CN103951756B - The fusion protein of TAT Yu PR domain and the application in preparing antitumor drug thereof - Google Patents

Abstract

The present invention relates to the fusion protein of TAT Yu PR domain and the application in preparing antitumor drug thereof.Specifically, construct the expression vector of TAT Yu PR domain fusion protein, and expression and purification obtains the fusion protein of TAT Yu PR domain, by experimental verification, it is on different stage meningioma primary cell propagation and the impact of apoptosis, result shows that fusion protein all exists depression effect for different stage meningioma primary cell propagation, has significant depression effect especially for high-level meningioma.The present invention is that the cancer suppressing action using PR domain provides effective way to treat the tumors such as high-level meningioma.

Description

The fusion protein of TAT Yu PR domain and the application in preparing antitumor drug thereof

[technical field]

The present invention relates to technical field of molecular biology, specifically, relate to the fusion protein of TAT Yu PR domain and the application in preparing antitumor drug thereof.

[background technology]

Benign meningioma (WHOI level) generally has good prognosis, but atypia and malignant meningioma (WHOII level and WHOIII level) prognosis relatively benign meningioma are poor.The prognosis of benign meningioma depends primarily on operative site, and high-level meningioma (WHOII level and WHOIII level) Postoperative recurrent rate is higher, and currently without effective chemotherapy target.

Antioncogene RIZ1 is positioned chromosome 1p36, all finds low expression or expression silencing in kinds of tumors, and the result of study of our early stage finds, along with the rising of meningioma rank, the expression of RIZ1 declines.PR-domain is 200 nucleotide sequences of RIZ1 aminopeptidase gene end, is proved to be RIZ1 gene at present and plays the main functional areas of cancer suppressing action.Therefore the cancer suppressing action of PR-domain can be used to treat high-level meningioma.

Cell-penetrating peptide (Cell-Penetrating Peptides, CPPs) can effectively pilot protein matter even nanoparticle enter cell, and can targeted cells core, its wear film ability be independent of classics endocytosis.TAT is the antisense activating polypeptide in HIV-1 virus, 86 aminoacid of total length, can mediate the polypeptide merged with it or albumen crosses over cell membrane or blood brain barrier.TAT protein transduction system is that PR domain albumen is applied to the treatment of high-level meningioma and provides a strong approach, is expected to promote that carrier wears film, plays and strengthen the cancer suppressing action of PR-domain.

Have not yet to see the report of cell-penetrating peptide and PR-domain combined treatment tumor.Can cell-penetrating peptide effectively guide foreign protein entrance nucleus to depend on cell-penetrating peptide and the conformation of foreign protein fusion protein, and the present invention is then devoted to the fusion protein of TAT Yu the PR domain that film can efficiently be worn by structure, cancer resistant effect is good.

[summary of the invention]

It is an object of the invention to for deficiency of the prior art, it is provided that the fusion protein of a kind of TAT Yu PR domain.

Another purpose of the present invention is to provide the nucleotide sequence of a kind of fusion protein encoding TAT Yu PR domain.

Another the purpose of the present invention is to provide a kind of expression vector.

Fourth object of the present invention is to provide a kind of host cell.

5th purpose of the present invention is to provide the fusion protein of above-mentioned TAT Yu PR domain, the coding nucleotide sequence of fusion protein of TAT Yu PR domain, expression vector, the purposes of host cell.

6th purpose of the present invention is to provide a kind of antitumor medicine composition.

For realizing above-mentioned first purpose, the present invention adopts the technical scheme that:

A kind of TAT and PR The fusion protein of domain, described TAT and PR The aminoacid sequence of the fusion protein of domain is as shown in SEQ ID NO.7 or SEQ ID NO.9.

For realizing above-mentioned second purpose, the present invention adopts the technical scheme that:

A kind of coding TAT and PR The nucleotide sequence of the fusion protein of domain, described nucleotide sequence is as shown in SEQ ID NO.8 or SEQ ID NO.10.

For realizing above-mentioned 3rd purpose, the present invention adopts the technical scheme that:

A kind of expression vector, described expression vector contains the nucleotide sequence of the fusion protein of coding TAT Yu PR domain as above.

For realizing above-mentioned 4th purpose, the present invention adopts the technical scheme that:

A kind of host cell, described host cell contains the nucleotide sequence of the fusion protein integrating coding TAT Yu the PR domain as above having external source in expression vector as above, or its genome.

Described host cell is the offspring of bacterial cell, fungal cell or these host cells.

For realizing above-mentioned 5th purpose, the present invention adopts the technical scheme that:

TAT and PR as mentioned above The fusion protein of domain, described nucleotide sequence, described expression vector or the application in preparing antitumor drug of the described host cell.

Described tumor is meningioma.

