CN108239656A - A kind of protein function switching system of small-molecule drug control - Google Patents

A kind of protein function switching system of small-molecule drug control Download PDF

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CN108239656A
CN108239656A CN201611223345.9A CN201611223345A CN108239656A CN 108239656 A CN108239656 A CN 108239656A CN 201611223345 A CN201611223345 A CN 201611223345A CN 108239656 A CN108239656 A CN 108239656A
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protease
ser
gly
leu
val
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邹庆剑
翟珍珍
高斌
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SHENZHEN BEIKE BIO-TECHNOLOGY Co.,Ltd.
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Tianjin Tian Rui Biotechnology Co Ltd
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Priority to CN201611223345.9A priority Critical patent/CN108239656A/en
Priority to PCT/CN2017/118970 priority patent/WO2018121602A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention discloses a kind of protein function switching systems of small-molecule drug control.The system includes the protease of a high special, can recognize that and cuts specific amino acid sequence in target protein.The synthesis in vivo of the protease can be regulated and controled by a small-molecule drug.The biological activity of target protein can be accurately adjusted using this small-molecule drug.Present invention discover that and confirm, target protein and virus protease amalgamation and expression, this regulator control system it is more efficient, reaction is faster.Virus protease can be that natural protease can also pass through genetic modification, reduce protease molecule amount or be connected into a degradation domain (degron), promote the decomposition of protease itself.The selection of hiv protease inhibitor is predominantly used to treat on the market or the drug into clinical trial.

Description

A kind of protein function switching system of small-molecule drug control
Technical field
The present invention relates to biotechnology, especially a kind of protein function switching system of small-molecule drug control.
Background technology
Intracellular different albumen exercises different functions, and to study its function, is typically interfered with certain methods It functions, and observes its reaction.Currently used method is to manipulate intracellular specific gene in genome or transcriptional level Expression, but it is directly relatively fewer in the study on regulation of protein level.Operation is directly carried out to the function of intracellular protein has spirit The advantages of quick, quick, reversible, controllability, simplicity etc..Regulate and control with reference to the specificity of genetic manipulation and based on small-molecule drug Method, cell or tissue specific protein accurately can be controlled over time and space, so as to reduce unnecessary secondary make With.Such as albumen positioning in the cell, activation or inhibition, the stabilization or degradation of protein molecular, intracellular signal specific access Activation or blocking etc..At present, protein function control methods are broadly divided into three classes：1. regulate and control two heterologous two using small molecule or light The combination or dissociation of aggressiveness；2. changed using small molecule regulation protein structure；3. utilize the stabilization of small molecule adjusting control albumen Property.The technology involved by each type protein function controlling is introduced separately below：
(1) combination or dissociation of two heterodimers are controlled using small molecule
In past 20 years, the chemical small molecule effective means as regulation protein-protein interaction. These compounds are referred to as the chemical inducer (chemical inducers of dimerization, CID) of dimerization, tool There is the ability in combination with two protein domains, so as to induce the close to each other of them.CID has been used to：1. promote or Inhibit the transcriptional activity of specific gene；2. target protein is raised to intracellular specific region；3. regulation protein assembles and depolymerization.
Liu et al. people in 1991 is found that first CID --- immune suppressant drug FK506.This small molecule passes through same When combine FK506 binding protein (FKBP12) and calcinerin, inhibit T cell receptor mediation signal transduction.In this base On plinth, by FK506 dimer synthons (being named as FK1012), FKBP12 dimerizations can be made.FKBP12 and T cell receptor The fusion of ζ chains can be achieved quickly, adjustable and reversible FK | and 012 signal transduction relied on is adjusted.And it studies most fill at present The CID divided is rapamycin (rapamycin), this is a kind of macrolide natural products, the FRB knots of mediation FKBP12 and mTOR Interaction between structure domain.It if, can be mould in thunder pa by two kinds of interested protein respectively with FKBP and FRB amalgamation and expressions Close to each other in the presence of element, rapamycin can quickly realize the combination of FKBP-FRB under the conditions of low dosage.However, Endogenous cellular FKBP and mTOR can lead to invalid interaction with FKBP and FRB fusion protein competitive binding rapamycins. The combination of rapamycin and mTOR also result in the stagnation of cell cycle.In order to mitigate its undershooting-effect, by by rapamycin Derivatization and FKBP or FRB structural domains carry out rite-directed mutagenesis, and optimization obtains the side of Rimiducid regulation and control FKBP-FRB dimerizations Method can effectively reduce CID undershooting-effects.
