CN101307094B - Novel nuclear factor -kappa B p65 subunit antagonizing polypeptide - Google Patents
Novel nuclear factor -kappa B p65 subunit antagonizing polypeptide Download PDFInfo
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Abstract
The invention relates to a novel nuclear factor -kB p65 subunit antagonism polypeptide, in particular to a nuclear factor -kB p65 subunit antagonism polypeptide for therapy. More specifically, the invention relates to a micromolecule peptide Arg Leu Arg Trp Arg which can peculiarly integrate with a nuclear factor-kB p65 subunit and inhibit the activity of the -kB p65 subunit. The invention also relates to the preparation and application of the nuclear factor -kB p65 subunit antagonism peptide.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to a kind of treatment with nuclear Factor-Kappa B (NF-κ B) p65 subunit antagonist peptide, more specifically, relate to and a kind ofly can and suppress its active small-molecular peptides with nuclear Factor-Kappa B p65 subunit specific combination.The invention still further relates to the preparation and the application of this nuclear Factor-Kappa B p65 subunit antagonist peptide.
Background technology
NF-κ B is a kind of transcription factor that is prevalent in nearly all cell cytosol, be the convergent point of multiple signal transduction pathway, have the function of the multiple participation immunne response of regulation and control, inflammatory reaction, virus replication, apoptosis and propagation and the related gene expression that grows.The protein dimer that NF-κ B system is made of two kinds of Rel family proteins.Difference according to aspects such as structure, function and synthesis modes, the Rel protein family can be divided into two classes: a class is precursor protein p105 and p100, its C-terminal contains ankyrin repeat (ankin repeat motif), can become sophisticated p50 and p52 by the proteolysis process cracking of ATP dependence.Such hypoproteinosis transactivation domain does not have the function that independent activated gene is transcribed; Another kind of is RelA (p65), Rel (c-Rel), v-Rel and RelB, and its C-terminal contains one or more trans-activation domains (transactivationdomain), has the function that independent activated gene is transcribed.
When cell is in quiescent condition, main and κ B arrestin (the I κ B of NF-κ B
s) in conjunction with forming tripolymer, be present in the tenuigenin in the non-activity mode.When cell is subjected to that multiple outer signals such as proinflammatory cytokine, virus, intracellular toxin, ultraviolet ray, oxygenant and radioactive rays stimulates and when starting the cell second messenger system, I κ B
sPhosphorylation and degraded take place under the effect of I kappa b kinase (IKK), and NF-κ B loses I κ B
sStrictness control and be activated, and displacement enters in the nucleus, its subunit forms ring texture and combines the expression of efficiently inducing pro-inflammatory cytokine (as TNF, GM-CSF, IL-1 β, IL-6, IL-11, IL-17 etc.), chemokine (as IL-8, RANTES, MIP-1 α, MCP-2 etc.), adhesion molecule (as ICAM-1, VCAM-1, ELAM-1 etc.), immunity receptor (as IL-2R) and participating in the plurality of enzymes (as NO synthetic enzyme, cyclooxygenase etc.) of inflammatory response cascade amplification with privileged site in the target gene promoters.NF-κ B institute some albumen of inductive can further reverse and activate NF-κ B, cause the expansion and the continuity of inflammatory reaction.Based on above-mentioned cognition, many scholars think that the transcriptional activity that suppresses NF-κ B will open up new approach for the treatment of inflammatory relative disease.In addition, NF-κ B also plays a significant role in apoptosis and propagation, it can suppress the apoptosis of cell by the expression of anti-apoptotic genes expressions such as direct upregulation of apoptosis supressor (c-IAP1, c-IAP2 and IXAP), TNF receptor associated factor (TRAF1 and TRAF2), zinc finger protein A20, super manganese oxide dismutase, Bcl-2 homologue A1/Bfl-1 and IEX-IL.In oncotherapy, chemotherapy and radiation can make the NF-kB activation, causes tumour cell to chemotherapy resistance and insensitive to radioactive rays.Therefore, suppress the transcriptional activity of NF-κ B, the anti-apoptosis capacity of reduction tumour cell be can yet be regarded as and improved the good plan of cancer drug therapy effect.