For realizing above-mentioned 6th purpose, the present invention adopts the technical scheme that:

A kind of antitumor medicine composition, described pharmaceutical composition comprises fusion protein and the conventional pharmaceutical carrier of TAT Yu PR domain described above.

The invention has the advantages that:

The present invention successfully constructs TAT and PR nullThe expression vector of the fusion protein of domain，And expression and purification obtains TAT Yu PR domain fusion protein，By experimental verification, it is on different stage meningioma primary cell propagation and the impact of apoptosis，Result shows that fusion protein all exists depression effect for different stage meningioma primary cell propagation，Especially for high-level meningioma, there is significant depression effect，M8(WHOIII level) and M12(WHOII level) meningioma primary cell is under 0.4 μM and 0.2 μM of TAT Yu PR domain fusion protein process，Within second day, propagation i.e. occurs significant difference compared with matched group，M8(WHOIII) under 0.4 μM of TAT Yu PR domain fusion protein processes，Within second day, even there is downward trend in growth curve.The present invention is that the cancer suppressing action using PR-domain provides effective way to treat high-level meningioma.

[accompanying drawing explanation]

Accompanying drawing 1 is pTAT-HA expression vector collection of illustrative plates.

Accompanying drawing 2 is the electrophoresis pattern of TAT-PR domain fusion protein.

Accompanying drawing 3 is that meningioma specimen MRI dyes with HE, Bar=50 μm.

Accompanying drawing 4 is the microphotograph that primary meningioma cell is cultivated, Bar=50 μm.

Accompanying drawing 5 is the Immunofluorescence test result of third generation meningioma primary cell, Bar=25 μm.

Accompanying drawing 6 is the Immunofluorescence test result of third generation meningioma primary cell, Bar=25 μm.

Accompanying drawing 7 is that RIZ1 is in intracellular positioning result, Bar=25 μm.

Accompanying drawing 8 is that TAT-PR domain is in intracellular positioning result, Bar=25 μm.

Accompanying drawing 9ab is WHOI (M9) growth curve.

Accompanying drawing 9cd is WHOII (M11) growth curve.

Accompanying drawing 9ef is WHOII (M12) growth curve.

Accompanying drawing 9gh is WHOIII (M8) growth curve.

Accompanying drawing 9ij is the detection of propagation target Ki67 Yu PCNA.

Accompanying drawing 10 is WHOI (M9)/WHOII (M12)/WHOIII (M8) 24 hour cell apoptosis ratio.

Accompanying drawing 11 is WHOI (M9)/WHOII (M12)/WHOIII (M8) apoptosis dose-response curve.

[detailed description of the invention]

The detailed description of the invention provided the present invention below in conjunction with the accompanying drawings elaborates.

Embodiment 1 TAT-HA-PR domain The expression plasmid of fusion protein builds and antitumor activity

One, TAT-HA-PR domain The expression plasmid of fusion protein builds and expression and purification

1 , material

Plasmid and antibody: pTAT-HA expression vector is given by Dowdy doctor S, pTAT-HA expression vector collection of illustrative plates is as shown in Figure 1, the nucleotide sequence of the pTAT-HA fragment in this expression vector is as shown in SEQ ID NO.1, comprising 6His sequence, TAT sequence, MCS multiple clone site, HA-tag etc. in pTAT-HA fragment, the nucleotide sequence of TAT is as shown in SEQ ID NO.2, aminoacid sequence such as SEQ Shown in ID NO.3, the nucleotide sequence of HA-tag such as SEQ Shown in ID NO.4, aminoacid sequence is as shown in SEQ ID NO.5.The cDNA total length plasmid of RIZ1 is purchased from GeneCopoeia company.

The main agents that molecular cloning uses is as follows: plasmid extraction test kit GenElute Plasmid Miniprep Kit is purchased from Sigma Company；Restricted enzyme, DNA reclaim test kit, DNA connects test kit purchased from Invitrogen company；Primer is synthesized by Invitrogen company.

Protein expression is as follows with purification related reagent: BL21 (DE3), Top10 Bacterial strain is purchased from TAKARA company；Peptone and yeast extract are purchased from OXOID company；Sodium ampicillin and isopropylthio-β-D-galactoside (Isopropyl β-D-1-thiogalactopyranoside, IPTG) purchased from through company of HTC of section；Nickel post is purchased from Roche company；PD-10 prepackage desalting column is purchased from GE company；10 × LA PCR Buffer I and dNTP Mixture is purchased from TaKaRa company.

, method

1), the expression plasmid of fusion protein builds

Carry out gene amplification with the full-length cDNA plasmid of RIZ1 for template and obtain PR-domain aim sequence (concrete nucleotide sequence is as shown in SEQ ID NO.6).

