CN104945485A - Mimic epitope peptide resistant to IL-6 receptor Tocilizumab and application of mimic epitope peptide - Google Patents

Abstract

The invention discloses mimic epitope peptide resistant to IL-6 receptor Tocilizumab and application of the mimic epitope peptide. The amino acid sequence of the mimic epitope peptide is shown in SEQ ID NO.1-4, the sequence of the mimic epitope peptide may be different from that of an IL-6 receptor, the mimic epitope peptide and the IL-6 receptor are similar in spatial structure, and the mimic epitope peptide and the IL-6 receptor have the same biological activity. Antibodies produced through the mimic epitope immunostimulation body can recognize mimic epitope and can also recognize the IL-6 receptor so that the activity of the IL-6 can be inhibited, the effect same as that of the Tocilizumab is generated, and meanwhile, the problem that passive immunity needs to be performed many times is solved. The mimic epitope peptide resistant to the IL-6 receptor Tocilizumab provides a new drug path for preventing and treating rheumatoid arthritis and other diseases.

Description

The analogue epi-peptide of anti-IL-6 acceptor Tocilizumab and application thereof

Technical field

The present invention relates to a peptide species, be specifically related to analogue epi-peptide and the application thereof of anti-IL-6 acceptor Tocilizumab.

Background technology

Rheumatoid arthritis (RA) is a kind of chronic, systemic, polydirectional Inflammatory Autoimmune disease, mainly involve brothers' Minor articulus, maximum feature is exactly the synovitis of persistence, symptom comprises the destruction of joint of arthralgia, arthroncus and progressivity, finally causes the deformity of patient.RA is found at European the Renaissance, and in current worldwide, the sickness rate of adult is 0.5 ~ 1%.

The paathogenic factor that Zvaifler proposes RA as far back as the seventies is Rheumatoid factors, polyclonal, in his theory, Rheumatoid factors, polyclonal defines immunocomplex and have activated complement pathway, inflammatory factor gathers around rheumatoid patient joint, activate and cause the destruction (Zvaifler in this joint, N. J. The immunopathology of joint inflammation in rheumatoid arthritis. Adv. Immunol.16,265 – 336,1973).And in 1976, it is relevant with mankind's specific white blood cells antigen (HLA)-DR gene that Stastny demonstrates rheumatoid arthritis, transcription product remains in MHC and by angtigen presentation, this research thinks that T cell has vital role (Stastny in rheumatoid arthritis morbidity, P. Mixed lymphocyte c μ ltures in rheumatoid arthritis. J. Clin. Invest.57,1148 – 1157,1976).To the late period of the eighties, the birth of new molecule clone technology and for the synovial membrane that detects Patients With Rheumatoid Arthritis and synovial fluid, thus provide evidence for the effect of T cell in rheumatoid arthritis morbidity.Thrilling discovery is that the concentration of product in the synovial fluid of rheumatoid arthritis of all T cell such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ) is quite low, and scavenger cell and fibrocellular product such as il-1 (IL-1), interleukin-6 (IL-6), interleukin-15 (IL-15), IL-18 (IL-18), tumor necrosis factor-alphas (TNF-α) etc. are quite abundant (Williams then, R. O., Mason, L. J., Feldmann, M. & Maini, R. N. Synergy between anti-CD4 and antitumor necrosis factor in the amelioration of established collagen-induced arthritis. Proc. Natl Acad. Sci. USA 91, 2762 – 2766, 1994, Lubberts, E. et al. IL-17 promotes bone erosion in murine collagen-induced arthritis thro μ gh loss of the receptor activator of NF-kB ligand/osteoprotegerin balance. J. Immunol.170,2655 – 2662,2003).The various cytokines that the synovial cell of the synovial cell of Mierocrystalline cellulose sample and macrophage-like produces activate they oneself or cell around by mesenchymal cell and antigen presenting cell, thus increase the weight of rheumatoid arthritis.In in the past 10 years, the resisting rheumatoid disease medicine (DMARDs) of all kinds of alleviation disease symptomses manufactured by biotechnology progressively enters the treatment of rheumatoid arthritis, these drug mains will to alleviate for the purpose of auto-inflammatory, delay the destruction of joint of patient, thus substantially increase result for the treatment of and the quality of life (Smolen of patient, J. S. et al. New therapies for the treatment of rheumatoid arthritis.Lancet 370,1861 – 1874,2007).The success of DMARDs is that they can act on proinflammatory factor TNF-α, and this excites the research interest of investigators to other cytokines.In these cytokines, interleukin-6 (IL-6) is shown one's talent gradually, becomes a study hotspot.

IL-6 is a polyphenic cytokine, be defined as the B cell differential factor of T cell origin the earliest, elicit B cell differentiation can become plasmocyte (Yoshizaki K et al. Isolation and characterization of B cell differentiation factor (BCDF) the secreted from a human B lymphoblastoid cell line. J Immunol 132:2948 – 2954 that can produce antibody, 1984), it is primarily of monocyte, synovial membrane fibrocyte, B cell and T cell secretion, in immunity system, the physiological functions such as inflammation and liver cell gross stress reaction play an important role in regulating.The disappearance of IL-6 regulatory function, may destroy immunologic function thus cause a series of self immune system inflammatory disease as rheumatoid arthritis, Juvenile idiopathic arthritis, Castelman syndromes and Crohn's disease etc.The excessive secretion of IL-6 can become a somatomedin or anti-apoptosis factor equally, thus causes some malignant diseases as multiple melanoma and kidney.The signal transduction of IL-6 forms primarily of a receptor system, comprise receptors bind membranin IL-6 acceptor (MIL-6 acceptor) and intracellular signaling membranin gp130, a kind of soluble form (SIL-6 acceptor) of IL-6 lacks endochylema structure also can cause the intracellular signaling (Nishimoto in downstream in conjunction with GP130, N. & Kishimoto, T. Interleukin 6:from bench to bedside. Nature Clin. Pract. Rheumatol.2,619 – 626,2006).

In recent years; different data has all been pointed out in the morbidity of IL-6 at rheumatoid arthritis and evolution has vital role; the IL-6 secretion of similar rheumatism patient synovial membrane increases; in patient's synovial fluid, the expression amount of IL-6 increases (Sack gradually; U. et al. Interleukin-6 in synovial fluid is closely associated with chronic synovitis in rheumatoid arthritis. Rheumatol. Int.13.45 – 51,1993; Madhok, R. et al. Serum interleukin 6 levels in rheumatoid arthritis:correlations with clinical and laboratory indices of disease activity. Ann. Rheum. Dis.52,232 – 234,1993).IL-6 is considered to have following functions in the development of rheumatoid arthritis: 1. activate endotheliocyte and produce chemokines and adhesion molecule, thus assemble white corpuscle (Romano at inflammation part, M., M. Sironi, C. Toniatti, N. Polentarutti, P. Fruscella, P. Ghezzi, R. Faggioni, W. Luini, V. van Hinsbergh, S. Sozzani, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity.6:315 – 325, 1997), 2. synovial membrane fibrocyte proliferation (Mihara is stimulated, M., Y. Moriya, T. Kishimoto, and Y. Ohs μ gi. Interleukin-6 (IL-6) induces the proliferation of synovial fibroblastic cells in the presence of soluble IL-6 receptor. Br. J.Rheumatol. 34:321 – 325, 1995) and the propagation (Tamura of osteoclast, T., N. Udagawa, N. Takahashi, C. Miyaura, S. Tanaka, Y. Yamada, Y. Koishihara, Y. Ohs μ gi, K. Kumaki, and T. Taga. Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proc. Natl. Acad. Sci. USA. 90:11924 – 11928, 1993), 3. the acute inflammatory reaction gene (Poli of liver is activated, V., and G. Ciliberto. Transcriptional reg μ lation of acute phase genes by IL-6 and related cytokines. In Liver Gene Expression. F. Tronche and M. Yaniv, editors. R.G. Landes Company Biomedical Publishers, Austin. 131 – 151,, thus cause the release of inflammatory factor 1994).

