CN104311670A - Fused polypeptide for IL-17 and TNF[alpha], and application thereof - Google Patents
Fused polypeptide for IL-17 and TNF[alpha], and application thereof Download PDFInfo
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- CN104311670A CN104311670A CN201410162984.3A CN201410162984A CN104311670A CN 104311670 A CN104311670 A CN 104311670A CN 201410162984 A CN201410162984 A CN 201410162984A CN 104311670 A CN104311670 A CN 104311670A
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- fusion polypeptide
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Abstract
The invention discloses a fused polypeptide for IL-17 and TNF[alpha], and an application thereof. The invention provides a fused polypeptide, specifically an IL-17scfv/sTNFR1 fused polypeptide. The fused polypeptide is composed of an A segment and a B segment, wherein the A segment is (a) protein composed of amino acid sequences shown as a sequence 1 in a sequence table or (b) protein that is derived from the sequence 1 by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequences shown as the sequence 1 and has the same functions; and the B segment is (c) protein composed of amino acid sequences shown as a sequence 3 in the sequence table or (d) protein that is derived from the sequence 3 by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequences shown as the sequence 3 and has the same functions. The fused protein provided by the invention can effectively alleviate clinical symptoms of rheumatoid arthritis (RA) and has significant application values for treating the rheumatoid arthritis (RA).
Description
Technical field
The present invention relates to a kind of genetically engineered novel bispecific Mutiple Targets fusion polypeptide with opposing Interleukin-17 and tumor necrosis factor alpha activity simultaneously, particularly relate to the dual specific Mutiple Targets fusion polypeptide of the inflammatory reaction blocking autoimmune disease.
Background technology
IL-17 is a kind of powerful Pro-inflammatory mediator, is also the fine setting factor (Fine-tuning cytokine) of inflammatory reaction.IL-17 can stimulate the cytokines such as keratinocyte, inoblast, epithelial cell and endotheliocyte release IL-6, IL-8, prostaglandin E2 (PGE2), metalloprotease-1 (MMP-1), granulocyte colony-stimulating factor (G-CSF) and monocyte chemo chemotactic protein (MCP)-1.Think that IL-17 promotes that in rheumatoid arthritis (RA), osteoclasia mechanism may comprise at present: the generation of induction TNF α and IL-1, make joint obtain destruction and strengthen; Increase the quantity of core stimulating factor receptor and core stimulating factor receptor, destroy OPG and both balances and increase the weight of osteoclasia; IL-17 also directly acts on osteoclast, stimulates differentiation and the activation of osteoclast.
IL-17 has the effect of powerful recruitment neutrophil leucocyte.When IL-17 and inoblast Dual culture, inoblast can continue to promote CD34
+the propagation of hemopoietic stem cell to neutrophil differentiation; The antibody of IL-6, IL-17, MIP-2 all has the effect suppressing neutrophil leucocyte to be assembled at air flue.Illustrate that IL-17 is the early promoter of T cell dependency inflammatory reaction, also play an important role in contact hematopoietic cells cytokine network and immunity system.
TNF α is the cytokine that a kind of scavenger cell primarily of activating produces, it is a kind of important biologically active factors, TNF α is except having stronger anti-tumor activity, also have biologic activity widely, it can activating macrophage and polymorphonuclear cell, and the generation of inducing various cytokines also acts synergistically, participate in immunomodulatory, promote the expression of histocompatibility complex, the anti-infection ability of enhancing body, participate in the absorption of osseous tissue and reinvent.But the generation of too much TNF α and release then can destroy the immunologic balance of body, cause serious inflammatory reaction, and heating, shock, multiple organ dysfunction syndrome can be caused even dead.
TNF α is combined mainly through the specific receptors TNFR on cytolemma and plays biologic activity, and TNFR has TNFRI and TNFRII two kinds, all can combine with TNF α and TNF β, but the biological function mediated is not quite similar.In vivo, some TNF membrane receptor extracellular region disintegrates down from cytolemma, dissociates in blood, becomes soluble TNF acceptor sTNFR.STNFR does not mediate the conduction of signal, but still can be combined with TNF α, in and the activity of TNF α, in vivo, the vitro inhibition TNF α cytotoxicity of inducing and autoimmune response, be the natural agonist of TNF α.
IL-1 and TNF α is the important inflammatory mediator in the RA disease of generally acknowledging at present, and IL-17 significantly can promote that inflammatory cell secretes IL-1 and TNF α.IL-17 and IL-1, TNF α share and can strengthen activator 21(AP21 in osteoblast-like cells) family member, early growth response gene (Egr-1), NF-κ B expression, and the gene 26(TSG26 that TNF α stimulates can be raised), the expression level of IL-6, IL-8.Effective synergy of IL-17 and other inflammatory cytokine, makes it the center being positioned at inflammatory network, thus promotes the development of RA inflammation.Can there is specific combination with interleukin-17 and tumor necrosis factor-alpha in the fusion polypeptide of the Neutralization effect of anti-interleukin-17 and tumor necrosis factor alpha, block its biologic activity, effectively slow down the development of inflammatory reaction, prevent the deterioration of the state of an illness.Fusion polypeptide for interleukin-17 and tumor necrosis factor alpha is not reported both at home and abroad.