Recently, the discovery of some new protein-ligands pair promotes the development of CID systems.For example, dexamethasone-first Aminopterin (Dex-Mtx) compound can induce mutual between glucocorticoid receptor (GR) and dihyrofolate reductase (DHFR) Effect.Regrettably, Mtx is the inhibitor of DHFR, this limits its application.After improvement, trimethoprim-SLF (TMP- SLF) compound can trigger the interaction between DHFR-FKBP12 fusions.The compound does not combine endogenous protein, but These small molecules are expensive and multistep are needed to synthesize, and which has limited their extensive uses.In recent years, scientist has found one A little plant hormones can Effective Regulation target protein combination and dissociation.Abscisic acid (ABA) is one of hormone for stimulating development of plants. It inhibits phosphoprotein phosphatase (PP2Cs) by combining pyribactin resistance (PYR)/PYR1-like (PYL).It will After PYL and PP2C structural domains merge respectively with DNA binding structural domains Gal4 and transcriptional activation domain VP64, Gal4 can be rebuild Transcriptional activity.Another Plant Hormone --- gibberellin (GA3) is also used as CID.GA3 can combine GID1, promote itself and GAI Between interaction.Plant hormone relatively economical safely and is quick on the draw, makes it have certain application value.
(2) complementation of small molecule regulation protein fragments is utilized
Interested protein is split into two inactive segments, specific compound small molecule can induce these segments Association, so as to restore the structure and function of target protein, this process is also referred to as fragment complementation.Muir and colleague develop a base In the conditionity protein splicing system of intein.Intein is divided into N- and C- ends two halves, during individualism, two inteins All without activity, but active structural domain is formed once once the two, by the protein splice at both ends together, form activity Albumen.Such as：Two sections the former merge between FKBP and maltose-binding protein (MBP), the latter merge in FRB and His labels Between.These fusions dimerization under rapamycin induction, includes peptide domain and approaches, restore intein montage activity simultaneously Lead to the formation of MBP that His- is marked.Intein montage is an irreversible process, the fragment complementation system based on intein Major defect be a lack of invertibity.
(3) stability of small molecule control albumen is utilized
Most said conditions protein degradation is completed using Ubiquitin-proteasome system, so utilizing small molecule regulation egg Links in white degradation process are, it can be achieved that protein stability regulates and controls.Degradation sub (degron) is a part for protein, It plays an important role in regulatory protein matter degradation rate.The degradation of majority degradation son mediation is degraded by Ubiquitin-proteasome What approach was completed, also have plenty of what is completed by autophagy approach.Many degradation subsequences have been identified at present, such as： DdFKBP is a unstable structure domain (DD domain), is degraded quickly after expression.But chemical small molecule shield-1 can be combined This structure makes it stablize without by fast degradation.Target protein with ddFKBP is combined, can effectively be adjusted by shield-1 Control the stabilization and degradation process of albumen.Similarly, ecDHFR can also by and the combination of trimethoprim regulate and control itself Stability.
On the contrary, certain structures domain itself is more stable, but then degraded under the effect of specific small molecule by quick ubiquitination. Such as：Plant hormone binding structural domain (AID) IAA17 can combine TIR1, and by ubiquitin ligase under the mediation of heteroauxin Identification, is degraded through a step by ubiquitination.
Invention content
The present invention by research and experiment, it was demonstrated that target protein (including：Functional protein, fusion protein or genetic engineering Albumen) in be inserted into one section of virus protease (or virus protease by transformation) identification sequence.By target protein and viral egg In white enzyme (or virus protease by transformation) expression vector cotransfection to mammalian cell, the expression of protease influences mesh The function of albumen is marked, and by adding and cancelling the small-molecule drug of corresponding protease inhibitors, the activity of controllable protease, So as to the function of indirect control target albumen.The small-molecule drug that the present invention is prepared for virus protease dependence is used for goal of regulation and control The switching system of protein function.It proposes a kind of quick, efficient, reversible, controllable, simple, economical and with applications well prospect Protein function regulation and control method.
The technical scheme is that：A kind of protein function switching system of small-molecule drug control, switchs by operon Original paper and sub- original paper is controlled to form, control by single virus protease or virus protease with protein degradation is sub melts It closes；The operon is located inside destination protein or between destination protein and protease-degradation minor structure, by egg White enzyme identification and cleavable one section connection peptide are formed；Whole system is used as out by the inhibitor compound of protease small molecule It closes, regulates and controls the stability of destination protein.