Because the powerful transcriptional control function of NF-κ B, the overactivity of NF-κ B is being played the part of important effect in the generation of numerous disease and evolution: as (class) rheumatic arthritis, asthma, chronic glomerulonephritis, helicobacter pylori related gastritis, chronic inflammatory diseases such as inflammatory bowel disease, Sepsis, septic shock, acute inflammation diseases such as acute glomerulonephritis, Alzheimer ' s disease, atherosclerosis, systemic erythema is all excessive or continuous activation is closely related in NF-κ B with development from the generation of body immunological disease such as lupus and cancer.In addition, NF-κ B has also participated in transcribing and duplicating in cells infected such as rhinovirus, influenza virus, Epstein-Barr virus, cytomegalovirus, adenovirus, HTL virus and HIV virus, plays an important role in the infection of these viruses and pathogenic course.
In view of generation and the developing latent effect of NF-κ B in above-mentioned disease, be target spot with the associated protein in NF-κ B and the signal transduction pathway thereof, the medicine of research and development NF-κ B relative disease has become the research focus in this area.At present external relevant be that the antagonism therapeutic strategy of target spot mainly comprises the following aspects: (1) anti-oxidant treatment: as antioxidant---tetramethyleneimine two thiocarbamates (Pyrrolidine dithiocarbamate, PDTC) transcriptional activity of non-special inhibition NF-κ B with NF-κ B; (2) formation and the activity of inhibition IKK: the interference peptide that antisense nucleic acid, expression IKK α dominant negative mutant and the application of interference IKK mixture of application IKK α forms etc. suppresses formation and the activation of IKK; (3) degraded of minimizing I κ B: the epoxomicin of some proteasome inhibitor such as MG101, MG115, MG132, PS-341, lactacystin and discovery recently etc. all can reach the purpose of antagonism NF-kB activity by suppressing the degraded of I κ B.(4) generation of promotion I κ B: use the adenovirus expression carrier of I κ B α, make I κ B α phenotype inactivation mutant overexpression, can obviously suppress nuclear displacement and the follow-up effect thereof of NF-κ B; (5) suppress the synthetic of NF-κ B: the antisense nucleic acid of design P50 and P65 and siRNA retardance NF-κ B's is synthetic; (6) traditional non-steroidal anti-inflammatory drug suppresses the activation of NF-κ B; (7) derive from the non-activation that suppress NF-κ B signal path specifically of compound such as curcumine, flavonoid compound and trans-resveratrol of Chinese medicine.
Above-mentioned is that the therapeutic strategy of target spot has shown curative effect in various degree with NF-κ B in external and experimentation on animals; the part therapeutic strategy has entered the clinic trial stage, and by registrations such as United States Patent and Trademark Office and European Patent Office and be subjected to patent protection.But there is the not good enough problem of specificity in above-mentioned part measure.As proteasome, it also has other many important function except degradable I κ B, so arrestin enzyme corpusculum activity will cause the potential toxic side effect.The sphere of action of antioxidant is more extensive, and toxic side effect is bigger.The measure of target IKK, I κ B is also not exclusively special to the intervention of NF-kB activity, is proved as the interference peptide that disturbs the IKK mixture to form, and it not only can suppress the activity of NF-κ B, also can suppress the activity of transcription factor AP-1 and NFAT.Compounds such as non-steroidal anti-inflammatory drug, curcumine, flavonoid compound and trans-resveratrol also have regulating and controlling effect to other signal path except suppressing NF-κ B signal path.Though and the synthesis strategy that directly suppresses NF-κ B is specific for NF-κ B, antisense nucleic acid and siRNA strategy enter clinical application and yet have many problems, and the synthesis strategy that therefore suppresses NF-κ B is not optimal selection.Being not difficult to find out that from the whole activated channel of NF-κ B NF-κ B is special with combining of DNA cis element, also is that NF-κ B starts the only way which must be passed that relevant target gene is transcribed.Therefore, have only NF-κ B is implemented direct intervening measure with combining of DNA cis element, just can reach specificity truly.The upstream pathway of NF-kB activation intervened then be difficult to reach this purpose.