, specific combination can be there is with interleukin-17 and TNF α, block its biologic activity in the dual specific Mutiple Targets fusion polypeptide that anti-interleukin-17 single-chain antibody and Soluble tumor necrosis factor receptor I are formed.Fusion rotein effectively can slow down the development of inflammatory reaction, prevents the deterioration of the state of an illness.All have report with TNF α, IL-17 etc. both at home and abroad using the single cell factor as the biostats of target at present, the biotechnological formulation simultaneously resisting two kinds of cytokines is not reported both at home and abroad.
Summary of the invention
The invention provides a kind of novel bispecific Mutiple Targets polypeptide drug (IL-17scfv/sTNFRI fusion polypeptide) being target with IL-17 and TNF α, there is the ability simultaneously blocking the inflammatory reaction caused by two kinds of cytokines.
The invention provides a kind of fusion polypeptide, be made up of A fragment and B fragment; Described A fragment is following (a) or (b): the protein that (a) is made up of the aminoacid sequence shown in sequence in sequence table 1; B the aminoacid sequence of sequence 1 is had the protein derived by sequence 1 of identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by (); Described B fragment is following (c) or (d): the protein that (c) is made up of the aminoacid sequence shown in sequence in sequence table 3; D the aminoacid sequence of sequence 3 is had the protein derived by sequence 3 of identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().
IL-17scfv/sTNFRI fusion polypeptide provided by the invention is the dual specific Mutiple Targets fusion polypeptide anti-human IL-17 single-chain antibody and Soluble tumor necrosis factor receptor I are connected to form by the hinge area of antibody.
After the preparation method of described fusion rotein comprises the steps: that the sequence 2 of sequence table is connected with the double chain DNA molecule over-lap PCR shown in sequence 4 by (1), insert the multiple clone site of pET-27b (+) carrier, obtain recombinant plasmid; (2) recombinant plasmid step (1) obtained imports e. coli bl21 (DE3), obtains recombinant bacterium; (3) recombinant bacterium that step (2) obtains is cultured to OD
600nm=0.5, adopt IPTG inducing culture 4h, collected by centrifugation thalline; (4) get the thalline that step (3) obtains, ultrasonication, collect inclusion body, then inclusion body sex change dissolved, (solvent is water, containing 2M urea, 0.1M NaH dropwise to add 50 milliliters of renaturation solutions
2pO
4with 0.01M Tris-HCl, pH=8.0) in, 4 DEG C of stationary incubation 12h, then dialyse, to obtain final product in the PBS damping fluid of pH7.4.
The present invention also protects a kind of DNA composition, is made up of the DNA molecular first of the described A fragment of coding and the DNA molecular second of the described B fragment of coding.Described DNA molecular first can be the DNA molecular of (1) or (2) or (3) as follows: the DNA molecular shown in sequence 2 in (1) sequence table; (2) DNA sequence dna limited with (1) is under strict conditions hybridized and the DNA molecular of described A fragment of encoding; (3) DNA sequence dna limited with (1) has more than 90% homology and the DNA molecular of described A fragment of encoding.The DNA molecular that described DNA molecular second is following (4) or (5) or (6): the DNA molecular shown in sequence 4 in (4) sequence table; (5) DNA sequence dna limited with (4) is under strict conditions hybridized and the DNA molecular of described B fragment of encoding; (6) DNA sequence dna limited with (4) has more than 90% homology and the DNA molecular of described B fragment of encoding.Above-mentioned stringent condition can be at 6 × SSC, in the solution of 0.5% SDS, hybridizes, then use 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS respectively to wash film once at 65 DEG C.
The present invention also protects a kind of plasmid composition, is made up of the recombinant plasmid first containing described DNA molecular first and the recombinant plasmid second containing described DNA molecular second.
The present invention also protects a kind of being used for the treatment of and/or the medicine of preventing autoimmune disease, and its activeconstituents is described IL-17scfv/sTNFRI fusion polypeptide.Described autoimmune disease is rheumatoid arthritis.The curative effect of described medicine is presented as the inflammatory reaction relating to and block rheumatoid arthritis.
The present invention also protects described fusion rotein preparing the application treated and/or prevented in the medicine of autoimmune disease.Described autoimmune disease is rheumatoid arthritis.The curative effect of described medicine is presented as the inflammatory reaction relating to and block rheumatoid arthritis.
