl structure and function
(1) Cbl contains a plurality of protein-protein binding domainss and a RING structural domain
Cbl albumen begins to contain respectively Tyrosylprotein kinase binding domains (TKB), Linker, RING structural domain, proline rich district, C-terminal several tyrosine phosphorylations site and leucine zipper by N-terminal.Wherein the TKB structural domain is made of the SH2 of 4H, EF and variation, identification and in conjunction with activated protein Tyrosylprotein kinase PTK.The RING mediation combines with ubiquitin binding enzyme E2.Linker has vital role for the best spatial location that keeps forming between bonded substrate, RING and the E2 three on the Cbl molecule.The proline rich domain and the tyrosine site of C end mediate Cbl molecule and other protein binding that contains SH3 or contain SH2 respectively.
(2) Cbl promotes multiple protein Tyrosylprotein kinase ubiquitinization, downward modulation as ubiquitin ligase, participates in the negative regulation of intracellular signal transduction
The multivalence binding ability of Cbl molecule has determined it to participate in signal transduction pathway as a kind of multivalence linkers on the one hand.On the other hand, it is a kind of ubiquitin ligase in itself that RING has given Cbl albumen, TKB in the molecule and other structural domains can be discerned and in conjunction with target protein, RING is responsible for raising general hitch synthase E2 and the ubiquitin that will be combined on the E2 is transferred on its bonded substrate molecule, thereby starts the ubiquitin degradation pathway.Cbl albumen promotes multiple receptor type protein tyrosine kinase (RTK) as EGFR, PDGFR α, β, CSF-1R, Met, Hck etc. part inductive ubiquitinization, degraded subsequently to take place by this way, participate in the negative regulation of intracellular signal transduction, this is significant for the homeostasis of keeping cell.And for many protein tyrosine kinases such as EGFR, Syk etc., Cbl N end truncate (comprising N-terminal to RING) is just be enough to impel substrate generation ubiquitinization.Can become cancer protein after the Cbl sudden change; Can escape after many carinogenicity RTK such as EGFRvV, CSF-1R, HER2 etc. the undergo mutation negative regulation effect of Cbl.These phenomenon promptings, the negative regulation effect of strengthening or rebuilding Cbl perhaps can suppress some tumor cell proliferation.
2. the recombinate application of chimeric 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 chimeric ubiquitin ligase of reorganization, are not some albumen of ubiquitin system natural substrate under the normal circumstances of successfully degrading.For example pass through to modify the chimeric F-box PROTEIN C FP of the substrate of ubiquitin ligase mixture Skpl-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.These studies show that the ubiquitin ligase that utilizes reorganization, target ground degraded some diseases associated molecule.But also do not use the relevant report that recombinant C bl ubiquitin ligase carries out gene therapy research at present.
Summary of the invention
In view of the SH2 of Cbl intracellular protein is responsible for identification and in conjunction with multiple activatory protein tyrosine kinase, for the carcinogenic associated protein HER2 of target ground degraded, the objective of the invention is to, structure can the carcinogenic associated protein HER2 of target degraded the chimeric Cbl ubiquitin ligase of reorganization eukaryon expression plasmid, and be applied to prepare the genomic medicine of anti-HER2 positive tumor.
The present invention with the SH2 of Cbl albumen n end part be replaced into can in conjunction with and the SH2 of the downstream signaling molecule Grb2 of mediation HER2 activation signals, make up recombinant chimeric Cbl-Grb2 (SH2)-RING, wherein metathetical SH2 structural domain is responsible for the combination of HER2 target, and RING is responsible for that the ubiquitin molecule is connected to substrate and to promote it ubiquitinization, degraded takes place.
To achieve these goals, the technical solution taked of the present invention is:
1) BamH I and EcoR v are introduced the two ends that Cbl N holds encoding gene SH2, make up pcDNA3.1 (+)-CblN (BamH I+EcoR V)
The present invention utilizes PCR and overlapping elongation technology, designs 3 pairs of primers respectively, carries out 3 and takes turns the PCR reaction, and the SH2 N end of at first BamH I being introduced the CblN gene promptly obtains CblN (BamH I).With CblN (BamH I) is template, with method the SH2 C end that EcoR V introduces the CblN gene is obtained CblN (BamH I+EcoR V).It is cloned into pGEM-T easy carrier checks order, utilizing the KpnI/XhoI enzyme to cut after order-checking is correct is inserted between the KpnI/XhoI restriction enzyme site of pcDNA3.1 (+) carrier for expression of eukaryon, promptly obtains pcDNA3.1 (+)-CblN (BamH I+EcoR V).
