CN106309447B - Purposes of the Tonzonium Bromide in the inhibitor that preparation can block the interaction of p97 and Npl4 - Google Patents

Abstract

The present invention relates to field of biotechnology, and in particular to purposes of the Tonzonium Bromide in the inhibitor that preparation can block the interaction of p97 and Npl4.Present invention firstly discovers that Tonzonium Bromide can effectively block the interaction of p97 and Npl4, the IFN β signal path for promoting RIG-I to mediate is to improve viral infection resisting ability and then play the role for the treatment of virus infection, great clinical and market value.

Description

Tonzonium Bromide is in the inhibitor of interaction that preparation can block p97 and Npl4 Purposes

Technical field

The present invention relates to field of biotechnology, and in particular to Tonzonium Bromide can block that p97's and Npl4 is mutual in preparation Purposes in the inhibitor of effect.

Background technique

The antiviral immunity reaction that virus infection can cause body strong, the innate immunity are that body resists virus infection First barrier, by the intracorporal cause of disease identification receptor of machine, including toll sample receptor (TLR), RIG-I sample receptor (RLRs) can be with Identify viral nucleic acid.The signal that the receptor being activated can star downstream transcription factor NF- κ B and interferon regulatory factor IRF turns Approach is led, a series of pro-inflammatory cytokines and I type interferon (type I interferons, type I IFN) are generated.I type The release of interferon can promote the transcription of downstream gene to effectively inhibit the further duplication of virus.Meanwhile I type interferes The secretion of element can also be activated including Dendritic Cells, NK cell, the participation inherent immunity including monocyte and macrophage The cell of response.In addition, the secretion of I type interferon also can directly activate T cell and B cell, and then generate adaptive immunity Responsing reaction.

The accuracy controlling of the innate immunity is for viral infection resisting and prevents the infection of autoimmune disease or body from generating Immune inflammation is crucial.Ubiquitination is the major regulatory mode of the receptor-mediated signal path of RLR, with its representative receptor For RIG-I, conformation change is generated in conjunction with viral RNA activation RIG-I, ubiquitin ligase is recruited, makes RIG-I that K63 chain occur general Elementization activates the transcription of downstream NF- κ B and IRF3, promotes the expression of proinflammatory factor and I type interferon, enhances its antiviral work Property.On the other hand, ubiquitin ligase c-Cbl and RNF125 makes RIG-I that K48 chain ubiquitination occur, and terminates the immune anti-of the access It answers, inhibits its antiviral activity.

P97 belongs to II type ATP enzyme family, it participates in including endoplasmic reticulum and the protein degradation that mitochondria participates in, autophagy, cell Film recombination, DNA are repaired, a series of physiology course such as cell cycle and Sex Determination.P97 and its confactor Npl4, Ufd1, p47, UBXD7 and FAF1 are combined, and are removed substrate from its cellular environment using its activity for separating enzyme, finally Majority enters proteasome and degrades.Existing research shows that p97 and its co-factor Npl4 composition compound participates in endoplasmic reticulum and line A series of physiology courses such as plastochondria degradation pathway, p97-Npl4 compound can promote TNF α to lure by mediating the degradation of I κ B α The NF- κ B signal access led.Recently also studies have found that p97 participates in the virus neutralization that TRIM21 is mediated.

Thonzonium bromide (thonzonium bromide), CAS number 553-08-2, molecular formula: C32H55N4O+.Br- is Single cationic detergent.Be currently known its biological function include for being coated with non-virus carrier, as it is potential, be used for the mankind Gene therapy, safe gene delivery mode；It is acidified yeast cells matter, inhibits the proton pump that ATP is relied on vesicular membrane, is destroyed The function of V-ATPase, yeast cell growth defect caused by causing pH sensitive.

But in the prior art and have no effect of the p97 in virus infection is immune.At the same time, in the prior art, Do not reported Tonzonium Bromide (Thonzonium Bromide, CAS number is 553-08-2) in virus infection is immune yet Effect.

Summary of the invention

It is an object of the invention to open Tonzonium Bromides can block the inhibitor of the interaction of p97 and Npl4 in preparation In purposes.

The nucleotides sequence of p97 gene is listed in the sequence of Serial No. NM_007126.3 in Genebank.

The present invention has found that Tonzonium Bromide can effectively block p97 and confactor Npl4 by extensive and in-depth research Between interaction, inhibit RNF125 mediate RIG-I K48 chain ubiquitination, and then inhibit RIG-I degradation, to promote The IFN β signal path that RIG-I is mediated finally plays the effect for improving viral infection resisting ability.

To achieve the purpose of the present invention and other related purposes, the invention adopts the following technical scheme:

The first aspect of the present invention, the suppression of the interaction of p97 and Npl4 can be blocked in preparation by providing Tonzonium Bromide Purposes in preparation.

Preferably, the inhibitor is able to suppress the K48 chain ubiquitination of the RIG-I of RNF-125 mediation.

Preferably, the inhibitor is able to suppress the proteasome degradation of RIG-I.

Preferably, the IFN β signal path that the inhibitor can promote RIG-I to mediate.

Preferably, the inhibitor can play the work for improving viral infection resisting ability and then playing treatment virus infection With.

Further, the second aspect of the present invention provides Tonzonium Bromide in the pharmaceutical composition of preparation treatment virus infection Purposes in object.

Preferably, the virus infection is picornavirus infection.

It is furthermore preferred that the virus infection is VSV virus infection or SeV virus infection.

Preferably, the Tonzonium Bromide is one of the effective component of pharmaceutical composition of the treatment virus infection.

It is furthermore preferred that the Tonzonium Bromide is unique effective component of the pharmaceutical composition of the treatment virus infection.

Preferably, the Tonzonium Bromide is the IFN β signal path by promoting RIG-I to mediate to improve antiviral sense Dye ability plays the role for the treatment of virus infection in turn.

Preferably, the Tonzonium Bromide is by inhibiting the proteasome degradation of RIG-I to promote RIG-I to mediate to play IFN β signal path effect.

Preferably, the Tonzonium Bromide is by inhibiting RNF-125 to mediate the K48 chain ubiquitination of RIG-I to play suppression The effect of the proteasome degradation of RIG-I processed.

Preferably, the Tonzonium Bromide is to play inhibition by inhibiting the interaction between RNF125 and RIG-I The effect of the K48 chain ubiquitination for the RIG-I that RNF-125 is mediated.

Preferably, the Tonzonium Bromide is to inhibit p97-Npl4 compound by blocking the interaction of p97 and Npl4 Formation, and then inhibit the interaction between RNF125 and RIG-I.

Further, the pharmaceutical composition for treating virus infection also includes pharmaceutically acceptable excipient.

Term "comprising" refers to that " containing " and " are made of ﹍ ", such as the composition of "comprising" X can be made of X completely, or Person can contain the substance except X, such as X-Y.The term as used herein " therapeutically effective amount " refers to therapeutic agent treatment, alleviation or pre- The amount of anti-target disease or situation, or show the detectable amount for treating or preventing effect.The effect for example can passing through Label or antigen levels are learned to detect.Therapeutic effect also includes the mitigation of physical symptoms.For the accurate effective of certain an object Dosage depends on the figure and health status of the object, the therapeutic agent and/or control that the property or degree of illness and selection are given Treat the combination of agent.Therefore, it is useless for preassigning accurate effective quantity.However, for the symptom that Mr. Yu gives, Ke Yiyong Routine experiment determines the effective quantity, and clinician can judge.

The pharmaceutically acceptable excipient (or carrier) refers to the excipient for Therapeutic Administration, itself and its Dosage should not induce the individual for receiving the composition to generate harmful antibody, and not have excessive toxicity after being administered.Pharmaceutically may be used The excipient of receiving generally includes nontoxic solid, semisolid or liquid filler, diluent, lapping or any conventional class The formulation auxiliary of type.Suitable excipient includes but are not limited to water, glucose, glycerol, salt water, ethyl alcohol or combinations thereof.Institute Stating excipient also includes other reagent such as wetting agents or emulsifier, pH buffer or the adjuvant for enhancing preparation effect.Other materials Material such as antioxidant, moisturizer, viscosity stabiliser and similar reagents can be added as needed.Liposome, which is also included within, pharmaceutically may be used In the definition of the excipient of receiving.

In general, pharmaceutical composition can be made to injectable agent, such as liquid solution or suspension；It may also be fabricated which before the injection It is suitble in supplying solution or suspension, the solid form of liquid-carrier.

The invention has the benefit that

Present invention firstly discovers that Tonzonium Bromide can effectively block the interaction of p97 and Npl4, RIG-I is promoted to mediate IFN β signal path to improve viral infection resisting ability and then play the role for the treatment of virus infection, great clinical and city Field value.

Detailed description of the invention

The antiviral response that Fig. 1 .p97 inhibits IFN β to mediate by its atpase activity.(A) it is transfected in HEK293T cell P97 siRNA or control RNA, after SeV stimulates 12h, IFN β (left side), PRDIII-I (in) and ISRE (right side) reporter gene Transcriptional activity.(B) in the cell that p97 is knocked out, after SeV stimulation, IFNB (left side), RANTES (in) and ISG56 (right side) Transcriptional level.(C-D) in the cell that p97 is knocked out, poly (I:C) reports base after poly (dA:dT), SeV or VSV-GFP processing Because of the result of (C) and Q-PCR (D).(E-F) glimmering after the MEF cell infection VSV-GFP virus for having transfected p97 siRNA Light phase contrast microscope detects (E) and cell streaming experimental result (F).(G-H) after p97 knocks out cell infection SeV, with p97 or Its mutant carries out rescue experiment, detects the transcriptional level (H) of IFN β transcriptional activation (G) and IFNB.N.c., indicate control siRNA；Scr indicates control shRNA；WT, wild type；PH differs micro- detection；FL, fluorescence microscopy detect (similarly hereinafter).Data Analysis uses: standard error (standard deviation of independent parallel experiment three times), variance analysis and t-test.N.s., become without significant Change；* P < 0.05 is represented；* represents P < 0.01；* * represents P < 0.001.

