CN106380522A - Eukaryotic recombinant protein for specifically inhibiting activity of STAT3, and preparation method and application thereof - Google Patents
Eukaryotic recombinant protein for specifically inhibiting activity of STAT3, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a eukaryotic recombinant protein for specifically inhibiting the activity of STAT3, and a preparation method and an application thereof, and belongs to the field of medicine bioengineering. The full length of the eukaryotic recombinant protein for specifically inhibiting the activity of STAT3 comprises 210 amino acids, the No.1-No.25 amino acids are an NLS sequence, the No.26-No.31 amino acids are a His sequence, the No.34-No.199 amino acids are an MH2 sequence, the No.200-No.210 amino acids are a TAT sequence, and the His label sequence and the MH2 sequence are connected through two amino acids of KL. A competitive binding principle blocks ubiquitylation and degradation of PIAS3 and promotes binding of the PIAS3 and the STAT3, so the problem of difficulty in reaching of the expected effect and the expected safety of inhibition of activation of the STAT3 by present target inhibitors, RNA interference, antisense oligodeoxynucleotide and decoy oligonucleotide from the signal pathway perspective is solved, thereby proliferation of tumor cells is effectively inhibited, and the sensitivity of tumor stem cells to chemotherapeutics is promoted.
Description
Technical field
The present invention relates to medical bioengineering field, the eucaryon restructuring of more particularly, to one species specificity suppression STAT3 activity
Albumen and its preparation method and application.
Background technology
Tumor stem cell (tumor stem cells, TSCs) theory increasingly receives publicity in recent years.Become at present
Work(is successfully separated in the Several Kinds of Malignancies such as brain tumor, lung cancer, liver cancer, breast cancer, prostate cancer, colorectal cancer, cutaneum carcinoma
Go out tumor stem cell.Tumor stem cell hypothesis thinks, tumor recurrence and transfer and tumor stem cell remaining after drug therapy
There is substantial connection.The proposition of tumor stem cell occurs for tumour and Mechanisms of recurrence provides reasonable dismissal, more effective for researching and developing
Antineoplastic and stem cell and RESEARCH ON CELL-BIOLOGY be respectively provided with important meaning.
Signal transduction and activating transcription factor (Signal Transducer and Activator of
Transcription, STAT) protein family is one group of GAP-associated protein GAP being activated by cytokine receptor, participate in propagation, differentiation,
The important biological processes such as apoptosis and immunological regulation.Being currently known STATs family has 6 members (STAT1-6), wherein
STAT3 unconventionality expression continuous activation in Several Kinds of Malignancy, directly or indirectly regulate and control multiple oncogenes thus affecting tumour
Occurrence and development, and the maintenance with tumor stem cell and self and chemicotherapy opposing in close relations.
The activated pathway of STAT3 signal path mainly has three classes:Classical JAK-STAT3 signal path；Receptor-independent
EGFR-TK (RTKs) approach；Non- receptor-independent EGFR-TK (No-receptor RTKs) approach.When being believed by upstream
After number activation phosphorylation, STAT3 aggregates into homology or heterodimer, enters karyon and ties with target gene promoters specific site
Close and promote its transcription.Normal STAT3 activation is quick and of short duration, only maintains several minutes to a few houres, but in kinds of tumors
Middle have STAT3 continuation overactivity and induce the gene unconventionality expression closely related with cell proliferation, differentiation, apoptosis.Grind
Study carefully the many oncogenic signals paths of discovery finally all to concentrate on a set of nuclear factor, targetting single transcription factor can block
The sustained activation that the change of a group upstream gene causes, transcription factor is the promising target of suppression cancer；STAT3 is the multiple crucial letter of tumour
" artis " in number path and gene regulation, therefore STAT3 has become one of most promising target spot in treatment of cancer.
However, the constitutively activated relying in non-part (EGF, IL-6 etc.) more than STAT3 in tumour.Existing targeted inhibition
Agent, RNA interference, ASON and decoy oligonucleotide etc. single from the suppression STAT3 activation of signal path angle, its effect and
Security all difficult to reach expections, therefore start with to study how to suppress exception tumour for the STAT3 from endogenous regulation mechanism
Transcriptional regulatory activity is increasingly subject to pay attention to.
Important endogenous negative regulators activation STAT3 supressor protein 3 (Protein in STAT3 path
Inhibitor ofactivated Stat3, PIAS3) it is STAT3 suppression albumen in special nucleus, it can be tied with STAT3
Merge blocking-up the latter to be combined with DNA and then suppress the expression of target gene, thus the signal path in STAT3 and transcriptional regulatory activity
The effect of upper performance " molecule gate ".Normal physiology and tissue homeostasis are most important for maintaining for the activity of PIAS3, and it passes through
To STAT3 interior multiple oncogenes or transcription factor activity suppression, thus playing cancer suppressing action.Research finds, works as PIAS3
After silence, STAT3 sustained activity increases, and proliferative activity o f tumor significantly improves；And PIAS3 raises the life that then can suppress tumour
Grow and strengthen the sensitiveness to chemotherapeutics.PIAS3 combine with EGFR inhibitor can produce synergistic antitumor effect.These all carry
Show that PIAS3 has tumor suppressor gene activity, its effect in tumour is also just increasingly subject to pay attention to.And, PIAS3 is multiple pernicious
Low expression and patient poor prognosis out of control with tumor cell proliferation is relevant in tumour, suppresses PIAS3 therefore in tumour cell
Degraded is conducive to maintaining its suppression to STAT3 activity in tumour, thus reaching the purpose of suppression tumour.