Further, control and operon merge into an entirety and protease identification are added between each original paper And cleavable sequence.
Further, specific protease is hepatitis C virus protease (NS3pro/4A).
Further, protease substrate is one of following structure sequences：
TRIF：Pro Ser Ser Thr Pro Cys Ser Ala His Leu；
MAVS：Glu Arg Glu Val Pro Cys His Arg Pro Ser Pro；
3-4A：Asp Leu Glu Val Val Thr Ser Thr Trp Val；
4A4B：Asp Glu Met Glu Glu Cys Ser Gln His Leu；
4B5A：Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp；
5A5B：Glu Asp Val Val Cys Cys Ser Met Ser Tyr.
Further, protease and molecule inhibitor compounds are one of following compounds：
Telaprevir(CAS Number：402957-28-2)；
Boceprevir(CAS Number：394730-60-0)；
Simeprevir(CAS Number：923604-59-5)；
Faldaprevir(CAS Number：801283-95-4)；
Asunaprevir(CAS Number：630420-16-5)；
Furaprevir(CAS Number：1435923-88-8).
Further, destination gene expression frame is fluorescin, drug resistant gene, antibody, cell factor and antigen recognizing Any one in molecule.
Further, degradation is NS3/4A unstable regions, ddFKBP unstable regions or ecDHFR ranges of instability Domain.
Further, protein expression switching system is in eukaryotic mammalian cell.
Further, the specific proteases are that have 70% homologous protease mutant or different with hepatitis C virus protease Structure enzyme.
The protein function switching system of the present invention includes three subsystems：1. the transformation of virus protease；2. target protein Transformation；3. the selection of hiv protease inhibitor.Target protein can separately be expressed with virus protease, can also merge table It reaches.
1. virus protease
The virus protease of the present invention is hepatitis C virus protease (NS3/4A).Under natural endowment, this albumen identification is simultaneously Specific amino acid sequence is cut, Hepatitis C Virus precursor protein is made to be broken down into multiple functional protein of small tool.This Hepatitis C virus protease (NS3/4A) is engineered by invention, removes NS3 RNA helicase sections in NS3/4A structures, is obtained NS3pro/4A structural regions.
Virus protease can also be connected into one section of degradation domain (degron), in the case where the two co-expresses, melt Hop protein can be by fast degradation, and can not function.Degradation domain can be NS4A degradation, melt with corresponding albumen After closing expression, degrade within the extremely short time, so as to influence protein function.Domain of degrading can also be by small molecule regulation Degradation, such as ddFKBP or exDHFR, under conditions of corresponding smaller ligand Shield-1 or trimethoprim is combined, Two structural domains can be stablized, and is maintained for a long time and is not degraded.After ligand is removed, the two structures are extremely unstable, expression Afterwards, it degrades within the extremely short time.
2. the transformation of target protein
Target protein can be the fusion protein of two or more structural domains or functional protein, in interleaving for each structural domain Enter virus protein enzyme recognition sequence.In some embodiments, target protein can be the fusion egg of two different fluorescins In vain or Chimeric antigen receptor (CAR), one section of virus protein enzyme recognition sequence is inserted.It is suppressed in proteinase activity In the case of, fluorescin amalgamation and expression understands common location in the cell；The each structural domain series connection of Chimeric antigen receptor, can be thin in T It is expressed in born of the same parents, and signal is transmitted with identification tumour antigen, activate the effect of T cell function.Under proteinase activity state, melt The fluorescin of expression is closed, is two albumen by proteolytic cleavage, separately expresses；The each structural domain of Chimeric antigen receptor is cut For two or more absolute construction, influence T cell identification tumour antigen and transmit signal.
Target protein can also can respectively be expressed with virus protease amalgamation and expression.Amalgamation and expression can make viral egg White enzyme and target protein spatial position are closer to quickly functioning.Separately expression can be reduced to mesh
Compared with prior art, the beneficial effects of the invention are as follows：Present invention discover that it and confirms, target protein and virus protein Enzyme amalgamation and expression, this regulator control system it is more efficient, reaction faster.Virus protease can be that natural protease can also be through Genetic modification is crossed, reduce protease molecule amount or is connected into a degradation domain (degron), promotes protease itself It decomposes.The selection of hiv protease inhibitor is predominantly used to treat on the market or the drug into clinical trial.