At present, the drug main being used for the treatment of rheumatoid arthritis will comprise hormone, NSAID (non-steroidal anti-inflammatory drug), improves the antirheumatic (DMARDs) of the state of an illness and emerging biotechnological formulation.Relative to conventional medicament, biotechnological formulation directly acts on main inflammatory factor, can more rapidly, relief of symptoms effectively, suppress articulation structure to destroy.At present, the biotechnological formulation being used for the treatment of RA has monoclonal antibody (monoclonal antibody), cytokine and antagonist, comprises anti-T lymphocytic cell surface differentiation antigen monoclonal antibody, anti-Interleukin 2 Receptor (IL-2R) monoclonal antibody, anti-CD54 receptor monoclonal antibody, anti-tumor necrosis factor (TNF)-Alpha antibodies, rHuIL-1Ra etc.

At the monoclonal antibody that Tocilizumab (ROACTEMRA) antibody through FDA approval in 2010 is a novel target IL-6 acceptor; also be that first clinical trial proves that alone curative effect is better than monoclonal antibody and MTX monoclonal antibody (the Jones G of anti-TNF; Sebba A; Gu J et al. Comparison of tocilizumab monotherapy versus methotrexate monotherapy in patients with moderate to severe rheumatoid arthritis:the AMBITION study. Ann Rheum Dis, 69:88; 96,2010; Kremer JM, et al. Tocilizumab inhibits structural joint damage in rheumatoid arthritis patients with inadequate responses to methotrexate:results from the double-blind treatment phase of a randomized placebo-controlled trial of tocilizumab safety and prevention of structural joint damage at one year. Arthritis Rheum. 63 (3): 609-21,2011).This antibody and DMARDs are with surmounting the alone result for the treatment of of DMARDs (Weinblatt M with also show it simultaneously, et al. Safety of Tocilizumab monotherapy and TCZ plus DMARDs in a US RA population with inadequate response to biologics or DMARDs:The ACT-STAR study. London:EULAR, 2011).Therefore Tocilizumab is considered to be in the new bio immunotherapy medicaments that can surmount anti-TNF therapy in the future.Clinical trial has demonstrated security and the validity of Tocilizumab.

Tocilizumab is a humanized monoclonal antibody, the framework being moved to human IgG1's antibody by the complementary determining region of the IL-6 receptor antibody by mouse-anti people reduces in the contingent immunogenic response of patient, from the binding site of antigen with suppress IL-6 function these two aspects, humanized antibody is the same with initial murine antibody with the mosaic antibody of anti-IL-6 acceptor.And the immunogenicity of humanized antibody is less and the transformation period is longer.Tocilizumab can be specific in conjunction with SIL-6 acceptor and MIL-6 acceptor, and regulate the intracellular signaling of SIL-6 and MIL-6, it obtains the medicine licence of Japan in 2005, be used for the treatment of Castelman syndromes, started to be used for the treatment of rheumatoid arthritis and Juvenile idiopathic arthritis in 2008.When clinical trial results display Tocilizumab and MTX two kinds of medicines of Tocilizumab use simultaneously, effectively can alleviate the symptom of rheumatoid arthritis, control the development of rheumatoid arthritis, efficiently in different experiments group, reach 50% and 70% (Maini RN et al. Efficacy of IL-6 receptor antagonist MRA in rheumatoid arthritis patients with an incomplete response to methotrexate (CHARISMA) [abstract #1704]. Arthritis Rheum 48 (Suppl): S652, 2003).Compared with traditional DMARDS medicine, Tocilizumab is efficient reaches 78%, and DMARD efficiently only have 34% (Nishimoto N et al. Blocking interleukin-6 (IL-6) by Tocilizumab (a humanized anti-interleukin-6 receptor monoclonal antibody) monotherapy reduces joint damage in active rheumatoid arthritis (RA): evidence from a X-ray reader-blinded randomised controlled trial [abstract #L27]. Arthritis Rheum 52 (Suppl), 2005).Tocilizumab, except have good curative effect in rheumatoid arthritis except, also has good curative effect in other autoimmune disorderss such as lupus.Be compared to the antibody of anti-tnf-alpha, its advantage, except better curative effect, is also that in the interference spleen that this antibody is less, IFN-γ produces, thus causes the immunizing power of patient to reduce, and increases the risk infected, and also can not cause drug induced hepatitis simultaneously.

Although the antibody of various anti-tnf-alpha and Tocilizumab monoclonal antibody advance the immunotherapy of rheumatoid arthritis, but monoclonal antibody still also exists many weak points in the immunotherapy of rheumatoid arthritis, as enough tiring for maintenance antibody in the treatment of passive antibody mediated immunity, immunity repeatedly must be carried out; Immunity causes the cost of use of antibody very expensive repeatedly simultaneously.In addition, the untoward reaction of antibody also limit its range of application.So the antibody needed for being produced by active immunity induction body becomes the new selection of investigator, and it can overcome passive immunization and need repeat with non-human portions shortcomings such as the immunity injuries of human body.

In view of this, special proposition the present invention.

Summary of the invention

In order to solve above-mentioned deficiency of the prior art, the invention provides the analogue epi-peptide of a kind of anti-IL-6 acceptor Tocilizumab.

The analogue epi-peptide of this anti-IL-6 acceptor Tocilizumab, aminoacid sequence is as follows shown in any one:

4A124：YHTTDKLFYMMR，SEQ ID NO.1；

4A125：YSAYEFEYILSS，SEQ ID NO.2；

4A126：KTMSAEEFDNWL，SEQ ID NO.3；

4A220：LTSHTYRSQADT，SEQ ID NO.4。

The present invention also provides one to have immunogenic material, and described material is connected with antigen, and described antigen is containing, for example one or more aminoacid sequence any in SEQ ID NO.1 ~ 4; Or described substance gives expression to antigen, described antigen is containing, for example one or more aminoacid sequence any in SEQ ID NO.1 ~ 4.

Immunogenicity (Immunogenicity), refers to the characteristic of energy induce immune response, refers to the characteristic that body can be stimulated to form immunologic effector substance (such as specific antibody) or primed lymphocyte.

Preferably, above-mentioned have immunogenic material, for such as the arbitrary shown aminoacid sequence in SEQ ID NO.1 ~ 4 is coupled to keyhole limpet hemocyanin.Also can select other carrier proteinss such as OVA(Protalbinic acid), BSA(bovine serum albumin) etc., but net effect is best with KLH.