The heterodimer that is formed by p50 and p65 two subunits is typical case's representative of NF-κ B, is most important a kind of in the NF-κ B form of ownership, almost is present in all cells in the body, and its content is far above other dimer.The N-terminal of this heterodimer can constitute 1 loop domain, is combined with the DNA base specific by its mediation.The crystalline diffraction analysis shows that p50/p65 heterodimer and immunoglobulin kappa chain genetic enhancer κ B sequence-specific bonded mode are: 5 of the Arg-54 of p50 subunit, Arg-56, Tyr-57, Glu-60, His-64 and Lys-241 amino-acid residue and κ B sequence 5 ' end '-G
-5G
-4G
-3A
-2C
-1-3 ' base series specific combination; The Arg-33 of p65 subunit, Arg-35, Arg-36, Glu-39 and Arg-187, with 5 of κ B sequence 3 ' end '-T
+ 1T
+ 2C
+ 3C
+ 4-3 ' 4 base pair specific combination.In addition, by the loop domain that the aminoterminal and the dimerization structural domain of NF-kB p50/p65 heterodimer constitutes jointly, also can non-ly discern DNA ribose phosphoric acid skeleton specifically.Promptly this loop domain that is made of jointly p50 and p65N end has formed the DNA binding domains of NF-κ B, mediates itself and the combining of cis-acting elements, and it should be noted that this specificity combination that is combined into.NF-κ B activation target gene at first needs the cis-acting elements in the DNA binding domains identification target gene promoters zone, and combination with it, and the back is down auxiliary the p65 trans-activation domain, the expression of activation target gene.Therefore, can antagonism p65 subunit and its cis-acting elements bonded polypeptide if we obtain, then certainly will be able to reach the effect of antagonism NF-κ B transcriptional activity, finally reach the purpose that suppresses NF-κ B regulate target gene expression and treatment acute or chronic inflammation relative disease and tumour.
Foundation, maturation of a kind of strong instrument of studying protein interaction that emerges along with the later stage eighties 20th century---yeast-two hybrid technique and developing finally becomes possibility to the screening operation of NF-κ B antagonism polypeptide.Yeast two-hybrid system has very high sensitivity, can detect faint and of short duration protein-protein interaction, and protein interactions betides in the yeast cell born of the same parents and/or in the nuclear in this system.Compare with prokaryotic cell prokaryocyte, the eucaryon yeast cell to proteinic translation and processing more near mammalian cell, with it is that engineering bacteria research protein interactions is with better physiological status near mammalian cell, promptly keep the natural folded state of protein, make protein-protein interaction more near the true horizon in the mammalian cell.Therefore, the interaction of using between this systematic study protein has obtained using more and more widely, and to the screening of target protein interaction protein or polypeptide in the born of the same parents, this system is more first-selected especially.NF-κ B is a nuclear factor, is present in the cytoplasm, but brings into play the transcriptional control function in nucleus.Therefore, we select yeast two-hybrid system for use, are bait protein with NF-κ B p65 DNA in conjunction with the territory, screen NF-κ B interaction polypeptide from random peptide library, and identify the function of these polypeptide.We have screened and have obtained 8 and have different sequence of N F-κ B p65 subunit antagonist peptides at present, and listed here is antagonism peptide is wherein seen patent 200510007479.2,200510069451.1 respectively for other 5.
Summary of the invention
The purpose of this invention is to provide can with single-minded combination of nuclear Factor-Kappa B p65 subunit, and can suppress the nuclear Factor-Kappa B antagonism peptide of nuclear Factor-Kappa B activity and institute's regulate gene expression thereof.
Another object of the present invention provides the pharmaceutical use of this nuclear Factor-Kappa B antagonism peptide.
Nuclear Factor-Kappa B p65 subunit antagonist peptide aminoacid sequence of the present invention is as follows:
Arg?Leu?Arg?Trp?Arg
The pharmaceutical use of antinuclear factor of the present invention-κ B is the medicine or the anti-tumor drug purposes of treatment inflammatory immune correlated disease.Wherein said inflammatory relative disease is Sepsis, septic shock, (class) rheumatic arthritis, asthma, acute and chronic glomerulonephritis, systemic lupus erythematous etc., and wherein said tumour is liver cancer, cancer of the stomach, esophagus cancer, breast tumor, bladder cancer, tumor of prostate etc.
The present invention also comprises the pharmaceutical composition that contains polypeptide of the present invention.Pharmaceutical composition of the present invention can contain the medicine acceptable carrier in case of necessity.Pharmaceutical composition of the present invention can exist by pharmaceutical dosage forms in any form.As the preparation that is fit to oral preparation or is fit to inject, preferably injection.It most preferably is freeze drying injection.
The present invention also comprises the preparation method of polypeptide of the present invention, and described method is selected from synthetic method, gene recombination method or chemical synthesis process in conventional solid phase synthesis process, the solution.
Have aminoacid sequence structure of the present invention polypeptide can with single-minded combination of nuclear Factor-Kappa B p65 subunit, and the combining of blocking-up nuclear Factor-Kappa B and its cis-acting elements, thereby suppress the biologic activity of nuclear Factor-Kappa B, promptly nuclear Factor-Kappa B is regulated and control the activity of various expression of target gene.This kind structural polypeptide both can be with the polypeptide form Individual existence, also can be warm with other albumen and polypeptide, or methylated, modification such as acetylize and amination; or carry out indivedual amino acid whose sudden changes or substitute; or insert the privileged site of protein surface, as the bulge loop district, or be connected with other material.No matter with what form exist, the material that contains this structural polypeptide may show with nuclear Factor-Kappa B p65 subunit and combine, and suppresses the active effect of nuclear Factor-Kappa B.