Smad6 belongs to one of suppressive Smads in Smad protein family.Research in recent years find Smad6 not only to BMP and
TGF signal beta path has inhibitory action, and to Wnt/ β-catenin, TLR4 and IL-1R/Toll sample acceptor, glucocorticoid
Receptor signaling pathways also have regulating and controlling effect.Therefore, Smad6 take part in the interaction between many A signal pathways and nucleoprotein, makes
Its effect in tumour is of increasing concern, and Smad6 is to the inhibitory action of transcription factor often through protein-interacting simultaneously
Starting ubiquitin protein enzyme system promotes its ubiquitination to degrade.Smad6C- terminal domains have mediated the knot of Smad6 and multiple protein
Close, and be that Smad6 induces the indispensable functional domain of associated proteins ubiquitination degraded.We have discovered that Smad6 in glioma
In rush cancer function may with its promote PIAS3 ubiquitination degrade thus promoting STAT3 abnormal activation relevant.Right further
Smad6 protein structure domain research display C-terminal MH2 functional domain has mediated SMAD6 and the combination with PIAS3, and is to promote PIAS3
The indispensable key element of ubiquitination degraded.And the expression vector individually building MH2 can block the combination of SMAD6 and PIAS3 and promote
Enter the stability of the latter (PIAS3) thus suppressing the activity of STAT3 in tumour cell.More importantly MH2 carrier is real in vivo
Its significantly suppression tumor effect is shown in testing.
Thus, with Smad6MH2 as core, the eucaryon recombinant protein being guiding using cell-penetrating peptide, targeted inhibition that can be special
STAT3 activity simultaneously shows more effective tumor inhibition effect, has higher practical value.
Content of the invention
For above-mentioned problem, the present invention provide the eucaryon recombinant protein that a species specificity suppresses STAT3 activity and
Its preparation method and application, to overcome existing targeted inhibition agent, RNA interference, ASON and decoy oligonucleotide etc.
List is from the suppression STAT3 activation of signal path angle, but problem expected from its effect and security all difficult to reaches, by competition
Property combination principle block PIAS3 ubiquitination degrade and promote the combination of PIAS3 and STAT3, thus effectively suppression tumour cell
Propagation, promote the sensitiveness to chemotherapeutics for the tumor stem cell.
To achieve these goals, the technical scheme that the present invention takes is：
One species specificity suppresses the eucaryon recombinant protein of STAT3 activity, wherein, the transcription factor that will be able to be combined with PIAS3
N-terminal 166 amino acid sequences of Smad6 are named as MH2, connect nuclear location peptide NLS in the N-terminal of MH2, in the C-terminal of MH2
Connect and wear film signal peptide TAT, add His label, the eucaryon restructuring of described specificity suppression STAT3 activity between NLS and MH2
The functional areas code name of albumen is：NLS-His-MH2-TAT；
210 amino acid of eucaryon recombinant protein total length of described specificity suppression STAT3 activity, wherein 1～25 is NLS sequence
Row, 26～31 be His sequence, 34～199 be MH2 sequence, 200～210 be TAT sequence, described His sequence label with described
It is attached with two amino acid of KL between MH2 sequence；
The amino acid sequence of eucaryon recombinant protein of described specificity suppression STAT3 activity is：
The eucaryon recombinant protein of above-mentioned specificity suppression STAT3 activity, wherein, described specificity suppression STAT3 activity
The corresponding base sequence of eucaryon recombinant protein be：
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, including：
(1) clone of NLS-His-MH2-TAT gene
166 ammonia containing Smad6 with pcDNA/Flag-Smad6 carrier for the amplification of template high-fidelity PCR amplification kit
The MH2 sequence of base acid, introduces His sequence and TAT sequence in upstream and downstream primer respectively, introduces NLS sequence in N-terminal, obtains just
Level PCR primer, with described primary PCR products for the final NLS-His-MH2-TAT sequence of template amplification, then final
NLS-His-MH2-TAT sequence two ends introduce restriction endonuclease Hind III and Kpn I restriction enzyme site respectively, obtain PCR primer；
With described restriction endonuclease Hind III and PCR primer described in Kpn I digestion, then reclaimed with glue reclaim kit, obtain
PCR digestion products；
With described restriction endonuclease Hind III and PCR primer described in Kpn I digestion, after 1% agarose gel electrophoresis, use matter
Grain extraction agent box reclaims, and obtains plasmid enzyme restriction product pcDNA3.1 plasmid；
Using T4DNA ligase by described PCR digestion products and described plasmid enzyme restriction in the environment of temperature is 3～5 DEG C
Product pcDNA3.1 plasmid connects overnight, then converts and is applied to containing 50 μ g/ml ammonia benzyls to E.coli Dh5 α competent cell
On the LB solid medium of penicillin, overnight incubation in the environment of temperature is 36～38 DEG C；
Picking single bacterium colony, is taken out by PCR primary dcreening operation positive colony, plasmid extraction kit after LB fluid nutrient medium incubated overnight
Carry, obtain positive plasmid：pcDNA3.1-NLS-His-MH2-TAT；
(2) transfection of HEK293 cell and expression
After pcDNA3.1-NLS-His-MH2-TAT is passed through lipofectamine transfected HEK 293 48 hours, plus
Enter neomycin and carry out pressurization screening, the HEK293 stable cell strain of construction expression NLS-His-MH2-TAT gene；
(3) the isolating and purifying of NLS-His-MH2-TAT recombinant protein
Described HEK293 stable cell strain is enlarged cultivate, then collects cell and extract total protein of cell, albumen
Extract is filtered with 0.45 μm of filter, purifies the recombinant protein with His label using Ni post；
Detailed process is as follows：Transfer resin, to Ni post, after precipitating completely, washs nickel affinity chromatography post with distilled water, and