Description of the drawings
Fig. 1 is the SwichOFF systems in the embodiment of the present invention 1 -- green fluorescence protein expression carrier collection of illustrative plates.
Fig. 2 is the small molecule ASV regulation and control egfp expressions in the embodiment of the present invention 1.Upper figure：It is green when being not added with ASV Color fluorescin stablizes expression；Figure below：Green fluorescent protein is degraded after adding in ASV, without expressing.
Fig. 3 is being switched fast for the fluorescent protein expression in the embodiment of the present invention 1.Upper figure：It is green 24 hours after addition ASV Color fluorescence weakens rapidly.Figure below：After removing ASV, the expression of fluorescence fast and stable.
Fig. 4 is the SwichON systems in the embodiment of the present invention 2 -- fusion fluorescence protein expression carrier collection of illustrative plates.
Fig. 5 is the positioning of SwichON system regulations fluorescin in the cell in the embodiment of the present invention 2.First is classified as Red fluorescence, second is classified as green fluorescence, and third is classified as fluorescence overlay chart.First row：ASV is not added with, green fluorescence is expressed in carefully Cytoplasm；Secondary series：ASV is added in, green fluorescence is expressed in mitochondria.
Fig. 6 is that the SwichON system regulations in the embodiment of the present invention 2 are switched fast fusion fluorescin determining in the cell Position.First is classified as red fluorescence, and second is classified as green fluorescence, and third is classified as fluorescence overlay chart.24 hours after addition ASV, green Fluorescence navigates to mitochondria from cytoplasm rapidly.
Fig. 7 is that the SwichON system regulations in the embodiment of the present invention 2 are switched fast fusion fluorescin determining in the cell Position (two).First is classified as red fluorescence, and second is classified as green fluorescence, and third is classified as fluorescence overlay chart.It removes after ASV 24 hours, Green fluorescence is discharged into cytoplasm from mitochondria rapidly.
Fig. 8 is the SwichOFF system regulations CAR expression schematic diagrams in the embodiment of the present invention 3.
Fig. 9 is the SwichOFF systems CAR regulation and control Vector maps in the embodiment of the present invention 3.
Figure 10 is the SwichON system regulation CAR schematic diagram of the function in the embodiment of the present invention 4.
Figure 11 is the SwichON systems CAR regulation and control Vector maps in the embodiment of the present invention 4.
Figure 12 is the SwichON system regulation CAR schematic diagram of the function (two) in the embodiment of the present invention 5.
Figure 13 is the SwichON systems CAR regulation and control Vector maps (two) in the embodiment of the present invention 5.
Figure 14 is the western blot detection Switch protein regulation systems in the embodiment of the present invention 6.Upper figure：fCAR- V1, fCAR-V2, fCAR-V3 structure diagram；Figure below：Western blot detect flag tag fusion protein sizes.fCAR- V1 groups：First row：When being not added with ASV, the Flag-CAR segments of one section of 63kd size are expressed.Secondary series：After adding in ASV, egg is merged White fast degradation.FCAR-V2 groups：First row：When being not added with ASV, the Flag-CAR1 albumen of size 45kd is detected.Secondary series：Add After entering ASV, the intact proteins of anti-flag antibody tests to 97kd.FCAR-V3 groups：First row：When being not added with ASV, size is expressed The Flag-CAR1 albumen of 33kd.Secondary series：After adding in ASV, the intact proteins of anti-flag antibody tests to 97kd.Due to The possible natural fracture in the hinge positions of CAR itself, so all groups of albumen that can detect a size 33kd.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawings and examples.
Embodiment 1：Small-molecule drug regulates and controls the activity (SwichOFF systems) of green fluorescent protein
In the present embodiment, green fluorescent protein in from the HCV protease fusions of degradation is expressed, is inhibited by HCV protease Agent ASV is by protease inhibition activity, to regulate and control the expression of green fluorescent protein.
1. vector construction：
SfGFP genes are synthesized, HCV virus protease recognition sequence 4A4B is connected into behind sfGFP genes： DEMEECSQHLP.N34d genes are synthesized, with NS3/4A sequences and one section of degradation subsequence.As shown in Figure 1, by three sections of sequences Series connection forms an amalgamation and expression gene, is inserted into slow virus carrier Pltr carriers.