Keyhole limpet hemocyanin (KLH, keyhole limpet hemocyanin) is the protein macromolecule with high degree of immunogenicity, is used for immunogenic preparation, strengthens their immunogenicity as carrier proteins.KLH forms (KLH1+KLH2) by 2 peptide chains are stranded, and about 1600 amino acid of every bar peptide chain, add up 3200, the about 400KD size of molecular weight, is to exist as a carrier proteins, and whole albumen is used for reinforced immunological.KLH can buy commodity or synthesize voluntarily, and aminoacid sequence can obtain from NCBI website: NCBI protein is numbered PDB:4BED_A; PDB:4BED_B; PDB:4BED_C; PDB:4BED_D.

Present invention also offers the encoding gene of the analogue epi-peptide of above-mentioned anti-IL-6 acceptor Tocilizumab.Preferred encoding gene nucleotide sequence is as follows:

4A124 encoding sequence: acg cat cat ata aaa caa ctt atc cgt cgt atg ata, SEQ ID NO.5;

4A125 encoding sequence: tat tct gct tat gag ttt gag tat att ttg tcg tcg, SEQ ID NO.6;

4A126 encoding sequence: aag acg atg agt gct gag gag ttt gat aat tgg ctg, SEQ ID NO.7;

4A220 encoding sequence: ctt acg agt cat act tat cgt tct cag gct gat act, SEQ ID NO.8.

The present invention also provides the biomaterial containing above-mentioned encoding gene, and described biomaterial is expression vector, transgenic cell line or Host Strains.

The present invention also provides the analogue epi-peptide of above-mentioned anti-IL-6 acceptor Tocilizumab as the application of antigen in the medicine preparing induction of antibodies generation.Described antibody can specific recognition IL-6 acceptor.

The present invention also provides above-mentioned and has immunogenic material as the application of antigen in the medicine preparing induction of antibodies generation.Described antibody can specific recognition IL-6 acceptor.

The present invention also provides a kind of pharmaceutical composition, the analogue epi-peptide containing above-mentioned anti-IL-6 acceptor Tocilizumab, or above-mentionedly has immunogenic material; And pharmaceutically acceptable carrier.

The present invention also provides a kind of antibody, is obtained by the analogue epi-peptide immune animal of above-mentioned anti-IL-6 acceptor Tocilizumab, or is obtained by the above-mentioned immunogenic material immune animal that has.The polyclonal antibody that this antibody can obtain for direct immunization animal, the monoclonal antibody also can prepared for immune mouse.

The present invention also provides another kind of pharmaceutical composition, and containing above-mentioned antibody, this pharmaceutical composition is used for preventing and/or treating rheumatoid arthritis; For preventing and/or treating tumour.

Preferably, in above-mentioned pharmaceutical composition, described tumour is histocytic lymphoma or cervical cancer.

The present invention compared with prior art has following beneficial effect:

The present invention screens the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab by display technique of bacteriophage, after mimic epitopes immune mouse, mouse is stimulated to produce antibody, the antibody produced can not only identify mimic epitopes, and IL-6 acceptor can be identified, thus suppress the activity of IL-6, produce and result for the treatment of like Tocilizumab (ROACTEMRA) antibody class.Because Tocilizumab is a kind of monoclonal antibody, its disease therapy is used to belong to passive immunization therapy, namely Toilizumab is directly in conjunction with target protein, thus stop it active, palliating a disease. Tocilizumab will go to beat according at least one moon of consumption and once control disease, not only increases that patient is painful but also medical expense is very expensive.The analogue epi-peptide of anti-IL-6 acceptor Tocilizumab of the present invention is then different, and use this polypeptide immune to belong to active immunity, body produces antibody for polypeptide, and this antibody goes in conjunction with target protein again, stops it active, is similar to vaccine.When early screening find that there is there is RA possible time, by this polypeptide of immunity, induction body produces antibody, in conjunction with target protein, suppresses self abnormal immune, thus prevention or alleviate RA.

Accompanying drawing explanation

Fig. 1 take Tocilizumab as target protein, and by the phage quantity enrichment result of phage display library screening analogue epi-peptide, X-coordinate is biopanning wheel number, and ordinate zou is eluted phage titre.

Fig. 2 be overall phage display library after 4 take turns elutriation, phage and Tocilizumab, normal human IgG and the normal human IgG of BSA(and BSA are negative control) detected result of binding ability.

Fig. 3 is the sequencing result of selected phage clone, and through checking order, the phage clone screened has 7, and figure is the DNA sequence dna of 7 clones.

Fig. 4 is the detected result of 7 phage clones and Tocilizumab and normal human IgG and BSA binding ability, and the phage quantity added is 1 × 10 ⁹in pfu/mL, figure, negative control is normal human IgG.

Fig. 5 is the detected result of 7 phage clones and Tocilizumab and normal human IgG and BSA binding ability, and the phage quantity added is 1 × 10 ⁷in pfu/mL, figure, Isotype control makes a comment or criticism ordinary person IgG.

Fig. 6 is the result of phage clone according to Fig. 4, Fig. 5 and Tocilizumab specific binding assays, wherein 4 clones and restructuring IL-6 acceptor titration competitive assay result.

Fig. 7 is 4 phage clones and restructuring IL-6 acceptor ELISA method competitive assay result.

Fig. 8 is 4 phage clones and the reverse competitive assay result of restructuring IL-6 acceptor ELISA method, and Fig. 8 A ~ D is respectively the experimental result of 4A124,4A125,4A126 and 4A220.

Fig. 9 is 4 energy and the protein sequence figure of the phage-displayed polypeptides of Tocilizumab specific binding, and they comparing with natural IL-6 receptor protein sequence.

Figure 10 is with after analogue epi-peptide immune animal, detects antiserum titre detected result respectively.

Figure 11 be detect mouse resisting anteserum whether can with the natural IL-6 receptors bind immunoblot experiment result on IL-6 receptor-expressing cells surface, swimming lane 1 is for adding Tocilizumab positive control, and swimming lane 2-7 is respectively the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

Figure 12 is immunoblotting competitive assay, experiment purpose be observe mouse resisting anteserum whether can with the natural IL-6 acceptor of Tocilizumab competition binding cell surface, indirect proof antiserum(antisera) and Tocilizumab have identical antigen binding site.Swimming lane 1-4 is that what add is the antiserum(antisera) of mimic epitopes inducing peptide, and swimming lane 5-8 is that add is antiserum(antisera) and the Tocilimab of mimic epitopes inducing peptide.

Figure 13 is cellular immunofluorescence experimental result, experiment purpose be from morphology observe mouse resisting anteserum whether can with the natural IL-6 receptors bind on IL-6 receptor-expressing cells surface.In figure, file A is for adding Tocilizumab positive control, and file B-G respectively is the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

Figure 14 is immunoblot experiment result, experiment purpose be mouse resisting anteserum whether can with the IL-6 receptors bind on the synovial cell surface of rheumatoid arthritis patients.Swimming lane 1 is for adding Tocilizumab positive control, and swimming lane 2-7 respectively is the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

Figure 15 is cellular immunofluorescence experimental result, experiment purpose be from morphology observe mouse resisting anteserum whether can with the IL-6 receptors bind on the synovial cell surface of rheumatoid arthritis patients.In figure, A is for adding Tocilizumab positive control, and in figure, B-G respectively is the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

Figure 16 is antibody dependent cellular cytotoxicity experiment (ADCC) result.Whether experiment purpose is for observing mouse resisting anteserum by the natural IL-6 receptors bind with HeLa, U937 and Jurkat cell surface, thus mediation scavenger cell, NK cell or other immunocytes produce lethal effect to HeLa, U937 and Jurkat cell.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described, to help understanding content of the present invention.