The present invention obtains and the novel polypeptide that combines of nuclear Factor-Kappa B p65 subunit by the yeast-two hybrid technique screening, and having confirmed that this polypeptide has the bioactive function of the nuclear Factor-Kappa B of inhibition, this medicine for design and development novel anti nuclear Factor-Kappa B is significant.
Result of study shows, the polypeptide that the present invention screens not only can competitive blocking-up nuclear Factor-Kappa B and the combining of its cis-acting elements, also can suppress the target gene expression that nuclear Factor-Kappa B is regulated and control, thereby can become new antinuclear factor-κ B medicine or prodrug, polypeptide of the present invention can be used for the treatment of various inflammation or immunity relative disease and tumour.In addition, nuclear Factor-Kappa B p65 subunit binding peptide of the present invention also has the potential using value as biotechnological formulation, as being used for the affinity purification of nuclear Factor-Kappa B as part, being used for detection of nuclear Factor-Kappa B or the like as probe.
The material that contains this aminoacid sequence can have various application modes, mainly comprise the activity that suppresses nuclear Factor-Kappa B in vitro and in vivo, realize that with various administering modes to suppress the nuclear Factor-Kappa B activity be the disease treatment of purpose, and the detection of nuclear Factor-Kappa B or purifying etc.
The material that contains aminoacid sequence of the present invention also comprises in the application of biomedical aspect:
1, passes through the mode of synthetic or gene recombination, to contain peptide section and other albumen or the peptide fusion of this sequence or carry out chemically modified, being used for to suppress the nuclear Factor-Kappa B activity is the treatment of various inflammatory relative diseases, autoimmune disease, tumour and other relative disease of purpose.
2, with after itself and the agarose coupling, be used for the affine separation of nuclear Factor-Kappa B.
3, with after itself and the indicating label coupling, be used for the evaluation of nuclear Factor-Kappa B.
4, polypeptide of the present invention is better than prior art on some indexs, as carried out p65 and analyzed comparative analysis in conjunction with polypeptide physicochemical characteristics constant, proof is more superior than several peptide species of previous exploitation, it is the shortest to be embodied in peptide sequence of the present invention, be convenient to synthesize, and have stronger wetting ability, be convenient to the preparation of preparation.See Table 1.
Table 1 p65 is in conjunction with polypeptide physicochemical characteristics constant analytical results
Annotate: a hydrophobic value high hydrophobicity more is strong more, and low more then wetting ability is strong more.
PT1：Arg?Leu?Arg?Trp?Arg
PT2：Glu?Gly?Gly?Val?Thr?Arg?Thr?Gln?Gly?Phe?Arg?Trp?Val?Val?Ser?Ile?Arg?Asn?Ser--Tyr?Arg?Asn?Glu?Ser?Ala?Ser?Asn?Gly?Arg?Cys?Leu?Leu?Leu?Ala?Ala?Gln?Gly
PT3：Val?Val?Met?Ile?Glu?Val?Val?Phe?Leu
PT4：Leu?Ala?Met?Val?Glu?Val?Thr?Val?Val?Leu?Ser?Trp?Gly?Phe?Thr
PT5：Pro?Ala?Met?Val?Glu?Val?Thr?Val?Val?Leu?Ser?Trp?Gly?Phe?Thr
PT6：Gln?Thr?Gln?Met?Glu?Val?Thr?Trp?Arg?Val?Glu?Cys?Cys?Leu?Phe?Leu
Description of drawings
Fig. 1 is polypeptide hydrophobicity analysis result.
Fig. 2 is a nuclear Factor-Kappa B p65 subunit and mixture 3D structure after peptide molecule docks.
Fig. 3 suppresses the result of experiment curve for the ELISA method detects polypeptide to nuclear Factor-Kappa B p65 subunit and the competition of nuclear Factor-Kappa B cis-acting elements bonded.