The antiviral response that Fig. 2 .Npl4 cooperates with p97 that IFN β is inhibited to mediate.(A-B) the cell sense of different siRNA has been transfected The transcriptional level (B) of transcriptional activity (A) IFNB of IFN β promoter after dye SeV.(C-D) the thin of various dose Npl4 has been transfected Born of the same parents infect SeV after IFN β (left side) and the reporter gene of ISRE (right side) promoter activity (C) and IFNB (left side) and The mRNA transcriptional level of ISG56 (right side).(E-F) in the cell that Npl4 is knocked out, after SeV stimulation, reporter gene (E) and Q-PCR (F) detection.(G) the MEF cell that Npl4 is knocked out, the rescue experiment after infecting VSV-GFP.(H) the cell transfer of p97 is knocked out After contaminating different plasmids, IFN β transcriptional activity and IFNB transcriptional level.

Fig. 3 .p97-Npl4 composite structure and mutation analysis.(A) the structural domain schematic diagram of Npl4 and p97.(B) drosophila The structure of Ter94-Npl4 compound.Residue in interaction interface is highlighted with ball-bar model, expression and fruit in bracket The amino acid of the corresponding source of people in the fly site.(C) critical sites of p97 (left side)-Npl4 (right side) interaction.(D) it has The Npl4pulldown of Strep label different p97 mutant.(E) p97pulldown with His label different Npl4 Mutant.(F) co-immunoprecipitation of Npl4 and p97 or p973A mutant.(G) p97 and Npl4 or Npl44A mutant is exempted from Epidemic disease co-precipitation.(H-I) transcriptional activity (H) and IFNB transcriptional level (I) of IFN β in the cell of different plasmids are transfected.3A generation Table p97-F52A/D55A/Y110A mutant；4A represents Npl4-I6A/R8A/R17A/R50A mutant.

Fig. 4 .p97-Npl4 compound promotes RIG-I to pass through proteasome Inhibition of degradation RIG-I signal path.(A)poly (I:C) stimulation p97 knocks out different time after cell, analyzes RIG-I, MAVS, IRF3 and IRF3 phosphorus by immunoblot experiment Acidification.(B) poly (I:C) stimulation is overexpressed the cell different time of Npl4, analyzes protein expression water by immunoblot experiment It is flat.(C) the cell different time that poly (I:C) stimulates silencing Npl4 to express analyzes RIG-I by immunoblot experiment, MAVS, p-TBK1, TBK1, IKK ε, IRF3 and IRF3 phosphorylation level.(D) MG132, which is handled, crosses table in the cell that p97 is knocked out Up to after p97, the protein level of RIG-I.(E) MG132 handle cell after be overexpressed Npl4 after RIG-I protein level.(F-G) After knocking out the cell SeV stimulation that p97 (F) knocks out Npl4 (G), the ubiquitination level of RIG-I.(H) it knocks out and is transfected in the cell of p97 After p97 wild type and 3A mutant, the ubiquitination level of RIG-I.(I) Npl4 wild type and 4A mutant have been transfected In cell, the ubiquitination level of RIG-I.

It is necessary that Fig. 5 .RNF125, which inhibits RIG-I signal path for p97-Npl4 compound,.(A) HEK293T cell Middle transfection control group knocks out RNF125, knocks out the Npl4 of c-Cbl or various dose, after SeV stimulation, IFN β luciferase report Accuse the activity of gene.(B) different RIG-I CARD structural domain and control are transfected in cell, knock out RNF125 or knock out c- After the Cbl and Npl4 of various dose, the activity of IFN β luciferase reporter gene.(C) in the cell for knocking out RNF125, turn In the case where contaminating or not transfecting Npl4, the protein level of RIG-I.(D) cell of cotransfection Npl4 and RNF125, SeV stimulation Afterwards, the ubiquitination level of RIG-I.(E) after cell transfecting Npl4, RNF125 of SeV infection.(F) cotransfection RNF125 and The mass spectral analysis in RIG-I ubiquitination site after HA-K48Ub.(G) cotransfection or respectively transfect Npl4, RNF125 cell in, transfection After wild type or mutant RIG-I, the immunoblotting assay of RIG-I.(H) in the cell of cotransfection Npl4 and RNF125, transfection After wild type or mutant RIG-I, the ubiquitination level of RIG-I.(I) in the cell of cotransfection Npl4 and RNF125, transfection is wild After raw type or mutant RIG-I, IFN β transcriptional activity.(J) knock out Npl4 and RNF125 cell in, transfect RIG-I or its After mutant, the fluorescence microscope result of VSV-GFP virus infected cell.

Fig. 6 .p97-Npl4 compound and RIG-I and RNF125 interact.(A) SeV stimulation and non-SeV stimulate situation Under, intracellular Npl4 and RIG-I co-immunoprecipitation.(B) in the case of SeV stimulation and non-SeV are stimulated, Npl4 and the total of RIG-I are determined Position.(C) interaction of external pulldown experimental verification Npl4 and RIG-I.(D-E) co-immunoprecipitation experiment detection Npl4 with The specific structure domain of RIG-I interaction.(F) phase of the CARD structural domain of external pulldown experiment detection Npl4 and RIG-I Interaction.(G) in the case of SeV stimulation and non-SeV are stimulated, the co-immunoprecipitation experiment of intracellular p97 and RNF-125.(H) body The interaction of outer pulldown experiment detection p97 and RNF125.(I) external pulldown experiment detection p97-Npl4 is compound Influence of the object to RIG-I and RNF125 interaction.

Fig. 7 .pulldown experimental verification small molecule compound destroys p97-Npl4 interaction.

Specific embodiment

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention.

The nucleotide sequence (NM_007126.3) and its amino acid sequence (NP_ of people's p97 gene can be obtained from Genebank 009057.1).Homologous gene herein refers to ortholog, originates from the one of a common ancestral gene in different plant species A little homologous genes, these genes generally remain identical or similar function.According to the website NCBI BLAST's as a result, people p97 Homologous gene includes mouse VCP gene (NM_009503.4) and drosophila TEAR94 gene (NM_001103780.2).

Alternatively, can " homology " Lai Dingyi the application homologous gene." homology " refers to two polynucleotides or two Percentage similarity between polypeptide portion.Two DNA or two polypeptide sequences in definite molecular length, when sequence is shown Have at least about 50%, preferably at least about 75%, more preferably at least about 80-85%, it is especially good be at least about 90%, it is best at least About 95-98% sequence similarity when, each other " substantially homologous ".As described herein, substantially it is homologous also refer to specific DNA or The identical sequence of polypeptide sequence.

Basic experiment method:

1. the clone of target gene: for required target gene design primer, with the method for PCR by the target gene from It clones and in the cDNA of plasmid comprising this target gene or Hela cell, there are two different for target gene fragment both sides band Restriction enzyme site.Whether the size with the method detection PCR product of agarose gel electrophoresis is in the same size with required target gene, And the segment is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit.It is added and PCR product both sides in glue recovery product The corresponding enzyme of restriction enzyme site and suitable buffer carry out double digestion, 37 DEG C of overnight incubations.Meanwhile with the double enzymes of identical enzyme Cut the segment carrier to be connected.The segment after digestion is recycled using common DNA product purification kit, utilizes fine jade Sepharose DNA QIAquick Gel Extraction Kit recycles the carrier after digestion, has then been connect segment with carrier with T4 ligase Come, and the connection product is converted into DH5 α.Using resistance screening, the clone of verifying and sequencing acquisition target gene.

2. the rite-directed mutagenesis of plasmid: designing pair of primers near mutational site, there is 15-20bp at the 5 ' ends of this two primers Reverse complemental region, mutational site should be located at this reverse complemental region centre.The size in the incomplementarity region at 3 ' ends is at least For 10bp.Using this pair of primers, plasmid is carried out by Inverse PCR amplification by the method for PCR.It is purified using common DNA product Kit recycles the product of PCR, and DpnI, 37 DEG C of incubation 2h, by the plasmid template of methylation are added in recovery product Removal.Then, the sample after DpnI being handled directly converts DH5 α.Gram of target gene is obtained using resistance screening and sequencing It is grand.

3. the expression and purification of albumen: prokaryotic expression plasmid being taken to convert BL21,37 DEG C of culture 12h.It is first inoculated with, that is, is chosen It is cloned into the LB culture medium that 7.5ml contains antibiotic, in 37 DEG C, 220rpm shake culture 10-12h.Then expand culture again, Above-mentioned 7.5ml bacterium solution is all added in the antibiotic TB culture medium of 750ml, 37 DEG C, 220rpm shake culture to OD600 For 0.6-0.8 (about 3-4h).At this point, shaking table temperature is adjusted to 16 DEG C, after bacterium solution is cooled to 16 DEG C completely, according to 1:2000 Ratio be added IPTG (final concentration of 0.4mM), continue shake culture 16-18h.Finally use the high speed refrigerated centrifuge of Beckman Machine collects thallus, i.e., 4 DEG C, 6000rpm is centrifuged 10min, abandons supernatant to the greatest extent, protein purification or preservation are carried out after thallus is weighed In -80 DEG C.