Primer sequence is as follows: PRDM2-F, SEQ ID NO.11:5 '-TTGCCATGGAGACCCTGGCTGAGGTACCC -3’；PRDM2-R, SEQ ID NO.12:5 '-ACAGAATTCTAACTTGTCTTCAGTTGTATGTC-3 ').

The composition of PCR reactant liquor is as follows: 2 × GC Buffer 25 μ l, dNTP Mixture 4 μ l, Forward Primer (50pmol/ μ l) 0.5 μ l, Reverse Primer (50pmol/ μ l) 0.5 μ l, template 5-10ng, self-produced KOD enzyme 0.5 μ l, supplies 50 μ l with water.

PCR response procedures is as follows: 94 DEG C, after 5min denaturation, expand by following bar: 94 DEG C of 30sec, 55 DEG C of 1min, 72 DEG C of 1min (adjusting according to the size of amplified fragments), after 5 circulations, 94 DEG C of 30sec, 62 DEG C of 1min (annealing temperature selects according to the Tm value of primer), 72 DEG C of 1min, after 18 circulations, 72 DEG C of 10min.

Sepharose electrophoresis detection pcr amplification product, cuts glue, utilizes Gel Extraction Kit (FOREGENE) reclaims test kit and reclaims.Pcr amplification product and pTAT-HA expression vector reclaim through NcoI and EcoRI double digestion rear electrophoresis respectively.PTAT-HA expression vector after recovery and the fragment of PR-domain mesh utilize DNA ligation Kit ver2.0 test kit to carry out 4 DEG C overnight to connect.Take connection product 10 μ l Transformed E .coil Top10 competence antibacterial, picking positive colony amplification culture, use GenElute Plasmid Miniprep Kit plasmid extraction test kit extracting plasmid, identify through double digestion after Ding Liang, serve Hai Yingjun company sequence verification, obtain connecting the expression plasmid of correct TAT-HA-PR domain fusion protein: TAT-HA-PR domain ,-20 DEG C save backup.

2), the expression identification of fusion protein

The expression plasmid of TAT-HA-PR domain fusion protein is converted DE3 competence, 35 DEG C of incubator overnight incubation, next day, picking positive colony was inoculated in the 5ml bacterial liquid culture medium containing certain antibiotics, 35 DEG C of shaking table overnight incubation, secondary daily culture medium continues to cultivate to OD after pressing 1:10 dilution proportion₆₀₀About=0.8-1.0, leaves and takes 1ml bacterium solution as not inducing comparison, adds IPTG to its final concentration of 100 μ g/ml, 16 DEG C, 180rpm abduction delivering 18h, take 1ml and induce bacterium solution in residue bacterium solution.The thalline do not induced and induce, after low-temperature centrifugation is collected, adds 100 μ l PBS ultrasonications, takes the full bacterium lysate sample preparation of part, and remainder takes cleer and peaceful precipitation, then sample preparation respectively after continuing to be centrifuged.All samples carries out the parallel coomassie brilliant blue staining of protein electrophoresis, finally determines whether protein expresses, the most solvable and be present in supernatant and still precipitate.

3), the purification of fusion protein

With above-mentioned protein expression step, after carrying out the amplification of large-scale DE3 antibacterial abduction delivering, low-temperature centrifugation collects antibacterial.Then with the 20mM of pre-cooling Imidazole buffer (25mM Tris-HCl, 300mM NaCl, pH8.0) resuspended thalline, adds the PMSF(Phenylmethanesulfonyl fluoride of 1mM), ultrasonication on ice (intensity 30%, 15min, each 5sec are spaced 5sec).After low-temperature centrifugation, take supernatant and cross nickel post 2 times (nickel post 20mM imidazole buffer pre-balance is good), then wash post 3 times with the 20mM imidazole buffer of 5 times of column volumes, finally with a small amount of 200mM Imidazole buffer eluting destination protein.The protein of eluting carries out desalting processing through PD-10 prepackage desalting column, and the protein solution after desalination is after filtration sterilization, and BCA protein concentration is quantitative, and subpackage is also positioned over-80 DEG C of preservations.Avoid protein multigelation to ensure its activity during use.

, result

The TAT-HA-PR domain fusion protein built is present in supernatant, and solvable.Seeing Fig. 2, electrophoresis finds TAT-HA-PR Domain molecular weight of albumen about 32KDa, and foreign protein amount is few, can be used for subsequent cell and processes experiment.TAT-HA-PR The aminoacid sequence of domain, as shown in SEQ ID NO.7, encodes the nucleotide sequence of this fusion protein as shown in SEQ ID NO.8.