Fig. 4 detects the retarding effect that polypeptide is expressed the reactive luciferase reporter gene of nuclear Factor-Kappa B for the luciferase reporter gene experiment.A, B, C, D, E, F, G, H, I are respectively U937, U937+LPS, U937+LPS+p4-kB-Luc, U937+LPS+p4-kB-Luc+9.375 μ mol/L, U937+LPS+p4-kB-Luc+18.75 μ mol/L polypeptide, U937+LPS+p4-kB-Luc+37.5 μ mol/L polypeptide, U937+LPS+p4-kB-Luc+75 μ mol/L polypeptide, U937+LPS+p4-kB-Luc+150 μ mol/L polypeptide, the negative peptide of U937+LPS+p4-kB-Luc+150 μ mol/L.
Fig. 5 is that polypeptide is to LOVO cells in vitro inhibition of proliferation experimental result.
Fig. 6 is polypeptide suppresses LOVO cells in vitro propagation to five Fluracils an enhanced sensitivity experimental result.
Fig. 7 is that polypeptide is to enhanced sensitivity experimental result in the body of five Fluracils inhibition tumor-bearing mice tumor growth.
Embodiment
Further specify the present invention by the following examples, but not as limitation of the present invention.
The aminoacid sequence of peptide molecule is pressed the desired form input of ANTHPROT V5.0 software, and the various physicochemical characteristics constants of peptide molecule are calculated in the back.
The results are shown in Table 2, Fig. 1
Table 2 p65 is in conjunction with polypeptide physicochemical characteristics constant analytical results
Embodiment 2, molecular docking prediction p65 is in conjunction with combining between polypeptide and p65
Use Biopolymer and Discover software module to make up and optimize the 3D structure of little peptide molecule.Method of steepest descent and method of conjugate gradient by standard are carried out the energy minimization processing with the energy minimization computing rule to the 3D structure of polypeptide.In order to simulate the interaction of micromolecule polypeptide and NF-κ B, use the molecular docking technology, promptly use the molecular docking that Docking software carries out the 3D structure of the 3D structure of peptide molecule and people p65 flexibility.Under the CVFF field of force, give corresponding electric charge to corresponding atom.Use the Discover module software and carry out the energy minimization processing, make the energy structure of p65-complex of polypeptides look like to be in optimization.In order to eliminate solvent effect, with 10
Molecular radius spherical TIP3P water molecules solvent is filled in the 3D structure of p65-complex of polypeptides.In order further to set forth interaction strength and the combining site between the p65-polypeptide, application Insight II branch submodule is built the Dephi module investigation NF-κ B surface property in the routine package.The Delphi module is a program of calculating molecule electrostatic feature (comprising solvent volume effect and ionic-strength effect), and it can be, and specific combination provides crucial data between ligand-receptor.
The result shows that polypeptide and p65 ask certain binding ability, points out polypeptide may have the ability of inhibition NF-κ B dna binding activity thus, the results are shown in Figure 2.
Embodiment 3, ELISA method detect polypeptide nuclear Factor-Kappa B p65 subunit are combined with the nuclear Factor-Kappa B cis-acting elements
Competition suppress experiment
The synthetic of peptide sequence synthesized purity＞95% by Shanghai Huada Tianyuan Biotechnology Co.ltd.In bag by 96 hole elisa plates of nuclear Factor-Kappa B cis-acting elements (available from the Mercury of U.S. Clontech company
TMTransfactor p65 kits) adds 150 μ l/ hole transcription factor confining liquids, incubated at room 15min in; Add the test sample of 50 μ l/ holes after abandoning the transcription factor confining liquid with the dilution of transcription factor confining liquid, sample be respectively the p65 standard protein that contains 10ng/ μ l as positive control, the p65 standard protein that contains 10ng/ μ l add the series concentration polypeptide simultaneously, the p65 standard protein that contains 10ng/ μ l adds the negative peptide of same train concentration simultaneously, incubated at room 1h, blank is for adding 50 μ l transcription factor confining liquids; Add transcription factor confining liquid 150 μ l/ holes washing 3 times, each 4min removes washings; Add with the p65 antibody 100 μ l/ holes of transcription factor confining liquid with dilution in 1: 500, incubated at room 1h; Add transcription factor confining liquid 150 μ l/ holes washing 3 times, each 4min removes washings; Add with the goat anti-rabbit igg-HRP two anti-100 μ l/ holes of transcription factor confining liquid with dilution in 1: 1000, incubated at room 30min; Add transcription factor damping fluid 250 μ l/ holes washing 4 times, each 4min removes washings; Add tmb substrate, incubated at room 10min, treat that liquid becomes basket after, with the stop buffer termination reaction in 100 μ l/ holes; Detect the OD value in 450nm and 630nm dual wavelength.The p65 binding peptide to p65 and its cis-acting elements bonded inhibition percentage ratio calculation formula is: (p65 standard protein positive control hole OD-polypeptide hole OD)/p65 standard protein positive control hole OD.
