CN101273060A

CN101273060A - Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain (PLAD) of tumor necrosis factor receptors

Info

Publication number: CN101273060A
Application number: CNA2006800298419A
Authority: CN
Inventors: M·莱纳多; G-M·邓; F·K-M·陈; L·郑
Original assignee: US Government
Current assignee: US Government
Priority date: 2005-06-24
Filing date: 2006-06-26
Publication date: 2008-09-24

Abstract

The present invention provides a polypeptide comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD) of a TNF receptor-like receptor. Also provided by this invention is a polypeptide comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD), wherein the PLAD is selected from the group consisting of: the PLAD of a TNF-R, the PLAD of p60, the PLAD of p80, the PLAD of Fas (CD95/APO-1), the PLAD of TRAIL receptors, the PLAD of LT/ssR, the PLAD of CD40, the PLAD of CD30, the PLAD of CD27, the PLAD of HVEM, the PLAD of OX40 and the PLAD of DR4. TNF-R, p60, p80, Fas, TRAIL receptor, LT/ssR, CD40, CD30, CD27, HVEM, OX40, DR4, TROY, EDAR, XEDAR, DCR3, AITR, 4-1BB, DR3, RANK, TACI, BCMA, DR6, DPG, DR5, DCRl AND DCR2 are all members of the TNF receptor superfamily or the TNF-like receptor family. The invention also provides the PLAD for other members of the TNF receptor superfamily. The polypeptides of the present invention can be utilized to inhibit oligomerization of members of theTNF receptor superfamily. These polypeptides can also be utilized to inhibit ligand binding to members of the TNF receptor superfamily. The present invention also provides a composition comprising an inhibitor of TNF receptor oligomerization. Further provided by this invention are members of the TNF receptor superfamily that are lacking a PLAD.

Description

Amelioration of inflammatory arthritis is come in preceding part assembling territory (PLAD) by the target tumor mecrosis factor receptors

The application requires the U.S. Provisional Application No.60/694 with submission on June 24th, 2005, the U.S. Provisional Application No.60/717 that on September 16th, 015 and 2005 submitted to, and 589 is basis for priority, its full text mode is by reference included this specification sheets in.

Technical field

The invention provides the new function of extracellular region conserved domain aspect the non-ligand dependent assembling of specificity of mediation acceptor oligomer of TNF acceptor (TNFR) superfamily member.

Background technology

The TNFR superfamily member contains 1 to 6 kytoplasm intracellular domain that is rich in the structural domain (CRD) of halfcystine, one membrane spaning domain and variable size of extracellular region usually.The member of this receptor family can be attached on the part by the defined TNF cytokine family of structure, function and sequence similarity usually.These acceptors form tripolymer with its active ligand combined (liganded state), and some members are contained the cytoplasmic structure territory that is called as death domain.

According to the present invention, the extracellular region of these acceptors can further characterize by new self-association or homotype association function, and described function is mediated by containing 1 preceding ligand receptor assembling territory (PLAD) of being rich in the structural domain of halfcystine at least.More particularly, the TNFR superfamily member comprises the TNFR of TRAIL acceptor 1, CD40,60kDa and the TNFR of 80kDa, has shown this homotype association function.Other the TNFR superfamily member that comprises Fas, LT β R, CD40, CD30, CD27, HVEM, RANK, OX40 and DR4 contains this PLAD.Described PLAD is that part combination and function of receptors are necessary.Therefore, the TNFR superfamily member seemingly transmits signal by different preformed complex bodys rather than by the single receptor subunit of part inductive crosslinked.Therefore, therefore PLAD can also be blocked function of receptors with the formation of blocking these preformed complex bodys by the pharmaceutical preparation target.

Determined the many microorganisms available strategy (79) of antagonism of all having evolved out at their immunne response.More particularly, some viruses and bacterium can be expressed and be used to regulate or the cell protein homologue of direct blocking immunity effector molecule effect, comprise cytokine and chemokine (80).Identified the viral homologue of the TNF acceptor sample acceptor (vTNFR) of some larger dna viruses, described larger dna virus comprises some poxvirus and human cytomegalic inclusion disease virus.Whether the vTNFR self-association of PLAD mediation or associate with the allos of TNF family receptors exists or its any effect is illustrated as yet.

Two kinds of main sacroiliitis are rheumatoid arthritis (RA) and septic arthritis (SA).RA is a kind of common human autoimmune disease (48) that is attended by chronic arthritis disease and carrying out property osteoclasia.Although do not understand the cause of disease and the pathogenesis of RA as yet fully, relate in the progression of disease cytokine for example TNF-α, IL-1, IL-6 and NF-κ B part receptor activation thing (RANKL) (56-60).Nuclear Factor-Kappa B (NF-κ B) is the key instrumentality (56-60) of these cytokines.TNF-α plays an important role in the pathogenesis of RA, and its antagonist for example etanercept (being also referred to as Enbrel), TNFR II immunoglobulin Fc fusion rotein and infliximab (being also referred to as Remicade), anti-TNF-alpha monoclonal antibodies can improve the clinical course (48) of RA.SA is a kind of the carrying out property fast of being brought out by infectation of bacteria and the joint disease of high-destruction, and wherein TNF-α has also played vital role (49).The experimental arthritis mouse model that is brought out by TNF-α (59), lipopolysaccharides (LPS), CpG-DNA (50,61) and collagen (62) has been used to test new methods of treatment.These reagent can bring out synovitis, pannus formation, bone and cartilage destruction and observed further feature in people RA and SA.

According to the present invention, the interior data of external and body show that TNFR PLAD albumen can suppress TNF-α and its effect consequence in experimental inflammatory arthritis effectively.

Summary of the invention

The preceding part that the invention provides a kind of TNF of comprising acceptor sample acceptor assembles the polypeptide of the isolating aminoacid sequence in territory (PLAD).

The present invention also provides a kind of polypeptide that comprises the isolating aminoacid sequence in preceding part assembling territory (PLAD), and wherein said PLAD is selected from: the PLAD of TNF-R, the PLAD of p60, the PLAD of p80, the PLAD of Fas (CD95/APO-1), the PLAD of TRAIL acceptor, the PLAD of LT β R, the PLAD of CD40, the PLAD of CD30, the PLAD of CD27, the PLAD of HVEM, the PLAD of OX40, the PLAD of DR4, the PLAD of NGFR, the PLAD of Troy, the PLAD of EDAR, the PLAD of XEDAR, the PLAD of DcR3, the PLAD of AITR, the PLAD of 4-1BB, the PLAD of DR3, the PLAD of RANK, the PLAD of TACI, the PLAD of BCMA, the PLAD of DR6, the PLAD of OPG, the PLAD of DRS, the PLAD of DcR1 and the PLAD of DcR2.TNF-R, p60TNFR, p80TNFR, Fas, TRAIL acceptor, LT β R, CD40, CD30, CD27, HVEM, OX40, DR4, NGFR, Troy, EDAR, XEDAR, DcR3, AITR, 4-1BB, DR3, RANK, TACI, BCMA, DR6, OPG, DRS, DcR1 and DcR2 are the member of TNF receptor superfamily (being also referred to as TNF acceptor sample receptor family in this article) entirely.The method that the present invention also provides other member's of TNF receptor superfamily PLAD and those skilled in the art how to identify it.

Polypeptide of the present invention can be used to suppress the self-association of PLAD and TNF receptor superfamily member's oligomerization.These polypeptide also can be used to suppress combining of part and TNF receptor superfamily member.

The present invention also provides a kind of composition of the TNF of comprising acceptor oligomerization inhibitor.The present invention further provides the TNF receptor superfamily member who lacks PLAD.

Description of drawings

Figure 1A shows the TNFR oligomer when lacking part.With the crosslinked H9T cell lymphoma of handling or handling without TNF α through TNF α of DTSSP (7).As shown in the figure, under non-reduced (swimming lane 1-4,9-12) property or reduction (swimming lane 5-8,13-16) property condition, full cellular lysate is carried out electrophoresis, and carry out trace at p60 or p80TNFR.The position of tripolymer (T) and monomer (M) indicated in bracket.Circle has been indicated the nonspecific proteins with anti--p80 antibody cross reaction.Described result has represented three independently experiments.

Figure 1B has illustrated specific p60 TNFR self-association.With p60 Δ CD-GFP-HA (swimming lane 1-3) or pEGFP-N1 (swimming lane 4-6), and with pcDNA3 (swimming lane 1,4) or p60 Δ CD-HA (swimming lane 2,5) or HVEM Δ CD-HA (swimming lane 3,6) transfection 293T cell.As shown in the figure, carry out immunoprecipitation (among superincumbent 2 figure of GFP IP) and carry out trace with anti--GFP antibody with anti--HA antibody (HA WB) or anti--GFP antibody (GFP WB).Last figure and middle figure show sedimentary p60 Δ CD-GFP-HA (or GFP) and p60 Δ CD-HA respectively.Illustrate p60 Δ CD-HA and HVEM Δ CD-HA albumen in the cellular lysate down.The result has represented five experiments.

Fig. 1 C has illustrated the definition in specific p80 self-association and preceding part assembling territory (PLAD).With the plasmid transfection 293T cell shown in the figure top.With the C-terminal specificity of only discerning total length p80 anti--p80 antibody (p80 IP) carries out immunoprecipitation (last figure and middle figure).The p80 of brachymemma or p60 are proteic in the lysate is expressed in shown in figure below.With anti--HA antibody (last figure and figure below) and C-terminal specificity resist-p80 antibody (middle figure) carries out western blotting.Empty circles is represented the p80 of glycosylation and non-glycosylated form.Solid circles is represented the Ig heavy chain.

Fig. 1 D has illustrated that PLAD is enough to satisfy the acceptor self-association.With the plasmid shown in p80 Δ CD-GFP-HA (swimming lane 1-5) and every swimming lane top transfection 293T cell together.Carry out immunoprecipitation and carry out western blotting with anti--GFP antibody with anti--HA antibody.The DCD albumen of co-precipitation and their expression in total cellular lysate are respectively shown in middle figure and figure below.On illustrate sedimentary p60 Δ CD-GFP-HA albumen.

Fig. 1 E has illustrated that PLAD is that TNF α is in conjunction with necessary.Histogram shows combine (25) that 293T transit cell in the transfection of construct shown in the usefulness dyes back receptor expression (by anti--HA dyeing) and they and TNF α.The x-axle is represented fluorescence intensity, and the y-axle is represented cell quantity.Shown in quantity for positive group that compares with the contrast of carrier transfection percentage ratio.

Fig. 2 A has illustrated that the residue among the displacement PLAD can prevent self-association.With shown in plasmid transfection 293T cell.Carry out immunoprecipitation with anti--GFP antibody according to the method among Fig. 1.Carry out western blotting with anti--HA antibody.Last figure and middle figure have shown sedimentary p60 Δ CD-GFP-HA (empty circles) and p60 Δ CD-HA mutain (bracket) respectively.Illustrate the expression of p60 Δ CD-HA mutant (bracket) and HVEM Δ CD-HA (solid circles) in the cellular lysate down.

Fig. 2 B has illustrated the p60 that proved by FRET (fluorescence resonance energy transfer) (FRET) and the homotype self-association of p80 TNFR.With shown in CFP (last figure) and YFP (figure below) plasmid to the histogram of the flow cytometry of the 293T cell of transfection.Dotted line represents to use separately the contrast of CFP transfection, and solid line is represented not use the FRET of TNF α and thick line represents to use the FRET of TNF α.X-axle and y-axle are represented FRET fluorescence intensity and cell quantity respectively.Analyze FRET in the positive group of CFP, all cells also all is the YFP male in the positive group of described CFP.FRET is defined as the fluorescent emission of the YFP that excited by CFP.This result has represented four independently experiments.

Fig. 3 A shows the sequence alignment of TNFR superfamily typical case member's CRD1 (CRD1 (SEQ ID NO:26) of CRD1 (SEQID NO:25), the HVEM of CRD1 (SEQ ID NO:24), the CD40 of CRD1 (SEQ ID NO:23), the LT β R of the CRD1 of p60 (SEQ ID NO:22), p80 and the CRD1 (SEQ ID NO:27) of CD30).The figure shows the halfcystine position of high conservative, described position is used for disulfide linkage and has defined the structural domain that is rich in halfcystine that can give the qualification of TNFR superfamily member.

Fig. 3 B has illustrated the acceptor self-association among other member of TNFR superfamily.With DR4 Δ CD-GFP-HA (swimming lane 1-4) or CD4 Δ CD-GFP-HA (swimming lane 5,6) with p80 Δ CD-HA (swimming lane 1,6), p60 Δ CD-HA (swimming lane 2), HVEM Δ CD-HA (swimming lane 3), DR4 Δ CD-HA (swimming lane 4) or CD40 Δ CD-HA (swimming lane 5) transfection 293T cell.Carry out immunoprecipitation and western blotting with anti--GFP and anti--HA antibody respectively.On illustrate protein precipitation in the immunocomplex, illustrate the DCD protein expression in the cellular lysate down.Solid circles is represented the GFP fusion rotein, and arrow is represented the DCD albumen in the immunocomplex.

Fig. 3 C shows by the DR4 of FRET proof and the associating flow cytometry of specific receptors of CD40.CFP (top) shown in using by the method among Fig. 2 B and YFP (bottom) plasmid are to carrying out transfection.FRET when the background FRET when dotted line is represented to use CFP separately, thick line represent to have CFP fusion rotein and YFP fusion rotein simultaneously.For each group, the x-axle is a FRET intensity, and the y-axle is a cell quantity.

Fig. 3 D shows two kinds of TNFR signal conduction models based on pre-association tripolymer complex body.Reset model (left side) for the pre-assembled chain, oval CRD (by to the direction of film near-end CRD being numbered 1-4), the spot box indicating cytoplasmic structure territory represented from the film far-end.This receptor is to observe from the angle perpendicular to plasma membrane.Roman number is represented the chain in the tripolymer complex body.For this tripolymer clustering model (right side), preassembled TNFR tripolymer on the grey symbolic representation cell surface, the triangle of band circle is represented trimeric TNF α.Numeral 1-3 represents three chains of the acceptor in the pre-assembled tripolymer complex body.This receptor is to observe by the angle of overlooking plasma membrane downwards.

Fig. 4 A shows not to be had under the part bonded situation, and pathogenicity bo Fas sudden change can cause dominant interference.The surface expression of wild-type (WT), Pt 2 (del 52-96) and Pt 6 (A241D) Fas molecule and in conjunction with feature.Left-hand column shows by at the terminal AU-1 epi-position label dyeing that exists of each receptor protein N-the time, transfection surface expression of 24 hours behind the 293T cell.Middle column shows with 10 μ g/ml and resists-the painted same cell of Fas antagonist antibody APO-1 (Kamiya).Right-hand column shows to be designed for by the leucine zipper of modifying comes trimerizing and with resisting-combination of the FasL that leucine zipper mAb (FasL dyestuff) develops by dyeing.Anti--mouse antibodies that antibodies is puted together with phycoerythrin develops.When bracket (bracket) expression is compared with the contrast of untransfected, dyeing is the percentage ratio of the cell of strong positive.In every width of cloth figure, thick line and fine rule are represented signal transfection and cell product untransfected respectively.All histograms have all been represented 10,000 incidents with relative cell counting (Y-axis) mapping of 4 decade scalings interval (4 decade) logarithm fluorescence scales (X-axis).On the FACScalibur flow cytometer that uses Flowjo software (Treestar software), collect data.

Fig. 4 B shows the dominance that the mutant Fas molecule with WT Fas cotransfection causes to be suppressed.With the expression vector shown in the 10 μ g or independent pCI carrier electroporation in the BW cell of the people Fas that does not contain (15) as discussed previously, with 5 μ g pEGFP-N1 (Clontech) mark transfections the cell of GFP.After 24 hours, the anti--Fas mAb APO-1 that measures shown in the adding and the soluble proteins A (Sigma) of 1/20 volume are so that the apoptosis induction maximization.Quantize apoptosis (15) by calculating GFP-male viable cell with flow cytometry and calculating cell loss.