Two, meningioma primitive cell culture and qualification

1 , material

Tumor specimen: meningiomas surgery Operated Specimens 4 example (seeing Fig. 3), WHOI level 1 example, WHOII level 2 example, WHOIII level 1 example, respectively numbered M9 (WHOI), M11 (WHOII), M12 (WHOII) and M8 (WHOIII).

It is as follows that cell cultivates the main agents used: 35mm, 60mm and 100mm culture dish, and 24 porocyte culture plates are purchased from Corning company；Cell base culture fluid (Dulbecco's Modified Eagle Medium, DMEM), hyclone (Fetal bovine serum, FBS), pancreatin (Trypsin-EDTA) and chain penicillin be purchased from Invitrogen company；Poly-D-lysine (PLL), without the PBS of calcium ions and magnesium ions purchased from Sigma company；Primary cell separation microinstrument；The disposable cell strainer of 40um Yu 70um is purchased from BIOLOGIX company.

Antibody: EMA mouse monoclonal antibody is purchased from Cell Signaling Technology company, Vimentin rabbit monoclonal antibodies is purchased from Cell Signaling Technology company, and Ki67 mouse monoclonal antibody is purchased from Cell Signaling Technology company, DAPI is purchased from green skies company.

, method

1), the cultivation of primary meningioma cell

Fresh tumor tissue is after removing blood vessel and arachnoidea, through the fragmentation of sharp property and pancreatin (0.25% Trypsin-EDTA, Gibco, 37 DEG C, 30 minutes) decompose after centrifugal (900 rpm/min, 4 DEG C), to abandon supernatant and be resuspended to 200 μ l PBS, the single cell suspension obtained filters through filter opening a diameter of 70um Yu 40um cell strainer the most respectively.Then this cell being inoculated in 100mm culture dish, inoculum density is 1 × 10⁶Cell/cm².Being removed by non-adherent cell after 6-8h, attached cell 3-4d changes liquid once.When adherent cell growth is fused to 80%, trypsinization Secondary Culture.Culture medium is selected containing 10% hyclone and 1% dual anti-DMEM in high glucose, and condition of culture is 37 DEG C, the carbon dioxide cell incubator of 5%.

2), the qualification of primary meningioma cell

Primary meningioma cell is cultivated to the third generation, in advance prior to using poly-D-lysine to process cell climbing sheet in 24 orifice plates, cell for dyeing is seeded in the cell climbing sheet of aseptic cleaning in advance, after cell is pooled to suitable density, by PBS quick wash 2-3 time of the attached cell on cell climbing sheet；4% paraformaldehyde room temperature of pre-cooling fixes 15min；PBS washs 4 times, last 5min；The ice methanol of-20 DEG C fixes 10min；PBS washs 4 times, last 5min；An anti-diluent containing 10% donkey serum, room temperature closes 30min；After washing confining liquid off, add containing the anti-anti-diluent of suitable dilution factor one, an anti-selection meningioma cell pathological diagnosis target: EMA(people's epithelial membrane antigen) and VIM(Vimentin).EMA mouse monoclonal antibody, dilution factor 1:100, VIM rabbit monoclonal antibodies, antibody dilution 1:100.Incubated at room 1h；PBS washs 4 times, last 5min；Add containing the anti-PBS of suitable dilution factor fluorescence two, incubated at room 45min-1h；PBS washs 4 times, last 5min；With 50% glycerol containing DAPI/PBS mounting, fluorescence microscope, shooting, often group cell counting is no less than 500 cells, calculates positive cell proportion.

, result

1), primary meningioma cell cultivation results

See Fig. 4.Primary meningioma separation and Culture is observation of cell well-grown after 24 hours, and adherent rate can reach 80%, and light Microscopic observation is visible, cell is bipolar or the arrangement of multipole shape, 12 hours form such as spindle cells, bipolar shape, 24 hours adherent growth, about 48 hour cells are assembled agglomerating, and the cell that can be observed to cultivate dramatically increases, and presents typical bipolarity and polygon, endochylema enriches, kernel is smaller, and within two days, cell can cover bottom culture dish, and in monolayer close-packed arrays.Passage cell class in early days is spherical, and after within 24 hours, most cells is adherent, bipolar and multipolar cell the phenomenon of cell becomes apparent from, Cytoplasm is more rich, the 5th generation cell, and cellular morphology substantially becomes big, bipolar or multipole grows, connecting in netted between cell, endochylema enriches, and core is big, kernel is high-visible, karyokinesis phenomenon is obvious, and cell proliferation is vigorous, is identified as meningioma cell.After 5 generations, cell proliferation slows down gradually, and old and feeble cellular morphology occurs, and benign meningioma is generally transferred to 7-8 generation, pernicious can pass more generation.