Prevent next step Ni2+ from precipitating；Washed with ddH2O, eliminate the air in matrix；The Binding buffer of 10 × column volume puts down
Weighing apparatus；Loading；The Binding buffer washing of 10 × column volume, collects efflux；Elution buffer elutes, and surpasses further
Filter obtains the eucaryon recombinant protein of described specificity suppression STAT3 activity after concentrating.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, described restriction endonuclease Hind
The corresponding base sequence in III digestion site is：AAGCTT.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, described restriction endonuclease Kpn
The corresponding base sequence of I restriction enzyme site is：GGTACC.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, in step (1), in temperature
Spend for adopting T4DNA ligase in the environment of 4 DEG C by described PCR digestion products and described plasmid enzyme restriction product pcDNA3.1 plasmid
Connect and overnight then convert to E.coli Dh5 α competent cell the LB solid training being applied to containing 50 μ g/ml ampicillins
On foster base, overnight incubation in the environment of temperature is 37 DEG C.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, in step (1),
To positive plasmid：After pcDNA3.1-NLS-His-MH2-TAT, described positive plasmid is sequenced, to verify gene cloning
Correctness.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, in step (2), in structure
After building the HEK293 stable cell strain of expression NLS-His-MH2-TAT gene, detect NLS-His- also by Western blot
The expression of MH2-TAT albumen.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, also includes：Will be described
Specificity suppresses the eucaryon recombinant protein of STAT3 activity after SDS-PAGE electrophoresis, transferring film, by His monoclonal antibody to described special
Property suppression STAT3 activity eucaryon recombinant protein identified.
A kind of application of the eucaryon recombinant protein of specificity suppression STAT3 activity described above, wherein, will be described special
Property suppression STAT3 activity eucaryon recombinant protein be used for suppress tumour cell propagation, promote tumor stem cell to chemotherapeutics
Sensitiveness.
Technique scheme has the advantage that or beneficial effect：
210 amino acid of eucaryon recombinant protein total length of the specificity suppression STAT3 activity that the present invention provides, wherein 1～
25 is NLS sequence, and 26～31 is His sequence, and 34～199 is MH2 sequence, and 200～210 is TAT sequence, described His label sequence
It is attached with two amino acid of KL between row and described MH2 sequence；This specificity suppresses the eucaryon recombinant protein of STAT3 activity
Degrade and promote the combination of PIAS3 and STAT3 by the ubiquitination that competitive binding principle blocks PIAS3, overcome existing
Targeted inhibition agent, RNA interference, ASON and decoy oligonucleotide etc. are single to be activated from signal path angle suppression STAT3,
But problem expected from its effect and security all difficult to reaches, thus the effectively propagation of suppression tumour cell, promotion Tumor Stem
The sensitiveness to chemotherapeutics for the cell.
Brief description
By reading the detailed description non-limiting example made with reference to the following drawings, the present invention and its feature, outward
Shape and advantage will become more apparent.
Fig. 1 is in the preparation method of eucaryon recombinant protein of specificity suppression STAT3 activity that the embodiment of the present invention 1 provides
The structure principle schematic of positive plasmid pcDNA3.1-NLS-His-MH2-TAT；
Fig. 2 is in the preparation method of eucaryon recombinant protein of specificity suppression STAT3 activity that the embodiment of the present invention 1 provides
The eucaryon recombinant protein of the specificity suppression STAT3 activity that step (3) is obtained carries out the knot of 12%SDS-PAGE electrophoresis detection
Fruit schematic diagram；
Fig. 3 is in the preparation method of eucaryon recombinant protein of specificity suppression STAT3 activity that the embodiment of the present invention 1 provides
The eucaryon weight of the specificity suppression STAT3 activity being obtained by Western blot authentication step (3) using His monoclonal antibody
The result schematic diagram of histone；
Fig. 4 is the eucaryon of employing immunofluorescence double dye display specificity suppression STAT3 activity that the embodiment of the present invention 2 provides
The cellular localization situation schematic diagram of recombinant protein；
Fig. 5 is that the eucaryon recombinant protein of the specificity suppression STAT3 activity that the embodiment of the present invention 3 provides suppresses Smad6 pair
The schematic diagram of PIAS3 proteins ubiquitinization degraded；
Fig. 6 is the eucaryon recombinant protein suppression STAT3SIE of the specificity suppression STAT3 activity that the embodiment of the present invention 3 provides
Activity schematic diagram；
Fig. 7 is that the eucaryon recombinant protein suppression gummed paper knurl of the specificity suppression STAT3 activity that the embodiment of the present invention 4 provides is done
The schematic diagram of the formation of cell ball；
Fig. 8 is that the eucaryon recombinant protein of the specificity suppression STAT3 activity that the embodiment of the present invention 4 provides promotes glioma to do
The schematic diagram expressing and suppressing STAT3 target protein CCND1, Sox2 expression of PIAS3 in cell；
Fig. 9 is the eucaryon recombinant protein suppression nude mice by subcutaneous of the specificity suppression STAT3 activity that the embodiment of the present invention 5 provides
The schematic diagram of the growth of glioma；
Figure 10 is that the eucaryon recombinant protein of the specificity suppression STAT3 activity that the embodiment of the present invention 5 provides is obviously prolonged cranium
The schematic diagram of the life cycle of interior glioma nude mice.