The result shows, but polypeptide specificity inhibition p65 of the present invention combines with its cis-acting elements, and this retarding effect has dose-effect relationship, and promptly restraining effect increases along with the rising of peptide concentration, negative peptide does not then influence combining of p65 and its cis-acting elements, as shown in Figure 3.
Embodiment 4, polypeptide are tested the inhibition of the reactive reporter gene expression of nuclear Factor-Kappa B.
1640 culture medium culturing U937 cells, transfection is adjusted into 0.5～2.5 * 10 with cell density the day before yesterday
5Individual/mL, incubated overnight; Get 1mL incubated overnight cell or with 0.5 * 10
5Individual cells/well cell concentration adds 24 well culture plates; Get 3 μ L GeneJuice transfection reagents and add in 100 μ L serum-frees, 1640 substratum, the vortex mixing, room temperature is placed 5min; Get 1 μ g p4kB-Luc plasmid and add in the GeneJuice/1640 substratum mixture, blow and beat mixing gently, room temperature is placed 5-15min; Said mixture is dropwise added in the perfect medium, and light shaking makes the abundant mixing of mixture and cell; 37 ℃, the 5%CO2 incubator is cultivated; Behind the transfection 5h, change with perfect medium and to wash cell 3 times with PBS after liquid is cultivated 1h, at last cell is resuspended in serum-free 1640 substratum of 100 μ L; With PBS polypeptide is diluted to 6 different concns (final concentration behind the adding substratum is respectively 0,9.375,18.75,37.5,75,150 μ mol/L), volume is the polypeptide liquid of 50 μ L, with PBS 2 μ L Chariot transfection reagents are diluted to 50 μ L simultaneously, the polypeptide of different concns is mixed with the Chariot transfection reagent, blow and beat mixing gently, room temperature is placed 30min.100 μ L polypeptide/Chariot transfection reagent mixed solution is dropwise added in the resuspended U937 cell culture fluid of above-mentioned 100 μ L serum-frees, 1640 substratum, and light shaking makes the abundant mixing of mixture and cell; 37 ℃, the 5%CO2 incubator is cultivated 1h; Remove transfection liquid, change substratum into perfect medium, the LPS that adds final concentration simultaneously and be 1 μ g/mL stimulates, and draws the centrifugal 5min of cell 500g after cultivating 2h, and the PBS washed cell once eliminates the PBS supernatant; Add 1 * CCLR cell lysis buffer solution with 100 μ l/ holes; Vortex EP manages 10-15s, and 4 ℃, the centrifugal 2min of 12000rpm gets supernatant ,-70 ℃ of activity of preserving or directly detecting luciferase; After in 96 hole check-out consoles, adding 100 μ L/ hole luciferase detection reagent, add behind the 100 μ L/ porocyte lysates reading at once again, and printing or record data.
The result shows, polypeptide of the present invention is inhibited to the expression of the reactive luciferase reporter gene of nuclear Factor-Kappa B, and this restraining effect has dose-effect relationship, and promptly restraining effect increases along with the rising of protein concentration, negative polypeptide does not then have this restraining effect, as shown in Figure 4.Explanation thus, the p65 binding peptide that we screen acquisition has the function that suppresses the nuclear Factor-Kappa B transcriptional activity, promptly has the function that suppresses genetic expressions such as nuclear Factor-Kappa B regulation and control inflammatory mediator.
When polypeptide is synthetic in this experiment, has increased at the N end and to have worn film sequence (TyrGlyArgLysLysArgArgGlnArgArgArg), full length sequence synthetic synthetic, purity＞95% by Shanghai Huada Tianyuan Biotechnology Co.ltd.Cultivate colon cancer cell line---LOVO cell with the RPMI1640 that contains 10% calf serum, the cell in the vegetative period of taking the logarithm is counted after 0.25% trysinization, with 2.5 * 10
4The cell concn of/ml is seeded in 96 orifice plates, and 100 μ l are inoculated in every hole, makes the cell inoculation amount be respectively 2.5 * 10
3/ hole.Treat that cell cultures is after adherent 4 hours, difference by grouping adds the positive polypeptide of series concentration, negative polypeptide 100 μ l respectively, final concentration is respectively 75 μ M, 37.5 μ M, 18.75 μ M, 9.375 μ M, establish the control group (adding RPMI-1640 100 μ l) that does not add polypeptide simultaneously, treat that cell cultures measures cell proliferation with mtt assay after 72 hours, adopt multi-functional plate reading machine under wavelength 570nm, to measure each hole OD value.