Screening and identification flow process comprises substantially:

1, with Tocilizumab monoclonal antibody for target protein carries out biopanning;

2, the phage display albumen application immunization experiment method screened is identified;

3, the DNA sequencing of phage positive colony is identified, infer its protein sequence;

4, use chemical synthesis to synthesize qualification albumen, this qualification albumen is the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab;

5, analogue epi-peptide immune animal is used;

6, the animal antiserum qualification of tiring;

7, the qualification of the ability of animal antiserum specific binding analogue epi-peptide and natural IL-6 acceptor.

Biomaterial and reagent source:

E.coli ER2738 bacterial strain: NEB company PH.D-12 phage library test kit;

PH.D-12 phage polypeptide display libraries: NEB company PH.D-12 phage library test kit;

Monoclonal antibody Tocilizumab:Roche company provides;

ABTS uses liquid: SIGMA company buys.

phage polypeptide display libraries screens the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab

(1)because copying of phage needs male intestinal bacteria, therefore we select E.coli ER2738 bacterial strain (hereinafter referred to as ER2738) as the carrier of phage replication.ER2738(has tetracyclin resistance) bacterium liquid adds 50 %(volume ratios) glycerine, be stored in-70 DEG C of refrigerators.

(2)in order to determine PH.D-12 phage polypeptide display libraries titre, first we carry out titer determination to it.

1. the picking single bacterium colony of ER2738 of newly recovering, is inoculated in 2mL LB substratum, is expanded to mid-log phase for subsequent use.

2. original PH.D-12 phage polypeptide display libraries (hereinafter referred to as former storehouse) is carried out 10 respectively ^{– 8}, 10 ^{– 9}, 10 ^-10, 10 ^-11dilution.

3. each extent of dilution adds above-mentioned bacterium liquid 200mL.

4. room temperature leaves standstill 5min, adds Top Agar [10g/L microbial culture Tryptones, 5g/L (0.5%) yeast extract, 5g/L (0.5%) NaCl, 1g/L (0.1%) MgCl of the pre-temperature of 4mL to 45 DEG C ₂6H ₂o, 7g/L (0.7%) agar] and 30mL IPTG/Xgal (50g/L IPTG, 40g/L Xgal).

5. mix rapidly, be poured over pre-temperature on the LB flat board of 37 DEG C.

6. rocking flat board makes Top Agar be coated with evenly.

7. cultivate after about 10 hours for 37 DEG C, count blue plaque, calculate PH.D-12 phage polypeptide display libraries titre.

(3)with the non-competing method phage library screening that monoclonal antibody Tocilizumab is screening target

1. with 100mL 0.1M NaHCO ₃(pH 8.6) bag is by 10mg monoclonal antibody Tocilizumab in 96 orifice plates, and 4 DEG C are spent the night.

2. incline coating buffer, and back-off, on clean filter paper, adds Blocking Buffer(Block buffer) 100mL, puts in wet box, places 2 hours for 37 DEG C.

3. incline deblocking damping fluid, and back-off is on clean filter paper, and TBST washes film damping fluid (hereinafter referred to as TBST) and washes 6 times.

4. 10mL former storehouse phage (1.5 × 10 is taken out ¹¹phage) dilute with the TBST of 100mL.Phage is added 96 hole microplates, place 2 hours for 37 DEG C.

5. incline unconjugated phage, and back-off is on clean filter paper.10 times are washed, each 3 minutes with TBST.

6. 100mL Elute Buffer A (Glycine-HCl pH 2.2) is added, shaking table jog 10 minutes.

7. collect supernatant, add 15mL neutralization buffer.

8. take a morsel (1mL) titration; All the other join in ER2738 bacterium and increase, and measure titre.

(4)the amplification of phage

1. by reclaim obtain have phage to be amplified, join 20mL and cultivate OD value and be about in the ER2738 bacterium liquid of 0.3,37 DEG C of shaking table shaking culture 5 hours.

2. collect bacterium liquid to centrifuge tube, 4 DEG C, centrifugal 10 minutes of 10000rpm, shift supernatant in new pipe.

3. 4 DEG C, 10000rpm is heavy centrifugal 10 minutes, carefully draws upper honest and upright and thrifty 80% in new pipe.

4. add 1/6 volume PEG/NaCl, 4 DEG C of placements are spent the night.

5. 4 DEG C, centrifugal 5 minutes of 10000rpm, remove supernatant, centrifugal, removes remaining supernatant.

6. 1mL TBS(Tris-HCl buffer salt solution is used) resuspended precipitation, transfer in 1.5mL Eppendorf pipe.

7. 4 DEG C, centrifugal 5 minutes of 10000rpm.Supernatant is transferred to new 1.5mL Eppendorf centrifuge tube.

8. again use 1/6 volume PEG/NaCl, hatch 60 minutes on ice.

9. 4 DEG C, centrifugal 5 minutes of 10000rpm, abandon supernatant, brief centrifugation, remove remaining supernatant.

10. use 200mL TBS dissolution precipitation, supernatant is moved in new 1.5mL Eppendorf pipe, be the elutriant of amplification.

Take a morsel (10mL) with 10 ^{– 8}, 10 ^{– 9}, 10 ^-10, 10 ^-11dilution titration.

Repeat (3) and (4) step, carry out second, third to take turns and fourth round screening, phage library screening proceeds to second when taking turns, taking turns from second, each is taken turns and all arranges the negative control that normal people's IgG antibody is target protein, phage after being increased by previous round adds negative control hole simultaneously, observes whether have significant difference, the results are shown in Figure 1.Result shows to obtain obvious enrichment in conjunction with the phage of Tocilizumab compared to the phage combining contrast IgG in phage library.

(5)the extracting of the amplification of phage mono-clonal and phage single-chain DNA

1. screen the phage titration that last takes turns (fourth round), method is as above-mentioned.Selected clone number, at the flat board of about 100, is about in the ER2738 bacterium liquid of 0.3 to 2mL jolting bacterium to OD value with the 10mL lancet choicest locus coeruleus of sterilizing.37 DEG C of shaking table shaking culture 4.5 hours.

2. collect bacterium liquid to 1.5mL Eppendorf centrifuge tube, centrifugal 3 minutes of 10000rpm, transfer supernatant is in new pipe.10000rpm is heavy centrifugal 10 minutes, and careful 500mL supernatant of drawing is in new pipe.Add 200mL PEG/NaCl, mixing, room temperature places 10 minutes.

3. centrifugal 10 minutes of 10000rpm, abandons supernatant, heavy centrifugal, carefully removes remaining supernatant.

4. 100mL Iodide Buffer(sodium iodide damping fluid is added) mixing, add 250mL ethanolic soln, room temperature places 10 minutes simultaneously.