Fig. 4 C has illustrated the self-association of Fas molecule.With expression vector and wild-type (WT) Fas and EC mutant Pt 2 Fas (del 52-96) cotransfection of the Fas of coding HA-mark, the C-end death domain of the Fas of described HA-mark is replaced by green fluorescent protein (HAFas 1-210:GFP).Control cells uses WT Fas and TNF receptor family member simplexvirus to invade the kytoplasm truncation type cotransfection of the HA-mark of medium (HVEM or HveA), and described kytoplasm truncation type has merged GFP (HA HVEM Δ CD:GFP).Press described in the embodiment, the lysing cell lysate carries out immunoprecipitation and carries out electrophoresis with anti--GFP.Survey the trace of protein precipitation with the total length Fas molecule of demonstration with the polyclonal antiserum (C20, Santa Cruz biotechnology) (anti--Fas CT) of anti-FasC-end with the albumen co-precipitation of GFP mark.Also can detect cellular lysate to measure these proteic total amounts by western blotting (WB) with anti--HA-HRP (Roche Molecular Biochemicals) and anti--Fas C20.Empty circles is represented the IgG heavy chain of immunoprecipitation species, and solid circles is represented WT Fas, and arrow is represented the Pt 2Fas albumen of brachymemma.Some represent glycosylated Fas with the top band in the swimming lane of anti--FasC20 trace.

Fig. 5 A shows expression and the function that lacks PLAD or part bonded Fas mutant.Utilize the APO-1 of the terminal Fas mutant of N-and the combination of FasL.As shown in Figure 1A (anti-except replacing with anti--HA-AU1), with the TNFR2 (TNFR2) of the HA mark of the terminal brachymemma of C-to shown in Fas mutant, R86S Fas mutant and the contrast transfection thing of HA mark dye with total expression of the surperficial every kind of mutant of showed cell.

The interaction that Fig. 5 B shows the Fas ectodomain depends on the structural domain in this albumen N-stub area.In swimming lane 1-4, with the Fas 1-210 of the AU-1 mark of death domain-containing not and shown in the Fas mutant or contrast TNF2 albumen (HA TNFR2 Δ CD) the cotransfection 293T cell of HA-mark.Lysate is surveyed to show the albumen of co-precipitation with anti--AU1 immunoprecipitation and with anti--HA.Proved the antibody that used anti-Fas N-end (WB is anti--FasN) contrast trace can be used to measure the proteic amount of AU-1 Fas 1-210 in the lysate.This result has represented three independently transfections.Swimming lane 5-7 show use with Fig. 1 C in used same step, utilize the WT Fas that HAFas1-210:GFP carries out and the co-precipitation of FasR86S mutant.Empty circles is represented the Ig heavy chain of immunoprecipitating antibody, and solid circles illustrates the position of the Fas of immunoprecipitation.

Fig. 5 C has illustrated that the Fas molecule that lacks the self-association structural domain can't be apoptosis-induced.The expression vector transfection BW5147 mouse thymoma cell of Fas molecule shown in being used for 10 μ g.Apoptosis-induced and undertaken quantitatively with 500 μ g/ml solubility APO-1 by the method among Figure 1B.

Fig. 5 D shows no part bonded R86S Fas mutant to apoptosis induced and restraining effect.With the every kind of Fas expression vector of 10 μ g and the GFP plasmid transfection BW cell of 5 μ g.Carry out apoptosis induction and quantitatively by the method among Fig. 2 C, except inducing the apoptosis in the sample of representing with hollow strips with APO-1 and in the sample of representing with solid bars, adding the FasL supernatant of 5%v/v.

Fig. 6 A shows the intermolecular FRET (fluorescence resonance energy transfer) of Fas.Relation between CFP, YFP and the FRET signal in cotransfection shown in point illustrates.Make up CFP and YFP fusion rotein and transfection in the 293T cell, on FACS vantage cell instrument, analyze.Numeral is for having the CFP of FRET signal or the percentage ratio (right upper quadrant) of YFP positive cell.

Fig. 6 B is the comparison of FRET signal between the Fas acceptor of total length and N-terminal deletion.The histogram of FRET signal is to produce in the cell of CFP fluorescence being established door.The YFP fluorescence of all transfectants all is suitable.Thick line is the signal of cotransfection cell, and fine rule is the signal of the independent transfection CFP of each centering construct.

Fig. 6 C show that micro-photobleaching by carrying out YFP on individual cells determines shown in CFP and the right FRET efficient (5 readings of 4-7 cell compartment) of YFP.This digitized representation right average E% and the standard error of each plasmid.

Fig. 7 A has illustrated the pre-association of endogenous Fas receptor chain.With 1 * 10 ⁷Individual H9 lymphoma cell with linking agent DTSSP handle (Pierce handled 30 minutes with 10mM under 4 ℃, and the Tris-Cl with 10mM pH8 stops 15 minutes then) and/or shown under the condition with the antagonist antibody APO-1 of 1 μ g or FasL stimulation 15 minutes.For anti--Fas immunoblotting, before electrophoresis, handle cellular lysate, and survey with anti--Fas C-terminal mAb B10 (Santa CruzBiotechnology) and anti--mouse IgG1-HRP (Southern Biotechnology) with N-glycanase-F (Roche MolecularBiochemicals).

Fig. 7 B after agent treated shown in the usefulness, carries out FADD and caspase-8 trace with lysis, immunoprecipitation and according to before described (11).Marked the position of the cleaved products (p43/41) of caspase-8 behind two kinds of caspase-8 proenzyme isotypes (p54/52) and the p11 caspase protein subunit enzymolysis with arrow.

Fig. 7 C shows the cutting of PARP.37 ℃ down with (A) and (B) used cell aliquots containig in addition cultivation 4 hours and with resist-PARP mAb (Research Diagnostics Inc) pair cell lysate carries out trace.The band of top is 115kD total length PARP, and the below band is the caspase cutting fragment of mark 85kD.This result has represented at least three independently experiments under every kind of condition.

Fig. 8 A has illustrated that dominant interference depends on the N-end of PLAD.Selected ALPS patient (Pt) the Fas sudden change from family that NIH studies is compared.The position of symbol " X " expression point mutation.As figure, show the dominance restraining effect (DI) of Fas inductive apoptosis in the studying of testing by co-precipitation with associating ability of wild-type Fas (SA) and cotransfection.Show the sequence of the coding dominant negative PLAD of patient #1 and #20, described PLAD contains by the sudden change encoded polypeptides.First amino acid open numbering behind the signal peptide.Tilted letter is represented the extra amino acid by the phase shift mutation introducing.

Fig. 8 B does not have dominant interference when showing no PLAD.Fas-susceptibility Jurkat T lymphoma cell is with construct shown in the 10 μ g and 2.5 μ g GFP reporter plasmid transfections.After the transfection 18 hours, the Apo-1 of amount shown in adding in continuous 6 hours also carried out quantitatively apoptosis by dyeing with annexin V-PE (Pharmingen).Percentage ratio is the per-cent of annexin V stained positive GFP (+) cell.These results have represented three independently transfections.

Fig. 9 shows and is used for determining that there are the immunoprecipitation analysis of situation in p80 Chimerical receptor or clipped form.With shown in plasmid transfection 293T cell and collecting cell so that carry out co-immunoprecipitation with the terminal specific antibody of anti--p80 COOH-.Use anti--HA antibody to analyze immunoprecipitate and have a situation (last figure) with what determine p80 Chimerical receptor or brachymemma by the western blot analysis method.Illustrate the expression of HA-labelled protein in the full cellular lysate down.

Figure 10 shows the proteic expression of recombinant bacteria PLAD.(a) how PLAD helps the model of assembling of acceptor tripolymer and part binding reactive.Thereby solubility PLAD albumen can associate, prevent trimeric receptor assembling and block ligand-inductive signal conduction with one receptor chain.(b) gel electrophoresis of the GST of purifying, P60PLAD-GST (P60) and P80 PLAD-GST (P80).The left side shows molecular weight marker, and its size is unit with the kilodalton.(c) western blot analysis of the monoclonal antibody (MAb) of use anti-P60 PLAD (last figure) or P80 PLAD (figure below).Showing P60 PLAD (left figure) or P80 PLAD (right figure) employed in initial gel electrophoresis is the titration of unit with μ g albumen.

Figure 11 shows the effect of PLAD albumen in the necrocytosis of TNF-α inductive.(a) (2ng), after TNF-α (2ng)+P60 PLAD (P60) (40 μ g) handles the L929 cell, utilize the necrocytosis of flow cytometry assessment with substratum, humanTNF-(hTNF).Illustration shows and differs Photomicrograph.The lower right corner has provided the percentage ratio of the viable cell of being established door.Y-axis is propidium iodide (PI) dyeing, and X-axis is FSC (forward angle light scatter distribution).Dead cell shows PI dyeing that increases and the FSC that reduces.(b) mouse TNF-α (mTNF) (2ng) or the protein induced cell loss of PLAD P60 (P60) of hTNF (2ng) and various dose.(c) extinction of the protein induced L929 cell of the PLAD CD40 (CD40) of mTNF (2ng) or hTNF (2ng) and various dose.(d) exist or do not exist under the situation of P60 PLAD (P60), P80 PLAD (P80) and GST, using TNF-α or anti--Fas to handle after 12 hours the extinction of 42.3 Jurkat cells.TNF-α (3ng), P60L are (low; 4.5 P60H (height μ g); 15 μ g), P80L is (low; 4.5 P80H (height μ g); 15 μ g), GST (15 μ g), anti--Fas (10ng).(e), measure caspase-8 activity in the L929 cell with the optical density(OD) (OD) of the substrate conversion of m TNF-α (4ng) processing by existing or do not exist under the situation of P60 PLAD (100 μ g), etanercept (25 μ g), infliximab (20 μ g).

Figure 12 shows P60 and P80 PLAD albumen to arthritic effect, and described sacroiliitis is by bringing out at the intra-articular injection TNF-α of BALB/c mouse and at the intra-articular injection bacterium CpGDNA of C3H/HeJ mouse.(a) the representative Photomicrograph of knee joint HE dyeing tissue slice shows: PBS; 45ng TNF-α; P60 PLAD (100 μ g) and 45ng TNF-α; P80 PLAD (100 μ g) and 45ngTNF-α; CpG DNA (1nM); CpG DNA (1mM) and P60 PLAD (100 μ g).Arrow is represented the concentrated kitchen range district of inflammation.Be labeled as: C (cartilage), JC (joint cavity), ST (synovial tissue), B (bone), and the coating of this arrow points synovial tissue.(b, c) uses TNF-α (TNF) separately or adds P60 PLAD albumen (P60) or analyze with synovitis, pannus, bone and cartilage erosive quantitative tissue that TNF-α adds the experimental group (n=5) that P80 PLAD albumen (P80) handles with TNF-α as shown in the figure.(d) synovitis of each experimental group (n=5), pannus, bone and cartilaginous tissue erosive quantitative tissue are analyzed.These analyses will repeat at least twice.Independent CpG DNA (CpG), CpG DNA add P60 PLAD (P60).Numerical value is mean+SD (s.d.), and * * is for comparing P＜0.01 of treatment group with control group.Intraarticular is inoculated back 3 days and is put to death mouse so that carry out histopathological examination.The result has represented three experiments.

Figure 13 shows the effect to CIA in the DBA/1J mouse body of P60 and P80 PLAD albumen.Blinded trial (a-f).(a) the sufficient pawl photo of the CIA mouse of handling with PBS or P60 PLAD.(b) the HE stained in the CIA joint in 75 days or intraperitoneal injection P60 PLAD 4 weeks of albumen behind the initial immunity of handling with PBS (on every Wendesdays time, each 100 μ g).(c) measurement and the body weight of arthritic severity, sufficient pawl thickness in the CIA mouse of usefulness PBS (n=13 mouse) (square), P60 PLAD albumen (P60) (n=12 mouse) (rhombus) and P80 PLAD albumen (P80) (n=13 mouse) (ellipse) processing.(d) arthritic sickness rate in PBS, P60 and the P80 treatment group.(e) method shown in pressing is handled synovitis, pannus, bone and the assessment of cartilage erosive in the CIA joint section in 4 weeks.(f) IL-1 and IL-6 level in the serum.*=compare P＜0.05 with contrast PBS group.(g) severity of peritoneal injection two all PBS (n=10 mouse) (square), P60PLAD albumen (P60) (n=10 mouse) (rhombus) and etanercept (n=10 mouse) (ellipse) back CIA mouse body intrinsic articulation inflammation.*=compare P＜0.05 with contrast PBS group.(h) the proteic effect of P60 PLAD in the CIA that is set up.The sacroiliitis severity of the CIA mouse that peritoneal injection two all PBS (n=9 mouse) (square), P60 PLAD albumen (P60,400 μ g every other day) (n=10 mouse) (rhombus) back are set up.*=compare P＜0.05 with contrast PBS group.

The expression, PLAD albumen that Figure 14 shows TNFR in the arthritis knuckle suppresses TNF-α bonded and the activation of NF-κ B.(a) handle the immunohistochemistry of TNFR1 and TNFR2 in the arthritis knuckle of the DBA/1J mouse that suffers from CIA of putting to death after 75 days with PBS or P60 PLAD.Brown represents to express the cell of TNFR.(b) after P60 PLAD albumen pre-treatment dyeing, carry out flow cytometry with biotinylated humanTNF-of 50ng (Bt-TNF-α) and various dose.On behalf of independent Bt-TNF-α, Bt-TNF-α, curve add 3 μ g P60 PLAD, Bt-TNF-α to add 15 μ g P60 PLAD, Bt-TNF-α and add 30 μ g P60 PLAD, humanTNF-or negative control.(c) the 50ng humanTNF-'s of etanercept (1 μ g), P60 (1 μ g) or P80 (1 μ g) PLAD albumen (test proteins) immunoprecipitation (IP) shown in the usefulness gel electrophoresis.The western blotting that uses anti-TNF-Alpha antibodies to carry out.(d) there be (+) or do not existing under the proteic situation of (-) P60 PLAD, use the radio-labeling oligonucleotide probe of NF-κ B (left arrow) or OCT1 (right arrow), the electrophoretic mobility change that the nuclear extract that makes from the A3 Jurkat cell of handling through TNF-α is carried out detects.Exist or do not exist under P60 (100 μ g) or P80 (100 μ g) the proteic situation of PLAD, handle at external use mTNF-α (2ng) and to knock out after (/-) isolating monocyte of mouse spleen, the quantitative analysis that transposition is carried out to the nuclear of the NF-κ B p65 in these cells from TNFR1 (e) or TNFR2 (f).*=compare P＜0.05 with control group; *=compare P＜0.01 with control group.Difference in each genetic background between P60 and the P80 treatment group all is significant difference (P＜0.01).

Figure 15 shows the expression that P60PLAD albumen can suppress osteoclast generation and RANK and RANK part (RANKL).(a) collagen initial immunity and handle the immunohistochemical representative Photomicrograph of Calcitonin Receptor in the arthritis knuckle of the DBA/1J mouse of putting to death after 75 days with PBS or P60 PLAD albumen.The light color hatched arrows has been indicated positive staining (being brown in the original color section).(b) effect of PLAD albumen in external TNF-α inductive osteoclast generates.The Photomicrograph that adds the positive osteoclast of the middle TRAP-of bone marrow macrophage (BMM) of 32 μ g P60 PLAD or PBS cultivation with M-CSF and mouse TNF-α (10 μ g), mouse TNF-α (10 μ g).The light color hatched arrows has been indicated the positive osteoclast of anti-tartaric acid phosphatase (TRAP).After the P60 PLAD albumen of various dose shown in the usefulness handled 7 days, the TRAP-positive cell in each hole of BMM culture quantitatively, use measurement microscope.(c) use in the arthritis knuckle of PBS, P60 PLAD albumen processing DBA/1J mouse of execution after 75 days the painted immunohistochemical Photomicrograph of RANK and RANKL then with the collagen initial immunity.Dark (being brown in the original color section) coloured portions is represented the positive staining cell.

Figure 16 shows the aminoacid sequence of people PLAD-GST fusion rotein of the single-letter code form of standard.(a) runic shown in and underscore (amino-acid residue 230-282) are P60 PLAD peptide sequence (SEQ IDNO:59).(b) runic shown in and underscore (amino-acid residue 230-274) are P80 PLAD peptide sequence (SEQID NO:60).What provide with the plain text form in these two portions is the GST protein sequence.

Figure 17 shows the effect of P60 PLAD albumen to TNF-α inductive necrocytosis in the L929 cell.With following agent treated 19 hours cell differ Photomicrograph: (a) independent substratum; (b) humanTNF-(2ng); (c) P60 PLAD (3 μ g)+hTNF-α (2ng); (d) P60 PLAD (30 μ g)+hTNF-α (2ng); (e) infliximab (1 μ g)+hTNF-α (2ng); (f) etanercept (1 μ g)+hTNF-α (2ng); (g) P80 PLAD (30 μ g)+hTNF-α (2ng).400 times of amplifications; (h) exist or do not exist under the situation of P60 PLAD (50 μ g), etanercept (1 μ g) and infliximab (1 μ g), by the extinction of hTNF (2ng) inductive L929 cell.