The invention discloses a kind of dual specific Mutiple Targets polypeptide drug for the treatment of rheumatoid arthritis, by antihuman interleukin-17(Interleukin-17) single-chain antibody scFv and human soluble tumor necrosis factor acceptor I (soluble tumor necrosis factor receptor I) merge and form.Fusion polypeptide provided by the invention can suppress the expression of collagen protein and multiple proinflammatory effectively, effectively alleviate the clinical symptom of rheumatoid arthritis, by directly application or Optimizing Reconstruction make up single target spot antibody drug of current Clinical practice or the deficiency of fusion rotein, or replace existing antibody drug or fusion rotein, become Mutiple Targets fusion rotein medicine of new generation, improve the result for the treatment of to rheumatoid arthritis, the treatment for rheumatoid arthritis has major application and is worth.
Accompanying drawing explanation
Fig. 1 is the structure composition schematic diagram of IL-17scfv/sTNFRI fusion polypeptide.
The Dot-blot result of conjugated antigen while that Fig. 2 being IL-17scfv/sTNFRI fusion polypeptide.
Fig. 3 is the MTT result that IL-17scfv/sTNFRI fusion polypeptide blocks hTNF α biologic activity.
Fig. 4 is the biologic activity Real time PCR result that IL-17scfv/sTNFRI fusion polypeptide blocks that hIL-17 produces IL-6.
Fig. 5 is the biologic activity Real time PCR result that IL-17scfv/sTNFRI fusion polypeptide blocks that hIL-17 produces IL-8.
Fig. 6 is the impact that IL-17scfv/sTNFRI fusion polypeptide is fallen ill on mouse RA.
Fig. 7 is the impact that IL-17scfv/sTNFRI fusion polypeptide produces CII antibody in RA mice serum.
Fig. 8 is the impact of IL-17scfv/sTNFRI fusion polypeptide on RA Mice Body based intracellular cvtokine IL-1 expression level.
Fig. 9 is the impact of IL-17scfv/sTNFRI fusion polypeptide on RA Mice Body based intracellular cvtokine IL-2 expression level.
Figure 10 is the impact of IL-17scfv/sTNFRI fusion polypeptide on RA Mice Body based intracellular cvtokine IL-6 expression level.
Figure 11 is the impact of IL-17scfv/sTNFRI fusion polypeptide on RA Mice Body based intracellular cvtokine IL-10 expression level.
Figure 12 is the impact of IL-17scfv/sTNFRI fusion polypeptide on RA Mice Body based intracellular cvtokine IL-17 expression level.
Figure 13 is the impact of IL-17scfv/sTNFRI fusion polypeptide on RA Mice Body based intracellular cvtokine TNF alpha expression level.
Figure 14 is the experiment RA mouse foot photo of the 49th day.
embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.PET-27b (+) carrier: Novagen, CB0471776.E. coli bl21 (DE3): TaKaRa company, 9126.Etanercept (sTNFRI-Fc): upper marine Xin Guojian medicine company stock company (Shanghai, 12.5mg/ props up)
the structrual description of embodiment 1, IL-17scfv/sTNFRI fusion polypeptide
Fusion polypeptide called after IL-17scfv/sTNFRI fusion polypeptide for hIL-17 and hTNF α provided by the invention, is made up of A fragment and B fragment.A fragment is made up of the variable region of heavy chain (VH) of anti-human IL-17 antibody, connection peptides (GS linker), the variable region of light chain (VL) of anti-human IL-17 antibody, the antibody hinge region (hinge region) be positioned between CH1 and CH2 to downstream successively from upstream.A fragment is as shown in the sequence 1 of sequence table, and its encoding gene is as shown in the sequence 2 of sequence table.B fragment is made up of Soluble tumor necrosis factor receptor I.B fragment is as shown in the sequence 3 of sequence table, and its encoding gene is as shown in the sequence 4 of sequence table.The structure composition of IL-17scfv/sTNFRI fusion polypeptide is shown in Fig. 1.
the preparation of embodiment 2, IL-17scfv/sTNFRI fusion polypeptide
1, after the sequence 2 of sequence table being connected with the double chain DNA molecule over-lap PCR shown in sequence 4, inserting the multiple clone site of pET-27b (+) carrier, obtain recombinant plasmid.
2, recombinant plasmid step 1 obtained imports e. coli bl21 (DE3), obtains recombinant bacterium.
3, the recombinant bacterium that step 2 obtains is seeded to the LB liquid nutrient medium containing 50 μ g/mL kantlex, 37 DEG C, 120r/min shaking culture is to OD
600nm=0.5, add IPTG and make its concentration be 0.5mmol/L, 37 DEG C, 120r/min shaking culture 4h, collected by centrifugation thalline.
4, the thalline that step 3 obtains is got, ultrasonication (20KHz, carries out 10 circulations, work in each circulation and stop 1 second in 10 seconds), centrifugal (12000r/min, 5min) collecting precipitation (i.e. inclusion body), with adding sex change liquid after washings (2M urea soln) washing, (solvent is water, containing 8M urea, 0.1M NaH
2pO
4with 0.01M Tris-HCl, pH=8.0) in, inclusion body sex change is dissolved, and obtains the denaturing soln that protein concentration is 2.5mg/mL.