5. centrifugal 10 minutes of 10000rpm, abandons supernatant.Wash once with 70% ethanol, precipitation, place 2 minutes for 37 DEG C, air-dry DNA.Add the abundant dissolving DNA of 30mL deionized water.

6. centrifugal 10 minutes of 10000 rpm, get supernatant, deliver to the order-checking of Shanghai Ying Jun company.

(6)the amplification of monoclonal phage

The ER2738 bacterium of 1. getting jolting of spending the night joins the LB substratum of 20mL with 1:100 ratio, add the phage supernatant [in step (5) for small portion that the bacterium liquid supernatant checked order is left and taken] of 5mL simultaneously, 37 DEG C of jolting culturing bacterium 5 hours.

2. centrifugal 10 minutes of 10000 rpm, carefully get supernatant in new pipe.Again centrifugal, centrifugal 10 minutes of 10000 rpm, get the supernatant of 80% in new pipe, add the PEG/NaCl of 1/6 volume, 4 DEG C of placements are spent the night.

3. centrifugal 15 minutes of 10000 rpm, remove supernatant, the heavy centrifugal supernatant as far as possible removing remnants.

4. use 1mL TBS resuspended, transfer in 1.5mL Eppendorf pipe.10000rpm is centrifugal, sucts clearly in pipe new in addition.Add the PEG/NaCl of 1/6 volume, place 1 hour on ice.

5. centrifugal 15 minutes of 10000 rpm, remove supernatant, the heavy centrifugal supernatant as far as possible removing remnants.

6. use 50mL TBS resuspended, titration, 4 DEG C save backup.

the specific binding assays of fourth round enrichment phage library and Tocilizumab

Specific ELISA

1. with 100mL 0.1M NaHCO ₃(pH 8.6) bag is by 1mg monoclonal antibody Tocilizumab in 96 hole microplates, and normal human IgG and bovine serum albumin (BSA) are negative control group.4 DEG C are spent the night.

2. incline coating buffer, and back-off, on clean filter paper, adds Block buffer 100mL, puts in wet box, places 2 hours for 37 DEG C.

3. incline deblocking damping fluid, and back-off is on clean filter paper, and TBST washes 6 times.

4. the phage library [phage library after the amplification that in 1, step (6) is 6. preserved] of whole fourth round enrichment is diluted with the TBST of 100mL.Phage is added 96 hole microplates, place 2 hours for 37 DEG C.

5. incline unconjugated phage, and back-off is on clean filter paper.6 times are washed with TBST.

6. with Block buffer with 1:5000 dilute HRP mark phage-resistance antibody and add 96 hole microplates, 37 DEG C place 1 hour.

7. incline unconjugated antibody, and back-off is on clean filter paper.6 times are washed with TBST.

8. every hole adds the ABTS use liquid of 50mL, places 20 minutes for 37 DEG C.

9. 96 hole microplates are put into Microplate Reader, Bio-rad Model 680 reads the light absorption value at OD405 place.

Result as shown in Figure 2.Elisa result illustrates the screening taken turns through 4, and the total phagocytosis physical efficiency in phage library, well in conjunction with IgG, comprises Tocilizumab and contrast IgG.

phage mono-clonal specific binding assays

Phage mono-clonal DNA sequencing result as shown in Figure 3.Wherein the nucleotide sequence of 4A13,4A113 and 4A133 is shown in sequence table SEQ ID NO:9 ~ 11.In order to identify the ability of phage mono-clonal in conjunction with Tocilizumab monoclonal antibody, we carry out specific ELISA detection equally.

1. with 100mL 0.1M NaHCO ₃(pH 8.6) wraps by 1mg monoclonal antibody Tocilizumab in 96 hole microplates.Normal human IgG and bovine serum albumin (BSA) are negative control group.4 DEG C are spent the night.

2. incline coating buffer, and back-off, on clean filter paper, adds Block buffer 100mL, puts in wet box, places 2 hours for 37 DEG C.

3. incline deblocking damping fluid, and back-off is on clean filter paper, and TBST washes 6 times.

4. by 7 different phage mono-clonals (1 × 10 ⁹pfu/mL) dilute with the TBST of 100mL.Phage is added 96 hole microplates, place 2 hours for 37 DEG C.

5. incline unconjugated phage, and back-off is on clean filter paper.10 times are washed, each 3 minutes with TBST.

6. with Block buffer with 1:5000 dilute HRP mark phage-resistance antibody and add 96 hole microplates, 37 DEG C place 1 hour.

7. incline unconjugated antibody, and back-off is on clean filter paper.6 times are washed with TBST.

8. every hole adds the ABTS use liquid of 50mL, places 20 minutes for 37 DEG C.

9. 96 hole microplates are put into Microplate Reader, Bio-rad Model 680 reads the light absorption value at OD405 place.

Result as shown in Figure 4.Point out in 7 phage clones through mono-clonal Binding experiment result, have 4 phage clone 4A124,4A125,4A126 and 4A220 energy specific in conjunction with Tocilizumab, and not in conjunction with contrast human IgG.The antigen binding site that polypeptide that 4 phage clones are shown is identical with Tocilizumab competition binding instead of other non-specific regions are described.

10. the phage added is worked as in the prompting of phage mono-clonal Binding experiment is 1 × 10 ⁹during pfu/mL, 4 clones are wherein had to show good specificity.Subsequent, with identical experiment step, the phage added is reduced to 1 × 10 by us ⁷, observe the power of these 4 clone's binding abilities, result display wherein has two to clone 4A125 and 4A126 to show stronger binding ability, and as shown in Figure 5, in figure, Isotype control's result makes a comment or criticism ordinary person IgG.

phage competion experiment (one)

More than in experiment, we have proved that phage clone that we screen can specific binding Tocilizumab, and not can be incorporated on normal people IgG, therefore phage clone is not be attached in the Fc section of IgG, subsequent we to prove phage clone whether with IL-6 Receptor Competition in conjunction with Tocilizumab, thus polypeptide entrained by proof phage clone is consistent with the space structure of IL-6 receptor epitope, and identify by Tocilizumab monoclonal antibody.

(1) with 100mL 0.1M NaHCO ₃(pH 8.6) bag is by 1mg monoclonal antibody Tocilizumab in four group of 96 hole microplate, and put in wet box, 4 DEG C are spent the night.

(2) incline coating buffer, and back-off, on clean filter paper, adds Block buffer 100mL, puts in wet box, places 2 hours for 37 DEG C.

(3) incline deblocking damping fluid, and back-off is on clean filter paper, and TBST washes 6 times.

(4) 4 are shown phage clone 4A124,4A125,4A126 and 4A220(equal 1 × 10 of good specificity and stronger binding ability ¹¹pfu/mL) add 96 hole microplates with after the TBST dilution of 100mL, often organize the clone that 96 hole microplates add a numbering, place 2 hours for 37 DEG C.

(5) incline unconjugated phage, and back-off is on clean filter paper.10 times are washed, each 3 minutes with TBST.

(6) in 96 hole microplates, add 100mL Elute Buffer A (Glycine-HCl pH 2.2), IL-6 acceptor and irrelevant polypeptide successively to be at war with.Elute Buffer A 37 DEG C places hypsokinesis in 10 minutes and goes out, and adds 15mL neutralization buffer, places 1 hour in 37 DEG C.