Figure 18 shows the effect to inflammatory arthritis of GST albumen and P80 PLAD albumen.(a) use separately TNF-α (45ng) (TNF) or TNF-α (45ng) add the quantitative tissue analysis of synovitis, pannus and bone and cartilaginous tissue in the BALB/c mouse experimental group (n=5) that GST albumen (GST, 100 μ g) handles; (b) in C3H/HeJ mouse, use separately CpG DNA (1nM) (CpG) or CpG DNA (1nM) add P80 PLAD albumen (P80,100 μ g).Numerical value is mean+SD.Inoculating back 3 days at intraarticular puts to death mouse so that carry out histopathological examination.The result has represented three experiments.(c) H﹠amp; The Photomicrograph of E stained shows the inflammation and the destruction in the CIA joint of having carried out behind the initial immunity of the peritoneal injection of the P80 PLAD albumen in 4 weeks (100 μ g) by a definite date 75 days.200 times of amplifications.(d) handle synovitis, pannus and bone and the analysis of cartilage erosive quantitative tissue among the CIA of DBA/1J mouse in 4 weeks with P80 PLAD albumen.Compare P＞0.05 with control group.

Figure 19 shows immunohistochemical Photomicrograph.(a) TNFR1 and the TNFR2 in the TNF-α transgenic mice arthritis knuckle of putting to death 6 months the time.Arrow among the TNFR1 figure has marked the deep colour dyeing part (being brown in the original color section) that shows expression of receptor.Arrow among the TNFR2 figure has indicated that the chondrocyte's in cartilage deep high TNFR2 expresses.(b) Calcitonin Receptor in the healthy joint in the arthritis knuckle of the TNF-α transgenic mice of putting to death when big in 6 months and contrast C57BL/6 mouse.(c) in the arthritis knuckle of the TNF-α transgenic mice of putting to death when big in 6 months and RANK and RANKL dyeing in the healthy joint of normal control C57BL/6 mouse.The positive tissue reaction of dark (in the original color section is brown) dyeing expression.300 times of amplifications.

Figure 20 shows proteic immunogenicity of P60PLAD and transformation period.(a) based on from handle the PLAD albumen of purifying the DBA/1 mice serum of putting to death in back 75 days, anti--PLAD P60 antibody horizontal of comparing with typical curve then with PBS, P60 or P80 PLAD albumen with the collagen initial immunity.GST ⁺Be meant and add GST so that from serum, remove GST antibody.(b) the proteic transformation period of P60 PLAD.(c) the proteic transformation period of P80 PLAD.

Figure 21 shows PLAD albumen can suppress TNF-α inductive IkB α degraded.Western blotting shows and exists or do not exist under the situation of P60 (10,50,100 μ g) or P80 (10,50,100 μ g), handle and to knock out (/-) 5 minutes (b) of the isolated monocyte of mouse spleen, 15 minutes (a) at external use mTNF-α (2ng) or exist or do not exist under the situation of GST (10,50,100 μ g) from TNFR1 or TNFR2, after handling these cells 1 hour (c) with mTNF-α (2ng), the IkB α degraded in these cells.

Figure 22 shows the gel electrophoresis that comes the 50ng humanTNF-of immunoprecipitation (IP) with the etanercept of different amounts and P60 PLAD albumen.(a) etanercept shown in (10 μ g) or P60 PLAD albumen (100 μ g) (test proteins).(b) etanercept shown in (0.1,1,10 μ g) or P60 PLAD albumen (0.1,1,10 μ g) (test proteins).Arrow has been indicated the position of TNF albumen on gel.Carry out western blotting with anti-TNF-Alpha antibodies (Ab).

Figure 23 shows the PLAD part of dimerization in the TNFR1 crystalline structure (PDB ID:1NCF).Dark-grey part and light gray are partly represented the dimeric single peptide chain of PLAD.Marked mirror image (mirror) imidazole ring of His-34 in each peptide in the circle.These pairs Histidine is fastened to each other in the interchain bag.

Figure 24 shows and proteic immunoprecipitation of etanercept blended P80 PLAD (IP) and western blotting (WB).

Figure 25 shows the MMP that P60-PLAD albumen can suppress among the CIA and expresses.Annotate: dark among the left figure (being brown in the original color section) coloured portions represents that MMP expresses.

Figure 26 shows the iNOS that P60-PLAD albumen can suppress among the CIA and expresses.Annotate: dark among the left figure (being brown in the original color section) coloured portions represents that iNOS expresses.

Embodiment

With reference to hereinafter about the preferred embodiment of the invention and the detailed description of contained embodiment wherein, and with reference to accompanying drawing and about they above and description hereinafter, can more easily understand the present invention.

Based on context, used " one " can represent one (kind) or a plurality of (kinds) in specification sheets and the claim.Be meant when therefore, for example, mentioning " a kind of nucleic acid " and used at least a nucleic acid.

Polypeptide

The invention provides a kind of polypeptide that comprises the isolating aminoacid sequence in preceding part assembling territory (PLAD).The present invention also provides a kind of polypeptide of being made up of the aminoacid sequence in preceding part assembling territory.PLAD of the present invention can be the PLAD of PLAD, DR4 of PLAD, OX40 of PLAD, HVEM of PLAD, CD27 of PLAD, CD30 of PLAD, CD40 of PLAD, LT β R of PLAD, TRAIL of PLAD, Fas (CD95/APO-1) of PLAD, p80 of PLAD, p60 of TNF-R or any other PLAD structural domain of TNFR superfamily member.Because the PLAD structural domain is a high conservative in the TNFR superfamily member, those skilled in the art can identify the PLAD structural domain of any TNF acceptor by the conservative motif of the described PLAD structural domain of search sign in available database.The evaluation in these zones compares to finish (for example, referring to Fig. 3) with conventional means by exemplary PLAD sequence provided herein and with them and the open sequence of other member of this family in the TNF acceptor sample acceptor.In addition, those skilled in the art also should be able to identify PLAD by functional examination (for example functional examination that is provided among the embodiment) is provided.In one embodiment, the described functional PLAD PLAD (83) that is not Fas/CD59.In another embodiment, described functional PLAD is not aminoterminal 49 amino acid of Fas/CD59 acceptor (83).

The PLAD that this paper provided can only comprise 38 amino acid of sophisticated TNF acceptor sample acceptor N-end.Sophisticated TNF acceptor sample acceptor is the TNF acceptor sample acceptor that does not contain signal sequence.Disclose the example of PLAD in the sequence table, this sequence table comprises the aminoacid sequence of the TNF acceptor sample acceptor example that contains its signal sequence.Can find the signal sequence residue of each acceptor with reference to the GenBank registration number of these TNF acceptor sample acceptors of listing in the table 1.Therefore, the sequence of mature T NF acceptor sample acceptor and corresponding PLAD thereof is disclosed in the sequence of being given.Table 3 provides the Additional Information about TNF acceptor sample acceptor disclosed herein and receptors ligand.It also provides about the isolating PLAD and the information of purposes that contains the polypeptide of isolating PLAD disclosed herein.Described PLAD can be used to study the meaning of the signal transduction path that disturbs the mediation of TNFR superfamily receptors.For example, if be known or be proved to be relevant, then can treat and obtain this disease of prevention by the preceding part assembling of polypeptide inhibition acceptor of the present invention with the disease approach via the signal conduction of TNFR superfamily receptors.For example, the disease that can be treated comprises cancer, heart trouble and inflammatory diseases.The modification of PLAD also can change the interactional avidity of ligand/receptor, and this can be used in vitro study, for example measures combination, receptor signal of part and acceptor etc.Fluorescently-labeled PLAD albumen also can be used as the reagent when determining specificity T NFR relative expression on the cell surface by flow cytometry or fluorescent microscopy.

The present invention also provides 38 to 125 amino acid whose polypeptide that contain separative PLAD.For example, described peptide can be 50 to 125 amino acid whose polypeptide that contain separative PLAD.In a further example, described polypeptide can comprise subsequence R ₁-TNF acceptor sample acceptor PLAD-R ₂, R wherein ₁And R ₂Choose wantonly, they can be H, acyl group, NH when existing ₂, amino acid or peptide.When existing, R ₁And/or R ₂Can be any amino acid.Work as R ₁And/or R ₂When being peptide, the length of this peptide is variable.For example, R ₁And/or R ₂Length can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or more a plurality of amino acid, be no more than 125 amino-acid residues as long as contain the whole peptide of separative TNF sample PLAD, and its length can be 38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124 or 125 amino acid.R1 and R2 also can be the sequences of TNF acceptor sample acceptor, described sequence generally is arranged in the side of naturally occurring TNF acceptor sample acceptor TNF acceptor sample acceptor PLAD, and the polypeptide of the wherein said TNF of containing sample acceptor PLAD is not the whole ectodomain of TNF acceptor sample acceptor.

The present invention further provides the polypeptide of any size, the isolating aminoacid sequence that comprises the preceding part assembling territory (PLAD) of TNF acceptor sample acceptor, wherein said polypeptide is R1-TNF acceptor sample acceptor PLAD-R2, and wherein R1 or R2 comprise and be not positioned at the lateral aminoacid sequence of TNF acceptor sample acceptor PLAD in the naturally occurring TNF acceptor sample acceptor.R1 or R2 (but being not both whiles) can be all or part of sequences of TNF acceptor sample acceptor, and described sequence is usually located at the side of TNF sample PLAD in the naturally occurring TNF acceptor sample acceptor.For example, PLAD can not be present in the described TNF acceptor sample acceptor or the aminoacid sequence in any other TNF acceptor sample acceptor from TNF acceptor sample acceptor and R1 or R2, and the TNF sample PLAD of described polypeptide comes from described TNF acceptor sample acceptor.R1 or R2 can be any aminoacid sequences, as long as R1-TNF sample PLAD-R2 is not naturally occurring total length TNF acceptor sample acceptor.In another example, PLAD can if present, can be the peptide sequence from another TNF acceptor sample acceptor from TNF acceptor sample acceptor and R1 or R2 or both.Therefore, those skilled in the art can combine the PLAD of a TNF acceptor sample acceptor to obtain this peptide species with R1 or R2 sequence from a different TNF acceptor sample acceptor.Because the sequence of known TNF acceptor sample acceptor can openly obtain,, but also be known and considered by this paper so the structure of the R1 of polypeptide of the present invention and R2 is a lot.Perhaps, R1 or R2 can be not with the relevant peptide sequence of any TNF acceptor sample receptor sequence.In one embodiment, the described polypeptide that contains separative PLAD is not a United States Patent (USP) 5,633,124 amino acid whose sequences of disclosed maturation (not containing signal sequence) TNF1 acceptor among 145 (the Feldman et al.).

The example that contains the polypeptide of above-mentioned subsequence comprises R ₁1-38, the 1-39 of-ripe p60,1-40,1-41,1-42,1-43,1-44,1-45,1-46,1-47,1-48,1-49,1-50,1-51,1-52,1-53 or 1-54 amino acids-R ₂(for example SEQ ID NO:1); R ₁10-48, the 10-49 of-ripe p80,10-50,10-51,10-52,10-53 or 10-54 amino acids-R ₂(SEQ ID NO:2); R ₁1-38, the 1-39 of-ripe Fas, 1-40,1-41,1-42 or 1-43 amino acids-R ₂(SEQ IDNO:3); R ₁1-38, the 1-39 of-ripe Fas, 1-40,1-41,1-42,1-43,1-44,1-45,1-46,1-47,1-48,1-49,1-50,1-51,1-52,1-53,1-54,1-55,1-56,1-57,1-58,1-59,1-60,1-61,1-62,1-63,1-64,1-65 or 1-66 amino acids-R ₂(SEQ ID NO:4); R ₁13-50 amino acids-R of-ripe Lt β R ₂(SEQ ID NO:5); R ₁6-39 amino acids-R of-ripe CD40 ₂(SEQ ID NO:6); R ₁11-49, the 11-50 of-ripe CD30 or 11-51 amino acids-R ₂(SEQ ID NO:7); R ₁7-42 amino acids-R of-ripe CD27 ₂(SEQ ID NO:8), R ₁6-37 amino acids-R of-ripe HVEM ₂(SEQ ID NO:9); R ₁3-36 amino acids-R of-ripe OX40 ₂(SEQ ID NO:10) and R ₁109-138 amino acids-R of-ripe DR4 ₂(SEQ ID NO:11).

Ripe p60 (TNFR1) polypeptide originates in the 30th of the total length p60 encoding sequence that is represented as SEQ ID NO:12.Therefore, the invention provides a kind of polypeptide (the 30-83 amino acids of SEQ ID NO:12) that contains the proteic 1-54 amino acids of ripe p60, a kind of polypeptide (the 30-82 amino acids of SEQ ID NO:12) that contains the proteic 1-53 amino acids of ripe p60, a kind of polypeptide (the 30-81 amino acids of SEQ ID NO:12) that contains the proteic 1-52 amino acids of ripe p60, a kind of polypeptide (the 30-80 amino acids of SEQ ID NO:12) that contains the proteic 1-51 amino acids of ripe p60, a kind of polypeptide (the 30-79 amino acids of SEQ ID NO:12) that contains the proteic 1-50 amino acids of ripe p60, a kind of polypeptide (the 30-78 amino acids of SEQ ID NO:12) that contains the proteic 1-49 amino acids of ripe p60, a kind of polypeptide (the 30-77 amino acids of SEQID NO:12) that contains the proteic 1-48 amino acids of ripe p60, a kind of polypeptide (the 30-76 amino acids of SEQ ID NO:12) that contains the proteic 1-47 amino acids of ripe p60, a kind of polypeptide (the 30-75 amino acids of SEQ ID NO:12) that contains the proteic 1-46 amino acids of ripe p60, a kind of polypeptide (the 30-74 amino acids of SEQ ID NO:12) that contains the proteic 1-45 amino acids of ripe p60, a kind of polypeptide (the 30-73 amino acids of SEQ ID NO:12) that contains the proteic 1-44 amino acids of ripe p60, a kind of polypeptide (the 30-72 amino acids of SEQ ID NO:12) that contains the proteic 1-43 amino acids of ripe p60, a kind of polypeptide (the 30-71 amino acids of SEQ ID NO:12) that contains the proteic 1-42 amino acids of ripe p60, a kind of polypeptide (the 30-70 amino acids of SEQID NO:12) that contains the proteic 1-41 amino acids of ripe p60, a kind of polypeptide (the 30-69 amino acids of SEQ ID NO:12) that contains the proteic 1-40 amino acids of ripe p60, a kind of polypeptide (the 30-68 amino acids of SEQ ID NO:12) that contains the proteic 1-39 amino acids of ripe p60 and other contain the proteic segmental polypeptide of the active 1-54 amino acids of PLAD that kept of ripe p60.

Ripe p80 (TNFR2) polypeptide originates in the 23rd of the total length p80 encoding sequence that is represented as SEQ ID NO:13.Therefore, the invention provides a kind of polypeptide (the 32-76 amino acids of SEQ ID NO:13) that contains the proteic 10-54 amino acids of ripe p80, a kind of polypeptide (the 32-75 amino acids of SEQ ID NO:13) that contains the proteic 10-53 amino acids of ripe p80, a kind of polypeptide (the 32-74 amino acids of SEQ ID NO:13) that contains the proteic 10-52 amino acids of ripe p80, a kind of polypeptide (the 32-73 amino acids of SEQ ID NO:13) that contains the proteic 10-51 amino acids of ripe p80, a kind of polypeptide (the 32-72 amino acids of SEQ ID NO:13) that contains the proteic 10-50 amino acids of ripe p80 and other contain the proteic segmental polypeptide of the active 10-54 amino acids of PLAD that kept of ripe p80.

Ripe Fas receptor polypeptides originates in the 17th of the total length Fas encoding sequence that is represented as SEQ ID NO:14.Therefore, the invention provides a kind of polypeptide (the 17-59 amino acids of SEQ ID NO:14) that contains the proteic 1-43 amino acids of ripe Fas, a kind of polypeptide (the 17-58 amino acids of SEQ ID NO:14) that contains the proteic 1-42 amino acids of ripe Fas, a kind of polypeptide (the 17-57 amino acids of SEQ ID NO:14) that contains the proteic 1-41 amino acids of ripe Fas, a kind of polypeptide (the 17-56 amino acids of SEQ ID NO:14) that contains the proteic 1-40 amino acids of ripe Fas, a kind of polypeptide (the 17-55 amino acids of SEQ ID NO:14) that contains the proteic 1-39 amino acids of ripe Fas and other contain the proteic segmental polypeptide of the active 1-43 amino acids of PLAD that kept of ripe Fas.