2), primary meningioma cell qualification result

Seeing Fig. 5, third generation meningioma primary cell carries out routine immunization fluoroscopic examination, and testing result finds, the equal overexpression of VIM, EMA, and every example counting is no less than 500 cells, and 4 example meningioma primary cell positive cell ratios are all higher than 90%.VIM is primarily targeted for kytoplasm, and EMA expresses can be positioned kytoplasm, after birth or karyon, and cellular morphology presents that typical endotheliocyte is multistage or bipolar state.Fig. 6, Ki67 positive cell ratio that sees is respectively 19.4%(M9), 25.9%(M11), 32.2%(M12) and 60.6%(M8).According to pathological diagnosis standard, it is believed that the meningioma primary cell of cultivation can be used for carrying out follow-up testing inspection.

Three, RIZ1 With PR-domain In intracellular location

1 , material

Cell derived: HEK293FT is purchased from Invitrogen company, and meningioma primary cell is from the meningioma cell original cuiture of operation meningioma Operated Specimens.

Transfection reagent and immunofluorescent staining reagent and antibody: X tremeGENE HP DNA transfection reagent is purchased from Roche company, and RIZ1 full-length cDNA plasmid is purchased from GeneCopoeia.HA-tag mouse monoclonal antibody is purchased from AbMart company.Needed for the cultivation of remaining cell and immunofluorescence, reagent is described above.

, method

1), confirm that RIZ1 is at intracellular site of action

HEK293FT cell creep plate in 24 orifice plates that poly-D-lysine processes in advance, uses X tremeGENE HP DNA transfection reagent to carry out the transfection of RIZ1 full-length cDNA plasmid after one hour.Change liquid after 6 hours, reduce the cytotoxic effect of transfection reagent.After transfection in 24 hours, row RIZ1 immunofluorescence dyes.Fluorescence staining step is as it has been described above, determine the cell location of RIZ1.

2) the TAT-HA-PR domain fusion protein site of action at meningioma primary cell, is confirmed

By four groups of meningioma primary cells row 24 orifice plate cell climbing sheet in advance, treat that cell is fused to 50%-60%, the protein concentration using 0.4 μM processes cell 2 hours, with HA-tag immunofluorescence dyeing at once, because carrying HA-tag label on expression vector pTAT-HA, TAT-HA-PR therefore can be determined by this method Domain fusion protein is in the location of meningioma primary cell.

, result

1), RIZ1 be considered as a kind of antioncogene, it can participate in the gene expression of Chromosome-encoded by methyl transferase activity, and PR-domain has been found to be the Main Function site of RIZ1 cancer suppressing action.Seeing Fig. 7, RIZ1 plasmid transfection experiment to find, 293T cell core occurs that strong positive dyes, and negative control group has no coloring, illustrates that the Main Function position of RIZ1 is at nucleus.

2), Fig. 8 is seen, HA-tag immunocytochemical stain found that, the coloring of strong positive occurs in the nucleus of meningioma primary cell, illustrate that TAT-HA-PR domain albumen location in meningioma primary cell is nucleus, this is completely the same with the apoptotic nueleolus of above-mentioned RIZ1, and TAT-HA-PR is described Domain albumen also produces biological action in nucleus.

Four, TAT-HA-PR domain The change of meningioma primary cell Proliferation and apoptosis after albumen process

1 , material

Cell derived: meningioma primary cell is from the original cuiture of meningiomas surgery Operated Specimens.

Apoptosis and cell proliferation detectable and instrument: Cell Proliferation Reagent WST-1 is purchased from Roche company, Annexin V-FITC/PI cell apoptosis detection kit is purchased from YEASEN company, and needed for cell cultivation, reagent material etc. are as described above, microplate reader: Thermo ELECTRON CORPORATION MULTISKAN MK3, flow cytometer: BD FACSCalibur.

Antibody: anti-Ki67 and PCNA is purchased from Cell Signaling Technology company, immunofluorescence dyeing reagent is as described above.

, method

1), the impact that the TAT-HA-PR domain albumen of research variable concentrations is bred for meningioma primary cell

Take the meningioma primary cell of the third generation, plant in 96 orifice plates with the cell density of 40%, within 24 hours, after cell is the most adherent, change with variable concentrations TAT- The culture fluid of HA-PR domain albumen, the amount of the total culture fluid in every hole is 100ul, and the activity of albumen is respectively 0 μM, 0.05 μM, 0.1 μM, 0.2 μM and 0.4 μM.Each concentration group sets six secondary orifices, culture fluid and albumen and changes once for every two days.Respectively at the 0th day, the 2nd day, the 4th day, within the 6th day, carry out the measurement of cell proliferation, finally draw out growth curve, parallel statistical analysis.Cell proliferation reagent is Roche Holding Ag's cell proliferation detectable WST-1.Operate according to explanation, during the detection of each microplate reader, be required in triplicate.