Specific embodiment
The present invention is further illustrated with specific embodiment below in conjunction with the accompanying drawings, but the limit not as the present invention
Fixed.
Embodiment 1：
The eucaryon recombinant protein of the specificity suppression STAT3 activity that the embodiment of the present invention 1 provides, by genetic manipulation, will
166 amino acid sequences of the aminoterminal (C-terminal) of transcription factor Smad6 being combined with PIAS3 are named as MH2, in the N of MH2
End connects nuclear location peptide NLS, connects in the C-terminal of MH2 and wears film signal peptide TAT, adds His label, specifically between NLS and MH2
Property suppression STAT3 activity the functional areas code name of eucaryon recombinant protein be：NLS-His-MH2-TAT；This specificity suppresses STAT3
210 amino acid of eucaryon recombinant protein total length of activity, wherein 1～25 is NLS sequence, and 26～31 is His sequence, 34～199
For MH2 sequence, 200～210 is TAT sequence, is attached with two amino acid of KL between His sequence label and MH2 sequence；Should
The amino acid sequence of eucaryon recombinant protein of specificity suppression STAT3 activity is：
The embodiment of the present invention 1 provide specificity suppression STAT3 activity the corresponding base sequence of eucaryon recombinant protein be：
In the eucaryon recombinant protein of specificity suppression STAT3 activity that the embodiment of the present invention 1 provides, comparison specificity suppression
The eucaryon recombinant protein of STAT3 activity, with the out of order control sequence of 16 amino acids before Smad6 PROTEIN C end
(ASVHRYYLRWTGERCV) replace MH2 sequence, other link solution are constant, are not easy to by plasmid table because sequence is less
Reach, therefore fusion protein is directly synthesized using polypeptide synthesis method, be named as NLS-His-MH2sc-TAT.
Fig. 1 is in the preparation method of eucaryon recombinant protein of specificity suppression STAT3 activity that the embodiment of the present invention 1 provides
The structure principle schematic of positive plasmid pcDNA3.1-NLS-His-MH2-TAT；Fig. 2 is the special of the embodiment of the present invention 1 offer
Property suppression STAT3 activity the preparation method of eucaryon recombinant protein in step (3) is obtained specificity suppression STAT3 activity
Eucaryon recombinant protein carries out the result schematic diagram of 12%SDS-PAGE electrophoresis detection；Fig. 3 is the special of the embodiment of the present invention 1 offer
Property suppression STAT3 activity the preparation method of eucaryon recombinant protein in reflected by Western blot using His monoclonal antibody
Determine the result schematic diagram of the eucaryon recombinant protein of specificity suppression STAT3 activity that step (3) obtains；As illustrated, it is a kind of special
The preparation method of the eucaryon recombinant protein of opposite sex suppression STAT3 activity, including：
(1) clone of NLS-His-MH2-TAT gene
166 ammonia containing Smad6 with pcDNA/Flag-Smad6 carrier for the amplification of template high-fidelity PCR amplification kit
The MH2 sequence of base acid, introduces His sequence and TAT sequence in upstream and downstream primer respectively, introduces NLS sequence in N-terminal, obtains just
Level PCR primer, with described primary PCR products for the final NLS-His-MH2-TAT sequence of template amplification, then final
NLS-His-MH2-TAT sequence two ends introduce restriction endonuclease Hind III and Kpn I restriction enzyme site (referring to Fig. 1) respectively, obtain PCR
Product；
With described restriction endonuclease Hind III and PCR primer described in Kpn I digestion, then reclaimed with glue reclaim kit, obtain
PCR digestion products；
With described restriction endonuclease Hind III and PCR primer described in Kpn I digestion, after 1% agarose gel electrophoresis, use matter
Grain extraction agent box reclaims, and obtains plasmid enzyme restriction product pcDNA3.1 plasmid；
Using T4DNA ligase by described PCR digestion products and described plasmid enzyme restriction product in the environment of temperature is 4 DEG C
PcDNA3.1 plasmid connects overnight, then converts to E.coli Dh5 α competent cell and is applied to containing 50 μ g/ml ammonia benzyl moulds
On the LB solid medium of element, overnight incubation in the environment of temperature is 37 DEG C；
Picking single bacterium colony, is taken out by PCR primary dcreening operation positive colony, plasmid extraction kit after LB fluid nutrient medium incubated overnight
Carry, obtain positive plasmid：pcDNA3.1-NLS-His-MH2-TAT；Obtaining positive plasmid：pcDNA3.1-NLS-His-MH2-
After TAT, positive plasmid is sequenced, to verify the correctness of gene cloning.