(7) collect supernatant, (1mL) titration that takes a morsel detects the quantity of observing by the phage competed.Result is (in figure, Glycine refers to Elute Buffer A, and IL-6R refers to IL-6 acceptor) as shown in Figure 6.This result further illustrate from the side polypeptide that 4 screened phage clones show can with Tocilizumab competition binding antigen binding site.

phage competion experiment (two)

(1) with 100mL 0.1M NaHCO ₃(pH 8.6) bag is by 10ng monoclonal antibody Tocilizumab in four group of 96 hole microplate, and 4 DEG C are spent the night.

(2) incline coating buffer, and back-off, on clean filter paper, adds Block buffer 100mL, puts in wet box, places 2 hours for 37 DEG C.

(3) incline deblocking damping fluid, and back-off is on clean filter paper, and TBST washes 6 times.

(4) 4 are shown phage clone 4A124,4A125,4A126 and 4A220(equal 1 × 10 of good specificity and stronger binding ability ¹¹pfu/mL) add 96 hole microplates with after the TBST dilution of 100mL, add IL-6 receptor protein 100ng simultaneously.The hole not adding IL-6 receptor protein is negative control.Often organize the clone that 96 hole microplates add a numbering, place 2 hours for 37 DEG C.

(5) incline and not combine or by the phage competed, back-off is on clean filter paper.6 times are washed, each 3 minutes with TBST.

(6) in 96 hole microplates, add the phage-resistance antibody of the HRP mark that 100mL 1:1000 dilutes.Place 1 hour for 37 DEG C.

(7) 6 times are washed with TBST, each 3 minutes.

(8) every hole adds the ABTS use liquid of 50mL, places 20 minutes for 37 DEG C.

(9) 96 hole microplates are put into Microplate Reader, Bio-rad Model 680 reads the light absorption value at OD405 place.

Result as shown in Figure 7.This result further illustrate from the side polypeptide that 4 screened phage clones show can with Tocilizumab competition binding antigen binding site.

phage competion experiment (three)

(1) with 100mL 0.1M NaHCO ₃(pH 8.6) bag is by 10ng IL-6 receptor protein in 96 hole microplates, and 4 DEG C are spent the night.

(2) incline coating buffer, and back-off, on clean filter paper, adds Block buffer 100mL, puts in wet box, places 2 hours for 37 DEG C.

(3) incline deblocking damping fluid, and back-off is on clean filter paper, and TBST washes 6 times.

(4) by phage clone 4A124,4A125,4A126 and 4A220(1 × 10 of 4 display good specificity and stronger binding ability ¹¹pfu/mL, 1 × 10 ¹⁰pfu/mL, 1 × 10 ⁹pfu/mL, 1 × 10 ⁸pfu/mL, 1 × 10 ⁷pfu/mL is totally 5 concentration gradients) add 96 hole microplates with after the TBST dilution of 100mL, add monoclonal antibody Tocilizumab 5ng simultaneously.The hole not adding monoclonal antibody Tocilizumab is negative control.Place 2 hours for 37 DEG C.

(5) incline and not combine or by the phage competed, back-off is on clean filter paper.6 times are washed, each 3 minutes with TBST.

(6) in 96 hole microplates, add the anti-human IgG antibodies of the HRP mark that 100mL 1:1000 dilutes.Place 1 hour for 37 DEG C.

(7) 6 times are washed with TBST, each 3 minutes.

(8) every hole adds the ABTS use liquid of 50mL, places 20 minutes for 37 DEG C.

(9) 96 hole microplates are put into Microplate Reader, Bio-rad Model 680 reads the light absorption value at OD405 place.

Result as shown in Figure 8.This result further illustrate from the side polypeptide that 4 screened phage clones show can with Tocilizumab competition binding antigen binding site.

phage-displayed polypeptides sequence compares with IL-6 receptor protein sequence

Phage mono-clonal DNA sequencing result as shown in Figure 3.An amino acid whose rule is translated into according to 3 bases, last DNA sequence dna is translated as 12 amino acid altogether, aminoacid sequence is shown in Fig. 9, the sequence of polypeptide is compared with natural IL-6 acceptor simultaneously, find all have amino acid to be the key amino acid affecting IL-6 and IL-6 receptors bind in 3 analogue epi-peptides, compare and see Fig. 9.IL-6R (aa, 261-290) is shown in sequence table SEQ ID NO.12, and IL-6R (aa, 211-240) is shown in sequence table SEQ ID NO.13.

analogue epi-peptide immune animal

Analogue epi-peptide is matched Bai Sheng company by Beijing and is synthesized, and coupling carrier keyhole limpet hemocyanin (KLH), purity is 95%.

The method of immune animal is as follows:

(1) preparation of immunizing antigen

The antigen of immunity is respectively polypeptide 4A124 (YHTTDKLFYMMR-KLH), polypeptide 4A125 (YSAYEFEYILSS-KLH), polypeptide 4A126 (KTMSAEEFDNWL-KLH), polypeptide 4A220 (LTSHTYRSQADT-KLH), control peptide KLH (CP-KLH) and KLH.The dosage of each immunity is 100mg albumen, and the adjuvant of application is completely not formula adjuvant (first time immunity) and freurd incomplete adjuvant (second and third, four immunity).

(2) preparation of BALB/c mouse

BALB/c mouse provides by tieing up company of tonneau China, and raising place is Department Of Medicine, Peking University's animal center, SPF level Balb/c mouse, 6 week age, female, be divided into 6 groups, respectively immune 4A124,4A125,4A126,4A220, control peptide KLH (CP-KLH) and KLH.

(3) immunity of BALB/c mouse

BALB/c mouse peritonaeum hemostasis immunity, immunity in the 1st, 22,43,65 day, totally 4 times, each immunity got mouse tail vein blood in latter 7 days.By mice serum packing after each immunity ,-20% preserves.

detect with the Antibody serum titer after analogue epi-peptide immune mouse

(1) preparation of BALB/c mouse serum

BALB/c mouse the 4th immunity gets the fresh mouse blood of about 50mL with root of the tail blood taking method afterwards, often organizes and extracts 3 immediately.Get upper serum after 4000rpm is centrifugal ,-20 DEG C save backup.

(2) ELISA method detects mice serum antibody titer

1. with 100mL 0.1M NaHCO ₃(pH 8.6) wraps respectively and is recombinated IL-6 receptor protein in 96 hole microplates by 1mg, and 4 DEG C are spent the night.

2. incline coating buffer, and back-off, on clean filter paper, adds Block buffer 100mL, puts in wet box, places 2 hours for 37 DEG C.

3. incline deblocking damping fluid, and back-off is on clean filter paper, and PBST washes 6 times.

4. add 96 orifice plates (diluting with Block buffer) by after 1:1000,1:2000,1:5000 gradient dilution serum, hatch 2 hours for 37 DEG C.

5. incline gradient dilution serum, and back-off is on clean filter paper.6 times are washed with PBST.

6. resist with the sheep anti-mouse igg two that Block buffer dilutes HRP mark with 1:1000 and add 96 hole microplates, placing 1 hour for 37 DEG C.

7. incline unconjugated antibody, and back-off is on clean filter paper.6 times are washed with PBST.