5, (solvent is water, containing 2M urea, 0.1M NaH the denaturing soln that 2 milliliters of steps 4 obtain dropwise to be added 50 milliliters of renaturation solutions
2pO
4with 0.01M Tris-HCl, pH=8.0) in, 4 DEG C of stationary incubation 12h, then dialyse, obtain the solution containing fusion polypeptide in the PBS damping fluid of pH7.4, are called for short IL-17scfv/sTNFRI fusion polypeptide solution.
The IL-17scfv/sTNFRI fusion polypeptide solution this step prepared is used for embodiment 3 and embodiment 4, by the concentration (concentration of fusion polypeptide is with the densitometer of protein) of physiological saline adjustment fusion polypeptide.
the biological assay of embodiment 3, IL-17scfv/sTNFRI fusion polypeptide and blocking-up Activity determination
One, the ability of IL-17scfv/sTNFRI fusion polypeptide simultaneously conjugated antigen
Directly put by the antigen protein hTNF α of maturation on NC film (negative control is BSA), confining liquid (containing 5% skim-milk) closes 2h, and the IL-17scfv/sTNFRI fusion polypeptide of then diluting with confining liquid 37 DEG C hatches 1h, and PBST washes film 3 times, each 10min; The ripe antigen protein h IL-17(diluted with confining liquid again purchased from OriGene Technologies company, LY400795), hatch 1h for 37 DEG C, PBST washes film 3 times, each 10min; Then mouse-anti human il-17 monoclonal antibody (purchased from eBioscience company, 14-7178) and sheep anti mouse two anti-(purchased from LifeSpan BioSciences company, LS-C60712-2000) is added successively.Finally, ECL chemoluminescence exposure.Dot-blot the results are shown in Figure 2, and result display IL-17scfv/sTNFRI fusion polypeptide has simultaneously in conjunction with the ability of hIL-17 and hTNF α.
Two, IL-17scfv/sTNFRI fusion polypeptide blocks the detection of hTNF α biologic activity
The L929 cell of taking the logarithm vegetative period, regulates cell density to be about 1.5 × 10 with RPMI1640 nutrient solution after 2.5g/L trysinization
5/ mL is also inoculated on 96 porocyte culture plates, every hole 100 μ L.Be placed in 37 DEG C, 5% CO
224 h are cultivated in incubator; Within 2nd day, by dilution for difference IL-17scfv/sTNFRI fusion polypeptide, (concentration is 100 μ g/mL in advance, 25 μ g/mL, 6.25 μ g/mL, 1.5625 μ g/mL) hatch 2h with equal-volume 100 μ g/mL TNF α in 4 DEG C, sucking-off culture supernatant after cell attachment, add the mixture of 100 μ L IL-17scfv/sTNFRI fusion polypeptide and TNF α, and establish IL-17scfv/sTNFRI fusion polypeptide group (only adding L929 cell and IL-17scfv/sTNFRI fusion polypeptide); TNF α group (only add L929 cell and TNF α, do not add IL-17scfv/sTNFRI fusion polypeptide).Often group establishes 4 multiple holes; It is 1 mg/mL dactinomycin that every hole adds final concentration again; 37 DEG C, 5% CO
2cultivate 12 ~ 14h; Add 10 μ L/ hole MTT (5 mg/mL), continue to cultivate 4h; Abandon supernatant, every hole adds DMSO 100 μ L; Absorbance A value is measured under 490 nm wavelength.
Fig. 3 is that IL-17scfv/sTNFRI fusion polypeptide suppresses TNF α to the MTT result of the lethal effect of L929 cell, and result display IL-17scfv/sTNFRI fusion polypeptide can effectively suppress TNF α to the lethal effect of L929 cell, and becomes dose-dependently.
Three, IL-17scfv/sTNFRI fusion polypeptide blocks the detection of hIL-17 biologic activity
In 10% calf serum (purchased from GIBCO company), Streptomycin sulphate 100 U/mL, penicillin 100 U/mL(is all purchased from TaKaRa company), 5% CO
2, 37 DEG C, cultivate Hela cell under saturated humidity condition.When Hela cell confluent monolayers, add different stimulator respectively in Hela Tissue Culture Flask.One bottle adds recombinant human IL-17 maturation protein to 50 μ g/L, one bottle adds recombinant human IL-17 maturation protein and IL-17scfv/sTNFRI is 50 μ g/L to final concentration simultaneously, one bottle adds IL-17scfv/sTNFRI to 50 μ g/L, and one bottle does not add any stimulator as negative control.5% CO
2, 37 DEG C, cultivate 20 h under saturated humidity condition.Collect cell after stimulating, utilize Trizol(purchased from Invitrogen company) method extraction cell total rna, reverse transcription becomes cDNA.Real time PCR method is utilized to detect the relative expression quantity of each group of IL-6, IL-8 cytokine.