(1) Ni column affinity chromatography:

Lysis buffer:20mM HEPES pH 7.0,500mM NaCl,20mM imidazole,1mM DTT；

Wash buffer:20mM HEPES pH 7.0,500mM NaCl,40mM imidazole,1mM DTT；

Elution buffer:20mM HEPES pH 7.0,500mM NaCl,500mM imidazole,1mM DTT；

It takes suitable beads to pour into chromatographic column, rinses beads with a large amount of ddH2O, generally (CV is represented for 10CV × 5 Column volume, similarly hereinafter).It is balanced with Lysis buffer: 10CV × 5.It is obtained after the beads balanced is moved into bacterial cell disruption Supernatant in, in Cool Room 4 DEG C Mix 1-2h.500g, 4 DEG C of centrifugation 2min collect supernatant and flow through liquid.50ml Wash is added Buffer, 500g, 4 DEG C of centrifugation 2min collect supernatant wash 1.It is repeated twice, the supernatant of collection is respectively 2 He of wash wash 3.Beads is poured into chromatographic column, continues to be washed once with Wash buffer.It is eluted with Elution buffer: 0.5CV × 10, eluent is collected respectively with EP pipe.SDS-PAGE electroresis appraisal.

(2) MBP column affinity chromatography:

Lysis buffer:20mM HEPES pH 7.0,500mM NaCl,1mM DTT；

Wash buffer:20mM HEPES pH 7.0,500mM NaCl,1mM DTT；

Elution buffer:20mM HEPES pH 7.0,500mM NaCl,10mM amylose,1mM DTT；

It takes suitable beads to pour into chromatographic column, rinses beads with a large amount of ddH2O, generally (CV is represented for 10CV × 5 Column volume, similarly hereinafter).It is balanced with Lysis buffer: 10CV × 5.It is obtained after the beads balanced is moved into bacterial cell disruption Supernatant in, in Cool Room 4 DEG C Mix 1-2h.500g, 4 DEG C of centrifugation 2min collect supernatant and flow through liquid.50ml Wash is added Buffer, 500g, 4 DEG C of centrifugation 2min, are repeated twice.Beads is poured into chromatographic column, continues to be washed once with Wash buffer. Eluted with Elution buffer: eluent is collected with EP pipe in 0.5CV × 10 respectively.SDS-PAGE electroresis appraisal.

(3) Strep Tactin column affinity chromatography:

Lysis buffer/Wash buffer:100mM Tris pH 8.0,150mM NaCl,10mMβ-ME；

Elution buffer:100mM Tris pH 8.0,150mM NaCl,2.5mM desthiobiotin,10mM β-ME；

Suitable beads is taken to pour into chromatography.It is balanced with Lysis buffer: 10CV × 5.By broken supernatant It pours into the chromatographic column equipped with beads, coutroi velocity is 1 drop/2s, and collection flows through liquid, it is primary to repeat loading.Use Wash Buffer is rinsed: washes is collected in 10CV × 5.Eluted with Elution buffer: 0.5CV × 6 are collected respectively with EP pipe and are washed De- liquid.SDS-PAGE electroresis appraisal.

(4) gel permeation chromatography (Superdex 75/200):

Buffer:20mM HEPES pH 7.0,100mM NaCl,1mM DTT；

DdH2O rinses 2CV: flow velocity: 1ml/min, upper pressure limit: 0.3MPa (similarly hereinafter).Buffer balances 2CV.Sample from The heart: 12000rpm, 4 DEG C of centrifugation 40min.Rinse loading ring: ddH2O washes 5 volumes, and Buffer washes 5 volumes.Loading: with note Emitter loading has to drain the bubble in syringe.After Buffer elutes about 40min, start to pay attention to observation ultraviolet absorption peak Appearance, once appearance immediately begins to collect.After sample collection, continue to be rinsed with Binding buffer until ultraviolet suction Receive baseline level.DdH2O rinses 2CV.20% ethyl alcohol suspends after rinsing 2CV, closes instrument.

(5) removal (TEV digestion) of affinity tag:

By the sample desalination after Ni column affinity chromatography into Lysis buffer.The concentration of albumen in eluent is measured, And go out the total amount of destination protein according to volume estimation.With the mass ratio of TEV and destination protein for 1:15 ratio in eluates It is added enough TEV, room temperature digestion 2 hours.Sample after digestion is hung back into Ni column, collects the liquid that flows through of outflow, removing is cut Under His label and addition TEV enzyme.

4. the parsing of crystal structure: using Single wavelength anomalous scattering method (Single wavelength anomalous Dispersion, SAD) structure of Ter94-N/Npl4-UBD compound is parsed.Utilize the anomalous scattering wavelength in seleniumIt is lower to collect obtained diffraction data, diffraction phase is obtained by the parsing of AutoSol program in Phenix software package Position.Then, the foundation that model is carried out using COOT software, utilizes the Refmac5 in the Refinement and CCP4 in Phenix Carry out the amendment of structure.

5.Pull-down experiment: the desired amount of affinity chromatography beads is taken, is washed 3 times with appropriate pull-down buffer. Two are mixed according to the ratio that molar ratio is 1:1 and is used for the albumen of pull-down, and will be mixed with pull-down buffer Protein solution final volume constant volume to 500 μ l.The pretreated affinity chromatography of 20 μ l is added in this mixed liquid of protein Beads, slowly rotation is incubated for 2h on the rotary mixer of Cool Room 4 DEG C.It is centrifuged 2min in 4 DEG C of speed with 500g, it will be affine Chromatography beads is centrifuged to EP tube bottom, abandons supernatant to the greatest extent.1ml pull-down buffer is added and cleans beads, at 4 DEG C with 500g Speed be centrifuged 2min, abandon to the greatest extent supernatant.Repeated washing is twice.40 μ l elution buffer are added to be eluted, are incorporated into Albumen wash-out on beads is got off.5 × SDS sample-loading buffer of 10 μ l, 100 DEG C of denaturation 10min are added in eluates.Benefit The combination situation of two albumen is analyzed with SDS-PAGE.

Pull-down buffer:

His pull-down buffer:100mM Tris pH 8.0,150mM NaCl, 40mM imidazole, 10mM β-ME；

His elution buffer:100mM Tris pH 8.0,150mM NaCl, 500mM imidazole, 10mM β-ME；

Strep pull-down buffer:100mM Tris pH 8.0,150mM NaCl, 0.1%Trition X- 100；

Strep elution buffer:100mM Tris pH 8.0,150mM NaCl, 2.5mM Desthiobiotin, 0.1%Trition X-100.

6. luciferase reporter gene is tested: luciferase reporter gene experiment is first to examine using fluorescein as substrate Survey the activity of firefly luciferase, after the activity of renilla luciferase is detected as substrate using coelenteron fluorescein, and rear The continuous substrate that renilla luciferase is added joined the substance for inhibiting firefly luciferase catalysis luciferin simultaneously, make subsequent Detection is only measured to the activity of renilla luciferase, realizes the detection of luciferase reporter gene.Firefly luciferase Luciferin can be catalyzed and be oxidized to oxyluciferin, during luciferin oxidation, bioluminescence can be issued.Sea pansy Luciferase can be catalyzed coelenteron fluorescein and be oxidized to coelenteramide, can also send out during the oxidation of coelenteron fluorescein Bioluminescence out.It may then pass through fluor tester and be also referred to as Chemiluminescence Apparatus or liquid flashing determining instrument and measure above-mentioned luciferase and urge The bioluminescence discharged in the oxidation process of change.Firefly luciferase is catalyzed the luminous most strong emission wavelength of luciferin 560nm.The most strong emission wavelength that renilla luciferase is catalyzed coelenteron fluorescein luminescence is 465nm.Pass through luciferase and its bottom This bioluminescence system of object, can extremely sensitive, the efficient expression for detecting gene.Usually interested genetic transcription Controlling element is cloned in the upstream or other places appropriate of firefly luciferase, is built into reporter plasmid, then turns Cell is contaminated, lytic cell after appropriate stimulation or processing measures uciferase activity.Thorn is judged by the height of uciferase activity Swash the influence of front and back or different stimulated to interested controlling element.Renilla luciferase is more used as the interior of transfection efficiency Ginseng, to eliminate the difference of cell quantity and transfection efficiency.

Experimental procedure: culture HEK293FT cell (or other aim cells), and be inoculated in 24 orifice plates, grow 12-24 Hour (80% convergence degree).It is glimmering with the luciferase reporter plasmid and sea pansy of lipo2000 transfection expression firefly luciferin Light element enzyme internal reference reporter plasmid and required destination gene expression plasmid.After 24-48 hours, cell cracking is added Liquid (5X lysis buffer is diluted with water to 1X, is purchased from promega): the every hole of 24 orifice plates adds 120 μ l, and the every hole of 48 orifice plates adds 60 μl.The plate is put into -80 DEG C of jelly 1h.Plate is taken out from -80 DEG C, after room temperature is melted, cell pyrolysis liquid is sucked out, is transferred to EP pipe In.Every pipe lysate takes 5 μ l to be added in 384 orifice plates.The reaction substrate of 10 μ l firefly luciferases is added, surveys reporter gene Fluorescent value.The reaction substrate of 10 μ l is added, surveys the fluorescent value of renilla luciferase.Reporter gene and sea pansy fluorescein fluorescence value Ratio be each sample relative fluorescence signal value.