The present invention also provides a kind of polypeptide (the 17-82 amino acids of SEQ IDNO:14) that contains the proteic 1-66 amino acids of ripe Fas, a kind of polypeptide (the 17-81 amino acids of SEQ ID NO:14) that contains the proteic 1-65 amino acids of ripe Fas, a kind of polypeptide (the 17-80 amino acids of SEQ ID NO:14) that contains the proteic 1-64 amino acids of ripe Fas, a kind of polypeptide (the 17-79 amino acids of SEQ ID NO:14) that contains the proteic 1-63 amino acids of ripe Fas, a kind of polypeptide (the 17-78 amino acids of SEQ ID NO:14) that contains the proteic 1-62 amino acids of ripe Fas and other contain the proteic segmental polypeptide of the active 1-66 amino acids of PLAD that kept of ripe Fas.

The present invention also provides a kind of polypeptide that contains the proteic 43-80 amino acids of total length LT β R that is represented as SEQ ID NO:15 and other maintenance that the contains SEQ ID NO:15 segmental polypeptide of the active 43-80 amino acids of PLAD.

The present invention also provides a kind of polypeptide that contains the proteic 26-59 amino acids of total length CD40 that is represented as SEQ ID NO:16 and other maintenance that the contains SEQ ID NO:16 segmental polypeptide of the active 26-59 amino acids of PLAD.

Ripe CD30 polypeptide originates in the 19th of the total length CD30 encoding sequence that is represented as SEQ ID NO:17.Therefore, the invention provides a kind of polypeptide (the 29-69 amino acids of SEQ ID NO:17) that contains the proteic 11-51 amino acids of ripe CD30, a kind of polypeptide (the 29-68 amino acids of SEQ ID NO:17) that contains the proteic 11-50 amino acids of ripe CD30, a kind of polypeptide (the 29-67 amino acids of SEQ ID NO:17) that contains the proteic 11-49 amino acids of ripe CD30, a kind of polypeptide (the 29-66 amino acids of SEQ ID NO:17) that contains the proteic 11-48 amino acids of ripe CD30, a kind of polypeptide (the 29-65 amino acids of SEQ ID NO:17) that contains the proteic 11-47 amino acids of ripe CD30 and other contain the proteic segmental polypeptide of the active 11-51 amino acids of PLAD that kept of ripe CD30.

The present invention also provides a kind of polypeptide that contains the proteic 27-62 amino acids of total length CD27 that is represented as SEQ ID NO:18 and other maintenance that the contains SEQ ID NO:18 segmental polypeptide of the active 27-62 amino acids of PLAD.

The maintenance that the present invention also provides a kind of polypeptide that contains the proteic 42-75 amino acids of total length HVEM that is represented as SEQ ID NO:19 and other to comprise to contain SEQ ID NO:19 the polypeptide of polypeptide fragment of the active 42-75 amino acids of PLAD.

Ripe OX40 polypeptide originates in the 29th of the total length OX40 encoding sequence that is represented as SEQ ID NO:20.Therefore, the invention provides a kind of polypeptide (the 31-64 amino acids of sEQ ID NO:20) that contains the proteic 3-36 amino acids of ripe OX40, a kind of polypeptide (the 31-63 amino acids of SEQ ID NO:20) that contains the proteic 3-35 amino acids of ripe OX40, a kind of polypeptide (the 31-62 amino acids of SEQ ID NO:20) that contains the proteic 3-34 amino acids of ripe OX40, a kind of polypeptide (the 31-61 amino acids of SEQ ID NO:20) that contains the proteic 3-33 amino acids of ripe 0X40, a kind of polypeptide (the 31-60 amino acids of SEQ ID NO:20) that contains the proteic 3-32 amino acids of ripe CD30 and other comprise and contain the proteic polypeptide that has kept the polypeptide fragment of the active 3-36 amino acids of PLAD of ripe OX40.

The maintenance that the present invention also provides a kind of polypeptide that contains the proteic 132-170 amino acids of total length DR4 that is represented as SEQ ID NO:21 and other to comprise to contain SEQ ID NO:21 the polypeptide of polypeptide fragment of the active 132-170 amino acids of PLAD.

Table 1 has been enumerated the example of the TNF acceptor sample acceptor that contains PLAD of the present invention.According to listing Nucleotide and the peptide sequence that the GenBank registration number can find these acceptors in the table 1.Include this specification sheets in according to the full content mode by reference of listing the shown nucleotide sequence of GenBank registration number, peptide sequence and any information (for example signal sequence and maturation protein residue number) in the table 1.For example, can find nucleotide sequence, peptide sequence and the Additional Information (for example signal sequence and maturation protein residue number) of p60 according to GenBank registration number M75866.Full content mode by reference according to these p60 sequences shown in the GenBank registration number M75866 and Additional Information is included this specification sheets in.Similarly, can find at the shown nucleotide sequence of p80, peptide sequence and Additional Information (for example signal sequence and maturation protein residue number) according to GenBank registration number M32315.Include this specification sheets in according to shown these p80 sequences of GenBank registration number M32315 and the whole modes by reference of Additional Information.By the GenBank registration number of listing in the access list 1, those skilled in the art can have access to the additional GenBank registration number wherein listed with acquisition and the signal sequence Additional Information relevant with the maturation protein sequence.For example, when visit GenBank registration number M75866, those skilled in the art can have access to the signal sequence of expression p60 and the GenBank registration number AAA61201 of maturation protein sequence information.This information also can find by direct visit GenBank registration number AAA51201 (p60), GenBank registration number AAA59929 (p80), GenBank registration number AAA63174 (Fas), GenBank registration number AAA36757 (LTBR), GenBank registration number CAA43045 (CD40), GenBank registration number AAA51947 (CD30), GenBank registration number AAA58411 (CD27), GenBank registration number AAB58354, GenBank registration number CAA53576 (OX40), GenBank registration number AAC51226 (DR4), and this information mode is by reference included this specification sheets in.

Table 1 also provides the Locus Link registration number of TNF sample acceptor.Now Locus Link registration number is equal to the Entrez Gene identifier (Gene ID number) that the American National biotechnology information center at u.s. national library of medicine can have access to.For example, those skilled in the art can obtain the Additional Information about p60 by Locus Link number 7132 (being the Gene ID7132 among the Entrez Gene now) in the visit EntrezGene database, comprise Nucleotide and protein sequence.Similarly, those skilled in the art can obtain the Additional Information about p80 by Locus Link number 7133 (being the Gene ID7133 among the EntrezGene now) in the visit Entrez Gene database, comprise Nucleotide and protein sequence.Therefore, those skilled in the art can obtain the information relevant with them at an easy rate by the corresponding Locus Link (Gene ID) of any TNF sample acceptor listed in the access list 1 in Entrez Gene number.All include this specification sheets in according to the full content mode by reference of the information that provides for the Locus Link that lists in the table 1 (Gene ID) number.

The polypeptide (SEQ IDNO:28-39) of the isolating aminoacid sequence that contains vTNFR albumen PLAD structural domain is provided.Can use the homology search that is used for TNFR1 and TNFR2 PLAD sequence of protein-protein BLAST database to identify other vTNFR PLAD.The full length amino acid sequence (SEQ ID NO:44-55) that also comprises every kind of vTNFR and modified protein thereof.Also provide can by those skilled in the art be used for to other vTNFR PLAD polypeptide identify, the method for production and function test etc.

VTNFR PLAD structural domain can interrupt the self-association of host TNFR and/or part subsequently exempts from the necrocytosis that TNF mediates in conjunction with the cell that infects with inhibition antiviral immunity and/or protection.The T cell that M-T2 albumen--a kind of TNF receptor-like protein by the myxoma virus coding--can protect myxoma to infect exempts from the death of TNF inductive and does not rely on the outer TNF binding ability (82) of its born of the same parents.Because at selecting with the evolution of host TNFR PLAD structural domain bonded higher affinity, vTNFR PLAD sequence can be as than P60 or the more effective TNF inducing action inhibitor of P80 PLAD itself.In this, isolating viral PLAD albumen has been represented the improvement reagent that is used to block the relevant clinical application of falling ill of the TNF-relevant with other autoimmune disorders with rheumatoid arthritis.

It should be understood that these technology for example as herein described, can be used to identify other microbial proteinous, the PLAD structural domain that exists in described albumen and other TNF acceptor sample acceptor (for example Fas) described herein has homology.For example disclose and contained and described Fas PLAD structural domain (SEQ ID NO.41-43,56-58) three examples of the microorganism polypeptide of homologous sequence.

" the isolating aminoacid sequence of PLAD " used herein is meant the sequence that is substantially free of at usually relevant with the described aminoacid sequence naturally occurring material of occurring in nature.Polypeptide of the present invention can comprise having the active PLAD structural domain of PLAD or its segmental whole aminoacid sequence.Polypeptide of the present invention or its fragment can be by separating from cell and the described polypeptide of purifying obtain, and wherein these polypeptide can natural generations or produced by the exogenous nucleic acid of expressing the PLAD that encodes.The fragment of PLAD can be by peptide chemosynthesis, by PLAD or contain PLAD polypeptide the proteolysis cutting and obtain by synthesizing with coding purpose nucleic acid partly.Described PLAD can comprise preservative replacement, and the amino acid that wherein naturally occurring amino acid is had similarity is replaced.This class preservative replacement can not change the function of this polypeptide.By producing various amino-acid substitutions and can finding the sudden change that can strengthen combination and validity they are tested described in the specification sheets in conjunction with the available technology of use those of ordinary skills in measuring.

Therefore, it should be understood that if desired, can in the nucleic acid of code book invention polypeptide and/or amino acid sequence of polypeptide of the present invention, modify and change and still obtain polypeptide with similar or required feature.This class change may reside in the natural isolate or can utilize rite-directed mutagenesis to introduce by synthesizing mean, and its method (as mispairing polymerase chain reaction (PCR)) is well known in the art.

For example, some amino acid in the polypeptide can not had tangible functionally active loss simultaneously by other amino-acid substitution.Therefore, should consider and in the aminoacid sequence (or its basic nucleotide sequence) of described PLAD, to make various changes, not have tangible biological effectiveness or loss of activity and these effectiveness or activity also may increase simultaneously.For example, the sudden change of the Q24A in the p60 TNFR native sequences, D49R sudden change and K19E sudden change can not diminish the self-association of PLAD.

These polypeptide also can obtain with any method in many methods well known in the art.A kind of method that produces polypeptide is to utilize the protein chemistry technology that two kinds of peptides or polypeptide are coupled together.For example, can pass through Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tertbutyloxycarbonyl) chemistry, utilize present obtainable Laboratory Instruments come chemical synthesising peptide or polypeptide (Applied Biosystems, Inc., Foster City, CA).Those skilled in the art can recognize at an easy rate and can synthesize peptide or the polypeptide that is equivalent to specific protein by the chemical reaction of standard.For example, can a kind of synthetic peptide or polypeptide and it not being scaled off from its synthetic resins, another fragment that can synthesize simultaneously a kind of T1249 also scales off it subsequently from described resin, thereby exposes on this another fragment by the end group of functional sealing.Utilize the peptide condensation reaction, can be respectively they be covalently boundly formed bigger polypeptide (Grant by these two segmental carboxyls and aminoterminal peptide bond, ASynthetic Peptides:A User Guide, W.H.Freeman and Co., N.Y. (1992) and Bodansky and Trost, Ed., Principles ofPeptide Synthesis, Springer-Verlag Inc., N.Y. (1993)).Perhaps, as mentioned above, can synthetic independently in vivo described peptide or polypeptide.In case separate, then can independently peptide or polypeptide couple together and form bigger albumen with these by similar peptide condensation reaction.

For example, the clone's or synthetic peptide section enzymatic connects and can make it possible to short relatively peptide fragment is coupled together to produce bigger peptide fragment, polypeptide or whole protein structural domain (Abrahmsen et al.Biochemistry, 30:4151 (1991)).Perhaps, can utilize the native chemical of synthetic peptide to connect to come by synthetic method by short bigger peptide or the polypeptide of peptide fragment structure.This method is formed (Dawson et al.A Synthesis of Proteins by Native Chemical Ligation, Science, 266:776-779 (1994)) by two step chemical reactions.The first step is that unshielded synthetic peptide-%-thioesters carries out chemical selective reaction to produce the thioesters connectivity intermediate product as initial covalency product with the another kind of unprotect peptide section that contains N-terminal Cys residue.Under the situation that does not change reaction conditions,, this intermediate product forms natural peptide bond at link position thereby having experienced spontaneous rapid molecular internal reaction.The application of this native chemical method of attachment in the complete synthesis process of protein molecular can illustrate (Clark-Lewis et al.FEBS Lett. by the preparation of human interleukin 8 (IL-8), 307:97 (1987), Clark-Lewis et al.J.Biol.Chem., 269:16075 (1994), Clark-Lewis et al.Biochemistry, 30:3128 (1991) and Rajarathnam et al.Biochemistry, 29:1689 (1994)).

Perhaps, unshielded peptide section can be that chemistry connects, and wherein the key that forms between described peptide section because chemistry connects is non-natural key (non-peptide bond) (Schnolzer et al.Science, 256:221 (1992)).This technology has been used to the analogue of synthetic proteins structural domain and has had bioactive fully purer albumen (deLisle Milton et al.ATechniques inProtein Chemistry IV relatively in a large number, Academic Press, New York, pp.257-267 (1992)).

The present invention also provides the peptide mimics that is used for disclosed polypeptide." peptide mimics " is defined as comprising compound or organic molecule or any other peptide mimics, and its structure is based on or derived from proteic calmodulin binding domain CaM.For example, people can set up the structure of the model of prediction chemical structure with simulation calmodulin binding domain CaM (as PLAD).Can use standard method to carry out this modeling.Perhaps, also can be to come from the combinatorial chemistry library, to select peptide mimics (Ostresh, J.M.et al., ProcNatl Acad Sci USA 1994 Nov 8 with the roughly the same mode of described peptide; 91 (23): 11138-42; Dorner, B.et al., BioorgMed Chem 1996 May; 4 (5): 709-15; Eichler, J.et al., Med Res Rev 1995Nov; 15 (6): 481-96; Blondelle, S.E.et al.Biochem J 1996 Jan 1; 313 (Pt1): 141-7; Perez-Paya, E.et al., J Biol Chem 1996 Feb23; 271 (8): 4120-6).Also can utilize functional examination to select peptide mimics.

Polypeptide of the present invention can for example nucleic acid, albumen, peptide, part, sugar moieties, viral protein, monoclonal antibody, polyclonal antibody or liposome be connected with another part.In addition, also the polypeptide of two or more PLAD of containing can be connected to each other.For example, can prepare the difunctional or multifunctional polypeptides that contains two or more different PLAD so that described polypeptide can be regulated the activity of multiple TNF acceptor sample acceptor.Described polypeptide also can contain two or more PLAD of identical TNF acceptor sample acceptor that are derived to increase the avidity of this peptide species to specific T NF acceptor sample acceptor.

The fusion constructs that contains PLAD

Herein disclosed is the fusion rotein and their nucleic acid of coding that contain PLAD.Described fusion rotein can comprise the TNF acceptor sample acceptor PLAD that has connected fusion tag disclosed herein.Described functional molecular can be antibody or its targeting moiety or other fusion tag.Because PLAD is on cell surface, the fusion rotein of the described PLAD of containing can comprise the integral part of PLAD target in the cell surface of the cell of expressing PLAD.For example, the mark bound fraction of the part of non-TNF acceptor sample mark can merge with PLAD, and described mark is positioned at the surface of specified target cell.Described PLAD can for example immunoglobulin (Ig) or other serum, soluble and/or stable albumen merge with various carrier proteinss.Described fusion tag can be that GST or other help the molecule of described fusion rotein purifying.The fusion rotein that contains PLAD can include and help recombinant expressed PLAD excretory signal sequence.