Fig. 4, Fig. 5 result display IL-17scfv/sTNFRI fusion polypeptide can in and the Hela cell of hIL-17 induction produce cytokine IL-6(Fig. 4) and IL-8(Fig. 5) ability.This result demonstrates the biologic activity that IL-17scfv/sTNFRI well can block hIL-17.
Detection primer sequence is as follows:
β-actin：
Upstream primer: 5 ' GCCAAAAGGGTCATCATCTC 3 '
Downstream primer: 5 ' GGCCATCCACAGTCTTCT 3 '
IL-6：
Upstream primer: 5 ' TTCGGTCCAGTTGCCTTCTCCCT 3 '
Downstream primer: 5 ' GCTCTGGCTTGTTCCTCACTACT 3 '
IL-8：
Upstream primer: 5 ' ACATACTCCAAACCTTTCCACC 3 '
Downstream primer: 5 ' AAAACTTCTCCACAACCCTCTG 3 '
the application of embodiment 4, IL-17scfv/sTNFRI fusion polypeptide
CII and chicken 2 Collagen Type VI: sigma company, article No. is 9007-43-5.
The mouse adopted in the present embodiment is the male Kunming mouse of SPF level in 6-8 age in week: purchased from Beijing dimension tonneau company of China (animal conformity certification number: SCXK (capital) 2012-0001).
CII is dissolved in 0.1mol/L aqueous acetic acid (stir at 4 DEG C and make it abundant dissolving), obtain the CII solution that CII concentration is 2g/L, 4 DEG C place after 12 hours mix with Freund's complete adjuvant equal-volume, emulsification, obtain CII emulsion (in CII emulsion, CII concentration is 1g/L).
One, mouse rheumatoid arthritis (RA) model is set up and administration of dividing into groups
Because Modling model and grouping administration are operate continuously, meter experiment number of days from Modling model.
1, mouse RA model is set up
Test the 1st day, by the mode CII emulsion immune mouse (every mouse immune 0.1mL CII emulsion, object is for causing inflammation) of right back sufficient intramuscular injection; Test the 8th day, the mode CII emulsion immune mouse (every mouse immune 0.1mL CII emulsion) injected by dorsal sc single-point; Test 15-20 days, observe mouse and mark to the arthroncus situation of mouse, total score is the mouse in 8 ± 0.5 scopes is model mice.
As follows to each sufficient standards of grading of mouse: 0 point: without red and swollen; 1 point: little toe redness and swelling of joints; 2 points: toe joint and toes swelling; 3 points: the sufficient pawl below ankle joint swells; 4 points: the whole sufficient pawl swelling comprising ankle joint.The scoring of mouse four limbs is added, is the total score (being up to 16 points) of this mouse.
2, the 21st day from experiment, model mice is divided into 5 groups (often organizing 10), is handled as follows respectively:
IL-17scfv/sTNFRI fusion polypeptide group: give Single cell fusion polypeptide (every kilogram of mouse gives 20nmol IL-17scfv/sTNFRI fusion polypeptide at every turn, and the administration volume of every mouse is 100 microlitres at every turn) by the mode of abdominal injection in every 2 days;
IL-1 β scfv/sTNFRI fusion polypeptide group: give Single cell fusion polypeptide (every kilogram of mouse gives 20nmol IL-1 β scfv/sTNFRI fusion polypeptide at every turn, and the administration volume of every mouse is 100 microlitres at every turn) by the mode of abdominal injection in every 2 days;
IL-17 scfv group: give an IL-17 scfv single-chain antibody (every kilogram of mouse gives 20nmol IL-17 scfv single-chain antibody at every turn, and the administration volume of every mouse is 100 microlitres at every turn) by the mode of abdominal injection in every 2 days;
Etanercept group: give an etanercept (every kilogram of mouse gives 20nmol etanercept at every turn, and the administration volume of every mouse is 100 microlitres at every turn) by the mode of abdominal injection in every 2 days;
CIA model group: give a physiological saline (the administration volume of every mouse is 100 microlitres at every turn) for every 2 days;
Normal group is set simultaneously, is made up of the male Kunming mouse of SPF level in 10 6-8 age in week, does not carry out any process.
Two, IL-17scfv/sTNFRI fusion polypeptide impact that mouse RA is fallen ill
From experiment the 21st day, the scoring of the arthroncus situation to mouse in every 4 days once.The results are shown in Figure 6(ordinate zou and represent total score, X-coordinate representative experiment the how many days; Result is the mean value of each group of mouse; * representative P < 0.01 compared with CIA model group).Result such as Fig. 6 shows, IL-17scfv/sTNFRI fusion polypeptide characterizes the polyarthritis of mouse has restraining effect significantly, and effect is better than univalent antibody, IL-1 β scfv/sTNFRI and soluble receptors.
Three, IL-17scfv/sTNFRI fusion polypeptide is on the impact of CII antibody generation in RA mice serum
Test the 49th day, get blood by eyeball, detect CII antibody horizontal in mice serum.