In the experiment that detection compound influences intracellular reporter gene expression, Flag is transfected in HEK293FT cell The RIG-I (residues 1-238) of label and the reporter gene of IFN β or ISRE, luciferase reporter gene.12 hours Afterwards, vitellophag is seeded in 384 orifice plates with 10000 cells/every hole density, and the cell culture medium of 50 μ l is added.7 is small Shi Hou, the compound that addition AlphaScreen experiment screening obtains, final concentration of 10 μM of compound.After 37 DEG C of culture 15h, Detect uciferase activity.

7. immunoblotting: preparing protein sample according to requirement of experiment, 100 DEG C are denaturalized 10 minutes, and 13200rpm is centrifuged 2 points Clock takes the supernatant of equivalent to be added in the loading hole of SDS-PAGE glue.Protein sample voltage when being concentrated in glue is 80V, is being separated Voltage is 120V when in glue.After electrophoresis, remove gel, membrane-transferring device be installed in the following order: (cathode), filter paper, gel, Nitrocellulose filter, filter paper, (anode).Membrane-transferring device is put into Cool Room 4 DEG C, 100V constant pressure transfers 1h.After the completion of transferring film, take out Film is immersed in 5% skim milk with TBST buffer preparation, is incubated on shaking table at room temperature by nitrocellulose filter 1h.Film, 5min × 3 time are washed with TBST buffer solution.

It is added and uses the diluted primary antibody of 5%BSA solution to scale, be incubated overnight on the shaking table of Cool Room 4 DEG C.Use TBST Buffer solution washes film, 10min × 3 time.It is added to scale with the diluted secondary antibody of 5% milk, is incubated on shaking table at room temperature 40-60min.Film, 10min × 3 time are washed with TBST buffer solution.Chromogenic substrate is covered on nitrocellulose filter, room temperature is aobvious Color 2 minutes.It is shot with LAS4000 cold light/bioluminescence image analyzers.

The preparation of required reagent: it 5%BSA solution: weighs 5g BSA powder and is dissolved in 1 × PBS solution of 100ml, add 0.02% Sodium azide is stored in 4 DEG C.10 × Western blot transferring film buffer: weighing 30.3g Tris alkali and 144 g are sweet 300ml methanol is added in propylhomoserin, then with deionized water constant volume to 1L.

The extracting of 8.RNA: the cell after transfection is discarded into culture medium, and is cleaned cell one time with the PBS of pre-cooling.Six holes 500 μ l Trizol Reagent are added in each hole of plate, and room temperature is handled 5 minutes.Cell pyrolysis liquid is transferred to 1.5ml In Eppendorf pipe.The chloroform of 100 μ l is added in every part of sample, and vortex low speed stands 5 minutes after mixing.4 DEG C, 12000g from The heart 15 minutes, the upper strata aqueous phase of 240 μ l was shifted into new Eppendorf pipe.The isopropanol of 240 μ l, top is added in every part of sample It mixes, is placed at room temperature for 10 minutes.4 DEG C, 12000g is centrifuged 15 minutes, is discarded supernatant, and leaves the RNA precipitate of white.Every part of sample 75% ethyl alcohol of 500 μ l is added in product, is mixed by inversion.4 DEG C, 12000g is centrifuged 5 minutes, abandons supernatant to the greatest extent.Room temperature is dried.It is added appropriate The processed deionized water of DEPC sufficiently dissolves.The concentration of extracted RNA is measured with NanoDrop ND1000,260nm's The ratio of the light absorption value of light absorption value and 280nm should maintain between 1.9-2.0.By extracted RNA be used for reverse transcription or directly Freeze to -80 DEG C and saves.

The preparation of required reagent: phosphate buffered saline solution (PBS): 800ml distilled water dissolves 0.2g KCl, 8g NaCl, 0.24g KH2PO4 and 1.44g Na2HPO4.The pH value for adjusting solution with HCl is settled to 1L to 7.4.High pressure steam sterilization or Filtration sterilization.

9. co-immunoprecipitation: the cell after transfection being discarded culture medium, and is cleaned cell one time with the PBS of pre-cooling.Add Enter suitable cell lysis buffer solution RIPA (containing protease inhibitors), cracks 30min on ice, by cell pyrolysis liquid in 4 DEG C, most Supernatant is taken after big revolving speed centrifugation 30min.The desired amount of protein A/G sepharose 4B is taken, is washed 3 times with appropriate lysis buffer. It takes a small amount of lysate to analyze in case of Western blot, the pretreated protein A/G fine jade of 20 μ l is added in remaining lysate Lipolysaccharide pearl and the 1 corresponding antibody of μ g, slowly rotation is incubated overnight on the rotator of Cool Room 4 DEG C.Immune precipitation is completed Afterwards, it is centrifuged 1min in 4 DEG C of speed with 7000rpm, sepharose 4B is centrifuged to EP tube bottom.Supernatant is carefully sucked, agarose Pearl is washed 2 times with 300 μ l lysis buffers, then is washed 2 times with 300 μ l PBS.It is eventually adding 2 × SDS sample-loading buffer of 20 μ l, 100 DEG C are boiled 10 minutes.The albumen in conjunction with antibody is analyzed using SDS-PAGE and Western blot

The preparation of required reagent: RIPA buffer:150mM NaCl, 100mM Tris pH 8.0,1%TritonX- 100,5 mM EDTA,10mM NaF.

10. cell culture: HEK293, HEK293T, MEF cell culture are in DMEM (Hyclone) culture solution, culture solution 10% serum of middle addition, 100ug/ml penicillin, 100 μ g/ml streptomysins.Cell is cultivated in 37 DEG C, and gas concentration lwevel is 5%.

11. real-time quantitative fluorescence PCR: real-time quantitative fluorescence PCR is real using Applied Biosystems company two-step method When PCR (Realtime-PCR) system, detect opposite CT value.Using quantitative fluorescence PCR premixed liquid, (Toyobo company is providedGreen Realtime PCR reagent) configuration reaction system detects and the expression of quantitative objective gene, GAPDH is as internal reference.

12. data are analyzed: being analyzed using SAS data software analysis bag (9.1.3) data, statistical data is averaged Number ± standard deviation.Single factor analysis of variance (ANOVA) and Student ' s t-test are for analyzing continuous variable.Confidence area Between be P < 0.05.

1 p97 negative regulation I type interferon signal path of embodiment is living dependent on its ATP enzyme

1. experiment purpose: determining whether p97 participates in the signal path of I type interferon response and antiviral immunity.

2. experimental method:

(1) cell infection is tested: people HEK293, HEK293T, mouse MEF cell is purchased from Chinese Academy of Sciences Shanghai life section Learn icm cell library.Cell culture processes are as described in basic experiment method 10.Sendai virus sendai virus (SeV) with And vesicular stomatitis virus (GFP-VSV) of the GFP-Vesicular stomatitis virus with green fluorescence is big by Wuhan It learns professor Shu Hongbing to grant, SeV and VSV-GFP are with commercially available, and VSV-GFP remodeling method is the same as Construction and expression of high titer pseudotyped GFP-retroviruses by cotransfection of VSV-G geneJournal of Shanghai Jiaotong University(Medical Science)》2007-07。 Poly (I:C), poly (dA:dT) are purchased from Sigma (St.Louis, MO), and poly (I:C) handles cell using rotaring transfecting mode, Poly (dA:dT), which is directly added into cell culture medium, handles cell.

(2) IFN β, IFN β control domain PRDIII and PRDI (PRDIII-I), the reporter gene of ISRE is by Chinese section Institute's Shanghai biochemistry is granted with cell institute professor Wang Chen, and above-mentioned Reporter gene vector is inserted into corresponding reporter gene member by commercial vector Part is transformed and is obtained, and carrier related information is detailed in TRIM21Is Essential to Sustain IFN Regulatory Factor 3 Activation during Antiviral Response2009vol.182no.63782-3792。

(3) detection of IFNB, RANTES and the ISG56 transcriptional level in cell: Cell extraction total serum IgE method is for example basic Described in experimental method 8, AMV reverse transcriptase synthesizes the first chain, and using it as template, with corresponding under the action of DNA taq enzyme Primer carries out PCR amplification；Reverse transcription system is shown in Table 1, and reverse transcription reaction program is shown in Table 2, and cell sample amplification PCR primer is shown in Table 3, Realtime-PCR reaction systems are shown in Table 4, Realtime-PCR amplification program and are shown in Table 5.

1 reverse transcription reaction system of table

2 reverse transcription process of table

37℃

15min

50℃

5min

95℃

5min

4℃

20min

3 primer sequence of table

4 Realtime-PCR reaction system of table

5 PCR reaction system program setting of table

(4) siRNA knocks out intracellular p97: for the siRNA and control siRNA of the code area p97 and noncoding region by upper The synthesis of Hai Jima Pharmaceutical Technology Inc., siRNA sequence are as shown in table 6.

6 siRNA sequence of table

(5) Rescue experimental verification p97 function: p97 wild type and p97QQ mutant are by Chinese Academy of Sciences's Shanghai biochemistry and cell Institute professor Zhao Yun grants, and above-mentioned carrier is inserted into p97 wild type or p97QQ mutant segment by commercial vector and is obtained, specific carrier Information is detailed in Ter94ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial degradation.Dev Cell.2013Jun 24；25(6):636-44.