2) the SH2 gene fragment of amplification Grb2 and displacement are gone into pcDNA3.1 (+)-CblN (BamH I+EcoR are v) obtained eukaryon expression plasmid pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING of recombinant chimeric Cbl-Grb2 (SH2)-RING
CDNA with the SK-BR-3 cell is a template, the design primer amplification obtains the Grb2SH2 gene fragment and is cloned in the pGEM-T easy carrier, enzyme is cut and is identified and after order-checking confirms that sequence is correct, with BamH I and EcoR v double digestion the Grb2SH2 gene fragment is cut out, and pcDNA3.1 (+)-CblN that will cut through same enzyme (BamH I+EcoR v) carrier segment reclaims respectively, two fragments are carried out ligation through the T4 ligase enzyme, connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation, extract plasmid, after BamH I and the evaluation of EcoR v double digestion, to the segmental carrier of insertion that obtains the expection size confirmation of checking order again, called after pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING, this is the eukaryon expression plasmid of recombinant chimeric Cbl-Grb2 (SH2)-RING.
3) Cbl-Grb2 (SH2)-RING promotes HER2 ubiquitinization and downward modulation, inhibition HER2 positive tumor cell to breed
With plasmid pcDNA3.1 (+)-Cbl-Grb2 (the SH2)-RING stable transfection HER2 male breast cancer cell line SK-BR-3 cell that successfully constructs, confirm that by immunoblotting, flow cytometer and cell experiment it by reducing the HER2 level, has effectively suppressed the tumor cell proliferation relevant with the HER2 overactivity.Further by can and promoting its ubiquitin degree to increase in conjunction with target protein HER2 in conjunction with experiment and ubiquitin experiment confirm recombinant chimeric Cbl-Grb2 (SH2)-RING in the body.
Description of drawings
Figure l is pcDNA3.1 (+) vector plasmid figure;
Fig. 2 cuts evaluation to the enzyme of pcDNA3.1 (+)-CblN (BamH I+EcoR V) carrier; Wherein:
1:pcDNA3.1 (+)-CblN (BamH I+EcoR V) is with Kpn I and Xho I double digestion;
2:pcDNA3.1 (+)-CblN is with Kpn I and Xho I double digestion;
3:pcDNA3.1 (+)-CblN (BamH I+EcoR V) is with BamH I and EcoR V double digestion;
4:pcDNA3.1 (+)-CblN is with BamH I and EcoR V double digestion;
M: known molecular amount standard substance.
Fig. 3 cuts evaluation to the enzyme of pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING carrier, wherein:
1:pcDNA3.1 (+)-CblN (BamH I+EcoR V) is with BamH I and EcoR V double digestion;
2:pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING is with BamH I and EcoR V double digestion;
M: known molecular amount standard substance.
Fig. 4 is the downward modulation effect of pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING recombinant plasmid to HER2.Stable transfection the SK-BR-3 cell strain cell extract of pcDNA3.1 (+) empty plasmid (vector), pcDNA3.1 (+)-CblN (being abbreviated as CblN) and pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING (writing a Chinese character in simplified form into CblN/Grb2) utilize the antibody of anti-HER2 and ACTIN to carry out immunoblotting assay respectively.CblN/Grb2 can reduce HER2 albumen.
Fig. 5 is the inhibited proliferation of Cbl-Grb2 (SH2)-RING to HER2 male breast cancer cell line SK-BR-3.Stable transfection the SK-BR-3 cell strain of pcDNA3.1 (+) empty plasmid (control) and CblN/Grb2, be inoculated in the 60mm culture dish with 200 cell count and cultivated 14 days, calculate cloning efficiency.CblN/Grb2 can suppress the clonal expansion ability of SK-BR-3 cell.