2. cell transfecting：
It recovers and cultivates 3T3 cells.The day before transfection adds in 5 × 10 in 24 orifice plates5A cell.Change liquid before transfection, one A hole adds in the culture medium without micromolecular inhibitor Asunaprevir (ASV), and a hole adds in the culture medium of the ASV containing 1uM. Transfection reagent is 3000 Reagent of Lipofectamine (Thermo Fisher).Transfection process is：1) two 1.5ml are taken EP is managed, and is separately added into 50uLThen culture medium adds in 3.0uL in a wherein branch pipe3000 reagents add in 2ug DNA plasmids in another branch pipe, respectively mixing.It 2) will be in two EP pipes Liquid is mixed into a pipe, and common 100uL gently blows and beats mixing 15 times, is stored at room temperature 5 minutes.3) 50uL mixtures is taken to be added to respectively In two holes, liquid is changed after 6h.
3. small-molecule drug regulates and controls：
In the present embodiment, using Asunaprevir (ASV) as small molecule regulation reagent, component will be tested on the day of transfection It it is two groups, one group is not added with ASV, the culture medium of the one group of ASV of addition containing 1uM.After transfection for 24 hours, the feelings of observation cell expression fluorescence Condition.
As shown in Figure 2：First row：It is not added with ASV, the fusion protein sfGFP-4A4B-NS3/4A-Degron of expression.Albumen Enzyme has cutting function, cuts off fusion protein at 4A4B.Then, Degron mediates NS3/4A protease fast degradations, and The function of sfGFP is not influenced.Second row：After adding in ASV, the similary expressed fusion protein sfGFP-4A4B-NS3/4A- of 3T3 cells Degron.But add in ASV and inhibit identification and cutting of the NS3/4A protease to 4A4B so that Degron is by entire fusion protein It brings Ubiquitin-Proteasome Pathway into, carries out fast degradation.SfGFP prevents cell from expressing green fluorescence egg by fast degradation In vain.
As shown in Figure 3：First row：It is not added with ASV, green fluorescent protein is expressed very high in cell, is added in after ASV for 24 hours, The expression quantity of green fluorescent protein drastically declines, and has arrived very faint level.Second row：Under conditions of having ASV in culture medium, Cell cannot express or only a small amount of cell expresses extremely weak green fluorescent protein.It removes after ASV for 24 hours, green fluorescent protein is thin Expression is restored to high level again in born of the same parents.
Embodiment 2：The positioning (SwichON systems) of small-molecule drug regulation and control fusion fluorescin
1. vector construction：
Dsred genes and release GFP genes are expanded from the plasmid that pCMV-IRES-DsRed EXPRESS are synthesized respectively, Add the mitochondrial membrane positioning signal of one section of Tom20 gene before DsRed：Met Val Gly Arg Asn Ser Ala Ile Ala Ala Gly Val Cys Gly Ala Leu Phe Ile Gly Tyr Cys Ile Tyr Phe Asp Arg Lys Arg Arg Ser Asp Pro Asn Phe Lys.NS3/4A protease genes are connected into behind sfGFP, between two genes It is connected into one section of flag sequence label：Asp Tyr Lys Asp Asp Asp Asp Lys.As shown in Figure 4：Several sections of sequences are connected, An amalgamation and expression gene is formed, is inserted into slow virus carrier Pltr carriers.
2. cell transfecting：
It recovers and cultivates 3T3 cells.The day before transfection adds in 5 × 10 in 24 orifice plates5A cell.Change liquid before transfection, one A hole adds in the culture medium without micromolecular inhibitor Asunaprevir (ASV), and a hole adds in the culture medium of the ASV containing 1uM. Transfection reagent is 3000 Reagent of Lipofectamine (Thermo Fisher), and transfection process is：1) two 1.5ml are taken EP is managed, and is separately added into 50uLThen culture medium adds in 3.0uL in a wherein branch pipe3000 reagents add in 2ug DNA plasmids in another branch pipe, respectively mixing.It 2) will be in two EP pipes Liquid is mixed into a pipe, and common 100uL gently blows and beats mixing 15 times, is stored at room temperature 5 minutes.3) 50uL mixtures is taken to be added to respectively In two holes, liquid is changed after 6h.
3. small-molecule drug regulates and controls：
In the present embodiment, using Asunaprevir (ASV) as small molecule regulation reagent, component will be tested on the day of transfection It it is two groups, one group is not added with ASV, the culture medium of the one group of ASV of addition containing 1uM.After transfection for 24 hours, the feelings of observation cell expression fluorescence Condition.