3. experimental result and analysis:

Transfect IFN β in HEK293 cell, the Regulatory domain PRDIII of IFN β, the Regulatory domain PRDI of IFN β, The reporter gene of ISRE, and the siRNA for the code area p97.SeV infection cell is used later, and inducing type I interferon signal is logical Road.After siRNA knocks out p97, IFN β, the obvious up-regulation (Figure 1A) of the activity of PRDIII-I and ISRE reporter gene.Equally, The mRNA level in-site of IFNB, RANTES and ISG56 constantly raise (Figure 1B) with the increase of SeV infection time.In people 293T In cell and mouse MEF cell, with poly (I:C), poly (dA:dT), VSV-GFP inducing type I interferon signal path, obtain Obtained similar result (Fig. 1 C-1D).These results suggest that knocking out p97 can be enhanced the signal path of I type interferon.

MEF cell is infected with the VSV-GFP that infection multiplicity is 2, the ability for knocking out the cell anti-virus infection of p97 is stronger, GFP is positive, i.e., the cell of virus infection is fewer than control group (Fig. 1 E).By cell flow cytometer detection, in the cell that p97 is knocked out, GFP is positive, i.e. the cell proportion of virus infection only accounts for 9%, and in the cell of control group, the cell proportion of virus infection is up to 93% (Fig. 1 F).In having transfected the cell for p97 noncoding region siRNA, the function of rescue experimental verification p97 is carried out. The activity for the IFN β reporter gene that the p97 for being overexpressed wild type inhibits Sev to induce and the transcription of IFNB, and it is made to lack ATP The p97QQ mutant of enzymatic activity does not have above-mentioned function (Fig. 1 G-1H).This shows p97 negative regulation antiviral immunity, the function according to Lai Yuqi atpase activity.

2 Npl4 of embodiment cooperates with p97 to regulate and control Immune responses of the antivirus

1. experiment purpose: p97 participates in the regulation of different signal paths, the present embodiment in conjunction with different confactors Purpose is the confactor determining in the regulation of Immune responses of the antivirus, p97 is combined.

2. experimental method:

(1) siRNA knocks out intracellular p97, Npl4, Ufd1, p47, FAF1: above-mentioned siRNA by Shanghai Ji Ma pharmaceutical technology Co., Ltd's synthesis, p97siRNA sequence is as shown in table 6, Npl4, Ufd1, p47, and FAF1 is as shown in table 7.

7 siRNA sequence of table

(2) detection of the transcriptional level of the activity and gene of reporter gene is the same as embodiment 1.

(3) the Npl4 vector construction being overexpressed in cell

In the method and system extracting cell total rna in embodiment 1, and reverse transcription is cDNA, in Takara high-fidelity DNA Polymerase PrimeSTARUnder the action of with corresponding primer carry out PCR amplification；PCR primer is shown in Table 8, PCR reaction system 9 are shown in Table, PCR amplification program is shown in Table 10.

8 primer sequence of table

9 PCR reaction system of table

10 PCR amplification program of table

The segment obtained is expanded, by 1% agarose gel electrophoresis, confirms that size is identical as purpose band, cuts glue, make (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP209) is recycled with Ago-Gel QIAquick Gel Extraction Kit.Npl4 segment uses Restriction enzyme NcoI and SalI (thermo scientific) carry out double digestion, and endonuclease reaction system is as shown in table 11, instead It should be carried out 2 hours at 37 degree.

11 fragment endonuclease reaction system of table

After digestion, by common product purification kit (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP204), Purifying obtains the segment after digestion.Segment (HindIII/NheI double digestion) and carrier pCDNA3 (the bis- enzymes of HindIII/NheI Cut), it is attached, shown in linked system table 12, connection reaction carries out 2 hours at (25 DEG C) of room temperature.Carrier double enzyme digestion reaction body As shown in table 13, reaction carries out 4 hours at 37 DEG C for system.It is obtained after the reaction was completed using the purifying of Tiangeng common product purification kit Carrier after digestion.Product after connection converts DH5 α competent cell, by resistance screening, the positive colony of acquisition, and surveys Sequence verifying.

12 coupled reaction system of table

13 carrier endonuclease reaction system of table

3. experimental result and analysis:

Different p97 confactors is knocked out in HEK293T cell, including after Npl4, Ufd1, p47 and FAF1, detect The activity of IFN β reporter gene and the transcriptional level of IFNB after SeV infection.Knock out Npl4 after, the transcriptional activity of IFN β and The transcriptional level of IFNB obviously raises, and can also raise the I type interferon that SeV infection mediates with the Npl4 Ufd1 for forming compound Signal path.Thus, in antiviral immunity signal path, Npl4-Ufd1 is the confactor (Fig. 2A -2B) of p97.We The Npl4 of various dose is transfected in the cell for having infected SeV, the results showed that the IFN β that Npl4 can inhibit SeV to induce swashs Living and ISRE reporter gene activity, and its inhibiting effect is presented metering and relies on effect (Fig. 2 C).Equally, it is overexpressed After Npl4, the mRNA level in-site of IFNB and ISG56 obviously lowers (Fig. 2 D) after SeV stimulation cell.It is knocked out with siRNA endogenous Npl4 can activate IFN β, the reporter gene (Fig. 2 E) of PRDIII-I and ISRE significantly.Equally, the cell knocked out in Npl4 In, the transcriptional level of IFNB, RANTES and ISG56 constantly raises (Fig. 2 F) with SeV stimulation time.Rescue tests table Bright, the ratio of cell infection VSV can be reduced by knocking out endogenous Npl4 with the siRNA for Npl4 noncoding region, be then overexpressed Npl4 can make cell more easy infection VSV (Fig. 2 G).After p97 in cell is knocked out, the p97 of cotransfection Npl4 and wild type or Npl4 and saltant type p97QQ, the only cell of cotransfection Npl4 and p97 wild type, the work of the IFN β reporter gene of SeV induction Property and the transcription of IFNB be suppressed, the corotation of individual Npl4 or Npl4 and p97QQ mutant cannot inhibit IFN β report Accuse the activity of gene and the transcription (Fig. 2 H) of IFNB.The above result shows that Npl4 participates in the interference of I type as the confactor of p97 The negative regulation of plain signal path and antiviral immunity reaction.

The structure elucidation of 3 p97-Npl4 compound of embodiment

1. experiment purpose: compound with p97, Npl4 albumen assembling that purification obtains by the method for structure biology Object parses its structure, studies protein-interacting mode.

2. experimental method:

(1) expression vector establishment of composite structure parsing:

HT- represents pET28a-HisTEV carrier, is the carrier that laboratory voluntarily constructs, with commercial vectors pET28a (Merck) it is transformed and obtains.Plasmid information is referring to (Shi, Z., et al.Structure of the MST4in complex With MO25provides insights into its activation mechanism.Structure 21,449- 461,2013)

Drosophila HT-Ter94-N (20-186), drosophila HT-Npl4-UBD (1-77) is respectively with drosophila Ter94 overall length, cell The cDNA of reverse transcription is template, in Takara high-fidelity DNA polymerase PrimeSTARUnder the action of with corresponding primer Carry out PCR amplification；PCR primer is shown in Table 14, and carrier construction method and system are the same as embodiment 2.The carrier of connection is above-mentioned PET28a-HisTEV, restriction enzyme site NcoI/XholI.Drosophila Ter94 overall length is by Chinese Academy of Sciences's Shanghai biochemistry and cell institute Professor Zhao Yun grants, and above-mentioned carrier is inserted into Ter94 segment by commercial vector and is obtained, and specific carrier information is detailed in Ter94 ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial degradation.Dev Cell.201324；25 (6): 636-44.The preparation method and system of the cDNA of cell reversal record is the same as real Apply example 1.

14 primer sequence of table

(2) purifying of Ter94-N/Npl4-UBD complex proteins:

It is purified by basic experiment method 3, has obtained the destination protein that purity is higher than 95%, then pass through a step gel mistake Filtering layer analyses uniform albumen.

The HT-Ter94-N of purifying and HT-Npl4-UBD is mixed, 4 DEG C of incubation 2h with the ratio that molar ratio is 1: 1, then used The method of gel permeation chromatography is purified, and single ultraviolet absorption peak, the position at the peak and independent purifying protein occurs UV absorption peak position is compared, and apparent offset has occurred to the left, illustrates that Ter94 and Npl4 can be with stoichiometric ratio for 1:1 Mode combine, form stable binary complex.

(3) structure elucidation of HT-Ter94-N/HT-Npl4-UBD compound:

HT-Ter94-N/HT-Npl4-UBD complex proteins are concentrated to 25mg/ml, it is raw to carry out crystal using sitting-drop methods The preliminary screening of elongate member.After three days, there is irregular in No. 69 condition of Index kit (Hampton Research) Shape crystal generate, constituent be 0.1M Tris pH 8.5,25%PEG 3350,0.2M ammonium sulfate, will be brilliant Body takes the collection that Shanghai synchrotron radiation light source biological macromolecule crystal light beam line station BL17U-MX carries out diffraction data to.