1, be the CII solution of 5 μ g/mL by the dilution of the CII solution PBS damping fluid of 2g/L.
2, with the CII solution coated elisa plate of 5 μ g/mL, 100 μ L/holes, 4 DEG C are spent the night, abandon supernatant.
3, after completing steps 2, confining liquid (1g skimming milk being dissolved in the PBS damping fluid of 20ml pH7.4, matching while using) sealase target, 37 DEG C, 2h, abandon supernatant.
4, after test serum sample PBS damping fluid being diluted to 50 times of volumes, add the enzyme plate of completing steps 3, every hole 100 μ l, hatches 1h for 37 DEG C, with PBST buffer solution 3 times.
5, after completing steps 4, it is anti-(purchased from LifeSpan BioSciences company that every hole adds 100 μ l sheep anti mouses two, LS-C60712-2000), hatch 1h for 37 DEG C, wash 3 times with PBST damping fluid (being the PBS damping fluid of the pH7.4 of 0.05% Tween20 containing volume ratio).
6, after completing steps 5, every hole adds 100 μ L TMB nitrite ions, hatches 5min for 37 DEG C, and every hole adds 50 μ L 2mol/L sulfuric acid termination reactions, reads OD450nm value.
The results are shown in Figure 7, result is the mean value of each group of mouse; * representative P < 0.01 compared with CIA model group.CII antibody horizontal in CIA model group mice serum is apparently higher than normal group.Compared with CIA model group, the CII antibody horizontal in treatment group mice serum all obviously reduces.CII antibody horizontal in IL-17scfv/sTNFRI fusion polypeptide group mice serum is significantly lower than IL-17scfv group, IL-1 β scfv/sTNFRI group and etanercept group mouse.
Four, IL-17scfv/sTNFRI fusion polypeptide is on the impact of RA Mice Body based intracellular cvtokine expression level
Test the 49th day, get mouse spleen, extract total serum IgE reverse transcription is cDNA, using cDNA as template, adopt β-actin gene as internal reference by Real time PCR() detect the relative expression quantity of each cytokine.
Primer for the identification of the relative expression quantity of IL-1 is as follows:
Upstream primer: 5 '-CCATGGCACATTCTGTTCAAA-3 ';
Downstream primer: 5 '-GCCCATCAGAGGCAAGGA-3 '.
Primer for the identification of the relative expression quantity of IL-2 is as follows:
Upstream primer: 5 '-GCACCCACTTCAAGCTCCA-3 ';
Downstream primer: 5 '-AAATTTGAAGGTGAGCATCCTG-3 '.
Primer for the identification of the relative expression quantity of IL-6 is as follows:
Upstream primer: 5 '-ACAACCACGGCCTTCCCTACTT-3 ';
Downstream primer: 5 '-CACGATTTCCCAGAGAACATGTG-3 '.
Primer for the identification of the relative expression quantity of IL-10 is as follows:
Upstream primer: 5 '-TGCCTTCAGCCAGGTGAAGACTTTC-3 ';
Downstream primer: 5 '-CTTGATTTCTGGGCCATGCTTCTCTG-3 '.
Primer for the identification of the relative expression quantity of IL-17 is as follows:
Upstream primer: 5 '-GGACTCTCCACCGCAATGA-3 ';
Downstream primer: 5 '-GACCAGGATCTCTTGCTGGA-3 '.
Primer for the identification of the relative expression quantity of TNF-α is as follows:
Upstream primer: 5 '-GGAAACCCAGAGGCATTGAC-3 ';
Downstream primer: 5 '-TCAGGATCTGGCCCTTGAAC-3 '.
Primer for the identification of the relative expression quantity of β-actin reference gene is as follows:
Upstream primer: 5 '-GAGACCTTCAACCCC-3 ';
Downstream primer: 5 '-GTGGTGGTGAAGCTGTAGCC-3 '.
The results are shown in Figure 8-13, result shows, compared with CIA model group, IL-17scfv/sTNFRI fusion polypeptide group, IL-17scfv group, IL-1 β scfv/sTNFRI fusion polypeptide group, IL-1(Fig. 8 in etanercept group mouse spleen), IL-2(Fig. 9), IL-6(Figure 10), IL-17(Figure 12) and the expression level of TNF α (Figure 13) all significantly reduce, and IL-10(Figure 11) expression level all significantly raise, reduction or the elevation amplitude of IL-17scfv/sTNFRI fusion polypeptide group are maximum, namely the result for the treatment of of IL-17scfv/sTNFRI fusion polypeptide is better than IL-17scfv, IL-1 β scfv/sTNFRI and etanercept, reach significant difference.Result is the mean value of each group of mouse; * representative P < 0.01 compared with CIA model group; # representative compares P < 0.05 with IL-17scfv group, IL-1 β scfv/sTNFRI group with etanercept group.