The result shows, polypeptide of the present invention can suppress the LOVO cell in external proliferative response, and this retarding effect has dose-effect relationship, and promptly restraining effect strengthens with the rising of peptide concentration, negative peptide does not then have obvious influence to the LOVO cell in external proliferative response, as shown in Figure 5.
Embodiment 6, polypeptide suppress the enhanced sensitivity experiment of LOVO cells in vitro propagation to five Fluracils.
Polypeptide is identical with embodiment 5 in this experiment.Five Fluracils (5-Fu) are purchased in Shanghai Xudong Hipu Medicine Co., Ltd (10ml:0.25g/ props up).Cultivate colon cancer cell line---LOVO cell with the RPMI1640 that contains 10% calf serum, the cell in the vegetative period of taking the logarithm is counted after 0.25% trysinization, with 2.5 * 10
4The cell concn of/ml is seeded in 96 orifice plates, and 100 μ l are inoculated in every hole, makes the cell inoculation amount be respectively 2.5 * 10
3/ hole.Treat that cell cultures is after adherent 4 hours, by following grouping application of sample: 5-Fu group, the positive peptide group of 5-Fu+, the negative peptide group of 5-Fu+, establish simultaneously do not add 5-Fu, only add positive peptide, only add negative peptide control group, the 5-Fu final concentration is respectively 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml, positive polypeptide, negative polypeptide final concentration are 37.5 μ M, treat that cell cultures measures cell proliferation with mtt assay after 72 hours, adopt multi-functional plate reading machine under wavelength 570nm, to measure each hole OD value.
The result shows that polypeptide of the present invention suppresses LOVO cells in vitro propagation to 5-Fu and has tangible sensitization when 37.5 μ M concentration, the enhanced sensitivity ratio is 132.50%, and negative peptide does not then have sensitization, as shown in Figure 6.
Embodiment 7, polypeptide are to enhanced sensitivity experiment in the body of five Fluracils inhibition tumor-bearing mice tumor growth.
Polypeptide is identical with embodiment 5 in this experiment.Nude mice (BALB/C strain, 4～6 ages in week, weight 18～20g, male and female half and half) is purchased in Beijing dimension tonneau China company.Raise in the ultra-clean Laminar Flow Room under the Third Military Medical University's big level ground SPF of animal housing of hospital condition.Get the LOVO cell that is in logarithmic phase, with 0.25% trysinization, physiological saline is adjusted cell concn to 1 * 10
7/ ml is inoculated under the season ribbed hide of nude mice right side by every 0.1ml.Treat that local tumor grows to 150～200mm
3The time, press following grouping administration: solvent control group, the positive peptide group of 5-Fu+, the negative peptide group of 5-Fu+.Wherein the solvent control group only gives not contain the solvent 100 μ l intratumor injections of polypeptide, the next day 1 time; Positive peptide group of 5-Fu+ and the negative peptide group of 5-Fu+, the next day of abdominal injection 5-Fu30mg/kg 1 time, get the positive peptide of 150 μ M simultaneously respectively, negative peptide 100 μ l make intratumor injection, put to death animal next day after administration the 6th time, dissect the knurl piece and weigh, calculate tumour inhibiting rate by following formula.
Tumour inhibiting rate (%)=(control group knurl average quality-medication group knurl average quality)/control group knurl average quality * 100%
The result shows that polypeptide of the present invention suppresses the tumor-bearing mice tumor growth to 5-Fu and has tangible sensitization, is contrast with negative peptide group when intratumor injection, the tumour inhibiting rate of positive peptide group is 63.54%, as shown in Figure 7.
Embodiment 8, Arg Leu Arg Trp Arg preparation method's operation steps.
Solid-phase polypeptide synthetic main operational steps is as follows: 1, resin swelling: the Wang-Resin resin is put into reaction tubes, add DMF (15ml/g), 30min.2, deprotection: remove DMF, add 20% piperidines DMF solution (15ml/g), 5min removes and adds 20% piperidines DMF solution (15ml/g), 15min again.3, detect: take out piperidine solution, get tens grainy resins, it is inferior to give a baby a bath on the third day after its birth with ethanol, adds triketohydrindene hydrate, KCN, and each one of phenol solution, 105 ℃～110 ℃ heating 5min deepen blue positive reaction.4, washing: DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g).5, condensation: it is excessive to add three times in amino acid of protection, and three times of activator HBTU are excessive, all with lack the DMF dissolving as far as possible, add reaction tubes, and it is excessive to add ten times of NMM at once, reacts 30min.6, the washing: DMF (10ml/g) once, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g).7, repeat two to six operations and connect amino acid (holding N end order from C: Arg, Trp, Arg, Leu, Arg), last washing: DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, DMF (10ml/g) twice, twice of DCM (10ml/g) successively.9, cracking: lysate (10ml/g)---TFA 94.5% in preparation; Water 2.5%; EDT 2.5%; TIS 1%, 120min.10, dry up washing: lysate is dried up with nitrogen as far as possible, wash six times with ether, normal temperature volatilizes then.11, HPLC purifying: do moving phase with water and acetonitrile, add 0.1% TFA in moving phase, gradient is used the C18 reversed-phase column from 5%--85%.