(4) composite structure parses:

In order to parse the phase of Ter94-N/Npl4-UBD compound, we are in the M9 culture medium containing selenomethionine Middle inducing expression HT-Ter94-N and HT-Npl4-UBD selenoprotein, then according to purification process identical with parent protein into The purifying of row selenoprotein.The compound is repeated using the Ter94-N/Npl4-UBD complex proteins that selenomethionine marks The growth conditions of parent crystal is Index-69 and Index-75 (Hampton Research),.By changing precipitating reagent PEG Type and concentration the seleno crystal grown in the two conditions is optimized respectively.It is same to take these seleno crystal to Beijing Walk the collection that radiating light source (BSRF) carries out the diffraction data, (peak under three different wavelengthinflectionAnd remote) have collected resolution ratio respectively and beWithDiffraction data, Space group is P212121.Using the diffraction data of peak, by the method for Single wavelength anomalous scattering (SAD) to Ter94-N/ Npl4-UBD compound has carried out the parsing of phase and the foundation of model, has finally obtained the crystal structure of the compound.It is compound The structure of object is shown: in the hydrophobic groove that two subdomains that the UBD structural domain of Npl4 has been incorporated in the end Ter94N are formed, Combination ratio in asymmetric cell is 1:1.

(5) detection of IFN β activation and IFNB transcription is the same as embodiment 1.

3. experimental result and analysis

There are four structural domains by Npl4: the UBD structural domain (1-80) of N-terminal is in conjunction with p97, zf-Npl4 structural domain (104- 246), Npl4 structural domain (248-557) and the NZF structural domain (580-608) of C-terminal in conjunction with ubiquitin (Fig. 3 A).Missing The IFN β activation and IFNB transcription that the Npl4 of UBD structural domain cannot inhibit SeV to induce, this further proves that Npl4 cooperates with p97 Inhibit antiviral immunity reaction.

We by the crystal structure of the drosophila Ter94-N/Npl4-UBD compound of parsing respectively with source of people p97N end structure The crystal structure (PDB number 3QQ7) in domain and the nuclear-magnetism structure (PDB number 2PJH) of source of mouse Npl4UBD structural domain are compared, We have found that the individual crystal structure of source of people p97-N can be good at being superimposed with the Ter94-N in drosophila compound, fold In addition the root-mean-square-deviation (rmsd) of C alpha atom is afterThis is consistent with sequence homology (78%) of their height. Still there is notable difference between another aspect drosophila Npl4-UBD and source of mouse Npl4-UBD the two structures.By to difference The Cdc48/Ter94/p97-N and Npl4-UBD of species carry out taking structure as the sequence alignment being oriented to, it has been found that on Ter94-N Key residues Q47, F49, R50, D52, K106, Y107 and Y140 with drosophila Npl4-UBD interaction is between different plant species It is all very conservative.The crystal structure for the drosophila Ter94-N/Npl4-UBD compound that we are parsed may be retouched more accurately The universal way and feature of p97 and Npl4 interaction are stated.

The Ter94/Npl4 composite structure and individual Ter94 that we parse, Npl4 is similar, the UBD structural domain of Npl4 It has been incorporated in the hydrophobic groove of two subdomains formation at the end Ter94N, the combination ratio in asymmetric cell is 1: 1 (Fig. 3 B).There are two the sites to interact for the heterodimer that Ter94/Npl4 compound is formed.First interaction The F49 of L7, R9 and R18 and the end Ter94N on first and second β lamella of site Npl4 form hydrophobic phase interaction With, while the R50 of R9, R18 and the H69 of Npl4 and Ter94, D52and Q47 form hydrogen bond and ionic bond.Second mutually The A13 and R49 of action site Npl4 is coupled with the Y107 at the end Ter94N and Y140 by hydrophobic interaction, Npl4's The Y107 of A13, Q11 and R49 and Ter94 are interacted by hydrogen bond and ionic bond.

In addition, the Y140 of the R49 and Ter94 of Npl4 form cation- π interaction.Above-mentioned these interact residual Base is all conservative in drosophila and people, thus the composite structure parsed with the Ter94-Npl4 of drosophila, equally applicable and people The structural analysis (Fig. 3 C) of p97-Npl4 compound.

The mutation research of 4 p97-Npl4 compound of embodiment

1. experiment purpose: by the interaction of mutation analysis research p97-Npl4 compound for it in antiviral signal The influence acted in access.

2. experimental method:

(1) vector construction of interaction detection carrier S trep-Npl4, His-Npl4, Strep-p97, p97-His:

Strep-Npl4, His-Npl4 are using HA-Npl4 carrier as template, in Takara high-fidelity DNA polymerase PrimeSTAR Under the action of with corresponding primer carry out PCR amplification；PCR primer is shown in Table 15, carrier construction method and body System is the same as embodiment 2.Strep-Npl4 is commercial vector using SEQ ID NO:31 in table 15,32 amplifications, the carrier of connection PET51b-Strep, His-Npl4 are voluntarily constructed using SEQ ID NO:31 in table 15,33 amplifications, the carrier of connection PET28a-HisTev, restriction enzyme site are NcoI/SalI.

15 primer sequence of table

P97-His, Strep-p97 are using V5-p97 carrier as template, in Takara high-fidelity DNA polymerase PrimeSTARUnder the action of with corresponding primer carry out PCR amplification；PCR primer is shown in Table 16, carrier construction method and the same embodiment of system 2.P97-His is using SEQ ID NO:34 in table 16, and the carrier of 35 amplifications, connection is commercial vector pET21a, and restriction enzyme site is equal For NdeI/SalI.Strep-p97 is commercial vector using SEQ ID NO:36 in table 16,37 amplifications, the carrier of connection PET51b-Strep, restriction enzyme site are BamHI/SalI.

16 primer sequence of table

(2) building of the mutational vector of p97 and Npl4:

P97 mutant F52A/D55A, Y110A, using p97-His as template, using SEQ ID NO:38-41 institute in table 17 After showing that primer carries out PCR amplification, unmutated wild type p97 template is removed using DpnI demethylase, amplification is had PCR product is converted DH5 α competent cell by the carrier segments with p97 gene of specific locus mutation, by resistance screening, The positive colony of acquisition, and sequence verification.

P97 mutant 3A, using SEQ ID NO:40 in table 17, draws using p97-His F52A/D55A as template shown in 41 After object carries out PCR amplification, unmutated p97-His F52A/D55A template is removed using DpnI demethylase, amplification obtains PCR product is converted DH5 α competent cell, passes through resistance by the carrier segments with p97 gene with specific locus mutation Screening, the positive colony of acquisition, and sequence verification.

17 primer sequence of table

Npl4 mutant I6A/R8A, R50A, using Npl4-His as template, using SEQ ID NO:42 in table 18,43, After primer shown in 46,47 carries out PCR amplification, unmutated wild type Npl4 template is removed using DpnI demethylase, is expanded The carrier segments with Npl4 gene for having specific locus mutation are obtained, PCR product is converted into DH5 α competent cell, is passed through Resistance screening, the positive colony of acquisition, and sequence verification.

Npl4 mutant I6A/R8A/R17A is using Npl4-His I6A/R8A as template, using SEQ ID NO in table 18: After primer shown in 44,45 carries out PCR amplification, unmutated Npl4-His I6A/R8A mould is removed using DpnI demethylase Plate, amplification obtain the carrier segments with Npl4 gene for having specific locus mutation, and PCR product conversion DH5 α competence is thin Born of the same parents, by resistance screening, the positive colony of acquisition, and sequence verification.

Npl4 mutant 4A is using Npl4-His I6A/R8A/R17A as template, using SEQ ID NO:46 in table 18,47 After shown primer carries out PCR amplification, unmutated Npl4-His I6A/R8A/R17A mould is removed using DpnI demethylase Plate, amplification obtain the carrier segments with Npl4 gene for having specific locus mutation, and PCR product conversion DH5 α competence is thin Born of the same parents, by resistance screening, the positive colony of acquisition, and sequence verification.

18 primer sequence of table

3. experimental result and analysis:

We construct the mutant of people p97 and Npl4 a series of according to structural information, are tested by pull down, We have found that (F52, Y110) (D55, the Y110) of p97 has conclusive effect for the combination of Npl4.The R50 of Npl4 with And the multipoint mutation of I6, R8, R17, R50, the interaction (Fig. 3 C-3E) of Npl4 and p97, co-immunoprecipitation experiment can be broken Also this result (Fig. 3 F-3G) is demonstrated.Thus, F52A/D55A/Y110p97 (3A) mutation and I6A/R8A/R17A/R50A Npl4 (4A) mutation be influence p97 and Npl4 interaction critical sites.In the HEK293T cell for knocking out endogenous p97, Under the stimulation of SeV, the p97 of transfected wild-type can inhibit the transcription of the transcriptional activation and IFNB of IFN β, transfection p973A mutation Body cannot (Fig. 3 H).Equally, the Npl4 of transfected wild-type can inhibit the activation of IFN β promoter and the transcription of IFNB, and Npl44A mutant cannot (Fig. 3 I).The above result shows that p97-Npl4 forms compound, played in antiviral signal path Effect.

The protein level of 5 p97-Npl4 compound negative regulation RIG-I of embodiment

1. experiment purpose: p97 is living with AAA ATP enzyme, can be with the stability of modulin.It is compound for research p97-Npl4 Whether object passes through the stability of the molecule in the antiviral signal path that regulation RLR is mediated, and it is compound that we have studied p97-Npl4 Regulation of the object to RIG-I protein level.