2) detection of target Ki67 Yu PCNA, is bred

Choose the protein concentration of 0.4 μM, before using immunofluorescence method detection albumen to process and after albumen process, each primary cell propagation target expression.Often at least 500 cells of group counting, calculate the expression of Ki67, PCNA respectively, and are depicted as bar figure.

3) apoptosis is detected after, TAT-HA-PR domain albumen processes meningioma primary cell

Each rank meningioma primary cell selects an example to carry out apoptosis test, take the meningioma primary cell of the third generation, cell density kind with 50% is in 60mm Tissue Culture Plate, 24 hours after cell is the most adherent, changing the culture fluid containing variable concentrations TAT-HA-PR domain albumen, protein concentration is respectively 0 μM, 0.05 μM, 0.1 μM, 0.2 μM and 0.4 μM.Hatch 24 hours, use the Annexin V-FITC/PI cell apoptosis detection kit of YEASEN company and the flow cytometer of BD company to carry out the detection of meningioma primary cell apoptosis.

, result

TAT-HA-PRdomain has anti-tumor activity in high-level meningioma (WHOII/WHOIII) primary cell.HA immunocytochemical stain result shows, TAT-HA-PR nullDomain is positioned the nucleus of primary meningioma cell，TAT-HA-PR domain albumen suppresses propagation (Fig. 9 ab of high-level meningioma primary cell、Fig. 9 cd、Fig. 9 ef、Fig. 9 gh): M8(WHOIII level) and M11、M12(WHOII level) meningioma primary cell is under the administration concentration of 0.4 μM and 0.2 μM，Propagation is suppressed，And M8(WHOIII level) and M12(WHOII level) meningioma primary cell propagation level change particularly evident，Under the concentration of 0.4 μM and 0.2 μM，Within second day, propagation is compared with matched group，Significant difference i.e. occurs，M8(WHOIII) under 0.4 μM，Within second day, even there is downward trend in growth curve，WHOI level meningioma primary cell，Experimental group is compared with matched group，Have no notable difference，The testing result (Fig. 9 ij) of propagation target Ki67 Yu PCNA supports above experimental result.As can be seen here, TAT-HA-PR domain albumen promotes the apoptosis of high-level meningioma primary cell.After albumen processes 24h, WHOIII and WHOII primary meningioma cell process group apoptosis rate is more significantly raised than matched group, and there is dose-dependant trend, and WHOI meningioma primary cell has no obvious apoptosis trend (Figure 10, Figure 11).

Embodiment 2 TAT-PR domain The expression plasmid of fusion protein builds and antitumor activity

According to the method for embodiment 1, build the TAT-PR not containing HA-tag label The expression plasmid of domain fusion protein: TAT-PR domain plasmid, expresses, identifies and purification TAT-PR domain fusion protein.TAT-PR The aminoacid sequence of domain fusion protein, as shown in SEQ ID NO.9, encodes the nucleotide sequence of this fusion protein as shown in SEQ ID NO.10.Electrophoresis finds TAT-PR domain molecular weight of albumen about 32KDa.

Those skilled in the art all know, HA-tag is merely possible to sequence label, the function of fusion protein will not be produced impact, TAT-PR Domain fusion protein is in intracellular location, and the impact on meningioma primary cell propagation and apoptosis should be with TAT-HA-PR domain fusion protein without significant difference.

Embodiment 3 TAT(9 Peptide )-HA-PR domain The expression plasmid of fusion protein builds and antitumor activity

The present embodiment investigates TAT cell-penetrating peptide and the PR of 9 peptides The antitumor action of the fusion protein that domain is constituted.Expression plasmid according to method structure TAT (9 the peptide)-HA-PR domain fusion protein of embodiment 1: TAT (9 peptide)-HA-PR Domain plasmid, the TAT sequence in this plasmid comprises 27 nucleotide (SEQ ID NO.13), and the TAT aminoacid sequence of coding is 9 amino acid residues (SEQ ID NO.14, RKKRRQRRR).Expressing, identify and purification TAT (9 peptide)-HA-PR domain fusion protein, electrophoresis finds TAT (9 peptide)-HA-PR domain molecular weight of albumen about 32KDa.Impact meningioma primary cell bred according still further to TAT (9 the peptide)-HA-PR domain albumen of the technique study variable concentrations of embodiment 1, detection propagation target Ki67 and PCNA.Result shows: M8(WHOIII level) and M11, M12(WHOII level) meningioma primary cell breeds under the administration concentration of 0.4 μM and be suppressed, M8(WHOIII level) and M12(WHOII level) meningioma primary cell propagation level change the most obvious, under 0.4 μM of concentration, within the 6th day, propagation starts significant difference occur compared with matched group；WHOI level meningioma primary cell, experimental group, compared with matched group, has no notable difference；The testing result of propagation target Ki67 Yu PCNA supports above experimental result.As can be seen here, the TAT cell-penetrating peptide of 9 peptides and PR The anti-tumor activity of the fusion protein that domain is constituted is significantly smaller than TAT-HA-PR domain fusion protein.