Claims (9)
1. one kind combines also the active polypeptide of antagonism nuclear Factor-Kappa B specifically specifically with nuclear Factor-Kappa B p65 subunit, and aminoacid sequence is as follows:
Arg?Leu?Arg?Trp?Arg。
2. the application of the polypeptide of claim 1 in the medicine of preparation antinuclear factor-κ B p65 subunit.
3. the application of claim 2, the medicine of described antinuclear factor-κ B p65 subunit are the medicine or the anti-tumor drugs of treatment inflammatory immune correlated disease.
4. the application of claim 3, wherein said inflammatory relative disease is Sepsis, septic shock, rheumatic arthritis, rheumatoid arthritis, asthma, acute and chronic glomerulonephritis, systemic lupus erythematous, and wherein said tumour is liver cancer, cancer of the stomach, esophagus cancer, breast tumor, bladder cancer, tumor of prostate.
5. a pharmaceutical composition is characterized in that, contains the described polypeptide of claim 1 in the composition.
6. the pharmaceutical composition of claim 5 is characterized in that, contains the medicine acceptable carrier in the composition.
7. the pharmaceutical composition of claim 5 is characterized in that, composition is an injection.
8. the pharmaceutical composition of claim 5 is characterized in that, composition is a freeze drying injection.
9. the preparation method of the polypeptide of claim 1 is characterized in that, described method is selected from synthetic method, gene recombination method or chemical synthesis process in conventional solid phase synthesis process, the solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101798336B (en) * | 2009-02-06 | 2012-02-29 | 中国人民解放军第三军医大学第三附属医院 | Anti-tumour compound with tracking function |
| CN101735306B (en) * | 2010-02-09 | 2012-03-07 | 中国人民解放军第三军医大学第三附属医院 | Anti-inflammatory peptide with membrane penetration effect |
| CN101974073B (en) * | 2010-11-05 | 2013-04-24 | 中国人民解放军第三军医大学第三附属医院 | Anti-inflammatory hexapeptide |
| CN103739671B (en) * | 2013-12-31 | 2015-08-26 | 郭向华 | A kind of polyethyleneglycol modified suppression nuclear Factor-Kappa B polypeptide and application thereof |
| CN103923188A (en) * | 2014-01-17 | 2014-07-16 | 南通诚信氨基酸有限公司 | Application of immunoglobulin kappa light chain gene enhancer profilin polypeptides in acute inflammation treatment drugs |
| CN103819540B (en) * | 2014-01-21 | 2015-08-12 | 南通诚信氨基酸有限公司 | Nf-KB peptide inhibitor and application thereof |
Citations (3)
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|---|---|---|---|---|
| CN1157828A (en) * | 1995-12-15 | 1997-08-27 | 弗·哈夫曼-拉罗切有限公司 | Truncated form of inhibitory kappa B protein (IKB) recombinant production and uses thereof |
| CN1339458A (en) * | 2000-08-23 | 2002-03-13 | 上海博德基因开发有限公司 | New polypeptide-nuclear factor kappa-B DNA binding subunit 10.23 and polynucleotide for encoding such polypeptide |
| US6673897B1 (en) * | 1998-05-06 | 2004-01-06 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Inhibitors of NF-κB Activation |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1157828A (en) * | 1995-12-15 | 1997-08-27 | 弗·哈夫曼-拉罗切有限公司 | Truncated form of inhibitory kappa B protein (IKB) recombinant production and uses thereof |
| US6673897B1 (en) * | 1998-05-06 | 2004-01-06 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Inhibitors of NF-κB Activation |
| CN1339458A (en) * | 2000-08-23 | 2002-03-13 | 上海博德基因开发有限公司 | New polypeptide-nuclear factor kappa-B DNA binding subunit 10.23 and polynucleotide for encoding such polypeptide |
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