Fig. 6 is that Cbl-Grb2 (SH2)-RING promotes that HER2 ubiquitin degree increases among the HER2 male breast cancer cell line SK-BR-3.Stable transfection SK-BR-3 cell strain transient transfection pcDNA3.1 (+)-3 * HA-Ub and give 25 μ M MG-132 and handle 4h respectively of pcDNA3.1 (+) empty plasmid (vector), CblN/Grb2 and pcDNA3.1 (+)-CHIP (, being abbreviated as CHIP) herein as positive control.Get the 1.5mg cell extract and carry out immunoprecipitation, carry out immunoblotting assay with anti-HA antibody (on Fig. 6) or Anti-HER 2 (under Fig. 6) with Anti-HER 2.CblN/Grb2 can promote HER2 ubiquitin degree to increase.
The present invention is described in further detail with experiment below in conjunction with accompanying drawing.
Embodiment
Below be that (structure of pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING) and evaluation and this plasmid are to the restraining effect of HER2 male growth of tumour cell for the chimeric Cbl ubiquitin ligase of reorganization Cbl-Grb2 (SH2)-RING eukaryon expression plasmid of providing of contriver.
1. the recombinate structure of chimeric Cbl ubiquitin ligase Cbl-Grb2 (SH2)-RING eukaryon expression plasmid
1.1 experiment material
Restriction enzyme, T4 ligase enzyme are all purchased the precious living biological company limited in Dalian, and recombinant mammalian expressing vector pcDNA3.1 (+) purchases the company in Invitrogen, and pcDNA3.1 (+) plasmid figure is referring to Fig. 1.Among the figure, PCMV:CMV promotor, 232-819 bit base, T7:T7 promotor/primer sites, 863-882 bit base; Polyclone restriction enzyme site: 895-1010 bit base, BGHpA: Trobest polyadenylation sequence, the 1028-1252 bit base, f1 ori:f1 initiation site, the 1296-1726 bit base, SV40 ori:SV40 initiation site, the 1731-2074 bit base, Neomycin: neomycin resistance gene, 2136-2930 bit base, the SV40pA:SV40 polyadenylation signal, the 3104-3234 bit base, pUC ori:pUC initiation site, 3617-4287 bit base, Amipicilin: penbritin drug resistance gene, 4432-5428 bit base.
1.2 recombinant chimeric Cbl-Grb2 (the SH2)-RING eukaryon expression plasmid (building process of pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING)
At first utilize PCR and overlapping elongation technology, BamH I and EcoR v are introduced the two ends that Cbl N holds encoding gene SH2, it is cloned into pGEM-T easy carrier checks order, utilize the KpnI/XhoI enzyme to cut after order-checking is correct and be inserted between the KpnI/XhoI restriction enzyme site of pcDNA3.1 (+) carrier for expression of eukaryon, be i.e. pcDNA3.1 (+)-CblN (BamH I+EcoR V).Utilize PCR to obtain the Grb2SH2 gene fragment and be cloned in the pGEM-T easy carrier, enzyme is cut and is identified and after order-checking confirms that sequence is correct, with BamH I and EcoR v double digestion Grb2 SH2 gene fragment is cut out, and with pcDNA3.1 (+)-CblN that cuts through same enzyme (BamH I+EcoR carries out ligation after v) the carrier segment reclaims respectively, connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation, extract plasmid, after BamH I and the evaluation of EcoR v double digestion, to the segmental carrier of insertion that obtains the expection size confirmation of checking order again, called after pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING, this is the eukaryon expression plasmid of recombinant chimeric Cbl-Grb2 (SH2)-RING.
2. the influence of on cell proliferation behind the positive breast cancer cell line SK-BR-3 of chimeric Cbl ubiquitin ligase Cbl-Grb2 (the SH2)-RING eukaryon expression plasmid stable transfection HER2 that recombinates
With the SK-BR-3 cell of logarithmic phase with every hole 2 * 10
5Cell inoculation is in 6 well culture plates, and nutrient solution is DMEM (GIBCO Co), and 10% calf serum (Hangzhou folium ilicis chinensis biological factory) is cultivated in CO2gas incubator (37 ℃).Next day is when growing to 90-95% when cell, according to Lipofectamine
TM2000 specification sheetss carry out liposome transfection.24h went down to posterity according to 1: 10 after transfection, and 48h begins to screen with the G418 of 400 μ g/mL, obtained the cell strain of stable transfection.Control cells is with same method transfection empty plasmid pcDNA3.1 (+) and screen stable cell line.Detect the influence of the expression of Cbl-Grb2 (SH2)-RING by immunoblotting, flow cytometer and cell experiment to HER2 protein level, cell proliferation.Further detect this recombinant chimeric and whether can and promote its ubiquitinization in conjunction with target protein HER2 by combination experiment and ubiquitin experiment in the body.
