CN102174550B - 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 restructuring 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 at first a kind of chaperone (co-chaperone) altogether.Owing to containing a U-box structural domain in its molecule, therefore belong to again in essence a class E3 ubiquitin ligase.CHIP plays an important role in protein quality control, stable regulation process by promoting ubiquitin, the degraded of protein.In breast cancer tissue, expression and the tumor grade of CHIP are negative correlation; Experimental evidence shows that also CHIP can suppress propagation and the transfer of tumour.The effect of these research prompting enhancings CHIP might be as a kind of new ideas of cancer therapy.
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 mediating proteins-protein-interacting can be identified and combination mutually with heat shock protein(HSP) Hsc70/Hsp70, Hsp90C end the folding process of regulating heat shock protein substrate/client's albumen; The function of Linker is still not clear, and only understanding it at present is essential for mutually combining of TPR dependence; It is active that the U-box structural domain of C end has been given the CHIP ubiquitin ligase, is responsible for raising ubiquitin binding enzyme E2 and in connection with on the mate molecule and substrate protein thereof that are transferred to the combination of TPR structural domain institute at the ubiquitin on E2, promotes these molecules to degrade in proteasome.
2) the CHIP molecule is as total molecule companion and a ubiquitin-like ligase enzyme, be intracellular protein folding-molecule hinge between refolding machine and these two kinds of paths of Ubiquitin-proteasome system, participate in the intracellular protein quality control
Cell dependent protein matter quality control system (protein quality control, QC) continues to monitor reparation or the degraded of the folding and misfolded protein of new polypeptide chain, to keep the stable state of cell.QC is comprised of molecular chaperones (comprising Hsp70, Hsp40 etc.) and Ubiquitin-proteasome system, and the former helps the 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, combination and the release of Molecular regulator companion Hsp70 family member and substrate protein; In addition, CHIP can stop the folding of Hsp70 after Hsp70 is combined, and relies 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 of inhibition tumor cell propagation 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 kytoplasm, also has small part to be present in core.Nearest studies show that, in human breast carcinoma tissue, and the expression level of CHIP and malignancy and classification inverse correlation.Mechanism Study and cell experiment show, CHIP is by promoting ubiquitin, the degraded of multiple carcinogenic protein such as HER2, ER-a, GR, SRC-3 etc., thereby suppress propagation and the transfer of tumour.
2, the application of restructuring ubiquitin ligase
Ubiquitin ligase E3 is responsible for specific recognition and bound substrates in the ubiquitin process.A plurality of study group use recombinant technology and build the restructuring ubiquitin ligase in recent years, and successfully degraded is not some albumen of ubiquitin system natural substrate under normal circumstances.The restructuring F-box PROTEIN C FP that for example consists of by the Binding Capacity F-box of subunit that modifies ubiquitin ligase mixture Skp1-Cullin1-F-box (SCF), but targeting degraded pRB and β-catenin.After recombinating from two RING of BRCA1 and BARD and proliferating cell nuclear antigen PCNA respectively, this recombinant molecule reduces the level of p57 albumen in the mode that proteasome relies on, and lowers its function.And the research of Li Xia etc. also shows, the restructuring ubiquitin ligase that utilizes Cb1 to build can effectively be lowered the HER2 of high expression level in breast cancer cell, thereby reaches the purpose that suppresses tumor growth.These studies confirm that the ubiquitin ligase that utilizes restructuring, but targeting ground degraded some diseases associated molecule.But also do not utilize the active restructuring ubiquitin ligase that builds 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 restructuring 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 ubiquitin and the degradation function of targeting, 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 restructuring 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 restructuring ubiquitin ligase PTB-U-box fusion gene is as shown in SEQ.ID.NO.1.
Described restructuring 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 restructuring ubiquitin ligase PTB-U-box fusion gene is cloned in destination carrier pAd/CMV/V5-DEST, obtains recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box.
Described restructuring ubiquitin ligase PTB-U-box fusion gene is applied to the preparation of anti-IGF-1R positive expression tumour medicine.
The application of the preparation of described antitumor drug is that the restructuring ubiquitin ligase PTB-U-box fusion gene is cloned into carrier for expression of eukaryon or adenovirus expression carrier.