Five, the monitoring of mice clinical symptoms
Normal group mouse is in each joint of duration of test four limbs without red and swollen phenomenon, and action nimbly and freely.Model group mouse about 10d after first immunisation, occurs that skin of sole of foot is congested, and hover height enough; After 12 ~ 16d, ankle, toe joint swelling are obviously, and toes are in drum Chui shape; The whole metapedes of about 21d, the swelling comprising ankle joint is more obvious, and skin is tight, and metapedes skin surface is congested, cannot bear a heavy burden, RA model modeling success.IL-17scfv/sTNFRI fusion polypeptide and each treatment group, typical arthritic symptom alleviates, and IL-17scfv/sTNFRI fusion polypeptide comparatively IL-1 β scfv/sTNFRI fusion polypeptide, IL-17scfv and etanercept result for the treatment of is obvious.The results are shown in Figure 14, this fusion polypeptide has significant therapeutic effect to mouse rheumatoid arthritis.
< 110 > Harbin Bo'ao Biopharmaceutical Technology Development Co., Ltd.
< 120 > is for the fusion polypeptide of IL-17 and TNF α and application thereof
＜160＞ 4
＜210＞ 1
＜211＞ 280
＜212＞ PRO
< 213 > A fragment amino acid sequence
＜400＞ 1
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
20 25 30
Asp Tyr His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
35 40 45
Glu Trp MET Gly Val Ile Asn Pro MET Tyr Gly Thr Thr Asp Tyr
50 55 60
Asn Gln Arg Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser
65 70 75
Thr Ser Thr Ala Tyr MET Glu Leu Ser Ser Leu Arg Ser Glu Asp
80 85 90
Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Asp Tyr Phe Thr Gly Thr
95 100 105
Gly Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly
110 115 120
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
125 130 135
Ile Val MET Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
140 145 150
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Val His
155 160 165
Ser Arg Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
170 175 180
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ile
185 190 195
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
200 205 210
Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
215 220 225
Tyr Cys Ser Gln Ser Thr His Leu Pro Phe Thr Phe Gly Gln Gly
230 235 240
Thr Lys Leu Glu Ile Lys Arg Thr Glu Pro Lys Ser Ser Lys Tyr
245 250 255
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
260 265 270
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
275 280
＜210＞2
＜211＞840
＜212＞ DNA
< 213 > A fragment-encoding gene sequence
＜400＞ 2
1 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt
60 tcctgcaagg catctggtta ctcattcact gactaccata ttcattgggt gcgacaggcc
120 cctggacaag ggcttgagtg gatgggagta attaatccta tgtatggtac tactgactac
180 aatcagaggt tcaagggcag agtcaccatt accgcggacg aatccacgag cacagcctac
240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagatatgat
300 tactttactg ggacgggtgt gtactggggc caaggaaccc tggtcaccgt ctcctcaggt
360 ggcggtggct ccggcggtgg tgggtcgggt ggcggcggat ctgatattgt gatgactcag
420 actccactct ccctgtccgt cacccctgga cagccggcct ccatctcctg cagatctagt
480 aggagccttg tacacagtcg tggaaacacc tatttacatt ggtacctgca gaagccaggc
540 caatctccac agctcctaat ttataaagtt tccaaccggt ttattggggt cccagacaga
600 ttcagcggca gtgggtcagg cacagatttc acactgaaaa tcagcagggt ggaggccgaa
660 gatgttgggg tttattactg ctctcaaagt acacatcttc cattcacgtt tggccaaggg
720 accaagctgg agatcaaacg aactgagccc aaatcttcca aatatggtcc cccatgccca
780 ccctgcccag cacctgagtt cctgggggga ccatcagtct tcctgttccc cccaaaaccc 840
＜210＞ 3
＜211＞ 160
＜212＞ PRO
< 213 > B fragment amino acid sequence
＜400＞ 3
Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His
1 5 10 15
Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr
20 25 30
Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys
35 40 45
Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu
50 55 60
Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu MET Gly Gln
65 70 75
Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly
80 85 90
Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe
95 100 105
Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu
110 115 120
Ser Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly
125 130 135
Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys
140 145 150
Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu
155 160
＜210＞ 4
＜211＞ 480
＜212＞ DNA
< 213 > B fragment-encoding gene sequence
＜400＞ 4
1 agggagaaga gagatagtgt gtgtccccaa ggaaaatata tccaccctca aaataattcg
60 atttgctgta ccaagtgcca caaaggaacc tacttgtaca atgactgtcc aggcccgggg
120 caggatacgg actgcaggga gtgtgagagc ggctccttca ccgcttcaga aaaccacctc
180 agacactgcc tcagctgctc caaatgccga aaggaaatgg gtcaggtgga gatctcttct
240 tgcacagtgg accgggacac cgtgtgtggc tgcaggaaga accagtaccg gcattattgg
300 agtgaaaacc ttttccagtg cttcaattgc agcctctgcc tcaatgggac cgtgcacctc
360 tcctgccagg agaaacagaa caccgtgtgc acctgccatg caggtttctt tctaagagaa
420 aacgagtgtg tctcctgtag taactgtaag aaaagcctgg agtgcacgaa gttgtgccta
480
Claims (10)
1. a fusion polypeptide, is made up of A fragment and B fragment;
Described A fragment is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
B the aminoacid sequence of sequence 1 is had the protein derived by sequence 1 of identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ();
Described B fragment is following (c) or (d):
C protein that () is made up of the aminoacid sequence shown in sequence in sequence table 3;
D the aminoacid sequence of sequence 3 is had the protein derived by sequence 3 of identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().