As shown in Figure 5：First row：It is not added with ASV, the fusion protein MTS-DsRed-4A4B-sfGFP-NS3/4A of expression.Egg White enzyme is active, can cut off fusion protein at 4A4B, forms two albumen of MTS-DsRed and sfGFP-NS3/4A.By In mitochondrial positioning signal, MTS-DsRed expression and localizations are on mitochondrial outer membrane, and sfGFP-NS3/4A fusion proteins are then wide It is general to be distributed in cytoplasm.Second row：After adding in ASV, 3T3 cell expressed fusion proteins MTS-DsRed-4A4B-sfGFP- NS3/4A.Since ASV inhibits identification and cutting of the NS3/4A protease to 4A4B, entire fusion protein is enable to be stabilized. Green fluorescence and red fluorescence is all influenced by mitochondria positioning signal and common location is in mitochondrial surface.
As shown in Figure 6：First row：Under conditions of no ASV, green fluorescent protein and red fluorescent protein separate table It reaches.Second row：Add in ASV, for 24 hours after, positioning of the green fluorescent protein in cell is gradually transferred to mitochondrial surface.Such as Fig. 7 It is shown：First row：At the beginning, under conditions of having ASV, green fluorescent protein and red fluorescent protein common location are expressed in line grain Body surface face.Second row：After revocation ASV, 24 small h, red fluorescent protein is still oriented towards mitochondrial surface, but green fluorescent protein Gradually Dispersed precipitate is in entire cytoplasm.
Embodiment 3：Chimeric antigen receptor activity regulation scheme one
In the present embodiment, the antigen behaviour source CD19 of Chimeric antigen receptor (CAR) identification, by the CAR and HCV from degradation Protease fusion is expressed, and protease site 4A4B is located between two albumen.HCV protease inhibitor ASV passes through inhibition Proteinase activity, to regulate and control the expression of CAR.As shown in Figure 8：Left figure：Under conditions of no ASV, NS3/4A cutting 4A4B sequences, CAR is separated with protease, and protease brings Ubiquitin-proteasome system by degron and degrades, and CAR expressed intact and can be exercised It identifies antigen and activates the function of T cell internal signal.At this point, switch is on states.Right figure：Under conditions of having ASV, NS3/4A is suppressed and cannot cut 4A4B sequences, CAR, protease and degron amalgamation and expressions, entire fusion protein quilt Degron brings Ubiquitin-proteasome system degradation into, and CAR is degraded, and T cell can not identify antigen by CAR, it is impossible to be swashed It is living.At this point, switch is in off states.
Vector construction：PCR amplification obtains CAR gene orders in pltr-CAR19 carriers, is connected into behind CAR genes HCV virus protease recognition sequence.It, will in the N34d genes (with NS3/4A sequences and one section of degradation subsequence) of access synthesis Three sections of sequence series connection, form an amalgamation and expression gene, are inserted into slow virus carrier Pltr carriers (as shown in Figure 9)
Embodiment 4：Chimeric antigen receptor activity regulation scheme two
In the present embodiment, the antigen behaviour source CD19 of Chimeric antigen receptor (CAR) identification melts CAR and HCV protease Expression is closed, protease site 4A4B is located between 4-1BB the and CD3zata structural domains of CAR.ASV is passed through by HCV protease To control the structure and function of CAR.
As shown in Figure 10：Left figure：Under conditions of no ASV, NS3/4A cutting 4A4B sequences, the CD3zeta in CAR albumen Structural domain is separated with albumen, can not transmit cell proliferation signals.Even if CAR-T cells can identify tumour antigen, but due to can not Proliferation, it is impossible to effectively kill corresponding tumour cell.At this point, switch is in off states.Right figure：Under conditions of having ASV, NS3/ 4A is suppressed and cannot cut 4A4B sequences, and CAR, protease fusion expression, CAR has complete structure, and it is anti-to exercise identification Former and activation T cell internal signal function.At this point, switch is on states.
Vector construction：It is consistent with the composition of embodiment three in the present embodiment, but 4A4B sequences are not in two fusion proteins Between, and in CAR active site of protein, between 4-1BB and CD3zata structural domains.By the amalgamation and expression gene of structure, it is inserted into slow In viral vectors Pltr carriers (as shown in figure 11)
Embodiment 5：Chimeric antigen receptor activity regulation scheme three
In the present embodiment, the antigen behaviour source CD19 of Chimeric antigen receptor (CAR) identification melts CAR and HCV protease Expression is closed, protease site 4A4B is located at the anti-CD19 scFv of CAR and CD8 transmembrane regions, 4-1BB and CD3zata are tied Between structure domain, 5A5B is between CD8 transmembrane regions and 4-1BB structural domains.The structure of CAR is controlled by ASV by HCV protease And function.