Compared with prior art, the present invention has following useful technique effect:
The present invention can identify and in conjunction with the PTB domain gene of the IRS-1 of IGF-1R with have the U-box domain gene of the CHIP of ubiquitin ligase activity, merge by PCR and consist of the restructuring ubiquitin ligase PTB-U-box fusion gene, can targeting after this fusion gene is expressed the carcinogenic associated protein IGF-1R of degraded, wherein, the PTB structural domain is responsible for the combination of IGF-1R targeting, and U-box is responsible for the ubiquitin molecule is connected to substrate to promote substrate generation ubiquitin 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, built restructuring ubiquitin ligase carrier for expression of eukaryon pFLAG-CMV4-PTB-U-box, this carrier is after transfection tumor cell, can lower the carcinogenic associated protein IGF-1R of host's tumour cell, 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 builds 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 is that immunoblotting detects the figure as a result that fusion gene PTB-U-box lowers host's tumour cell IGF-1R albumen;
Fig. 5 is the cell proliferation graphic representation of the Hela cell of MTT colorimetric determination transfection different carriers;
Fig. 6 is the Giemsa coloration result figure of the Hela Cell clonality of transfection different carriers;
Fig. 7 is the column analytical results figure of the Hela cell clonal formation rate of transfection different carriers;
Fig. 8 is the cell invasion visual field observations figure of the Hela cell of transfection different carriers;
Fig. 9 is that the cell invasion of the Hela cell of transfection different carriers is counted column figure as a result.
Embodiment
The present invention utilizes the PTB structural domain (Phosphotyrosine Binding domain) of linkers IRS-1 (Insulin Receptor Substrate 1) can identify and bound insulin like growth factor acceptor (Insulin-like Growth Factor Receptor 1, IGF-1R) characteristic and the ubiquitin ligase of CHIP are active, the restructuring ubiquitin ligase PTB-U-box that structure can the carcinogenic associated protein IGF-1R of targeting degraded.Constructed fusion gene PTB-U-box clones respectively the PTB domain gene of IRS-1 and has the U-box domain gene of the CHIP of ubiquitin ligase activity, adopts recombinant PCR to merge; Wherein, the PTB structural domain is responsible for the combination of IGF-1R targeting, and U-box is responsible for the ubiquitin molecule is connected to substrate to promote substrate generation ubiquitin, 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 restructuring ubiquitin ligase PTB-U-box fusion gene
1.1PTB and the clone of U-box domain gene
According to the PTB gene order of IRS-1 in Genbank, the U-box gene order difference design of amplification primers of CHIP, P is used for amplification PTB gene order in contrast, amplimer is used for the restructuring amplification of fusion gene to P1, P2, and specific design is:
Primer pair P:
Upstream primer: ggcgaattca atggaggctg gggaggactt gag 33;
Downstream primer: ggcgatatcc tagcgagggc ggaactcatc ac 32;
Primer pair 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 in 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, introduce restriction enzyme site EcoR I in the upstream primer sequence Up of amplification PTB, introduce restriction enzyme site EcoR V in the downstream primer sequence D own of amplification U-box.
Then, take the cDNA of Hela cell as template, respectively take primer pair P, P1 (Up, M1) as amplimer pair, pcr amplification PTB domain gene, the pcr amplification program is: 94 ℃ of 5min, 94 ℃ of 30s, 57 ℃ of 45s, 72 ℃ of 30s, 35cycle;
Take the cDNA of Hela cell as template, take primer pair P2 (M2, Down) as amplimer pair, pcr amplification U-box domain gene, pcr amplification program are 94 ℃ of 5min, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, 35cycle.
Collect pcr amplification product, obtain PTB domain gene and U-box domain gene after 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:
Be template with the PTB domain gene of primer pair P1 amplification with the U-box domain gene equal amount of mixture (mixing at 1: 1) that primer pair P2 increases, with PTB upstream primer Up, U-box downstream primer Down as primer pair, 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 after 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, 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 respectively double digestion respectively, after reclaiming 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 completed, connect product and transform DH5 α competent cell, after 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 at last sequence verification; With constructed successful recombinant molecule called after pFLAG-CMV4-PTB-U-box, this is restructuring 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 cuts PTB domain gene insertion vector to detect 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, can find out all two fragments of digested one-tenth size of two carriers, 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 as 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 PTB domain gene and the PTB-U-box fusion gene of amplification, utilize restriction enzyme EcoR I, EcoR V and T4 ligase enzyme, respectively this two bar segment is inserted into entry vector pENTR
TMIn 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 destination carrier pAd/CMV/V5-DEST from entry vector.Then add Proteinase K, 10 minutes termination reactions of 37 ℃ of effects, get recombinant products 1 μ L and transform the Top10 competent cell, the picking mono-clonal, shake bacterium and cultivate and use the penbritin LB nutrient solution negativity screening 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 pENTR-PTB carrier in contrast, Fig. 3 b is the double digestion qualification result of pENTR-PTB-U-box entry vector, can find out all two fragments of digested one-tenth size of two carriers, 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 completed, it is infected human embryo kidney (HEK) 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 restructuring ubiquitin ligase in the animal level detection.