8. every hole adds the ABTS use liquid of 50mL, places 20 minutes for 37 DEG C.

9. 96 hole microplates are put into Microplate Reader, Bio-rad Model 680 reads the light absorption value at OD405 place.Result as shown in Figure 10.This result illustrates use immunogenic epitope peptide, control peptide KLH(CP-KLH) and KLH immune animal after, analogue epi-peptide can generation antibody in induced animal body, and this antibody can be recombinated IL-6 acceptor in conjunction with people

10. the serum antibody after Western Blot detection mouse immune and HeLa, U937, the binding ability of Jurkat cell surface IL-6 receptor protein

(1) extracting of albumen

Get HeLa, the U937 of a plate well-grown (6cm) respectively, Jurkat cell removes nutrient solution, with the PBS washed cell of appropriate precooling, in culture dish, add 600mL PBS, cell is scraped with cell, collect in Eppendorf pipe, centrifugal 1 minute of 8000rpm, abandons supernatant; Precipitation adds the cell pyrolysis liquid 100 μ L now joined, and 1:100 adds proteinase inhibitor (Roch, USA) by volume, and piping and druming suspends and precipitates, and places 30 min on ice; Centrifugal 15 minutes of 12000 rpm, collect supernatant, are total protein.BCA method surveys protein concentration.

(2) PAGE electrophoresis glue is prepared

10% separation gel preparation

3.9% spacer gel preparation

(3) loading

The each cell extraction albumen getting 30 μ g respectively adds the mixing of 6mL 6 × protein electrophoresis sample solution, boiling water bath process 3 minutes.Centrifugal 5 minutes of 10000rpm, gets 5mL loading and carries out protein electrophoresis, add the protein standard substance of identical amount simultaneously.

(4) electrophoresis

Constant voltage 80V used in spacer gel, 150V in separation gel, use Tris-Glycine(pH 8.0) damping fluid.Electrophoresis is run out of outside glue to bromine Finland.

(5) transferring film

Get the filter paper onesize with glue and pvdf membrane in advance ready.Pvdf membrane first soaks 15 seconds in methyl alcohol, washing, then turns in damping fluid at electricity and soak 5min; It is wetting that filter paper also turns immersion in damping fluid at electricity.Place filter paper, glue, pvdf membrane, 400mA, electricity turns 70min.

(6) Western trace

Close with Block buffer 4 DEG C and spend the night.Second day, pvdf membrane added the mice serum after the immunity of 1:1000 dilution, and diluent is PBST(PBS damping fluid/0.5% Tween-20, the v/v of the skim-milk containing 0.5%), hatch 2 hours for 37 DEG C; PBST washes film 3 times, each 15min; The sheep anti-mouse igg two anti-(LiCOR company) of the IRDye800 mark of 1:10000 is prepared, incubated at room temperature 1h with the PBST of the skim-milk containing 0.5%; PBST washes film 3 times, each 15min.Band is read on infrared imaging scanner.Result as shown in figure 11.Blotting experiments result shows the antibody capable that produced by the analogue epi-peptide induced animal natural IL-6 acceptor in conjunction with HeLa, U937 and Jurkat cell surface expression.Swimming lane 1 is for adding Tocilizumab positive control, and swimming lane 2-7 is respectively the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

detect the ability of the serum antibody after mouse immune and Tocilizumab competition binding HeLa cell surface IL-6 receptor protein

Method is with step 10.Because the structure of IL-6 receptor protein in HeLa, U937 and Jurkat cell system is identical, therefore this experiment only with HeLa cell representatively, extracts HeLa cell protein and test.Experiment is changed as follows, in step 10(3) in, carry out 8 swimming lane electrophoresis, in step 10(6) in, pvdf membrane (swimming lane 1-4) adds serum with 1:1000, and pvdf membrane (swimming lane 5-8) swimming lane adds Tocilizumab(10 μ g/mL while adding serum).Result as shown in figure 12.Figure 12 shows the antiserum(antisera) competition HeLa cytolemma IL-6 acceptor of Tocilizumab energy and 4a220,4a124,4A125 and 4A126, and the antiserum(antisera) competition Hela cell solubility IL-6 acceptor of 4A220,4A125 and 4A126.The antiserum(antisera) of indirect proof these four clone has identical antigen binding site with Tocilizumab.

serum antibody after Immunofluorescence test mouse immune and HeLa, U937, the binding ability of the surperficial IL-6 receptor protein of Jurkat cell

(1) reagent preparation: 1:4% paraformaldehyde, dissolves paraformaldehyde and stirs until dissolve in 65 DEG C of water, dissolve if incomplete dissolving can add appropriate NaOH.1 week can be preserved.2:10% sheep blood serum.

(2) HeLa taken the logarithm vegetative period, U937, Jurkat cell is inoculated on the cover glass that diameter is 10cm, 37 DEG C of 5% CO ₂incubated overnight.

(3) 3 times are washed with PBS, each 10 minutes.

(4) cell 4% paraformaldehyde fixes 15 ~ 30 minutes in 4 DEG C.

(5) 3 times are washed with PBS, each 10 minutes.

(6) with 10% sheep blood serum (being dissolved in PBS/pH 7.3), room temperature closes 2 hours.

(7) 3 times are washed with PBST, each 10 min.

(8) add positive control antibodies Tocilizumab(and be diluted in PBS with 10mg), test group and negative control group

(9) mice serum (being diluted in PBS/pH 7.3 with 1:1000), incubated at room 1 h.

(10) 3 times × 10 min are washed with PBST.

(11) add sheep anti-mouse igg (1:50) (Sigma company) and the DAPI (1:1000) (Roche company) of FITC mark, be all diluted in PBS, incubated at room 30 min.

(12) 3 times are washed with PBS, each 15 min.

(13) with prolong anti-fade mountant (molecular probe) mounting.

(14) with confocal fluorescent microscope or common fluorescent microscopic examination, take a picture.Result as shown in figure 13.Cellular immunofluorescence result observed the antibody capable that produced by the analogue epi-peptide induced animal natural IL-6 acceptor in conjunction with HeLa, U937 and Jurkat cell surface expression from view of morphology.Picture A is for adding Tocilizumab positive control, and picture B-G is respectively the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

detect the binding ability of the serum antibody after mouse immune and rheumatoid arthritis patients fibroblast-like synoviocyte surface IL-6 receptor protein

Method is with step 10, and from rheumatoid arthritis patients fibroblast-like synoviocyte, extract proteins detects.Result as shown in figure 14.Blotting experiments result shows the antibody capable that produced by the analogue epi-peptide induced animal natural IL-6 acceptor in conjunction with rheumatoid patient fibroblast-like synoviocyte surface expression.Swimming lane 1 is for adding Tocilizumab positive control, and swimming lane 2-7 is respectively the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

the binding ability of the serum antibody after Immunofluorescence test mouse immune and rheumatoid arthritis patients fibroblast-like synoviocyte surface IL-6 receptor protein

Method is with step 11, and from rheumatoid arthritis patients fibroblast-like synoviocyte, extract proteins detects.Result as shown in figure 15.Cellular immunofluorescence result observed the antibody capable that produced by the analogue epi-peptide induced animal natural IL-6 acceptor in conjunction with rheumatoid patient fibroblast-like synoviocyte surface expression from view of morphology.Picture A is for adding Tocilizumab positive control, and picture B-G is respectively the serum after adding mouse immune 4A220,4A124,4A125,4A126, CP-KLH and KLH.

the antiserum(antisera) produced after antibody dependent cellular cytotoxicity experimental observation mouse immune whether can mediated immunity cell to the lethal effect of HeLa, U937 and Jurkat cell.