(2) transfection of HEK293 cell and expression
After pcDNA3.1-NLS-His-MH2-TAT is passed through lipofectamine transfected HEK 293 48 hours, plus
Enter neomycin and carry out pressurization screening, the HEK293 stable cell strain of construction expression NLS-His-MH2-TAT gene；In construction expression
After the HEK293 stable cell strain of NLS-His-MH2-TAT gene, detect NLS-His-MH2-TAT also by Western blot
The expression of albumen.
(3) the isolating and purifying of NLS-His-MH2-TAT recombinant protein
HEK293 stable cell strain is enlarged cultivate, then collects cell and extract total protein of cell, Protein Extraction
Liquid is filtered with 0.45 μm of filter, purifies the recombinant protein with His label using Ni post；
Detailed process is as follows：Transfer resin, to Ni post, after precipitating completely, washs nickel affinity chromatography post with distilled water, and
Prevent next step Ni2+ from precipitating；Washed with ddH2O, eliminate the air in matrix；The Binding buffer of 10 × column volume puts down
Weighing apparatus；Loading；The Binding buffer washing of 10 × column volume, collects efflux；Elution buffer elutes, and surpasses further
Filter obtains the eucaryon recombinant protein of specificity suppression STAT3 activity after concentrating；It is named as：NLS-His-MH2-TAT is (referring to figure
2).
(4) the Western blot identification of NLS-His-MH2-TAT eucaryon recombinant protein
By the eucaryon recombinant protein of specificity suppression STAT3 activity after SDS-PAGE electrophoresis, transferring film, by His monoclonal antibody
, being identified, qualification result is referring to Fig. 3 to the eucaryon recombinant protein of specificity suppression STAT3 activity.
In the preparation method of the eucaryon recombinant protein of the specificity suppression STAT3 activity that the embodiment of the present invention 1 provides, interior
The corresponding base sequence in enzyme cutting Hind III digestion site is：AAGCTT；The corresponding base sequence of restriction endonuclease Kpn I restriction enzyme site
For：GGTACC.
The preparation method of the above-mentioned eucaryon recombinant protein of specificity suppression STAT3 activity, wherein, by described specificity
The eucaryon recombinant protein of suppression STAT3 activity is used for suppressing the propagation of tumour cell, promotes tumor stem cell to chemotherapeutics
Sensitiveness.
Embodiment 2：
Eucaryon recombinant protein N LS- of 500 μM of specificity suppression STAT3 activity is added in the 293T cell of culture
Fixed in case follow-up immunization fluorescent staining after His-MH2-TAT, 6h with 4% paraformaldehyde；
Carry out the double dye of immunofluorescence using anti-more than anti-His mouse monoclonal and anti-tubulin rabbit, referring to Fig. 4, Fig. 4 is this
The cell of the eucaryon recombinant protein of employing immunofluorescence double dye display specificity suppression STAT3 activity that bright embodiment 2 provides is fixed
Position situation schematic diagram, eucaryon recombinant protein N LS-His-MH2-TAT of result display specificity suppression STAT3 activity can not only be entered
Enter cell, and be smoothly positioned nucleus.
Embodiment 3：
HA-PIAS3, Flag-Smad6-NLS and Myc-Ub cotransfection in the 293T cell of culture, and special with variable concentrations
The eucaryon recombinant protein N LS-His-MH2-TAT coprocessing cell of opposite sex suppression STAT3 activity, after 24 hours, with 10 μM
MG132 reprocesses 4 hours, collects detection HA-PIAS3 ubiquitination degraded change after albumen；Referring to Fig. 5, Fig. 5 is that the present invention is implemented
The eucaryon recombinant protein suppression Smad6 of the specificity suppression STAT3 activity that example 3 provides shows to PIAS3 proteins ubiquitin degraded
It is intended to, the eucaryon recombinant protein N LS-His-MH2-TAT suppression Smad6 of result display specificity suppression STAT3 activity is to PIAS3
Proteins ubiquitinization degraded.
In the 293T cell of culture, HA-PIAS3, Flag-Smad6-NLS and SIE plasmid (STAT3 reporter plasmid) is common
Transfection, and the eucaryon recombinant protein N LS-His-MH2-TAT coprocessing cell with variable concentrations specificity suppression STAT3 activity,
After 24 hours, reprocessed 6 hours with 25ng/ml IL-6, collect detection SIE activity after albumen；Referring to Fig. 6, Fig. 6 is the present invention
The eucaryon recombinant protein of the specificity suppression STAT3 activity that embodiment 3 provides suppresses the schematic diagram of the activity of STAT3SIE, result
It is right that the eucaryon recombinant protein N LS-His-MH2-TAT antagonism Smad6 of display specificity suppression STAT3 activity is degraded by PIAS3
The facilitation of STAT3 activity.
Embodiment 4：
Cultivate human glioma cells U87 and Shg44 with Culture of neural stem cells liquid (D/F12+EGF+bFGF+N2), simultaneously
Add specificity suppression STAT3 activity eucaryon recombinant protein N LS-His-MH2-TAT and reference protein TAT-NLS, cultivate to
The Forming ability of glioma stem cells ball is observed, part Cell extraction albumen is used for carrying out Western blot detection during 5d
The expression of STAT3 target gene CCND1, Sox2 and the expression of endogenous PIAS, reference protein TAT-NLS therein adopts
Before Smad6 PROTEIN C end, the out of order control sequence of 16 amino acids (ASVHRYYLRWTGERCV) replaces MH2 sequence, other side of link
Case is constant, is not easy to by plasmid expression because sequence is less, therefore is directly synthesized fusion protein using polypeptide synthesis method.