The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, on the premise of without departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be regarded as protection scope of the present invention.

SEQUENCE LISTING

<110>Second Military Medical University, PLA

<120>fusion protein of TAT Yu PR domain and application in preparing antitumor drug thereof

<130> /

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<170> PatentIn version 3.3

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Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr

1 5 10 15

Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp

20 25 30

Arg Trp Gly Ser Lys Leu Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg

35 40 45

Arg Arg Gly Gly Ser Thr Met Ser Gly Tyr Pro Tyr Asp Val Pro Asp

50 55 60

Tyr Ala Gly Ser Met Glu Thr Leu Ala Glu Val Pro Glu His Val Leu

65 70 75 80

Arg Gly Leu Pro Glu Glu Val Arg Leu Phe Pro Ser Ala Val Asp Lys

85 90 95

Thr Arg Ile Gly Val Trp Ala Thr Lys Pro Ile Leu Lys Gly Lys Lys

100 105 110

Phe Gly Pro Phe Val Gly Asp Lys Lys Lys Arg Ser Gln Val Lys Asn

115 120 125

Asn Val Tyr Met Trp Glu Val Tyr Tyr Pro Asn Leu Gly Trp Met Cys

130 135 140

Ile Asp Ala Thr Asp Pro Glu Lys Gly Asn Trp Leu Arg Tyr Val Asn

145 150 155 160

Trp Ala Cys Ser Gly Glu Glu Gln Asn Leu Phe Pro Leu Glu Ile Asn

165 170 175

Arg Ala Ile Tyr Tyr Lys Thr Leu Lys Pro Ile Ala Pro Gly Glu Glu

180 185 190

Leu Leu Val Trp Tyr Asn Gly Glu Asp Asn Pro Glu Ile Ala Ala Ala

195 200 205

Ile Glu Glu Glu Arg Ala Ser Ala Arg Ser Lys Arg Ser Ser Pro Lys

210 215 220

Ser Arg Lys Gly Lys Lys Lys Ser Gln Glu Asn Lys Asn Lys Gly Asn

225 230 235 240

Lys Ile Gln Asp Ile Gln Leu Lys Thr Ser

245 250

<210> 8

<211> 1354

<212> DNA

<213>artificial sequence

<400> 8

atattttgtt tactttagaa ggagatatac atatgcgggg ttctcatcat catcatcatc 60

atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg 120

ataaggatcg atggggatcc aagcttggct acggccgcaa gaaacgccgc cagcgccgcc 180

gcggtggatc caccatgtcc ggctatccat atgacgtccc agactatgct ggctccatgg 240

agaccctggc tgaggtaccc gaacatgtgc tgcgaggact tccggaggaa gtgaggcttt 300

tcccttctgc tgttgacaag acccggattg gtgtctgggc cactaaacca attttaaaag 360

gcaaaaaatt tgggccattt gttggtgata agaaaaaaag atctcaggtt aagaataatg 420

tatacatgtg ggaggtgtat tacccaaatt tgggatggat gtgcattgat gccactgatc 480

cagagaaggg aaactggctg cgatatgtga attgggcttg ctcaggagaa gagcaaaatt 540

tattcccact ggaaatcaac agagccattt actataaaac tttaaagcca atcgcgccgg 600

gcgaggagct cctggtctgg tacaatgggg aagacaaccc tgagatagca gctgcgattg 660

aggaagagcg agccagcgcc cggagcaagc ggagctcccc caagagccgg aaagggaaga 720

aaaaatccca ggaaaataaa aacaaaggaa acaaaatcca agacatacaa ctgaagacaa 780

gttagaattc gaagcttgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 840

ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 900

gttttttgct gaaaggagga actatatccg gatctggcgt aatagcgaag aggcccgcac 960

cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg 1020

cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 1080

cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 1140

ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 1200

cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 1260

ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 1320

tggaacaaca ctcaacccta tctcggtcta ttct 1354

<210> 9

<211> 241

<212> PRT

<213>artificial sequence

<400> 9

Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr

1 5 10 15

Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp

20 25 30

Arg Trp Gly Ser Lys Leu Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg

35 40 45

Arg Arg Gly Gly Ser Thr Met Ser Gly Gly Ser Met Glu Thr Leu Ala

50 55 60

Glu Val Pro Glu His Val Leu Arg Gly Leu Pro Glu Glu Val Arg Leu

65 70 75 80

Phe Pro Ser Ala Val Asp Lys Thr Arg Ile Gly Val Trp Ala Thr Lys

85 90 95

Pro Ile Leu Lys Gly Lys Lys Phe Gly Pro Phe Val Gly Asp Lys Lys

100 105 110

Lys Arg Ser Gln Val Lys Asn Asn Val Tyr Met Trp Glu Val Tyr Tyr