(1) by HeLa, U937 and Jurkat cell (2.0 × 10 ³-1.0 × 10 ⁴) be inoculated in 96 hole microplates, 37 DEG C of 5%CO ₂incubated overnight.

(2) prepare 30mL serum-free RPMI1640 substratum be positioned over 37 DEG C for subsequent use.

(3) with 4A124, after 4A125,4A126 and 4A220 immune mouse, eyeball depletion method is taked to put to death mouse, alcohol infiltrates, put on Bechtop, in abdominal injection 5mL plasma-free DMEM medium, extrude belly 5-8 time gently, recovery mononuclear macrophage as much as possible, is transferred to 50mL conical centrifuge tube these cells.

(5) minicell is ground filter screen and be placed in 50mL conical centrifuge tube top.After breastbone gently win thymus gland, thymus gland put into grinding filter screen on.The spleen of careful taking-up mouse, be positioned over equally on grinding filter screen, crush and grind Thymus and spleen, makes these cells be entered in 50mL conical centrifuge tube by filter screen.

(6) with 20mL plasma-free DMEM medium flushing filtering net, centrifugal 15 minutes of room temperature 1500rpm, cell needs to be resuspended in plasma-free DMEM medium depending on test.Supernatant is got for subsequent use after mouse blood is centrifugal.

(7) substratum inclined in 96 orifice plates, add the target cell (effect target is than being 100:1) be resuspended in serum-free RPMI1640 substratum, add Tocilizumab(10 μ g/mL) simultaneously, the mice serum (being diluted in substratum with 1:100) of experimental group and negative control group, 37 DEG C of 5% CO ₂cultivate 72 hours.

(8) carefully remove substratum with pipettor, add counting cell kit-8(and be diluted in serum-free RPMI1640 substratum with 1:10), 37 DEG C of 5% CO ₂place 1 hour.

(9) 96 orifice plates are put into Microplate Reader, Bio-rad Model 680 reads the light absorption value at OD450 place.Result display 4A124,4A125 and 4A126 these 3 analogue epi-peptides antiserum(antisera) of inducing can mediated immunity cell to the lethal effect of HeLa cell.These 2 analogue epi-peptides of 4A125 and 4A126 antibody of inducing can mediated immunity cell to the lethal effect of U937 cell.4 analogue epi-peptides antibody of inducing all can not mediated immunity cell to the lethal effect of Jurkat cell.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

SEQUENCE LISTING

 

<110> poplar, unicorn

 

The analogue epi-peptide of <120> anti-IL-6 acceptor Tocilizumab and application thereof

 

<130> P20150054

 

<160> 13

 

<170> PatentIn version 3.3

 

<210> 1

<211> 12

<212> PRT

<213> artificial sequence 4A124

 

<400> 1

 

Tyr His Thr Thr Asp Lys Leu Phe Tyr Met Met Arg

1 5 10

 

 

<210> 2

<211> 12

<212> PRT

<213> artificial sequence 4A125

 

<400> 2

 

Tyr Ser Ala Tyr Glu Phe Glu Tyr Ile Leu Ser Ser

1 5 10

 

 

<210> 3

<211> 12

<212> PRT

<213> artificial sequence 4A126

 

<400> 3

 

Lys Thr Met Ser Ala Glu Glu Phe Asp Asn Trp Leu

1 5 10

 

 

<210> 4

<211> 12

<212> PRT

<213> artificial sequence 4A220

 

<400> 4

 

Leu Thr Ser His Thr Tyr Arg Ser Gln Ala Asp Thr

1 5 10

 

 

<210> 5

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 5

acgcatcata taaaacaact tatccgtcgt atgata 36

 

 

<210> 6

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 6

tattctgctt atgagtttga gtatattttg tcgtcg 36

 

 

<210> 7

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 7

aagacgatga gtgctgagga gtttgataat tggctg 36

 

 

<210> 8

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 8

cttacgagtc atacttatcg ttctcaggct gatact 36

 

 

<210> 9

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 9

agtcagctga ttaggattcc accaagacca atgcca 36

 

 

<210> 10

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 10

attcctatac ccatgcacat tcatctcatg cgacat 36

 

 

<210> 11

<211> 36

<212> DNA

<213> artificial sequence

 

<400> 11

ccttagcagc ggcctctgaa tactactaag aacagt 36

 

 

<210> 12

<211> 30

<212> PRT

<213> artificial sequence

 

<400> 12

 

His Asp Ala Trp Ser Gly Leu Arg His Val Val Gln Leu Arg Ala Gln

1 5 10 15

 

 

Glu Glu Phe Gly Gln Gly Glu Trp Ser Glu Trp Ser Pro Glu

20 25 30

 

 

<210> 13

<211> 30

<212> PRT

<213> artificial sequence

 

<400> 13

 

Asn Pro Arg Trp Leu Ser Val Thr Trp Gly Asp Pro His Ser Trp Asn

1 5 10 15

 

 

Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg Tyr Arg Ala

20 25 30

 

 

Claims (10)

1. the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab, is characterized in that, aminoacid sequence is as follows shown in any one:

4A124：YHTTDKLFYMMR，SEQ ID NO.1；

4A125：YSAYEFEYILSS，SEQ ID NO.2；

4A126：KTMSAEEFDNWL，SEQ ID NO.3；

4A220：LTSHTYRSQADT，SEQ ID NO.4。

2. have an immunogenic material, it is characterized in that, described material is connected with antigen, and described antigen is containing, for example one or more aminoacid sequence any in SEQ ID NO.1 ~ 4; Or described substance gives expression to antigen, described antigen is containing, for example one or more aminoacid sequence any in SEQ ID NO.1 ~ 4.

3. according to claim 2 have immunogenic material, it is characterized in that, for such as the arbitrary shown aminoacid sequence in SEQ ID NO.1 ~ 4 is coupled to keyhole limpet hemocyanin.

4. the encoding gene of the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab according to claim 1.

5. the biomaterial containing encoding gene according to claim 4, described biomaterial is expression vector, transgenic cell line or Host Strains.

6. the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab according to claim 1 is as the application of antigen in the medicine preparing induction of antibodies generation.

7. described in Claims 2 or 3 have immunogenic material as antigen prepare induction of antibodies produce medicine in application.

8. a pharmaceutical composition, is characterized in that, the analogue epi-peptide containing anti-IL-6 acceptor Tocilizumab according to claim 1, or claim 2 ~ 3 is arbitrary described has immunogenic material; And pharmaceutically acceptable carrier.

9. an antibody, by the analogue epi-peptide of anti-IL-6 acceptor Tocilizumab according to claim 1, or is obtained by the immunogenic material immune animal that has described in Claims 2 or 3.

10. a pharmaceutical composition, is characterized in that, containing antibody according to claim 9, this pharmaceutical composition is used for preventing and/or treating rheumatoid arthritis; For preventing and/or treating tumour.