Cultivate human glioma cells U87 and Shg44 with Culture of neural stem cells liquid, be simultaneously introduced specificity suppression STAT3
Eucaryon recombinant protein N LS-His-MH2-TAT of activity and reference protein TAT-NLS, concentration is 500 μM, after cultivating 5 days, referring to
Fig. 7, Fig. 7 are the eucaryon recombinant protein suppression gummed paper knurl stem cells of the specificity suppression STAT3 activity that the embodiment of the present invention 4 provides
The schematic diagram of the formation of ball；It is found that eucaryon recombinant protein N LS-His-MH2-TAT of specificity suppression STAT3 activity can
Suppression human glioma cells U87 and the formation of Shg44 stem cell sphere.
Cultivate human glioma cells U87 and Shg44 with Culture of neural stem cells liquid, be simultaneously introduced the specificity of variable concentrations
Eucaryon recombinant protein N LS-His-MH2-TAT of suppression STAT3 activity, is done to the glioma collected by Western blot
In cell, endogenous PIAS3 and STAT3 target protein CCND1, Sox2 are detected, with actin and GAPDH as internal reference, referring to figure
8, Fig. 8 is in the eucaryon recombinant protein promotion glioma stem cells of specificity suppression STAT3 activity that the embodiment of the present invention 4 provides
The schematic diagram expressing and suppressing STAT3 target protein CCND1, Sox2 expression of PIAS3；Result shows that specificity suppression STAT3 lives
Eucaryon recombinant protein N LS-His-MH2-TAT of property can remarkably promote expressing and suppressing in tumor stem cell of endogenous PIAS3
The expression of STAT3 target protein CCND1, Sox2.
Embodiment 5：
By every balb/c nude mice 2x106/100ul concentration hypodermic injection U87 glioma cell, set up nude mice by subcutaneous mould
Type.After the obvious tumor mass of subcutaneous formation after 4 days, random packet：(1)NLS-His-MH2sc-TAT；(2)NLS-His-MH2-
TAT, gives lumbar injection NLS-His-MH2sc-TAT and NLS-His-MH2-TAT eucaryon recombinant protein (10 respectively by every group 6
Only, 4 days are once, continue 20 days for μ g/), the size of every 4 days measurement tumours, put to death nude mice when raising 24 days and weigh tumour
Size.Referring to Fig. 9, Fig. 9 is that the eucaryon recombinant protein suppression of the specificity suppression STAT3 activity that the embodiment of the present invention 5 provides is naked
The schematic diagram of the growth of the subcutaneous glioma of mouse；Result display NLS-His-MH2-TAT eucaryon recombinant protein can significantly suppress
The growth of nude mice by subcutaneous glioma.
Set up glioma stem cells nude mice encephalic model by every balb/c nude mice 2x106/100ul concentration intracranial injection.
After encephalic model sets up 4 days, random packet：(1)NLS-His-MH2sc-TAT；(2) NLS-His-MH2-TAT, every group 10
Only, lumbar injection NLS-His-MH2sc-TAT and NLS-His-MH2-TAT eucaryon recombinant protein are given respectively (10 μ g/ only, 4 days
Once dead to nude mice), the life cycle of record nude mice.Referring to Figure 10, Figure 10 is the specificity suppression that the embodiment of the present invention 5 provides
The eucaryon recombinant protein of STAT3 activity is obviously prolonged the schematic diagram of the life cycle of Intracranial Gliomas nude mice；Result shows NLS-
His-MH2-TAT can be obviously prolonged the life cycle (p of nude mice<0.001).
In sum, the eucaryon recombinant protein total length 210 of specificity suppression STAT3 activity provided in an embodiment of the present invention
Amino acid, wherein 1～25 is NLS sequence, and 26～31 is His sequence, and 34～199 is MH2 sequence, and 200～210 is TAT sequence,
It is attached with two amino acid of KL between described His sequence label and described MH2 sequence；This specificity suppression STAT3 activity
Eucaryon recombinant protein by competitive binding principle block PIAS3 ubiquitination degrade and promote the knot of PIAS3 and STAT3
Close, overcome the lists such as existing targeted inhibition agent, RNA interference, ASON and decoy oligonucleotide from signal path angle
Degree suppression STAT3 activation, but problem expected from its effect and security all difficult to reaches, thus effectively suppress tumour cell
Propagation, the promotion sensitiveness to chemotherapeutics for the tumor stem cell.
It should be appreciated by those skilled in the art that those skilled in the art combine prior art and above-described embodiment can be real
Existing described change case, will not be described here.Such change case has no effect on the flesh and blood of the present invention, will not be described here.
Above presently preferred embodiments of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation；Any those of ordinary skill in the art, are making many possible changes without departing from technical solution of the present invention
Move and modify, or be revised as the Equivalent embodiments of equivalent variations, this has no effect on the flesh and blood of the present invention.Therefore, every not
Depart from technical solution of the present invention content, according to the present invention technical spirit to any simple modification made for any of the above embodiments,
Equivalent variations and modification, all still fall within the range of technical solution of the present invention protection.