2.DNA composition, is made up of with the DNA molecular second of the described B fragment in coding claim 1 the DNA molecular first of the described A fragment in coding claim 1.
3. DNA composition as claimed in claim 2, is characterized in that:
Described DNA molecular first is the DNA molecular of following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA sequence dna limited with (1) is under strict conditions hybridized and the DNA molecular of described A fragment of encoding;
(3) DNA sequence dna limited with (1) has more than 90% homology and the DNA molecular of described A fragment of encoding;
Described DNA molecular second is the DNA molecular of following (4) or (5) or (6):
(4) DNA molecular shown in sequence 4 in sequence table;
(5) DNA sequence dna limited with (4) is under strict conditions hybridized and the DNA molecular of described B fragment of encoding;
(6) DNA sequence dna limited with (4) has more than 90% homology and the DNA molecular of described B fragment of encoding.
4. plasmid composition, is made up of the recombinant plasmid first containing the described DNA molecular first in Claims 2 or 3 and the recombinant plasmid second containing the described DNA molecular second in Claims 2 or 3.
5. one kind is used for the treatment of and/or the medicine of preventing autoimmune disease, its activeconstituents is fusion polypeptide according to claim 1, it is characterized in that: the dual specific Mutiple Targets fusion polypeptide anti-human IL-17 single-chain antibody and Soluble tumor necrosis factor receptor I are connected to form by the hinge area of antibody, i.e. IL-17scfv/sTNFRI, can simultaneously in conjunction with human interleukin-17 and huamn tumor necrosis factory alpha.
6. medicine as claimed in claim 5, is characterized in that: described autoimmune disease is rheumatoid arthritis.
7. medicine as claimed in claim 6, is characterized in that: the curative effect of described medicine is presented as the inflammatory reaction relating to and block rheumatoid arthritis.
8. fusion polypeptide as claimed in claim 1 is preparing the application treated and/or prevented in the medicine of autoimmune disease.
9. apply as claimed in claim 8, it is characterized in that: described autoimmune disease is rheumatoid arthritis.
10. apply as claimed in claim 9, it is characterized in that: the curative effect of described medicine is presented as the inflammatory reaction relating to and block rheumatoid arthritis.
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| CN107857818A (en) * | 2017-08-07 | 2018-03-30 | 上海科新生物技术股份有限公司 | A kind of bispecific fusion protein for IL 17 and TNF α |
| CN109796534A (en) * | 2017-11-16 | 2019-05-24 | 北京比洋生物技术有限公司 | Anti- IL-17 antibody/TNFR ECD fusion protein and application thereof |
| CN116425836A (en) * | 2022-12-16 | 2023-07-14 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | A+ polypeptides/proteins and uses thereof |
| WO2024125445A1 (en) * | 2022-12-12 | 2024-06-20 | 江苏康缘瑞翱生物医药科技有限公司 | BISPECIFIC FUSION PROTEIN TARGETING TNF-α AND IL-17A, AND USE THEREOF |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107857818A (en) * | 2017-08-07 | 2018-03-30 | 上海科新生物技术股份有限公司 | A kind of bispecific fusion protein for IL 17 and TNF α |
| CN109796534A (en) * | 2017-11-16 | 2019-05-24 | 北京比洋生物技术有限公司 | Anti- IL-17 antibody/TNFR ECD fusion protein and application thereof |
| CN109796534B (en) * | 2017-11-16 | 2021-08-10 | 北京比洋生物技术有限公司 | anti-IL-17 antibody/TNFR ECD fusion protein and application thereof |
| US11667704B2 (en) | 2017-11-16 | 2023-06-06 | Beijing Beyond Biotechnology Co., Ltd | Anti-IL-17 antibody/TNFR ECD fusion protein and use thereof |
| WO2024125445A1 (en) * | 2022-12-12 | 2024-06-20 | 江苏康缘瑞翱生物医药科技有限公司 | BISPECIFIC FUSION PROTEIN TARGETING TNF-α AND IL-17A, AND USE THEREOF |
| CN116425836A (en) * | 2022-12-16 | 2023-07-14 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | A+ polypeptides/proteins and uses thereof |
| CN116425836B (en) * | 2022-12-16 | 2024-04-16 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | A+ polypeptides/proteins and uses thereof |
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