115 120 125

Pro Asn Leu Gly Trp Met Cys Ile Asp Ala Thr Asp Pro Glu Lys Gly

130 135 140

Asn Trp Leu Arg Tyr Val Asn Trp Ala Cys Ser Gly Glu Glu Gln Asn

145 150 155 160

Leu Phe Pro Leu Glu Ile Asn Arg Ala Ile Tyr Tyr Lys Thr Leu Lys

165 170 175

Pro Ile Ala Pro Gly Glu Glu Leu Leu Val Trp Tyr Asn Gly Glu Asp

180 185 190

Asn Pro Glu Ile Ala Ala Ala Ile Glu Glu Glu Arg Ala Ser Ala Arg

195 200 205

Ser Lys Arg Ser Ser Pro Lys Ser Arg Lys Gly Lys Lys Lys Ser Gln

210 215 220

Glu Asn Lys Asn Lys Gly Asn Lys Ile Gln Asp Ile Gln Leu Lys Thr

225 230 235 240

Ser

<210> 10

<211> 1327

<212> DNA

<213>artificial sequence

<400> 10

atattttgtt tactttagaa ggagatatac atatgcgggg ttctcatcat catcatcatc 60

atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg 120

ataaggatcg atggggatcc aagcttggct acggccgcaa gaaacgccgc cagcgccgcc 180

gcggtggatc caccatgtcc ggcggctcca tggagaccct ggctgaggta cccgaacatg 240

tgctgcgagg acttccggag gaagtgaggc ttttcccttc tgctgttgac aagacccgga 300

ttggtgtctg ggccactaaa ccaattttaa aaggcaaaaa atttgggcca tttgttggtg 360

ataagaaaaa aagatctcag gttaagaata atgtatacat gtgggaggtg tattacccaa 420

atttgggatg gatgtgcatt gatgccactg atccagagaa gggaaactgg ctgcgatatg 480

tgaattgggc ttgctcagga gaagagcaaa atttattccc actggaaatc aacagagcca 540

tttactataa aactttaaag ccaatcgcgc cgggcgagga gctcctggtc tggtacaatg 600

gggaagacaa ccctgagata gcagctgcga ttgaggaaga gcgagccagc gcccggagca 660

agcggagctc ccccaagagc cggaaaggga agaaaaaatc ccaggaaaat aaaaacaaag 720

gaaacaaaat ccaagacata caactgaaga caagttagaa ttcgaagctt gatccggctg 780

ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat 840

aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat 900

ccggatctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 960

cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 1020

tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 1080

cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 1140

tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 1200

tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 1260

cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 1320

ctattct 1327

<210> 11

<211> 29

<212> DNA

<213>artificial sequence

<400> 11

ttgccatgga gaccctggct gaggtaccc 29

<210> 12

<211> 32

<212> DNA

<213>artificial sequence

<400> 12

acagaattct aacttgtctt cagttgtatg tc 32

<210> 13

<211> 27

<212> DNA

<213>artificial sequence

<400> 13

cgcaagaaac gccgccagcg ccgccgc 27

<210> 14

<211> 9

<212> PRT

<213>artificial sequence

<400> 14

Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5

Claims (7)

1. the fusion protein of TAT Yu a PR domain, it is characterised in that the aminoacid sequence of the fusion protein of described TAT Yu PR domain is as shown in SEQ ID NO.7 or SEQ ID NO.9.

2. the nucleotide sequence of the fusion protein encoding TAT Yu PR domain, it is characterised in that described nucleotide sequence is as shown in SEQ ID NO.8 or SEQ ID NO.10.

3. an expression vector, it is characterised in that described expression vector contains the nucleotide sequence described in claim 2.

4. a host cell, it is characterised in that described host cell contains to integrate in the expression vector described in claim 3, or its genome the nucleotide sequence as claimed in claim 2 of external source.

Host cell the most according to claim 4, it is characterised in that described host cell is the offspring of bacterial cell, fungal cell or these host cells.

6. host cell application in preparing anti-meningioma medicine described in expression vector described in nucleotide sequence, claim 3 described in the fusion protein of TAT Yu PR domain, claim 2 described in claim 1 or claim 4.

7. the pharmaceutical composition of an anti-meningioma, it is characterised in that described pharmaceutical composition comprises fusion protein and the conventional pharmaceutical carrier of TAT Yu PR domain described in claim 1.