The nucleic acid of the polypeptide that also coding can be comprised PLAD or be made up of PLAD is connected with other nucleic acid function ground with the coding immune conglutinin.For purposes of the invention, term " immune conglutinin " is defined as comprising any polypeptide by nucleic acid encoding, at least a portion coupling of the nucleic acid of at least a portion of the nucleic acid of the NIg molecule of wherein encoding (for example PLAD) and coding heavy chain immunoglobulin polypeptide (for example IgG).The Fc district of IgG2, IgG3, IgM, IgA, IgE also can be used to make up immune conglutinin.In a concrete example, described fusion rotein comprises the part with Ig Fc district--particularly the Fc district part of the Ig γ 4--PLAD of fusion.Described coupling be by a kind of guarantee described nucleic acid fragment can carry out functional transcribe and translate and provide from the mode of the information that wherein obtains respectively realize.

Can express the PLAD polypeptide amalgamation protein by the instantaneous or stable transfection in the cell of various mammalian host cells and baculovirus infection.Expressed fusion protein can be come purifying according to standard method.With antibody class seemingly, the IgG immune conglutinin can come out by step albumin A or Protein G affinity chromatography purifying from the substratum of having been secreted this immune conglutinin.

Transgenosis

The transgenosis of coding PLAD is provided." transgenosis " is meant and is inserted in the cell artificially and becomes this cell and the nucleotide sequence of the genomic part of daughter cell.For described cell, this transgenosis can be (but not necessarily) partially or completely allogenic (for example, from different species).Term " transgenosis " broadly refers to be directed to any nucleic acid in the animal gene group, includes but not limited to contain the gene that may not be present in the sequence in the described genome usually or DNA, exists in given genome but can not be transcribed and translate the gene of (expression) or people usually and wish to import to any other gene or DNA in the described genome.This can comprise and may be present in usually in the non-transgenic genome but people wish to change its expression or people wish with the gene that changes or variant form imports.Transgenosis can comprise one or more transcription regulating nucleotide sequences and may be required any other nucleic acid of optimum expression of selected nucleic acid, for example intron.Transgenosis can have only two Nucleotide long, but preferably long or even longer at least about 50,100,150,200,250,300,350,400 or 500 Nucleotide.Transgenosis can be coding or non-coding sequence or its combination.Transgenosis generally includes the controlling element that can impel one or more transgene expressions under proper condition.

Antibody

The present invention also provides the PLAD specificity bonded antibody with TNF acceptor sample acceptor.For example, antibody of the present invention can be with the PLAD specificity bonded antibody of TNF acceptor, with the PLAD specificity bonded antibody of FAS or with the PLAD specificity bonded antibody of DR4, only lift these and be example.What any polypeptide of being considered that described antibody (polyclone or mono-clonal) can be meant that this paper provides, the polypeptide and the immunogenic fragment thereof of naturally occurring and reorganization.In technology such as described antibody can be used to for example diagnose, treatment or vaccine inoculation or the method.Also considered anti--idiotype antibody and affinity maturation antibody.

Can prepare antibody (for example, referring to Harlow and Lane, " Antibodies by many methods of knowing; A Laboratory Manual " Cold Spring Harbor Laboratory, ColdSpring Harbor, New York, (1988)).In brief, with the amount that is enough to draw immunne response with at interval the antigen of purifying is expelled in the animal body.Antibody can be direct purification or splenocyte can in animal body, obtain.Then can be with described cell and immortal cell line fusion and screening antibody-secreting.Described antibody can be used to screen the nucleic acid clone library that can secrete this antigenic cell.Then to these positive colonies check order (for example, referring to Kelly et al.Bio/Technology, 10:163-167 (1992); Bebbington et al.Bio/Technology, 10:169-175 (1992)).The present invention has also considered humanized antibody and chimeric antibody.Can prepare heterologous antibody (for example, referring to United States Patent (USP) 5545806,5569825,5625126,5633425,5661016,5770429,5789650 and 5814318) by the method for knowing.

Term is meant to determine whether there is described proteic a kind of association reaction in a group heterologous protein and other biological goods with polypeptide " specificity combines ".Therefore, under specified immunoassay condition, with specific protein bonded specific antibodies not with sample in other albumen combine with significant quantity.Under this class condition, may need the antibody that the specificity of specific protein is selected according to it with the selective binding of antibody.Can use various immunoassay forms select can with the antibody of specific protein selective binding.For example, use solid phase ELISA immunoassay to select optionally to carry out immunoreactive antibody usually with albumen.About the immunoassay form that can be used to determine selective binding and the description of condition see also Harlow and Lane " Antibodies, A Laboratory Manual " Cold Spring HarborPublications, New York, (1988).

Nucleic acid

The present invention also provides coding to reach the nucleic acid of 125 amino acid whose polypeptide most, and described polypeptide contains the PLAD of TNF acceptor sample acceptor; And the nucleic acid that the polypeptide that coding is made up of TNF acceptor sample acceptor PLAD is provided.

The present invention also provides coding to reach the nucleic acid of 125 amino acid whose polypeptide most, and described polypeptide contains separative PLAD, and wherein said polypeptide contains subsequence R ₁-PLAD-R ₂, R wherein ₁And R ₂Choose wantonly, and when existing, they can be H, acyl group, NH ₂, amino acid and peptide.

The present invention further provides the nucleic acid of such peptide species of encoding, described polypeptide contains the isolating aminoacid sequence in the preceding part assembling territory (PLAD) of TNF acceptor sample acceptor, and wherein said polypeptide is R ₁-TNF acceptor sample acceptor PLAD-R ₂, R wherein ₁Or R ₂Comprise and be not positioned at the lateral aminoacid sequence of TNF acceptor sample acceptor PLAD in the naturally occurring TNF acceptor sample acceptor.

Term used herein " nucleic acid " is meant it can is the strand or the multichain molecule of DNA or RNA or its any combination, comprises the modification to these nucleic acid.Described nucleic acid can be represented coding strand or its complementary strand or its any combination.The sequence of nucleic acid can exist sequence identical or can comprise coding and the natural the same amino acid whose alternative codon of finding in the sequence of amino acid that exists with any new gene discussed in this article natural.Also can modify the typical structure of these nucleic acid.These modifications include but not limited to methylated nucleic acid, the not bridge joint oxygen on the phosphoric acid residue are replaced with sulphur (producing the thiophosphoric acid deoxynucleotide), selenium (producing seleno phosphoric acid (phosphorselenoate) deoxynucleotide) or methyl group (producing the methyl-phosphorous acid deoxynucleotide).

Can be with its separation from the organism of the nucleic acid molecule of common existence coding PLAD.For example, can make up genomic dna or cDNA library and screen purpose nucleic acid and whether exist.The method that makes up and screen this class library is as known in the art, the test kit that is used to carry out described structure and screening step be commercially available (for example, Stratagene Cloning Systems, La Jolla, CA).In case separate, just described nucleic acid directly can be cloned in the appropriate carriers, perhaps if necessary, can modify to help follow-up clone's step it.These modification steps are conventional, and an example is at the terminal oligonucleotide joint that contains restriction site that adds of described nucleic acid.Sambrook et al., " MolecularCloning, a Laboratory Manual " have set forth general method among the Cold Spring Harbor Laboratory Press (1989).The present invention has also considered the nucleic acid that does not contain ligand-binding site point of coding PLAD.

In case obtained the nucleotide sequence of required PLAD, just can utilize technology as known in the art the sequence of coding specific amino acids is modified or to change at any concrete amino acid position.For example, can design cross over described one or more amino acid positions and any amino acid can be by the PCR primer of another kind of amino-acid substitution.Then can amplification of nucleic acid and be inserted in the wild-type PLAD encoding sequence to obtain any locational amino acid whose many any that may make up at described PLAD.Perhaps, those skilled in the art can introduce specific sudden change by any site of point mutation technology in specific nucleic acid sequence.Smith, M. " In vitro mutagenesis " Ann.Rev.Gen., 19:423-462 (1985) and Zoller, M.J. " New molecular biology methods for protein engineering " Curr.Opin.Struct.Biol., 1:605-610 has set forth general method in (1991).These technology can be used to change encoding sequence and not change coded aminoacid sequence.

Another example of the method for the dna molecular of a kind of PLAD that obtains to encode is the recombinant DNA molecules of the described PLAD of composite coding.For example, the oligopeptides synthetic method is being conventional in this area, and the oligonucleotide that is used for the encode specific protein zone is easy to synthesize by automated DNA and obtains.Can synthesize the nucleic acid of a chain of duplex molecule and make it and the hybridization of its complementary strand.People can design these oligonucleotide and make the duplex molecule of gained have the internal limitations site or contain 5 ' or 3 ' suitable overhang endways to be cloned in the appropriate carriers.By the following method relatively large proteic duplex molecule of composite coding at an easy rate: at first make up the duplex molecule of several different encoding said proteins specific regions, afterwards these dna moleculars are linked together.For example, Cunningham, et al., " Receptor andAntibody Epitopes in Human Growth Hormone Identified byHomolog-Scanning Mutagenesis; " Science, 243:1330-1336 (1989) thus by at first making up overlapping and complementary synthetic oligonucleotide and these fragments being coupled together the synthetic gene that has made up the coding human growth hormone gene.Also can be referring to Ferretti, et al., Proc.Nat.Acad.Sci.82:599-603 (1986) wherein discloses the synthetic ox rhodopsin gene that synthesizes 1057 base pairs with the synthetic oligonucleotide.By making up PLAD by this way, those skilled in the art can obtain any specific PLAD at an easy rate, have amino acid needed on any one or a plurality of specific position of described PLAD in PLAD.Also referring to United States Patent (USP) 5,503,995, described patent has been described a kind of enzyme template reaction method for preparing synthetic gene.This class technology is conventional in the art and exquisite detail is arranged.Then can according to hereinafter described in vivo these nucleic acid of vivoexpression or the coding PLAD nucleic acid fragment.

The present invention also provides the isolating nucleic acid of coding PLAD, and described nucleic acid is arranged in the carrier that is suitable for expressing it.In case after a zone of the nucleic acid of the specific purpose PLAD that encodes or this nucleic acid made up, modifies or separate, just can be in appropriate carriers with this nucleic acid clone, described carrier can instruct this wild-type and/or modify interior or external the synthesizing of body of back PLAD.Considered that described carrier should have guidance and regulate and control to insert gene or the required functional element of transcribed nucleic acid.These functional element include but not limited to promotor, the active enhanser of described promoter transcription for example may be regulated and control in the upstream and downstream zone of described promotor, replication orgin, the suitable restriction site that helps near the insertion fragment cloning of described promotor, antibiotics resistance gene maybe can be used for screening and contain described carrier or described other mark that contains the cell that inserts segmental carrier, the RNA splice junction, transcription termination region or can be with helping any other district (usually referring to Sambrook et al.) that described insertion gene or heterozygous genes are expressed.

Existing many those of ordinary skills are known to be used to express described nucleic acid and to insert segmental E.coli (colon bacillus (Escherichia coli)) expression vector.Other microorganism host that be fit to use comprises for example Bacillus subtilus (Bacillus Substilis) of bacillus, and other enterobacteria (Bacillus) for example Salmonellas (Salmonella), Serratia (Serratia) and various pseudomonas (Pseudomonas) kind.In these prokaryotic hosts, people also can prepare expression vector, and described expression vector contains the expression control sequenc compatible with described host cell (for example replication orgin) usually.In addition, should there be any amount of various known promotor, for example the promoter systems of lactose promoter systems, tryptophane (Trp) promoter systems, β-Nei Xiananmei promoter systems or lambda particles phage.Described promotor can be controlled expression usually, randomly has operon sequence; And should contain the ribosome bind site sequence, for example be used for starting and finishing and transcribe and translate.If necessary, can provide the N-terminal methionine(Met) by inserting 5 ' end Met codon and meeting insertion downstream, frame ground nucleic acid inset.Also can use the oligonucleotide mutation method of standard to remove nucleic acid and insert segmental C-terminal extension.

In addition, can use yeast expression.Yeast expression system has multiple advantage.At first, evidence suggests that albumen that the yeast secretary system produces presents and has correct disulfide linkage pairing.Secondly, post-translational glycosylation carries out effectively by the yeast secretary system.Preceding-former-alpha factor the leader of yeast saccharomyces cerevisiae (Saccharomycescerevisiae) (by MF " 1 genes encoding) can be used to instruct zymic protein excretion (Brake; et al.; Alpha-Factor-Directed Synthesis andSecretion of Mature Foreign Proteins in Saccharomyces cerevisiae.Proc.Nat.Acad.Sci., 81:4642-4646 (1984)) usually.Before-leader of former-alpha factor contains signal peptide and former section, and described former section comprises the recognition sequence by the endotrypsin of KEX2 genes encoding: this kind of enzyme can cut the precursor protein on the carboxyl side of Lys-Arg dipeptides cutoff signal sequence.Described nucleic acid coding sequence can be in frame with described before-leader of former-alpha factor merges.Then this construct is placed strong transcripting promoter for example under the control of ethanol dehydrogenase I promotor or glycolysis-promotor.After the described nucleic acid coding sequence is translation stop codon, is transcription termination signal after described translation stop codon.Perhaps, described nucleic acid coding sequence can merge with another kind of albumen coded sequence (as Sj26 or beta-galactosidase enzymes) so that come fusion rotein as described in the purifying by affinity chromatography.For the construct that is used for yeast expression, it is feasible that each integral part of minute isolated fusion protein is come in insertion proteolytic enzyme cutting site.Also can in rhabdovirus system, realize effective post-translational glycosylation and expression of recombinant proteins.

Mammalian cell can be in the environment that helps important posttranslational modification (for example folding and halfcystine pairing) expressing protein, add complicated sugared structure and secretion activity albumen.Being used at the proteic carrier of mammalian cell expression activity is feature to have inserted albumen coded sequence between strong virus promotor and polyadenylation signal.Described carrier can contain the gene of having given the hygromycin resistance, gentamicin or the G418 resistance that are used for gene amplification, but perhaps other is fit to be used as the gene or the phenotype of selection markers, methotrexate resistant gene perhaps.Use the selection markers that methotrexate resistance code carrier can import to described encoded chimeric protein sequence in Chinese hamster ovary (CHO) clone or use is fit to that it is imported in other clones.Can prove conclusively whether in cell transformed, there is described carrier DNA by the southern blotting technique analysis.Can confirm to have produced corresponding to the RNA that inserts the fragment coding sequence by the RNA trace.Develop the many host cell systems that can secrete complete human protein that other is fit in this area, comprised Chinese hamster ovary celI system, Hela cell, myeloma cell line, Jurkat cell etc.The expression vector that is used for these cells can comprise expression control sequenc, for example replication orgin, promotor, enhanser and essential information processing site, for example ribosome bind site, RNA splice site, polyadenylation site and transcription termination sequence.Preferred expression control sequenc is the promotor from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus etc.The carrier that contains the purpose nucleic acid segment can be transferred in the host cell by known method, these methods can change according to the type of cell host.For example, calcium chloride transforms and is normally used for prokaryotic cell prokaryocyte, and the transfection or the electroporation of calcium phosphate, deae dextran or lipofection reagent (lipofectin) mediation can be used to other eukaryotic cell host.

Can adopt the alternative carrier that is used at mammalian cell expressing gene or nucleic acid, those carriers be developed that to be used for the carrier of expressing human gamma-interferon, tissue plasmin incitant, blood coagulation factor VIII, hepatitis B virus surface antigen, protease N exinl and the main basic protein of eosinophilic granulocyte similar.In addition, described carrier can comprise the CMV promoter sequence and the polyadenylation signal that can be used for expressing at mammalian cell (as COS-7) insertion nucleic acid.

Insect cell also can be expressed mammalian proteins.The recombinant protein of producing in containing the insect cell of baculovirus vector can be through being similar to the posttranslational modification of wild-type protein.In brief, the baculovirus vector that is used at the expressed in insect cells activated protein is a feature with the albumen coded sequence that has inserted autographa california (Autographicacalifornica) nuclear polyhedrosis virus (AcNPV) promotor downstream, and described promotor is at the main inclusion body protein of coding--the gene of polyhedrin.For example covet noctuid (Spodopterafrugiperda) clone and described daughter of virus is carried out flat board cultivate in the meadow with the insect cell that the mixture transfection of viral DNA and plasmid DNA is cultivated.The disappearance of polyhedron gene or insertion inactivation can cause producing the negative virus of inclusion body, and described virus can formation and the visibly different plaque of wild-type inclusion body positive-virus.These characteristic plaque forms allow visually to screen the chosen recombinant virus that heterozygous genes replaced of AcNPV gene.