As shown in figure 12：Left figure：Under conditions of no ASV, NS3/4A cutting 4A4B sequences, CAR albumen is cut to four A individual structural domain, causes corresponding T cell, and None- identified antigen can not transmit cell-stimulating and proliferation signal, it is impossible to Kill corresponding tumour cell.At this point, switch is in off states.Right figure：Under conditions of having ASV, NS3/4A is suppressed and cannot 4A4B sequences are cut, CAR, protease fusion expression, CAR has complete structure, and exercises in identification antigen and activation T cell The function of portion's signal.At this point, switch is on states.
Vector construction：As shown in figure 13：It is consistent with example IV principle in the present embodiment, but protease cog region increases Be 3, respectively positioned at scFv and CD8 transmembrane regions, CD8 transmembrane regions and 4-1BB structural domains, 4-1BB and CD3zata structural domains it Between.The amalgamation and expression gene of structure is inserted into slow virus carrier Pltr carriers.
Embodiment 6：In 293 cell upregulation control Chimeric antigen receptors activity
1. vector construction：
By the carrier designed in three schemes of Chimeric antigen receptor activity regulation be named as successively CAR-V1, CAR-V2, CAR-V3.It is connected into one section of sequence label Flag respectively in the N-terminal of three albumen：Asp Tyr Lys Asp Asp Asp Asp Lys is respectively designated as fCAR-V1, fCAR-V2, fCAR-V3.
2. cell transfecting：
It recovers and cultivates 293T cells.The day before transfection adds in 5 × 10 in 12 orifice plates5A cell.Liquid is changed before transfection, One hole adds in the culture medium without micromolecular inhibitor Asunaprevir (ASV), and a hole adds in the culture of the ASV containing 1uM Base.Transfection reagent is 3000 Reagent of Lipofectamine (Thermo Fisher), and transfection process is：1) two are taken 1.5ml EP are managed, and are separately added into 100uLThen culture medium adds in 6.0uL in a wherein branch pipe3000 reagents add in 4ug DNA plasmids in another branch pipe, respectively mixing.It 2) will be in two EP pipes Liquid is mixed into a pipe, and common 200uL gently blows and beats mixing 15 times, is stored at room temperature 5 minutes.3) 100uL mixtures is taken to be added to respectively In two holes, liquid is changed after 6h.
3. small-molecule drug regulates and controls：
In the present embodiment, using Asunaprevir (ASV) as small molecule regulation reagent, component will be tested on the day of transfection It it is two groups, one group is not added with ASV, the culture medium of the one group of ASV of addition containing 1uM.After transfection for 24 hours, culture solution is discarded, cell is added in and splits Liquid is solved, lytic cell collects albumen.Utilize the size of western blot technologies detection label protein containing flag.
As shown in figure 14：Upper figure：FCAR-V1, fCAR-V2, fCAR-V3 structure diagram.Figure below：Western blot are examined Survey flag tag fusion protein sizes.FCAR-V1 groups：First row：When being not added with ASV, the fusion protein F lag-CAR- of expression 4A4B-NS3/4A-Degron.Protease has cutting function, cuts off fusion protein at 4A4B.Then, Degron is mediated NS3/4A protease fast degradations form the Flag-CAR segments of one section of 63kd size.Secondary series：After adding in ASV, 293 cells Similary expressed fusion protein Flag-CAR-4A4B-NS3/4A-Degron.But it adds in ASV and inhibits NS3/4A protease to 4A4B Identification and cutting so that Degron brings entire fusion protein into Ubiquitin-Proteasome Pathway, carries out fast degradation.fCAR- V2 groups：First row：When being not added with ASV, the fusion protein F lag-CAR1-4A4B-CAR2-NS3/4A of expression.Protease, which has, lives Property, fusion protein can be cut off at 4A4B, form two eggs of CAR2-NS3/4A of the Flag-CAR1 and 52kd of size 45kd In vain.The Flag-CAR1 of 45kd is arrived by anti-flag antibody tests.Secondary series：After adding in ASV, 293 cells expression fusion egg White Flag-CAR-4A4B-CAR2-NS3/4A.Since ASV inhibits identification and cutting of the NS3/4A protease to 4A4B, make whole A fusion protein can be stabilized, and the size of anti-flag antibody tests to albumen is 97kd.FCAR-V3 groups：First row：No When adding ASV, the fusion protein F lag-CAR1-4A4B-CAR2-5A5B-CAR3-4A4B-CAR4-NS3/4A of expression.Protease has It is active, fusion protein can be cut off at 4A4B and 5A5B, form CAR2,5kd of Flag-CAR1,7kd of size 33kd Two albumen of CAR4-NS3/4A of CAR3 and 52kd.The Flag-CAR1 of 33kd is arrived by anti-flag antibody tests.Second Row：After adding in ASV, 293 cell expressed fusion protein Flag-CAR1-4A4B-CAR2-5A5B-CAR3-4A4B-CAR4-NS3/ 4A.Since ASV inhibits identification and cutting of the NS3/4A protease to 4A4B, entire fusion protein is enable to be stabilized, anti- The size of flag antibody tests to albumen is 97kd.Due to the possible natural fracture in the hinge positions itself of CAR, so all groups It can detect the albumen of a size 33kd.