3, eukaryotic expression vector transfection IGF-1R positive tumor cell is Hela, promotes the degraded of IGF-1R, and suppresses cell proliferation and invasion and attack
IGF-1R is expressed the Hela tumour cell of positive logarithmic phase 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 Growth of Cells to 80%, 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 downward of host's tumour cell IGF-1R albumen
Collect the restructuring 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 content of IGF-1R albumen 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, pFLAG-CMV4-PTB-U-box carrier, shows that with anti-FLAG tag antibody detected result purpose fragment PTB or the PTB-U-box of transfection all correspondingly expressed 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, be also that its IGF-1R α, β content reduce accordingly, and its immunoblotting detection display of Anti-tubulin in contrast is consistent, without considerable change; This explanation is under identical transfection conditions, and the expression of fusion gene PTB-U-box has produced the targeting degraded to IGF-1R albumen in the Hela tumour cell, makes its IGF-1R albumen lower.
3.2MTT colorimetric experiment and colony formation detect fusion gene PTB-U-box expression to the impact of 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 MTT colorimetric determination rear 1～5 day every day in inoculating, the cell proliferation curve is drawn in the relatively cell proliferation of three kinds of transfectional cells.Result as shown in Figure 5, wherein X-coordinate is the number of days after transfection, ordinate zou is the absorbance of reaction viable count, can find out, from the 2nd day, the propagation situation of the tumour cell of transfection empty carrier and PTB is basically identical, 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 simultaneously G418 to final concentration 600 μ g/ml, cultivated 14 days, cell is carried out the dyeing of Giemsa dye liquor, takes a picture and calculate cloning efficiency, and the plate clone that detects transfectional cell forms ability.Photo after 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 is without 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, obviously suppress the clonality of Hela cell, further illustrate fusion gene and express rear inhibition to tumor cell proliferation.
3.3Transwell experiment detects fusion gene PTB-U-box expression to the impact of 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), carry out the transwell Matrigel respectively, and cell dilution is become 10
4Individual/ml, each transwell cell (spreading in advance matrigel 80 μ l) inoculation 200 μ l, fully wipe matrigel in cell with cotton swab after 24h, carry out cell dyeing with the Giemsa dye liquor, under inverted microscope, (200 *) are observed and are taken a picture, and carry out cell counting, and count at random 10 visuals field, 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 cell 24h of the same quantity of transwell cell inoculation, the cell quantity of invasion and attack occurs without significant difference in the Hela cell of transfection empty carrier and PTB, 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, the invasive ability that the expression of fusion gene PTB-U-box can inhibition tumor cell is described.
Through above-mentioned checking surface: the eukaryon expression plasmid pFLAG-CMV4-PTB-U-box of restructuring ubiquitin ligase PTB-U-box, after transfection IGF-1R positive tumor cell is Hela, by promoting IGF-1R to lower, can effectively suppress propagation and the invasion and attack of IGF-1R positive cell, have effect and the purposes of anti-IGF-1R positive tumor.
Claims (5)
1. a restructuring 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;
The nucleotide sequence of described restructuring ubiquitin ligase PTB-U-box fusion gene is as shown in SEQ.ID.NO.1.
2. restructuring ubiquitin ligase PTB-U-box fusion gene as claimed in claim 1, it is characterized in that, described restructuring 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.
3. restructuring ubiquitin ligase PTB-U-box fusion gene as claimed in claim 1, it is characterized in that, described restructuring ubiquitin ligase PTB-U-box fusion gene is cloned in destination carrier pAd/CMV/V5-DEST, obtains recombinant adenoviral vector pAd/CMV/V5-DEST-PTB-U-box.
4. restructuring ubiquitin ligase PTB-U-box fusion gene claimed in claim 1 is applied to the preparation of anti-IGF-1R positive expression tumour medicine.
5. the application of the preparation of antitumor drug as claimed in claim 4, is characterized in that, the ubiquitin ligase PTB-U-box fusion gene of recombinating is cloned into carrier for expression of eukaryon or adenovirus expression carrier.
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| CN100590129C (en) * | 2006-08-21 | 2010-02-17 | 中国医学科学院基础医学研究所 | Human ring protein application |
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| CN100590129C (en) * | 2006-08-21 | 2010-02-17 | 中国医学科学院基础医学研究所 | Human ring protein application |
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