The present invention also provides to contain and has expected the carrier of nucleic acid, and described carrier is arranged in the host who is suitable for expressing described nucleic acid.The carrier that contains the purpose nucleic acid segment can be transferred in the host cell by known method, these methods can change according to the type of cell host.For example, calcium chloride conversion, transduction and electroporation are normally used for prokaryotic cell prokaryocyte, and the transfection or the electroporation of calcium phosphate, deae dextran or lipofection reagent (lipofectin) mediation can be used to other cell host.

Perhaps, nucleic acid of the present invention can be operably connected with the functional element of one or more guidances and regulation and control insertion transcribed nucleic acid, and described nucleic acid can be expressed.For example, nucleic acid can be operably connected with bacterium or phage promoter and be used to instruct the in-vitro transcription of described nucleic acid.Further example be included in link coupled transcribe-use nucleic acid provided herein in the translation system, wherein said nucleic acid guided transcribe and consequent RNA can be used as the template of translation to produce polypeptide.The product that it will be understood by those skilled in the art that these reactions can be used in many purposes (for example applying marking RNA as probe and use polypeptide to produce antibody) or be used in described polypeptide is given to cell or experimenter's method.

The expression of nucleic acid that carries out in conjunction with carrier can be in vivo or external.Synthesize in the body and comprise that conversion can be used as the protokaryon or the eukaryotic cell of the host cell of described carrier.Perhaps, described expression of nucleic acids may reside in the vivoexpression system.For example, the in-vitro transcription system that is used to the mRNA of synthetic relatively large amount usually is commercially available.In this class in-vitro transcription system, the nucleic acid of coding PLAD can be cloned in the expression vector and contiguous transcripting promoter.For example, described Bluescript II cloning vector and expression vector contain a plurality of cloning sites that the side is strong protokaryon transcripting promoter (Stratagene Cloning Systems, La Jolla, CA).Comprise be useful on the test kit of external required reagent (as Bluescript vector) according to the synthetic RNA of dna profiling be can obtain (Stratagene Cloning Systems, LaJolla, CA).Then the external RNA that produces by this system can external translation produce required PLAD polypeptide (Stratagene Cloning Systems, La Jolla, CA).

Gene therapy method

By can the encode nucleic acid of the polypeptide that contains PLAD or form by PLAD of gene therapy method.The nucleic acid of polypeptide of the present invention of encoding can be placed in the carrier and in vivo or under the isolated condition and by standard method it be sent in the cell that is delivered to the experimenter.

Nucleic acid encoding polypeptide of the present invention can functionally be connected to can be used for this polypeptide excretory specificity leading peptide specifically.For example described peptide can have signal sequence, mouse Ig-λ signal sequence (Blezinger et al.Nat.Biotechnol.17:343-8 for example, 1999), rat insulin leader sequence (Fakhral et al.J.Immunother.20:437-8,1997), FGF-4 signal sequence (Ueno et al.Aterioscler.Thromb.Vasc.Biol., 17:2453-2460,1997), human growth hormone signal peptide (Rade et al.Gene Ther.6:385-92,1999), beta lactamase signal sequence (Hughes et al.Hum.Gene Ther.5:1445-55,1994), ox prolactin antagonist signal sequence (Gorman et al.Bran Res.Mol.Brain Res.44:143-146,1997) and other similar signal sequence.

For vivo medicine-feeding, described cell can be can give by pharmaceutically acceptable carrier at the intravital and described nucleic acid of experimenter.The experimenter can be any need be in cell the animal of selective expression's nucleic acid.In a preferred embodiment, animal of the present invention is the people.In addition, can include but not limited to by the non-human animal that method of the present invention is treated cat, dog, bird, horse, milk cow, goat, sheep, cavy, hamster, gerbil jird and rabbit and any other can be according to the animal of methods described herein selective expression's nucleic acid in cell.

Introduce in the above-mentioned experimenter's of being included in cell in the method for foreign DNA (being gene transfer or transfection), nucleic acid of the present invention can be that naked DNA form or described nucleic acid can be arranged in to be used for described nucleic acid sent and is delivered to cell so that at the carrier of this nucleic acid of cell inner expression.Described carrier can be commercially available goods, for example adenovirus carrier (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada)).Can described nucleic acid or carrier be sent by various mechanism and be delivered in the cell.As an example, can send by liposome and pass, for example use commercially available liposome product

(GIBCO-BRL, Inc., Gaithersburg, MD),

(Qiagen, Inc.Hilden, Germany) and (Promega Biotec, Inc., Madison, WI), and other is according to the liposome of the standard method exploitation of this area.In addition, can pass through electroporation and Sonoporation instrument (ImaRx Pharmaceutical Corp., Tucson, AZ) send in vivo and pass nucleic acid of the present invention and carrier, the technology that is used for electroporation can be from Genetronics, and (SanDiego CA) obtains Inc..

As an example, can carry out carrier by viral system and send and pass, for example can pack the retroviral vector system of recombinant chou reverse transcription virus gene group.Thereby described then recombinant chou retrovirus can be used to infect nucleic acid sent and be delivered in the infected cells.Importing in the mammalian cell described nucleic acid really, blanking method is to be not limited to use retroviral vector certainly.Other technology that can be widely used in this process comprises uses adenovirus carrier, adeno associated virus (AAV) carrier, lentiviral vectors, pseudo-type retrovirus vector and poxvirus vector, for example vaccinia virus vector.Also can use physics transduction technology, for example liposome send the mechanism of passing, receptor-mediated mechanism and other endocytosis mechanism.The present invention can use with any these or other normally used gene transfer method.

Nucleic acid of the present invention and nucleic acid send pass vehicle (for example virus; Liposome, plasmid, carrier) can be arranged in pharmaceutically acceptable carrier and give to the experimenter in vivo being used for." pharmaceutically acceptable " is meant not can be biologically or have the material of undesirable action aspect other, promptly can together give the experimenter, can not cause any bad biological impact simultaneously or with deleterious mode and any other component interaction that contains its pharmaceutical composition with described material and described nucleic acid or vehicle.Should so select to make any degraded of activeconstituents to drop to minimum and make the intravital any adverse side effect of experimenter reduce to minimum carrier, this is well-known to those skilled in the art.

Described nucleic acid or vehicle administration in the following manner: oral, parenteral (for example intravenously), by intramuscularly, by peritoneal injection, through skin, external, part etc.The exact amount of needed nucleic acid or carrier can change with experimenter's difference, and this depends on the severity of experimenter's species, age, body weight and overall state, any illness for the treatment of or mechanism, used concrete nucleic acid or vehicle, its mode of administration etc.

PLAD self-association inhibitor

The present invention further provides the composition of a kind of PLAD of comprising self-association inhibitor or TNF sample acceptor oligomerization inhibitor." inhibitor " is defined as a kind ofly can or a kind ofly can combining and stop the active compound of PLAD (comprising antibody) with the PLAD of target with PLAD bonded compound.In case combine with PLAD, described inhibitor can interrupt or prevent the PLAD self-association, therefore suppresses the oligomerization of TNF acceptor sample acceptor.TNF sample acceptor oligomerization inhibitor can be PLAD specificity bonded antibody (polyclone or mono-clonal), with PLAD bonded part, with PLAD bonded polypeptide, with PLAD or based on the peptide mimics bonded compound of PLAD.For example, comprise PLAD or the polypeptide be made up of PLAD can associate with the PLAD of naturally occurring TNF acceptor sample acceptor, therefore prevent or suppress this TNF acceptor sample acceptor and other naturally occurring TNF acceptor sample acceptor self-association.The described PLAD of comprising or can be solubility PLAD by the polypeptide that PLAD forms.Anti-id AB and affinity maturation antibody have also been considered.Other inhibitor comprises but is not limited to be designed for molecule or the compound of blocking the PLAD self-association.Described inhibitor can be whole protein or the protein fragments that suppresses the PLAD self-association, has therefore prevented the oligomerization of TNF acceptor sample acceptor.Described inhibitor can be the organic molecule of identifying according to methods described herein.The crystalline structure of TNF acceptor and their oligomerization complex body can be used to design the molecule that can interrupt the PLAD self-association.Also can analyze the molecule that described crystalline structure can be simulated PLAD and interrupt the PLAD self-association with design.

Therefore, provide a kind of method for preparing micromolecular inhibitor, produced this inhibitor by this method.Interference TNFR assembling and TNF bonded small molecules (SM) and PLAD-peptide derivant are provided.According to the dimeric crystalline structure of TNFR1 albumen, carried out the search of PLAD association mating surface.Combine the above-mentioned search of conservative homology search and can identify some potential interchain association sites based on the data of this paper PLAD sudden change are feasible.For example, as if shown in the PLAD dimeric structure (Figure 23), two Histidine rings that every peptide chain is the 34th are the fixing mutual mirror lock of mirror image (mirror-lock) each other in the interchain bag.Because the imidazoles of histidine residues is the good candidate of interrupting the PLAD self-association, so imidazoles and imdazole derivatives can be effective receptor-specific blocking-up thing and the disease that can be used to treat the TNF mediation.

Herein disclosed is and to disturb TNFR assembling and TNF bonded compound.In general, the inhibitor of Shi Heing is that those can enter the interchain bag of described PLAD dimeric structure and can disturb interactional compound between two Histidine rings of the 34th.In this sense, preferably do not contain the inhibitor than large-substituent, described substituting group can hinder and enter into described interchain bag or stop interactional interruption between two Histidines of the 34th.On the contrary, preferably contain the substituent inhibitor with following character, described substituting group makes that enter described interchain bag is easy to and can promotes to insert and the interactional interruption between two Histidines of the 34th subsequently.In addition, even more preferably contain the substituent inhibitor with following character, described substituting group has higher avidity with respect to other residue in the described interchain bag, therefore can promote and/or strengthen the combination of inhibitor in the PLAD dimeric structure.Whether those skilled in the art can be with the inhibitor of computer for analysis supposition assess some substituting group and can strengthen or the combining of obstruction and described PLAD dimeric structure.

Term used herein " substituted " should be believed to comprise the organic compound substituting group of all permissions.In a broad aspect, the substituting group of described permission comprises acyclic and cyclic, side chain and non-side chain, isocyclic and heterocyclic and fragrance and organic compound substituting group non-fragrance.Exemplary substituting group for example comprises substituting group hereinafter described.For suitable organic compound, the substituting group of described permission can be one or more and can be identical or different.For the disclosure, described heteroatoms (as nitrogen) can have the substituting group of any permission of hydrogen substituting group and/or organic compound described herein, and described substituting group satisfies heteroatomic coordination valence.The disclosure is not intended to be subjected to the restriction of the substituent any way of organic compound of described permission.Term " replacement " or " using ... replace " also comprise implied condition, be that this replacement should be stable compound according to what be substituted atom and substituent permission coordination valence and described replacement generation, for example can spontaneously not experience the compound of conversion, for example rearrangement of described conversion, cyclisation, elimination etc.

" A herein ¹", " A ²", " A ³" and " A ⁴" be used as general symbol(s) to represent each concrete substituting group.These symbols can be any substituting groups, be not limited to disclosed herein those, and when they are restricted to some substituting group in one case, they can be defined as some other substituting group in another case.

Term used herein " alkyl " is the side chain or the non-branched saturated hydrocarbon group of 1 to 24 carbon atom, for example methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl etc.Described alkyl also can be substituted or unsubstituted.Can replace described alkyl with one or more groups, described group includes but not limited to alkyl as described below, haloalkyl, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halogenide, hydroxyl, ketone, nitro, silyl, sulfo-oxo (sulfo-oxo), alkylsulfonyl, sulfone, sulfoxide or sulfydryl.

In this manual, " alkyl " generally is used to refer to unsubstituted alkyl group and substituted alkyl group; But one or more specified substituent that also can be tested and appraised herein on the alkyl group are refered in particular to described substituted alkyl group.For example, term " haloalkyl " refers in particular to the alkyl group that replaces with one or more halogenide (for example fluorine, chlorine, bromine or iodine).As described below, term " alkoxyalkyl " refers in particular to the alkyl group that replaces with one or more alkoxy bases.As described below, term " alkylamino " refers in particular to alkyl group that replaces with one or more amino groups or the like.When using specific term as " alkyl alcohol " in another case, be not to be intended to hint that term " alkyl " can't also refer to specific term for example " alkyl alcohol " etc. when using " alkyl " in one case.

This practice also can be used to other group as herein described.That is, be meant unsubstituted as " cycloalkyl " and during substituted cycloalkyl moiety, the part of this replacement also can be indicated especially when term; For example a kind of concrete substituted cycloalkyl can be called as for example " alkyl-cycloalkyl ".Similarly, a kind of substituted alkoxyl group can be called specifically, and for example " halogenated alkoxy ", a kind of concrete substituted thiazolinyl can be, for example: " alkenyl alcohol " etc.Equally, use generic term for example " cycloalkyl " and the next term for example the practice of " alkyl-cycloalkyl " be not to be intended to hint that this generic term does not comprise this next term yet.

Term used herein " alkoxyl group " is by independent terminal ether key bonded alkyl; That is, " alkoxyl group " group can be defined as-OA ¹, A wherein ¹It is aforesaid alkyl.

Term alkoxyalkyl used herein is to contain alkoxy substituent and can be defined as-A ¹-O-A ²Alkyl group, A wherein ¹And A ²Be alkyl group.

Term used herein " thiazolinyl " is for containing the alkyl with 2 to 24 carbon atoms of a carbon-to-carbon double bond at least in the structural formula.Unsymmetrical structure is (as (A ¹A ²) C=C (A ³A ⁴)) be intended to comprise E and Z type isomer.This point can be inferred according to the structural formula of this paper, wherein has asymmetric alkene, perhaps can indicate clearly by keysym C=C.Described thiazolinyl can be replaced by one or more group, and described group includes but not limited to alkyl as mentioned below, haloalkyl, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halogenide, hydroxyl, ketone, nitro, silyl, sulfo-oxo, alkylsulfonyl, sulfone, sulfoxide or sulfydryl.

Term used herein " alkynyl " is for containing the alkyl with 2 to 24 carbon atoms of a carbon-to-carbon triple bond at least in the structural formula.Described alkynyl group can be replaced by one or more group, and described group includes but not limited to alkyl as described below, haloalkyl, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halogenide, hydroxyl, ketone, nitro, silyl, sulfo-oxo, alkylsulfonyl, sulfone, sulfoxide or sulfydryl.

Term used herein " aryl " is for containing the group of any aromatic group based on carbon, and described aromatic group includes but not limited to benzene, naphthalene, phenyl, xenyl, phenoxy group benzene etc.Term " aryl " also comprises " heteroaryl ", and described heteroaryl is defined as containing aromatic group and has one at least and mixes the heteroatomic group of described aromatic group intra-annular.Heteroatomic example includes but not limited to nitrogen, oxygen, sulphur and phosphorus.Equally, term " non-heteroaryl " also is included in the term " aryl ", and it is defined as contains the group that does not contain heteroatomic aromatic group.Described aromatic yl group can be substituted or unsubstituted.Described aromatic yl group can be replaced by one or more groups, and described group includes but not limited to alkyl as described herein, haloalkyl, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halides, hydroxyl, ketone, nitro, silyl, sulfo-oxo, alkylsulfonyl, sulfone, sulfoxide or sulfydryl.Term " dibenzyl " is a kind of aromatic yl group of particular type and is included in the definition of aryl.Dibenzyl is meant two aromatic yl groups (as in naphthalene) that combine by the condensed ring structure or two aromatic yl groups (as in xenyl) that connect by one or more C-Cs.

Term used herein " cycloalkyl " is the non-aromatic carbocyclic that is made of at least three carbon atoms.The example of group of naphthene base includes but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl etc.Term " Heterocyclylalkyl " is that wherein at least one ring is gone up the group of naphthene base as mentioned above that carbon atom is replaced by heteroatoms, and described heteroatoms is such as but not limited to nitrogen, oxygen, sulphur or phosphorus.Described cycloalkyl and heterocycloalkyl can be substituted or unsubstituted.Described cycloalkyl and heterocycloalkyl can be replaced by one or more group, and described group includes but not limited to alkyl as described herein, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halogenide, hydroxyl, ketone, nitro, silyl, sulfo-oxo, alkylsulfonyl, sulfone, sulfoxide or sulfydryl.