When applying to tumour patient, plan this plasmid with liposome after the injection of the local knurl body of row; Perhaps further Cbl-Grb2 (SH2)-RING is building up in the adenovirus carrier, obtains to utilize the recombinant adenovirus of suitable titre to carry out local knurl body injection behind Cbl-Grb2 (SH2)-RING recombinant adenovirus.
Technique effect of the present invention is:
1. made up eukaryon expression plasmid pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING of recombinant chimeric Cbl-Grb2 (SH2)-RING, cut evaluation (Fig. 2,3) and determined dna sequence proves that sequence is entirely true through enzyme.
2. the influence of chimeric Cbl ubiquitin ligase Cbl-Grb2 (SH2)-RING of recombinating to HER2 male breast cancer cell line SK-BR-3 cell proliferation
2.1 HER2 albumen in the downward modulation SK-BR-3 cell
Immunoblotting assay shows, with stable transfection pcDNA3.1 (+) empty plasmid (vector) and CblN the control cells strain relatively, HER2 reduces (Fig. 4) in the SK-BR-3 cell of stably express Cbl-Grb2 (SH2)-RING (CblN/Grb2).Indirect IF staining and flow cytometry analysis cell surface HER2 expression prove that also latter HER2 positive rate is reduced to 28.4% (transfection the control cells strain of empty carrier be 89.6%).
2.2 suppress the propagation of SK-BR-3 stable transfected cells strain
The clone forms experiment and shows, compares with the control cells strain (control) of stable transfection pcDNA3.1 (+), and the SK-BR-3 cloning efficiency of stably express Cbl-Grb2 (SH2)-RING (CblN/Grb2) reduces by 66% (Fig. 5).
2.3 in conjunction with and promote the HER2 ubiquitinization
Show in conjunction with experiment in the body, cross chimeric recombinant molecule Cbl-Grb2 (the SH2)-RING and the HER2 that express in the SK-BR-3 cell and have interaction, show that this chimeric recombinant molecule can be in vivo in conjunction with HER2.The SK-BR-3 cell of the chimeric recombinant molecule Cbl-Grb2 of stably express (SH2)-RING and stable transfection control cells strain transient transfection pcDNA3.1 (+)-3 * HA-Ub respectively of pcDNA3.1 (+), and give MG-132 and handle and to degrade with arrestin, cell pyrolysis liquid carries out immunoprecipitation with the antibody of anti-HER2, carry out immunoblotting with the antibody of anti-HA and detect, the result shows that the HER2 ubiquitinization obviously increases than control cells in the SK-BR-3 cell of stably express recombinant molecule Cbl-Grb2 (SH2)-RING.(Fig. 5).
Experimental results show that through above-mentioned: the chimeric Cbl ubiquitin ligase of reorganization Cbl-Grb2 (SH2)-RING eukaryon expression plasmid pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING that the present invention makes up, behind the positive breast cancer cell line SK-BR-3 of transfection HER2, by with the HER2 protein binding and promote its ubiquitinization, downward modulation, can effectively suppress the propagation of the cell relevant, thereby have the purposes of anti-HER2 positive tumor with the HER2 overactivity.
Claims (3)
1. recombinant chimeric Cbl-Grb2 (SH2)-RING Construction of eukaryotic method is characterized in that:
At first BamH I is introduced the SH2 N end of CblN gene, obtain CblN (BamH I); With CblN (BamH I) is template, with method the SH2 C end that EcoR V introduces the CblN gene is obtained CblN (BamH I+EcoR V);
CblN (BamH I+EcoR V) is cloned into pGEM-T easy carrier to check order, utilize the KpnI/XhoI enzyme to cut and be inserted between the KpnI/XhoI restriction enzyme site of pcDNA3.1 (+) carrier for expression of eukaryon after order-checking is correct, promptly obtain pcDNA3.1 (+)-CblN (BamH I+EcoR V);
CDNA with the SK-BR-3 cell is a template again, the design primer amplification obtains Grb2 SH2 gene fragment and is cloned in the pGEM-T easy carrier, enzyme is cut and is identified and after order-checking confirms that sequence is correct, with BamHI and EcoR v double digestion Grb2 SH2 gene fragment is cut out, and pcDNA3.1 (+)-CblN that will cut through same enzyme (BamH I+EcoR v) carrier segment reclaims respectively, two fragments are carried out ligation through the T4 ligase enzyme, connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation, extract plasmid, after BamH I and the evaluation of EcoR v double digestion, to the segmental carrier of insertion that obtains the expection size confirmation of checking order again, called after pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING is the eukaryon expression plasmid of recombinant chimeric Cbl-Grb2 (SH2)-RING.