2. experimental method:

(1) slow virus recombination shRNA knocks out intracellular p97, Npl4, Ufd1, p47

The target sequence (shRNA) of one section of 21 base is designed, which can form the hair fastener shape structure of reverse complemental, sink Silent related gene expression.The shRNA of corresponding gene and control shRNA (scramble) are connected to slow virus carrier pLKO.1- puro.ShRNA (scramble) is control.ShRNA sequence is as shown in table 19.

19 shRNA sequence of table

The forward and reverse DNA chain of 0.01M mixes, and is diluted to 50ul, by reacting as follows:

95℃

5min

70℃

10min

55℃

30min

37℃

30min

Annealing is connected as double-strand.Double-stranded DNA is connect with pLKO.1-puro (EcoRI/AgeI double digestion) carrier, converts DH5 α, the positive colony identified extract plasmid after sequence verification.By 293 cell of plasmid transfection, with 2 μ g/ml puromycin The cell of screening-gene group stable integration shRNA sequence, after 48-72 hours, uses Vivaspin 20ml (Sartorius Stedim Biotech, Aubagne, France) with the centrifugal force of 3000g, it is centrifuged 50min, concentrating virus liquid.Virus infection Afterwards, it can get the cell for knocking out corresponding gene.PLKO.1-puro plasmid is biological from Shanghai Inst. of Life Science, CAS institute Chemistry is located to obtain with Institute of Cell Biology Ji Hongbin researcher, can be from for the commercially available common carrier for constructing RNAi Addgene company buys.

(3) vector construction of Flag label RIG-I, Flag label RNF125:

In the method and system extracting cell total rna in embodiment 1, and reverse transcription is cDNA, in Takara high-fidelity Archaeal dna polymerase PrimeSTARUnder the action of with corresponding primer carry out PCR amplification；PCR primer is shown in Table 20, vector construction Method and system are the same as embodiment 2.Flag-RIG-I is used using SEQ ID NO:60 in table 20,61 amplifications, Flag-RNF125 The carrier of SEQ ID NO:62 in table 20,63 amplifications, connection are commercial vector pCDNA3.1-Flag, and restriction enzyme site is HindIII/XholI。

20 primer sequence of table

(4) RIG-I K181 mutation construction:

Using Flag-RIG-I as template, after carrying out PCR amplification using primer shown in table 21, DpnI demethylase is used Unmutated template is removed, amplification obtains the carrier segments with Npl4 gene for having specific locus mutation, and PCR product is turned Change DH5 α competent cell, by resistance screening, the positive colony of acquisition, and sequence verification.

21 primer sequence of table

(5) antibody detection protein expression: experimental method is the same as basic experiment method 7.Detect Flag, Myc and β- The antibody of actin is purchased from Sigma (St.Louis, MO).Detect p97, RIG-I, MAVS, p-TBK, TBK, IKK ε, p-IRF3, The antibody of IRF3 and K48Ub is purchased from Cell Signaling (Danvers, MA).HA, Ubiquitin are detected, RNF125's Antibody is purchased from Santa Cruz (Santa Cruz, CA).The antibody for detecting Npl4 (IF) is purchased from Novus Biologicals (Littleton, CO).It detects Npl4 (IP and WB) and is purchased from Abcam (Cambridge, UK).

3. experimental result and analysis:

We have detected the protein level (Fig. 4 A) of RIG-I in the cell for transfected shp97, MAVS and IRF3.It knocks out P97 can dramatically increase the RIG-I expression of poly (I:C) induction, and the protein level of same MAVS also increases, total egg of IRF3 White level does not change, but its phosphorylation level obviously increases.In turn, overexpression Npl4 significantly reduces poly (I:C) and lures The expression of the RIG-I and MAVS that lead, the phosphorylation level of IRF3 are significantly reduced (Fig. 4 B).In addition, knocking out Npl4 can show Write the RIG-I for increasing poly (I:C) induction, the expression of MAVS enhances the phosphorylation level of IRF3, and TBK1, IKK ε and The protein level of IRF3 does not change (Fig. 4 C).Equally, p47 is knocked out, another confactor of p97 is antiviral to RIG-I logical There is no regulating and controlling effects on road.Thus the regulating and controlling effect of p97 signal path antiviral for RIG-I depends on Npl4.It is overexpressed Npl4, can't cause poly (I:C) induce RIG-I mRNA level variation, and with proteasome inhibitor MG132 at The RIG-I protein level decline that p97 and Npl4 is mediated after reason cell is also suppressed (Fig. 4 D-4E).This illustrates that p97-Npl4 is multiple Close the protein level that object passes through proteasome degrading and regulating RIG-I.P97 or Npl4 are knocked out into the cell in HEK293, then are transfected The K48 chain ubiquitination level of Flag label RIG-I, RIG-I are decreased obviously (Fig. 4 F-4G).On the contrary, in the HEK293 for knocking out p97 Flag label RIG-I is transfected simultaneously in cell and the K48 chain ubiquitination level of wild type p97, RIG-I obviously increase, and it is same When transfection Flag-RIG-I and the ubiquitination level of RIG-I cannot not be influenced with the p973A mutant in conjunction with Npl4 (Fig. 4 H).Equally, the Npl4 for being overexpressed wild type can increase the K48 ubiquitination level of RIG-I, and being overexpressed cannot be with p97 In conjunction with Npl44A mutant (Fig. 4 I) is not influenced on the ubiquitination level of RIG-I.These results suggest that p97-Npl4 is compound Object makes it through proteasome degradation, inhibits antiviral immunity signal path by the K48 chain ubiquitination of promotion RIG-I. P97-Npl4 compound by mediate RIG-I the antiviral signal path of degrading and regulating, and before research shows that E3 ubiquitin connect The K48 chain ubiquitination that enzyme c-Cbl and RNF125 participate in RIG-I is connect, we have studied participate in the regulation of p97-Npl4 compound The ubiquitin ligase of RIG-I degradation.We have separately designed the shRNA of RNF-125 and c-Cbl, demonstrate it by Q-PCR Knock out efficiency.Double luciferase element Reporter Gene Experiments show to knock out endogenous RNF125, and Npl4 can not lower the IFN of SeV induction The activation of β promoter, and knock out c-Cbl and do not act on (Fig. 5 A).Equally, in the cell for knocking out RNF-125, Npl4 cannot The transcriptional activation for the IFN β for inhibiting the card structural domain for being overexpressed RIG-I to mediate, in the cell for knocking out c-Cbl, the suppression of Npl4 It makes of unaffected (Fig. 5 B).In the cell for knocking out endogenous RNF125, Npl4 can not reduce the protein level of RIG-I (Fig. 5 C).These results suggest that RNF125 is that p97-Npl4 compound inhibits E3 special in the antiviral signal path of RIG-I general Plain ligase.We have detected the ubiquitination level of total RIG-I in the cell of transfection Npl4 or RNF125, and it is significant to be overexpressed Npl4 The K48 chain ubiquitination level of RIG-I is increased, and its ubiquitin can be further increased by being overexpressed Npl4 and RNF125 simultaneously Change horizontal (Fig. 5 D), this shows that Nlp4 and RNF125 acts synergistically, and promotes the K48 chain ubiquitination of RIG-I.QPCR tests also table Bright cotransfection Npl4 and RNF125 transfects Npl4 or RNF125 (Fig. 5 E) much higher than independent for the transcripting suppressioning action of IFN β.I Be overexpressed RNF125 in cell, and not to be overexpressed the cell of RNF125 as control, be catalyzed by mass spectral analysis RNF125 RIG-I ubiquitination site, the results showed that its ubiquitination betides 181 lysines (Fig. 5 F).Pass through point mutation means structure 181 are built not by the RIG-I mutant K181 of ubiquitination, the RIG-I and Npl4 of cotransfection wild type or mutant in cell Or after RNF125, SeV stimulation, pass through the protein level of immune-blotting method RIG-I, the results showed that Npl4 or RNF125 can be shown The protein level for reducing the RIG-I of wild type is write, but (Fig. 5 G) is not influenced on the protein level of mutant K181R, this shows 181 lysines are the major sites that p97-Npl4 compound regulates and controls the relevant antiviral access of RIG-I by RNF125.This Outside, Npl4 can increase the K48 chain ubiquitination level of RIG-I, but not have shadow to the K48 chain ubiquitination level of K181R mutant It rings (Fig. 5 H).Equally, the activity for the IFN β promoter that RNF125 and Npl4 can obviously inhibit wild type RIG-I to mediate, But (Fig. 5 I) is not influenced on K181R mutant.After knocking out Npl4 or RNF125 in cell, the RIG-I of transfected wild-type can To inhibit VSV to infect (infection multiplicity MOI=20), transfection K181R mutant cannot generate antivirus action (Fig. 5 J).We The structure feature of the 181st lysine of RIG-I is further analyzed, which is structurally close 172 lysines, i.e., RIG-I activates phosphorylation site the 170th of the relevant chain ubiquitination site key K63 and negative regulation RIG-I signal path Threonine, thus, the 181st lysine of RIG-I is the key that the RIG-I ubiquitination position that RNF125/p97-Npl4 is mediated Point.

6 p97-Npl4 compound of embodiment is in conjunction with RIG-I and RNF125

1. experiment purpose: whether detection p97-Npl4 compound binds directly RIG-I and RNF125.