Term used herein " cycloalkenyl group " is the non-aromatic carbocyclic that is constituted and contained at least one two key (being C=C) by at least three carbon atoms.The example of cycloalkenyl group includes but not limited to encircle propenyl, cyclobutene base, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl etc.Term " heterocycloalkenyl " be a class as hereinbefore defined cycloalkenyl groups and be included in the implication of term " cycloalkenyl group ", at least one carbon atom of wherein said ring is replaced by heteroatoms, described heteroatoms is such as but not limited to nitrogen, oxygen, sulphur or phosphorus.Described cycloalkenyl groups and heterocycloalkenyl group can be substituted or unsubstituted.Described cycloalkenyl groups and heterocycloalkenyl group can be replaced by one or more group, and described group includes but not limited to alkyl as described herein, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halogenide, hydroxyl, ketone, nitro, silyl, sulfo-oxo, alkylsulfonyl, sulfone, sulfoxide or sulfydryl.

Term used herein " cyclic group " is meant aromatic yl group, non-aromatic yl group (being cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl group) or both.Cyclic group has one or more substituted or unsubstituted loop systems.Cyclic group can contain one or more aromatic yl groups, one or more non-aromatic yl group or one or more aromatic yl group and one or more non-aromatic yl group.

Available formula-the C of term used herein " aldehyde " (O) H represents.This specification sheets in the whole text in, " C (O) " is the dummy suffix notation of C=O.

Term used herein " amine " or " amino " available formula NA ¹A ²A ³Represent, wherein A ¹, A ²And A ³Can be aforesaid hydrogen, alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group independently.

Available formula-the C of term used herein " carboxylic acid " (O) OH represents." carboxylate radical " used herein available formula-C (O) O ^-Represent.

Available formula-the OC of term used herein " ester " (O) A ¹Or-C (O) OA ¹Represent, wherein A ¹Can be aforesaid alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group.

The available formula A of term used herein " ether " ¹OA ²Represent, wherein A ¹And A ²Can be aforesaid alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group independently.

The available formula A of term used herein " ketone " ¹C (O) A ²Represent, wherein A ¹And A ²Can be aforesaid alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group independently.

Term used herein " halogenide " is meant halogens fluorine, chlorine, bromine and iodine.

Available formula-the OH of term used herein " hydroxyl " represents.

Available formula-the NO of term used herein " nitrogen " ₂Represent.

Available formula-the SiA of term used herein " silyl " ¹A ²A ³Represent, wherein A ¹, A ²And A ³Can be aforesaid hydrogen, alkyl, haloalkyl, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group independently.

Term used herein " sulfo-oxo " available formula-S (O) A ¹,-S (O) ₂A ¹,-OS (O) ₂A ¹Or-OS (O) ₂OA ¹Represent, wherein A ¹Can be aforesaid hydrogen, alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group.This specification sheets in the whole text in, " S (O) " is the dummy suffix notation of S=O.

Term used herein " alkylsulfonyl " is meant available formula-S (O) ₂A ¹The sulfo-oxo group of representing, wherein A ¹Can be aforesaid hydrogen, alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group.

Term used herein " sulfonamido " or " sulfamyl " available formula-S (O) ₂NH-represents.

The available formula A of term used herein " sulfone " ¹S (O) ₂A ²Represent, wherein A ¹And A ²Can be aforesaid alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group independently.

The available formula A of term used herein " sulfoxide " ¹S (O) A ²Represent, wherein A ¹And A ²Can be aforesaid alkyl, haloalkyl, thiazolinyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl group, Heterocyclylalkyl or heterocycloalkenyl group independently.

Available formula-the SH of term used herein " sulfydryl " represents.

" R used herein ¹", " R ²" " R ³", " R ⁿ", (wherein n is an integer) can have one or more above-mentioned groups independently.For example, if R ¹Be the straight chained alkyl group, a hydrogen atom of so described alkyl group randomly can be replaced by oh group, alkoxy base, alkyl group, halogenide etc.According to selected group, one first group can be incorporated in second group or can be to described second group described first group suspention (promptly connecting).For example, for phrase " alkyl group of amido-containing group ", described amino group can be incorporated on the skeleton of alkyl group.Perhaps, described amino group can be connected on the skeleton of described alkyl group.The character of selected group will determine described first group to be embedded into or to be connected on described second group.

Unless otherwise mentioned, the chemical bond that contains has not only been considered every kind of possible isomer with wedge shape or the chemical formula that is shown in dotted line with solid line,, for example every kind of enantiomer and diastereomer and mixture of isomers are as racemize or non-racemization chirality (scalemic) mixture.

A kind of imidazoles or imdazole derivatives method of suppressing part and TNF acceptor sample receptors bind by giving significant quantity is provided.

The example of the inhibitor that this paper considered is generally 5 member heterocyclic ring containing nitrogens, and described heterocycle works by various substituting groups.The example of this class inhibitor illustrates hereinafter, and generally determines (that is R wherein, by the title of unsubstituted basic heterocycle structure ⁿBe H).

Imidazoles Bi Zuo oxazole thiazole

1,2,3-

triazoles

1,2,4-triazole pyrroles

Isoxazole isothiazole

In many examples of inhibitor with said structure, R ¹, R ², R ³, R ⁴And R ⁵If exist, then be H, alkyl, alkoxyl group, thiazolinyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halogenide, hydroxyl, ketone, nitro, silyl, sulfo-oxo, alkylsulfonyl, sulfone, sulfoxide or sulfydryl independently.In concrete example, R ^1-5Be C independently ₁-C ₆Alkyl, C ₁-C ₆Alkoxyalkyl or C ₁-C ₆Alkoxy base.Exemplary C ₁-C ₆Alkyl group and C ₁-C ₄Alkyl group comprises methyl, ethyl, propyl group, sec.-propyl, butyl, sec-butyl, isobutyl-, amyl group, isopentyl, hexyl, 2-ethyl-butyl, 2-methyl amyl etc.Corresponding C ₁-C ₆Alkoxyl group contains the above-mentioned C with the Sauerstoffatom bonding ₁-C ₆Alkyl, described Sauerstoffatom also close with described positively charged ion ring key.Alkoxy-alkyl group contains with alkyl linked ether and contains six carbon atom at most altogether.Should be noted that the 1,2,3-triazoles of two kinds of isomeries.

The present invention has also considered other zone of target TNF acceptor sample acceptor, makes with this zone in case after combining, the conformation of PLAD will be destroyed in the described acceptor, thereby stops it and another PLAD association.For example, the CRD3 that those skilled in the art can target p60TNFR makes the CRD3 district of p60 TNFR in case combined, and the conformation of described acceptor will be changed, thereby prevents the PLAD and another PLAD association of p60 TNFR.

Therefore, the present invention also provides a kind of method that suppresses TNF acceptor sample acceptor oligomerization in the cell by the TNF acceptor sample acceptor oligomerization inhibitor that gives significant quantity.

The present invention has also considered by strengthening the effect that the PLAD self-association comes enhance TNF acceptor sample acceptor.For example, there is the situation that needs the conduction of enhance TNF R signal.Under this class situation, PLAD self-association agonist can be used to the responsive cell of the paired TNFR effect of cell transformation that effect has resistance to TNFR because weak PLAD interacts, and for example some can combine and have the antibody or the molecule of the special properties that strengthens the PLAD self-association to described agonist with PLAD.Combination of this class enhanced PLAD self-association can raising part and signal conduction.Need the example of the interactional morbid state of this class enhanced PLAD to include but not limited to autoimmunity lymphadenosis syndrome (ALPS) and high IgM syndrome.

The present invention also provides and has utilized PLAD as targeting moiety biological reagent to be sent to be delivered in the cell.For example, the PLAD that links to each other with toxin can be sent and is delivered in the cell, and naturally occurring PLAD one feasible and on the TNF-R combines, oligomerization just is suppressed, and described naturally occurring TNF-R one internalization, the PLAD that is connected with toxin will be by internalization, is delivered in the cell thereby described toxin sent.

Used in the whole text " the TNF acceptor sample acceptor " of this paper is meant any member of TNF receptor superfamily, includes but not limited to TNF-R, p60 (being also referred to as p55 and TNFR1), p80 (being also referred to as p75, TNFR2), Fas (CD95/APO-1), TRAIL acceptor, LT β R, CD40, CD30, CD27, HVEM, OX40, DR4, TROY, EDAR, XEDAR, DCR3, AITR, 4-1BB, DR3, RANK, TACI, BCMA, DR6, DPG, DR5, DCR1 and DCR2 (referring to table 1).The full content mode by reference of nucleotide sequence, peptide sequence and any information of listing according to the GenBank registration number that provides in the table 1 (for example signal sequence and maturation protein residue number) is included this specification sheets in.

As discussed previously, the inhibitor of TNF acceptor sample acceptor oligomerization comprises and PLAD specificity bonded antibody, part, peptide mimics, compound and polypeptide.These polypeptide comprise the polypeptide that contains PLAD or be made up of isolating (for example soluble) PLAD.

The present invention also provides a kind of method that suppresses part and TNF acceptor sample receptors bind by the TNF acceptor sample acceptor oligomerization inhibitor that gives significant quantity.For example,, can suppress TNF acceptor oligomerization, thereby prevent TNF-α and TNF receptors bind and reduce the deleterious effect of TNF-α by giving a kind of inhibitor (as comprising TNFR-PLAD or the polypeptide of forming by TNFR-PLAD).Similarly, the polypeptide that contains CD40 acceptor-PLAD (CD40R-PLAD) can suppress the CD40R oligomerization, thereby prevents that the CD40 part from combining with CD40R and reduce the deleterious effect of CD40R to the state of an illness (as allograft rejection, rheumatoid arthritis and systemic lupus erythematosus).Suppress part and TNF acceptor sample receptors bind and can cause suppressing signal transduction by means of TNF acceptor sample acceptor, thus the method that provides a kind of adjusting to conduct by means of the signal of TNF acceptor sample acceptor.In addition, the present invention has confirmed that TNF acceptor sample acceptor associates binding partner and conducted signal by homotype, and promptly TNFR-PLAD and TNFR-PLAD interact; Fas-PLAD and Fas-PLAD interact; CD40-PLAD and CD40-PLAD interaction etc.Therefore, utilize the PLAD self-association to disturb the treatment of peptide and peptide mimics can guarantee the receptor-specific treatment, only can associate by described PLAD and himself because the present invention has proved every kind of acceptor.For example disturb the TNF-R1 function and do not influence TNF-R2 and be better than present non-selective treatment.Similarly, specificity is disturbed specific TNF acceptor sample function of receptors and do not influenced other TNF acceptor sample function of receptors is unusual ideal, and can be provided by this paper instruction.

The protein for treatment method

The present invention also provides a kind of method for the treatment of the intravital inflammation of experimenter by the PLAD self-association inhibitor that gives significant quantity.The present invention also provides a kind of method for the treatment of inflammation relevant with autoimmune disorder in the subject by the PLAD self-association inhibitor that gives significant quantity.This class disease includes but not limited to periodic fever syndrome, sepsis syndrome and adult respiratory distress syndrome.Therefore, providing a kind of wherein said inflammation and described inhibitor relevant with septic arthritis is the method for the solubility PLAD of TNF acceptor sample acceptor.

In the present invention, described experimenter can be any Mammals, preferred people, and can include but not limited to mouse, rat, cavy, hamster, rabbit, cat, dog, goat, monkey, horse and orangutan.

" treatment " used herein described the improvement of patient's clinical state.Described improvement comprises from alleviating inflammatory response to improving inflammatory diseases fully.

The health that " autoimmune disorder " used herein described a kind of experimenter has produced morbid state or syndrome with the dysfunction immunne response of deleterious effect to himself body composition.The generation that this can comprise the generation of B cell or can comprise the T cell, described B cell can produce specific antibody at all antigens, allergen or main histocompatibility (MHC) antigen, and described T cell has the acceptor that can discern the cytokine that self component and generation can cause inflammation.The example of autoimmune disorder includes but not limited to ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, septic arthritis, diabetes, pernicious anemia, autoimmunity gastritis, psoriatic, behcets disease, the Wei Genashi granulomatosis, sarcoidosis, autoimmune thyroiditis, autoimmune oophoritis, bullous pemphigoid, pemphigus, the multiple endocrine glands disease, this Ti Leershi disease, Lambert-Eton myasthenic syndrome, myasthenia gravis, Goodpasture's syndrome, autoimmunity orchitis, the autoimmunity uveitis, systemic lupus erythematosus, siogren's syndrome and ankylosing spondylitis.

Be subjected to know from experience the oligomerization effect that depends on because some TNFR acceptor (as HVEA) is virus receptor and these, the present invention has also considered by stoping PLAD to assemble blocking virus to enter.

Used optimal dose should change according to the individual and used inhibitor of being treated.The amount of inhibitor also should change according to age of individuality, stature, body weight, situation etc.Those skilled in the art will recognize that the dosage that the medical practitioner optimizes is best, and for example, described the method that is used for definite dosage and therapeutic regimen and prepares formulation among Remington ' the sPharmaceutical Sciences.For example, by with at present in the treatment of inflammation or autoimmune disorder or prevention used medicament relatively can determine the dosage and the dosage regimen that are fit to.

Usually, can come orally give or parenteral to give inhibitor of the present invention by the dosage range of 0.1 to 100mg/kg body weight according to the clinical response that will obtain.For example, when described inhibitor is solubility PLAD or when containing the PLAD compound, can give described inhibitor, for example 5mg/kg with 0.5 to 100mg/kg amount.In a further example,, can give described PLAD with the dosage in each joint 0.5 to 100mg when described inhibitor is solubility (isolating) p60PLAD and described disease when being sacroiliitis (for example septic arthritis).For example, to give dosage be that the p60 PLAD that 4mg/kg or parenteral give 16mg/kg can treat septic arthritis effectively to intraarticular.Disease sign and symptom long-term relax or stable after can stop the administration of inhibitor fully and can rechallenge behind described disease sign or severity of symptoms or after the immunological status considerable change, this can follow up a case by regular visits to immune Research by the known routine of this area doctor and determine.

Determine the effect of inhibitor aspect treatment inflammation as herein described or autoimmune disorder of the given dose that given by the concrete aspect of assessment medical history, sign, symptom and objective lab investigation, have proof property purposes aspect the pathologic, physiologic of the particular disorder that described objective lab investigation are treated in assessment is active.These signs, symptom and objective lab investigation can change according to the concrete illness of being treated, and this is that any doctor in this area is known.For example, according to the comparison of suitable control group and about the knowledge of the normal procedure of described disease in general crowd or particular individual, if 1) proved that experimenter's recurrence frequency or severity have obtained improvement; 2) it is stable to have proved that disease or illness process have obtained; 3) reduced the demand of using other immunosuppression medicament, can think that then a kind of specific treatment is effective.

In case confirm by a kind of special inhibitor can significantly improve or the stable disease activity after, just can according to simplification or discontinuous treatment plan assess specific sign, symptom or lab investigation.According to the standard method of assessment specific sign as herein described, symptom or objective lab investigation, if disease activity recurrence, begin treatment so once more.

In addition, can determine the effect of peptide part aspect prevention experimenter autoimmune disorder of the given dose that given by long-term evaluation criteria sign, symptom and objective lab investigation (well known by persons skilled in the art), described experimenter does not suffer from autoimmune disorder but the danger that develops autoimmune disorder is arranged.This timed interval can very long (that is several years/many decades).Can define the patient of developing the autoimmune disorder risk according to the knowledge of the known danger factor of the specified disease of being familiar with about this area doctor and researchist at present (as strong especially disease family history or be exposed to or obtained to cause producing the factor or the condition of autoimmune disorder).

" pharmaceutically acceptable " is meant not can be biologically or have the material of undesirable action aspect other, promptly can together give individuality, can not cause simultaneously any bad biological effect or with bad mode and any other component interaction that contains its pharmaceutical composition with described material and selected compound.Medication and concrete patient are depended in the selection of carrier.Medication can be oral, the hypogloeeis, mucous membrane, that suck, that absorb or pass through injection.Also should note not being that all methods that gives TNF acceptor sample acceptor oligomerization inhibitor described herein all need pharmaceutically acceptable carrier.

Among the present invention, can orally give or parenteral give at the PLAD of the pharmaceutically acceptable carrier that is used for people experimenter self-association or TNF sample oligomerization inhibitor.The carrier that is suitable for oral or inhalation can comprise that one or more are used for pharmaceutically acceptable carrier of people experimenter.Be suitable for oral carrier and comprise that one or more also can be used as seasonings, lubricant, suspending agent or protectant material.The solid-state carrier that is fit to comprises calcium phosphate, lime carbonate, Magnesium Stearate, sugar, starch, gelatin, Mierocrystalline cellulose, carboxylic polymethylene (carboxypolymethylene) or cyclodextrin.The liquid carrier that is fit to comprises the mixture of water, the salt solution that does not contain pyrogen, acceptable oil or any of these.Described liquid also can contain other medicated premix that is fit to for example buffer reagent, sanitas, seasonings, viscosity or Osmolyte regulator, stablizer or suspending agent.The example of the liquid carrier that is fit to comprises the water that contains or do not contain various additives, and described additive comprises the carboxypolymethylene of regulating glue as ph-.Described inhibitor can be contained in the enteric coated capsule, and described enteric coated capsule can discharge polypeptide in the enteron aisle to avoid destruction in the stomach.In order to give antagonist through parenteral, with the prepared in saline sterile solution that can contain additive (as ethyl oleate or isopropyl myristic acid ester) or suspension and it can be expelled to for example subcutaneous or the intramuscular tissue in and intravenously.