The amino acid sequence of eucaryon recombinant protein of specificity suppression STAT3 activity is：
1 MDPKKKRKVD PKKKRKVDPK KKRKVHHHHH HKLWCSVAYW
41 EHRTRVGRLY AVYDQAVSIF YDLPQGSGFC LGQLNLEQRS
81 ESVRRTRSKI GFGILLSKEP DGVWAYNRGE HPIFVNSPTL
121 DAPGGRALVV RKVPPGYSIK VFDFERSGLQ HAPEPDAADG
161 PYDPNSVRIS FAKGWGPCYS RQFITSCPCW LEILLNNPRY
201 GRKKRRQRRR.
Specificity suppression STAT3 activity the corresponding base sequence of eucaryon recombinant protein be：
1 GCCACCATGG ATCCAAAAAA GAAGAGAAAG GTAGATCCAA
41 AAAAGAAGAG AAAGGTAGAT CCAAAAAAGA AGAGAAAGGT
81 ACACCATCAC CATCACCACA AGCTTTGGTG CAGCGTGGCG
121 TACTGGGAGC ACCGGACGCG CGTGGGCCGC CTCTATGCGG
161 TGTACGACCA GGCCGTCAGC ATCTTCTACG ACCTACCTCA
201 GGGCAGCGGC TTCTGCCTGG GCCAGCTCAA CCTGGAGCAG
241 CGCAGCGAGT CGGTGCGGCG AACGCGCAGC AAGATCGGCT
281 TCGGCATCCT GCTCAGCAAG GAGCCCGACG GCGTGTGGGC
321 CTACAACCGC GGCGAGCACC CCATCTTCGT CAACTCCCCG
361 ACGCTGGACG CGCCCGGCGG CCGCGCCCTG GTCGTGCGCA
401 AGGTGCCCCC CGGCTACTCC ATCAAGGTGT TCGACTTCGA
441 GCGCTCGGGC CTGCAGCACG CGCCCGAGCC CGACGCCGCC
481 GACGGCCCCT ACGACCCCAA CAGCGTCCGC ATCAGCTTCG
521 CCAAGGGCTG GGGGCCCTGC TACTCCCGGC AGTTCATCAC
561 CTCCTGCCCC TGCTGGCTGG AGATCCTCCT CAACAACCCC
601 AGATACGGCC GCAAGAAACG CCGCCAGCGC CGCCGCTAAG
641 CTT.
The corresponding base sequence in restriction endonuclease HIND III digestion site is：AAGCTT.
The corresponding base sequence of restriction endonuclease KPN I restriction enzyme site is：GGTACC.
Before Smad6 PROTEIN C end, the out of order control sequence of 16 amino acids is：ASVHRYYLRWTGERCV.
Claims (10)
1. a species specificity suppresses the eucaryon recombinant protein of STAT3 activity it is characterised in that the transcription that will be able to be combined with PIAS3
N-terminal 166 amino acid sequences of factor S mad6 are named as MH2, connect nuclear location peptide NLS in the N-terminal of MH2, MH2's
C-terminal connects wears film signal peptide TAT, adds His label, the eucaryon of described specificity suppression STAT3 activity between NLS and MH2
The functional areas code name of recombinant protein is：NLS-His-MH2-TAT；
210 amino acid of eucaryon recombinant protein total length of described specificity suppression STAT3 activity, wherein 1～25 is NLS sequence,
26～31 is His sequence, and 34～199 is MH2 sequence, and 200～210 is TAT sequence, described His sequence label and described MH2 sequence
It is attached with two amino acid of KL between row；
The amino acid sequence of eucaryon recombinant protein of described specificity suppression STAT3 activity is：
2. the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 1 is it is characterised in that described special
Property suppression STAT3 activity the corresponding base sequence of eucaryon recombinant protein be：
3. a kind of preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 1, its feature
It is, including：
(1) clone of NLS-His-MH2-TAT gene
166 amino acid containing Smad6 with pcDNA/Flag-Smad6 carrier for the amplification of template high-fidelity PCR amplification kit
MH2 sequence, upstream and downstream primer introduces His sequence and TAT sequence respectively, N-terminal introduce NLS sequence, obtain primary PCR
Product, with described primary PCR products for the final NLS-His-MH2-TAT sequence of template amplification, then in final NLS-
His-MH2-TAT sequence two ends introduce restriction endonuclease Hind III and Kpn I restriction enzyme site respectively, obtain PCR primer；
With described restriction endonuclease Hind III and PCR primer described in Kpn I digestion, then reclaimed with glue reclaim kit, obtain PCR enzyme
Cut product；
With described restriction endonuclease Hind III and PCR primer described in Kpn I digestion, taken out with plasmid after 1% agarose gel electrophoresis
Extraction reagent kit reclaims, and obtains plasmid enzyme restriction product pcDNA3.1 plasmid；
Using T4DNA ligase by described PCR digestion products and described plasmid enzyme restriction product in the environment of temperature is 3～5 DEG C
PcDNA3.1 plasmid connects overnight, then converts to E.coli Dh5 α competent cell and is applied to containing 50 μ g/ml ammonia benzyl moulds
On the LB solid medium of element, overnight incubation in the environment of temperature is 36～38 DEG C；
Picking single bacterium colony, passes through PCR primary dcreening operation positive colony, plasmid extraction kit extracts after LB fluid nutrient medium incubated overnight,
Obtain positive plasmid：pcDNA3.1-NLS-His-MH2-TAT；
(2) transfection of HEK293 cell and expression
After pcDNA3.1-NLS-His-MH2-TAT is passed through lipofectamine transfected HEK 293 48 hours, add new
Mycin carries out pressurization screening, the HEK293 stable cell strain of construction expression NLS-His-MH2-TAT gene；
(3) the isolating and purifying of NLS-His-MH2-TAT recombinant protein
Described HEK293 stable cell strain is enlarged cultivate, then collects cell and extract total protein of cell, Protein Extraction
Liquid is filtered with 0.45 μm of filter, purifies the recombinant protein with His label using Ni post；
Detailed process is as follows：Transfer resin, to Ni post, after precipitating completely, washs nickel affinity chromatography post, and prevents with distilled water
Next step Ni2+ precipitates；Washed with ddH2O, eliminate the air in matrix；The Binding buffer balance of 10 × column volume；On
Sample；The Binding buffer washing of 10 × column volume, collects efflux；Elution buffer elutes, and further ultrafiltration is dense
The eucaryon recombinant protein of described specificity suppression STAT3 activity is obtained after contracting.