2. experimental method:

(1) vector construction of Flag label RIG-I 1-238,200-803,804-925:

Using Flag-RIG-I as template, in Takara high-fidelity DNA polymerase PrimeSTARUnder the action of with pair The primer answered carries out PCR amplification；PCR primer is shown in Table 20,22, and carrier construction method and system are the same as embodiment 2.The carrier of connection is Commercial vector pCDNA3.1-Flag, restriction enzyme site are HindIII/XholI.(" Flag label RIG-I 1-238 amplimer For 20 primer 60 of table, 22 primer 66 of table；Flag label RIG-I 200-803 amplimer is 22 primer 67,68 of table；Flag mark Label RIG-I 804-925 amplimer is 22 primer 69 of table, 20 primer 61 " of table)

22 primer sequence of table

(2) HA-Npl4 overall length, Δ NZF 1-579, Δ UBD (81-608), and individually UBD vector construction: with HA- Npl4 is template, in Takara high-fidelity DNA polymerase PrimeSTARUnder the action of with corresponding primer carry out PCR Amplification；PCR primer is shown in Table 8,23, and carrier construction method and system are the same as embodiment 2.The carrier of connection is commercial vector pCDNA3, Restriction enzyme site is HindIII/NheI.(" HA-Npl4 Δ NZF 1-579 amplimer is 8 primer 25 of table, 23 primer 70 of table； HA-Npl4 Δ UBD (81-608) amplimer is 23 primer 71 of table, 8 primer 26 of table；HA-Npl4 UBD amplimer is that table 8 draws Object 25,23 primer 72 " of table)

23 primer sequence of table

(3) vector construction of MBP-RIG-I CARD, MBP-RNF 125: MBP-RIG-I using Flag-RIG-I as template, MBP-RNF125 is using Flag-RNF125 as template, in Takara high-fidelity DNA polymerase PrimeSTARUnder the action of use Corresponding primer carries out PCR amplification；PCR primer is shown in Table 24, and carrier construction method and system are the same as embodiment 2.The carrier of connection is (vector construction information is referring to Shi, Z., et al.Structure of the by the carrier pET28aMBPTev voluntarily constructed MST4in complex with MO25provides insights into its activation Mechanism.Structure 21,449-461,2013)), restriction enzyme site is NcoI/XholI.

24 primer sequence of table

3. experimental result and analysis:

We carry out co-immunoprecipitation experiment in the HEK293T cell of SeV stimulation, it is found that endogenous RIG-I can be with Endogenous Npl4 is combined, can also be in conjunction with RNF125 (Fig. 6 A).Meanwhile laser confocal microscope detection also demonstrates, Under SeV stimulation, there are common location (Fig. 6 B) with RIG-I by Npl4.Npl4 and the RIG-I recombinant protein of purification are in pulldown It is further proved in experiment, the two albumen can be with direct interaction (Fig. 6 C).We intercepted different RIG-I and The structural domain of Npl4 studies the specific region of its interaction, the Npl4 cotransfection HEK293T of different RIG-I segments and overall length Cell, co-immunoprecipitation experiment show that the CRAD structural domain (amino acid 1-238) of RIG-I and Npl4 interact, Helicase structural domain and CTD structural domain are not involved in its interaction (Fig. 6 D) with Npl4.Equally, the deletion mutant of Npl4 With the RIG-I cotransfection cells of overall length, co-immunoprecipitation experiment shows that the mutant for lacking the NZF structural domain of Npl4 loses completely The ability in conjunction with RIG-I is lost, and the Npl4 for lacking UBD structural domain still can be in conjunction with RIG-I (Fig. 6 E), i.e., Npl4 passes through NZF structural domain is in conjunction with RIG-I.External pulldown experiment further prove Npl4 can directly with the CARD structure of RIG-I Domain combines (Fig. 6 F).Endogenous co-immunoprecipitation experiment shows that RNF125 (can not only can also scheme in conjunction with RIG-I in conjunction with p97 6G).Utilize the albumen of purification, it has been found that p97 can have direct interaction in conjunction with RNF125 between the two (Fig. 6 H).In addition, RNF125 can bind directly RIG-I and p97-Npl4 compound, p97-Npl4 compound can be into one Step promotes the interaction (Fig. 6 I) between RNF125 and RIG-I.The above result shows that p97-Npl4 is multiple under the stimulation of SeV Closing object can be in conjunction with RIG-I and RNF125, and RNF-125 mediates the K48 chain ubiquitination of RIG-I, and then promotes RIG-I's Proteasome degradation, inhibits the antiviral signal path of RIG-I.

The screening of 7 p97-Npl4 complex inhibitor of embodiment and its effect in antiviral signal path

1. experiment purpose: determination can with specific inhibition p97-Npl4 interact small molecule compound and detect chemical combination Influence of the object to the p97-Npl4 antiviral signal path mediated.

2. experimental method:

(1) AlphaScreen is tested: in 384 shallow bore hole plates (PerkinElmer), 2.5 μ l are added with His label Npl4, the p97 of 2.5 μ l biotin labelings, add reaction buffer (20mM Hepes pH 7.5,100mM NaCl, 0.1% BSA), every hole final volume is 11 μ l, and the final concentration of 125nM of the Npl4 with His label, the p97 of biotin labeling are final concentration of 31.25nM, by reaction system in incubation at room temperature 60 minutes.With dilution buffer (20mM Hepes, 100mM NaCl, pH 7.5) compound (10mM) being dissolved in DMSO is diluted to 100 μM, add 1 μ l to reaction system the compound after dilution In, room temperature is protected from light incubation 60 minutes.2.5 μ l nickel chelating acceptor beads (PerkinElmer) is added in reaction system, Final concentration of 10 μ g/ml, room temperature are protected from light incubation after sixty minutes, add the donor of the streptavidin crosslinking of equivalent Beads (PerkinElmer), room temperature are protected from light incubation 60 minutes.In instrument EnVision Multilabel Plate Reader (PerkinElmer) reading numerical values have positive control and negative control on every piece of 384 orifice plates.

(2) the RIG-I CARD structural domain of Flag label intracellular Reporter Gene Experiments: is transfected in HEK293FT cell The reporter gene of (1-238) and IFN β or ISRE, luciferase reporter gene.After 12 hours, vitellophag, with 10000 Cell/every hole density is seeded in 384 orifice plates, and the cell culture medium of 50 μ l is added.After 7 hours, AlphaScreen is added The compound that experiment screening obtains, final concentration of 10 μM of compound.After 37 DEG C of culture 15h, uciferase activity is detected.

3. experimental result:

The interaction of p97 and Npl4 be for the antiviral signal path that IFN β mediates it is necessary, we pass through This screening means of AlphaScreen form compound library with 8120 biologically active compounds and carry out high-throughput sieve The inhibitor that can break p97-Npl4 interaction is found in choosing.Optimized in preliminary experiment by a series of experiment flow, it is real The Z factor tested reaches 0.7, shows that experiment condition or measurement method can be applied in next high-throughput experiment.Right After 8120 compounds are screened, compound 132 of inhibiting rate > 50% are obtained.By biotin labeling Npl4, screening is false Positive condition, we determined that 40 inhibiting rate > 30%, compound that p97-Npl4 compound can be inhibited to be formed.Pass through Intracellular Reporter Gene Experiments, we have detected this effect of 40 compounds in antiviral signal path, wherein three changes Object is closed for activation effect > 30% of reporter gene IFN β and ISRE, i.e. Thonzonium Bromide (TB), Epirubicin Hydrochloride (EH) and Purpurin.TB is 102.6% for the activation multiplying power of IFN β, to ISER Activation multiplying power be 107.6%；EH is 89.5% for the activation multiplying power of IFN β, and the activation multiplying power to ISER is 207%, thus The two Compounds Against viral signal accesses have apparent activation.

It is further the influence of experimental verification TB, EH for p97-Npl4 compound by pulldown, using embodiment 4 The pulldown system of middle p97, Npl4 interaction detection, is separately added into contrast solution and compound, detection two in system Interaction between person uses gray analysis to electrophoresis result, shows that both small molecule compounds have slackened the two significantly It interacts (Fig. 7).The result shows that the small molecule compound that above-mentioned screening obtains can be by destroying p97-Npl4 compound It is formed, promotes the relevant antiviral signal path of RIG-I.

Claims (6)

1. Tonzonium Bromide is in preparation treatment sendai virus virus infection or the pharmaceutical composition for the treatment of vesicular stomatitis virus infection In purposes.

2. purposes according to claim 1, which is characterized in that the Tonzonium Bromide is the IFN by promoting RIG-I to mediate Signal beta access is to improve viral infection resisting ability and then play the role for the treatment of virus infection.

3. purposes according to claim 2, which is characterized in that the Tonzonium Bromide is the protease by inhibiting RIG-I Body degradation is to play the role of the IFN β signal path for promoting RIG-I to mediate.

4. purposes according to claim 3, which is characterized in that the Tonzonium Bromide is by inhibiting RNF-125 to mediate What the K48 chain ubiquitination of RIG-I was degraded to play the role of the proteasome of inhibition RIG-I.

5. purposes according to claim 4, which is characterized in that the Tonzonium Bromide is by inhibiting RNF125 and RIG-I Between interaction to play the role of the K48 chain ubiquitination for inhibiting RNF-125 to mediate RIG-I.

6. purposes according to claim 5, which is characterized in that the Tonzonium Bromide is the phase by blocking p97 and Npl4 Interaction inhibits the formation of p97-Npl4 compound, and then inhibits interaction between RNF125 and RIG-I.