Using above-mentioned desirable embodiment according to the present invention as enlightenment, by above-mentioned description, relevant staff is complete Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention Property range is not limited to the content on specification, it is necessary to determine its technical scope according to right.
<110>Tianjin Tian Rui bio tech ltd
<120>A kind of protein function switching system of small-molecule drug control
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Claims (10)

1. a kind of protein function switching system of small-molecule drug control, which is characterized in that switch by operon original paper and control Sub- original paper is formed, control by single virus protease or virus protease with protein degradation is sub merges；It is described Operon is located inside destination protein or between destination protein and protease-degradation minor structure, by protease identification and can One section of connection peptide of cutting is formed；Whole system is used as switch by the inhibitor compound of protease small molecule, regulates and controls purpose The stability of albumen.
2. the protein function switching system of a kind of small-molecule drug control according to claim 1, which is characterized in that described Control and operon merge into an entirety and protease identification and cleavable sequence are added between each original paper.
3. the protein function switching system of a kind of small-molecule drug control according to claim 1, which is characterized in that special Property protease is hepatitis C virus protease (NS3pro/4A).
4. the protein function switching system of a kind of small-molecule drug control according to claim 3, which is characterized in that described Specific proteases are that have more than 70% homologous protease mutant or isomerase with hepatitis C virus protease.
A kind of 5. protein function switching system of small-molecule drug control according to claim 1, which is characterized in that albumen Zymolyte is one of following structure sequences：
TRIF：Pro Ser Ser Thr Pro Cys Ser Ala His Leu；
MAVS：Glu Arg Glu Val Pro Cys His Arg Pro Ser Pro；
3-4A：Asp Leu Glu Val Val Thr Ser Thr Trp Val：
4A4B：Asp Glu Met Glu Glu Cys Ser Gln His Leu；
4B5A：Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp；
5A5B：Glu Asp Val Val Cys Cys Ser Met Ser Tyr.
A kind of 6. protein function switching system of small-molecule drug control according to claim 1, which is characterized in that albumen Enzyme is one of following compounds with molecule inhibitor compounds：
Telaprevir(CAS Number：402957-28-2)；
Boceprevir(CAS Number：394730-60-0)；
Simeprevir(CAS Number：923604-59-5)；
Faldaprevir(CAS Number：801283-95-4)；
Asunaprevir(CAS Number：630420-16-5)；
Furaprevir(CAS Number：1435923-88-8).
A kind of 7. protein function switching system of small-molecule drug control according to claim 1, which is characterized in that purpose Gene expression construct is any one in fluorescin, drug resistant gene, antibody, cell factor and antigen recognition molecule.
A kind of 8. protein function switching system of small-molecule drug control according to claim 1, which is characterized in that degradation Son is NS3/4A unstable regions, ddFKBP unstable regions or ecDHFR unstable regions.
A kind of 9. protein function switching system of small-molecule drug control according to claim 1, which is characterized in that albumen Switching system is expressed in eukaryotic mammalian cell.
10. the nucleic acid molecules of the expression protein function switching system in claim 1, plasmid, cell line and include above-mentioned member The kit that element is assembled.
CN201611223345.9A 2016-12-27 2016-12-27 A kind of protein function switching system of small-molecule drug control Pending CN108239656A (en)

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