4. the preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 3, its feature exists
In the corresponding base sequence in described restriction endonuclease Hind III digestion site is：AAGCTT.
5. the preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 3, its feature exists
In the corresponding base sequence of described restriction endonuclease Kpn I restriction enzyme site is：GGTACC.
6. the preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 3, its feature exists
In in step (1), using T4DNA ligase by described PCR digestion products and described plasmid enzyme in the environment of temperature is 4 DEG C
Cut product pcDNA3.1 plasmid and connect and overnight then convert and be applied to containing 50 μ g/ml ammonia to E.coli Dh5 α competent cell
On the LB solid medium of parasiticin, overnight incubation in the environment of temperature is 37 DEG C.
7. the preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 3, its feature exists
In, in step (1), obtaining positive plasmid：After pcDNA3.1-NLS-His-MH2-TAT, described positive plasmid is surveyed
Sequence, to verify the correctness of gene cloning.
8. the preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 3, its feature exists
In in step (2), after the HEK293 stable cell strain of construction expression NLS-His-MH2-TAT gene, also by Western
Blot detects the expression of NLS-His-MH2-TAT albumen.
9. the preparation method of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 3, its feature exists
In also including：By the eucaryon recombinant protein of described specificity suppression STAT3 activity after SDS-PAGE electrophoresis, transferring film, pass through
His monoclonal antibody is identified to the eucaryon recombinant protein of described specificity suppression STAT3 activity.
10. a kind of application of the eucaryon recombinant protein of specificity suppression STAT3 activity as claimed in claim 1, its feature exists
In the eucaryon recombinant protein that described specificity is suppressed STAT3 activity is used for suppressing the propagation of tumour cell, promotes Tumor Stem
The sensitiveness to chemotherapeutics for the cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822898A (en) * | 2017-02-17 | 2017-06-13 | 南京九寿堂医药科技有限公司 | Application of the STAT3 gene expressions in terms of adenocarcinoma of lung chemosensitivity is improved is lowered in targeting |
| CN107144618A (en) * | 2017-06-20 | 2017-09-08 | 孙丽洲 | A kind of methods and applications of the protein activation levels of utilization DNA probe part Electrochemical Detection STAT 3 |
| CN113238053A (en) * | 2021-04-30 | 2021-08-10 | 四川大学华西医院 | Plasmid for detecting STAT3 dimerization |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333251A (en) * | 2008-01-30 | 2008-12-31 | 中国人民解放军第三军医大学 | Artificial zinc finger protein transcription factor capable of starting A20 gene expression and use |
-
2016
- 2016-08-30 CN CN201610790673.0A patent/CN106380522B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333251A (en) * | 2008-01-30 | 2008-12-31 | 中国人民解放军第三军医大学 | Artificial zinc finger protein transcription factor capable of starting A20 gene expression and use |
Non-Patent Citations (2)
| Title |
|---|
| 孙少飞，等: "重组蛋白TAT-NLS-Nkx6.2 在大肠杆菌的表达纯化及活性测定", 《中国生物工程杂志》 * |
| 李曼，等: "含蛋白转导域的SARA／SBD 融合蛋白的原核表达、纯化及生物学活性鉴定", 《生物技术通讯》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822898A (en) * | 2017-02-17 | 2017-06-13 | 南京九寿堂医药科技有限公司 | Application of the STAT3 gene expressions in terms of adenocarcinoma of lung chemosensitivity is improved is lowered in targeting |
| CN107144618A (en) * | 2017-06-20 | 2017-09-08 | 孙丽洲 | A kind of methods and applications of the protein activation levels of utilization DNA probe part Electrochemical Detection STAT 3 |
| CN113238053A (en) * | 2021-04-30 | 2021-08-10 | 四川大学华西医院 | Plasmid for detecting STAT3 dimerization |
| CN113238053B (en) * | 2021-04-30 | 2022-05-13 | 四川大学华西医院 | Plasmid for detecting STAT3 dimerization |
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