2. eukaryon expression plasmid pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING of the resulting recombinant chimeric Cbl-Grb2 of the described recombinant chimeric Cbl-Grb2 of claim 1 (SH2)-RING Construction of eukaryotic method (SH2)-RING is used to prepare the purposes of the genomic medicine of anti-HER2 positive tumor.
3. purposes as claimed in claim 2, it is characterized in that, behind the positive breast cancer cell line SK-BR-3 of recombinant chimeric Cbl-Grb2 (SH2)-RING eukaryon expression plasmid pcDNA3.1 (+)-Cbl-Grb2 (SH2)-RING transfection HER2, by with the HER2 protein binding and promote its ubiquitinization, downward modulation, suppress the propagation of the cell relevant with the HER2 overactivity.
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| CN103012566B (en) * | 2011-09-26 | 2015-04-08 | 中国医学科学院基础医学研究所 | Non-phosphorylation ligand combined with Grb7 protein SH2 structural domain and pharmaceutical application thereof |
| CN103319569B (en) * | 2013-07-05 | 2016-01-20 | 成都医学院 | The high throughput screening system of the potential drug being target spot with c-Cbl albumen ring-type functional domain and screening method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000027865A1 (en) * | 1998-11-06 | 2000-05-18 | The Brigham And Women's Hospital, Inc. | CHARACTERIZATION OF NOVEL GENE cbl-SL |
| WO2000073326A2 (en) * | 1999-06-02 | 2000-12-07 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
| WO2002036142A2 (en) * | 2000-11-03 | 2002-05-10 | University Of Vermont And State Agricultural College | Compositions for inhibiting grb7 |
| WO2002042457A1 (en) * | 2000-11-22 | 2002-05-30 | Bristol-Myers Squibb Company | Cloning and expression of human slap-2: a novel sh2/sh3 domain-containing human slap homologue having immune cell-specific expression |
| JP2003070479A (en) * | 2001-09-04 | 2003-03-11 | Japan Science & Technology Corp | SH2 DOMAIN VARIANT OF Grb2 |
-
2005
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000027865A1 (en) * | 1998-11-06 | 2000-05-18 | The Brigham And Women's Hospital, Inc. | CHARACTERIZATION OF NOVEL GENE cbl-SL |
| WO2000073326A2 (en) * | 1999-06-02 | 2000-12-07 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
| WO2000073326A3 (en) * | 1999-06-02 | 2001-05-25 | Us Gov Health & Human Serv | Redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use |
| WO2002036142A2 (en) * | 2000-11-03 | 2002-05-10 | University Of Vermont And State Agricultural College | Compositions for inhibiting grb7 |
| WO2002042457A1 (en) * | 2000-11-22 | 2002-05-30 | Bristol-Myers Squibb Company | Cloning and expression of human slap-2: a novel sh2/sh3 domain-containing human slap homologue having immune cell-specific expression |
| JP2003070479A (en) * | 2001-09-04 | 2003-03-11 | Japan Science & Technology Corp | SH2 DOMAIN VARIANT OF Grb2 |
Non-Patent Citations (3)
| Title |
|---|
| c-Cbl Is a Suppressor of the Neu Oncogene. Gil Levkowitz,et al.THE JOURNAL OF BIOLOGICAL CHEMISTRY,Vol.275 No.45. 2000 * |
| 人 cbl 基因真核表达载体的构建及表达 李霞，等.第四军医大学学报，第25卷第24期 2004 * |
| 以EGFR家族受体酪氨酸激酶为靶点的抗肿瘤治疗研究进展 孔维佳，将建东.中国药理学通报，第19卷第8期 2003 * |
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