Screening method

A kind of method of screening PLAD association inhibitor, comprise: a) with following two kinds of same cells of plasmid transfection, wherein a kind of plasmid contains the nucleic acid of the nucleotide sequence that comprises a kind of isolating PLAD that encodes, and another kind of plasmid contains the nucleotide sequence of the another kind of isolating PLAD that encodes; B) described cell is contacted with the inhibitor of supposition; And c) measure the self-association of PLAD, wherein with not with cell that described supposition inhibitor contact in the PLAD association compare, the associating minimizing of PLAD has shown and has had PLAD association inhibitor in the cell of step b).

An example of this screening method is a kind of method of the PLAD-of screening association inhibitor, comprise: a) with following two kinds of same cells of plasmid transfection, the nucleic acid that wherein a kind of plasmid contains comprises coding and the nucleotide sequence of the functional isolating PLAD that is connected of fluorescence donor, and another kind of plasmid contains contains the nucleotide sequence of encoding with the functional isolating PLAD that is connected of fluorescent receptor; B) described cell is contacted with described inhibitor; And c) measure FRET, wherein with not with cell that described inhibitor contacts in the FRET measurement compare, the decline of FRET shows and has PLAD association inhibitor.

The present invention also provides a kind of method of the PLAD of screening association agonist, comprise: a) with following two kinds of same cells of plasmid transfection, wherein a kind of plasmid contains the nucleic acid of the nucleotide sequence that comprises a kind of isolating PLAD that encodes, and another kind of plasmid contains the nucleotide sequence of the another kind of isolating PLAD that encodes; B) described cell is contacted with putative agonist; C) measure the self-association of PLAD, wherein with not with cell that described putative agonist contact in the PLAD association compare, the associating increase of PLAD shows and has PLAD association agonist in the cell of step b).

Following example understands that for example using FRET to measure PLAD associates.In addition, when carrying out above-mentioned screening method, can use single plasmid to send to pass the nucleic acid of a plurality of coding PLAD.In comprising the method that FRET analyzes, can use single plasmid to send to pass the nucleic acid of a plurality of coding PLAD, described PLAD is connected with fluorescence donor or function of receptors ground.

Those skilled in the art also can utilize the yeast two-hybrid screening method to screen PLAD association inhibitor or excited anti-agent.Also can identify associating inhibitor of PLAD or agonist by using raji cell assay Raji, described raji cell assay Raji can include but not limited to that apoptosis-inducing, NF-KB induce, lymphocyte maturation or activation and necrosis induction (3,4,8,15,29,33,42,45).

More specifically described the present invention among the following embodiment, described embodiment only is intended to illustrate, because many modifications and variations wherein are apparent to those skilled in the art.

Embodiment 1

Washing H9 lymphoma cell also is resuspended among the PBS.Then at 4 ℃ of people's recombinant chou TNF α (R﹠amp that use 100ng/ml down; D Systems) the rotation incubated cell is 1 hour.Use 2mM linking agent DTSSP (Pierce) to handle cell 30 minutes then and with 20mM Tris.Cl[pH 7.5] stop described reaction 15 minutes on ice.With the 150mM NaCl, the 20mM Tris.Cl[pH 7.5 that have added proteinase inhibitor (Boehringer Mannheim)], 1mM EDTA, 30mM NaF, 2mM b-glycerophosphate and 1mM sodium orthovanadate lysing cell.Under the condition of non-reduced (not containing beta-mercaptoethanol) or reduction (containing the 280mM beta-mercaptoethanol), analyze p60 and p80 complex body (19) with the lysate electrophoresis of equivalent and with specific antibody.Carry out photodensitometry with Kodak Image Station 440.

Find that the shown molecular size of p80 complex body is about three times of cell size, meet glycosylation and glycosylation tripolymer (Figure 1A) not.Surprisingly, under the situation that has or do not exist TNF α, all can capture p80 complex body (recording the 65-70% that accounts for chain by photodensitometry, Figure 1A, swimming lane 3 and 4) effectively.Be present in the golgi body and can't touch linking agent (7) although the fact is most of p60, no matter whether add TNF α, all there is the p60 chain of similar 15-20% to be cross-linked into tangible tripolymer and discontinuous higher complex body (Figure 1A, swimming

lane

11 and 12).The control experiment demonstration does not have detectable endogenous TNF α and does not demonstrate other albumen (as p80) crosslinked with the p60 complex body.Western blot analysis confirms not contain TNF α in the lysate.But the immunoprecipitation that uses the crosslinked complex body of anti--p60 antibody shows the p80 that does not have detection level in the p60 complex body in the western blotting.

By described complex body being resolved into monomer (Figure 1A, swimming

lane

7,8,15 and 16) with beta-mercaptoethanol cutting linking agent.Therefore, these results show at part in conjunction with preceding p60 and p80 chain meeting self-association.

For verifying the possibility of non-ligand dependent self-assembly, identified the structural domain of the TNFR that mediates this phenomenon.Determined to have served as when expressing, the kytoplasm death domain of p60 can self-association and can be triggered apoptosis (8).But,, be present in outside the cytoplasmic region so people suppose described assembling territory because observed pre-assembled complex body obviously is that non-signal is conductive.It is found that in yeast two-hybrid interacts mensuration the N-terminal district self-association specifically (9) of the ECD of p60 and p80.

Can produce the various clipped forms of p60, p80, HVEM, DR4 and CD40 and mutant form and with they order-checkings by polymerase chain reaction (PCR).In brief, the leader sequence of amplification p80 and preceding 10 amino-acid residues make and can comprise that HA epi-position label is to produce the HA label at the N-of acceptor end at 3 ' end.Digest described PCR product and it is cloned among the pcDNA3 with BamH I and EcoR I.The PCR fragment that will contain receptor fragments by EcoRI and XhoI site is directed in this plasmid then.For described GFP/CFP/YFP mosaic, can meet among the Xho I and Xba I site of frame ground importing p60 Δ CD-HA by the described fragment of pcr amplification and with it.According to the specification sheets of each manufacturers Fugene6 (Boehringer Mannheim) transfection 293T cell.With 150mM NaCl, 20mM Tris.Cl[pH7.5], 1mM EDTA, 30mM NaF, 2mM β-glycerophosphate, 1mM sodium orthovanadate, 5mM iodo-acid amide, 2mM dithiothreitol (DTT) (DTT), 1%TRITON X-100 and proteinase inhibitor (Boehringer Mannheim) lysing cell.With behind Protein G agarose microbeads (BoehringerMannheim) and the normal mouse IgG preclearing, with 2mg resist-GFP and Protein G agarose microbeads get off albumen immunoprecipitation from described lysate., wash three times with conventional lysis buffer then described immunocomplex washed twice with the lysis buffer that contains 0.5M NaCl.Go up the discrimination immunocomplex at Tris/ glycine glue (Novex).The transfection of Jurkat cell has shown similar result.In mammalian cell, it is found that chimeric p60 is subjected to know from experience and anury p60 (p60 Δ CD-HA) interacts consumingly and can not invade regulatory factor (HVEM Δ CD-HA) interaction with TNFR sample acceptor simplexvirus, the cytoplasmic structure territory of described chimeric p60 acceptor is replaced (Figure 1B, relatively swimming lane 2 and 3) by green fluorescent protein (GFP).

GFP can't be separately and p60 Δ CD-HA associate (Figure 1B, swimming lane 5).Observe similar born of the same parents' outside part homotype interaction (Fig. 1 C, swimming lane 6) is arranged between total length p80 and the anury p80.

In addition, only remove the 10-54 amino acids (a.a.) of p80, promptly overlapping CRD1 can stop the non-ligand dependent association (Fig. 1 C, swimming lane 7) with complete p80 fully.Can eliminate self-association (vide infra) by in p60, similarly lacking (a.a.1-54).

Append on the p60 acceptor by the N-terminal portions (a.a.10-54) with p80, described then p60 acceptor and total length p80 interact, can further specify the N-terminal portions of this p80 importance (Fig. 1 D, relatively swimming lane 3 and

swimming lane

4 and 5, Fig. 9).Therefore, this structural domain is enough to mediate the specificity association of heteroreceptor.This association is non-ligand dependent, because chimeric p80 _10-5460 _55-211(R1) described restriction site is inserted in the joint of p80 and p60 sequence by EcoRI restriction site amino acids coding to have two, and described amino acid has been ended the combination (Fig. 1 E, picture k and 1) of TNF α.Therefore, lacking under the situation of part, a kind of new functional domain different with the ligand binding pocket of TNFR-ECD mediated self-assembly.Since then, part assembling territory (PLAD) before this structural domain is called as.

The described PLAD of disappearance can eliminate part fully in conjunction with (table 2 and Fig. 1 E, relatively picture d, f and h) among p60 or the p80.Yet the PLAD that adds p80 can make the p60 of disappearance PLAD to combine with TNF α, but as mentioned above, can stop this combination (Fig. 1 E, picture j and 1) by inserting two by EcoRI site amino acids coding.Therefore, TNFR and TNF α effective combines the self-assembly of depending on acceptor.Perhaps, remove PLAD and can destroy whole ECD structure.Remove correct folding (13) that CRD1 can not destroy p60 ECD because previous result shows, a kind of possibility is impossible after the institute.Yet,, in the CRD 1-3 of p60 Δ CD-HA, introduce amino-acid substitution and measure them the interactional influence of p60 Δ CD-GFP-HA for differentiating these possibilities clearly.Specification sheets according to each manufacturers uses Quikchange method (Stratagene) to carry out mutagenesis.Confirm described sudden change by dna sequencing.Except that K19, all metathetical amino acid all are conservative propertys among p60 and the p80.Residue corresponding among the p80 is E22.Among the PLAD two kinds displacement KY19/20AA and K32A (5) eliminated self-association (Fig. 2 A, relatively swimming lane 3 and 5 with swimming lane 2) and got rid of TNF α and combined (table 2), described displacement not desire is interrupted direct part and is contacted.The displacement of another residue among the PLAD--Q24A does not influence self-association or TNF α combination (Fig. 2 A, swimming lane 4 and table 2).The residue number of indication is based on the mature sequence of p60 in the table 2.Two kinds of other displacements in the PLAD outside in the CRD2 ligand binding pocket--E57A and N66F can interrupt TNF α combination but the acceptor self-association almost do not influenced (table 2 and Fig. 2 A, swimming lane 6 and 7).The mutant receptors that people also find to lack cytoplasmic tail is relevant with the ability of the association of wild-type ECD and its dominant interference p60 inductive apoptosis aspect, shown described mutant receptors enter by PLAD in source functional p60 acceptor complex body (table 2).But these results show PLAD itself different with part contact structures territory be still for effective TNF α combination and function of receptors necessary.

Different with the monomeric acceptor chain, estimate that the kytoplasm part of receptor chain in the pre-assembled acceptor complex body is extremely approaching each other.TNF α causes the cytoplasmic structure territory to associate more closely in conjunction with meeting like this, thereby has caused raising of signal conductive protein.For assessing this hypothesis, adopted the novel fluidic cell method described in the example II (11) to analyze two kinds of FRET (fluorescence resonance energy transfer) (FRET) between the GFP spectrum variant, cyan fluorescent protein (CFP) is as the fluorescence donor, and yellow fluorescence protein (YFP) is as fluorescent receptor (12).FRET is the method for interaction of molecules in a kind of effective measurement viable cell.Because energy shifts and weakens rapidly when distance increases between the fluorophore, so the FRET between the GFP variant allows to detect

Interior molecular interaction.

Produced chimeric protein, wherein with the cytoplasmic region of CFP or YFP displacement p60 and p80 and test with determine different acceptors between whether the energy transfer has taken place.Carry out FRET with two laser FACSvantage instruments, excite YFP albumen, excite CFP albumen at the 413nm place then at the 514nm place.Detect the energy transfer of CFP by the emission that detects the 546nm place then to YFP.With coming transfectional cell more than the proteic YFP albumen of CFP far away.Use Flowjo program (Treestar Inc.) to analyze the positive group's of CFP FRET then.

The energy of having observed between p60 Δ CD-CFP and the p60 Δ CD-YFP shifts, and described energy shifts and increases after adding TNF α basically that (Fig. 2 B, picture a).Stop this FRET (Fig. 2 B, picture b and c) by disappearance PLAD or by preventing that the associating K32A of PLAD from suddenling change.Similarly p80 Δ CD-CFP:p80 Δ CD-YFP has shown the strong FRET signal (Fig. 2 B, picture d) that increases once more by adding TNF α to analysis.Use p60 Δ CD-YFP to show there is not FRET (Fig. 2 B, picture e and f) as the contrast of donor as acceptor and p80 Δ CD-CFP or CFP-p80 Δ CD (with the terminal CFP that merges of the N-of ectodomain).These data have proved together in viable cell, p60 and p80 chain self all extremely near and part can induce the variation of complex body, described variation can cause CFP and the YFP in the kytoplasm partly to take place to associate closely.

Conservative property (Fig. 3 A, the Y23 among the p80, P36, G37 and T50) in the PLAD of p60 and p80 and the TNFR family outside the halfcystine that relatively demonstrates the disulfide linkage support that forms described structural domain of a CRD of many acceptors.The conservative property that some other TNFR sample acceptor that contains DR4 and Fas demonstrates CRD1 is relatively poor, has produced the query that whether has PLAD spline structure territory in these acceptors thus.The present invention has probed into whether there is non-ligand dependent self-association among other member of TNFR superfamily.

It is shocking that the ECD of TRAIL acceptor 1 (DR4) and CD40 can self-association, but with the ECD of other TNFR sample acceptor interact not significantly (Fig. 3 B).These mosaics have also shown isotypic specificity FRET (Fig. 3 C).Described in example II, Fas (CD95/APO-1) also can with himself specificity associate (11).Therefore, the self-assembly by PLAD is a conservative property feature of TNFR family.

The present invention has shown and has lacked under the situation of part, but p60 becomes the function sex camplex with p80TNFR by the novel N-end structure territory pre-assembled that is called as PLAD.This has disclosed CRD1 is (10) that how play an important role in the part combination of p60 and p80 and receptor signal conduction.Up to the present, the key concept of carrying out the signal conduction by the TNFR superfamily member is that part can make the monomeric acceptor chain be attached on three times of complex bodys, and this can cause raise (1,3,5,6) of kytoplasm signal transducer.This model is based on the crystalline structure of part compound p60 to a great extent, and this crystalline structure shows that three receptor chains are around the subunit inter-drain of described trimerization part and keep separately at least

Described part contacts with the CRD3 structural domain with the CRD2 of extension, and the CRD1 structural domain is not with ligand interaction or do not interact each other (5).Recently the description about the DR5/TRAIL complex structure has demonstrated similar receptor-ligand binding (13).As if yet the described structure that combines part can't reflect that part is in conjunction with preceding receptor structure.People have known p60 and the p80 meeting self-association on the cell surface now, if disappearance PLAD, then they only exist with monomeric form.Crosslinked endogenous p60 and p80 acceptor show that tripolymer is the conformation of preponderating, but also can have other oligomerization complex body.

Owing to clear preceding association now is that TNF α is in conjunction with required, so how to receive great concern with pre-association acceptor complex body interaction about part.One of them that utilize two big class models can be explained the conduction of TNFR signal, and described model is: 1) chain rotation and resetting; With 2) super cluster (supercluster) formation model (Fig. 3 D).In chain rotation with reset in the model, part embeds in the preformed acceptor tripolymer, causes the PLAD contact to be interrupted and chain is rotated and is rearranged into tripolymer, and described tripolymer is only stabilized by contacting with the part tripolymer.Perhaps, preassembled TNFR is trimerical to cluster part in conjunction with triggering, and wherein said PLAD contact is not interrupted fully.The importance of the signal conduction of being undertaken by this receptor extended familys has been illustrated in the trimerical existence of preassembled TNFR of PLAD-mediation again, and many members are vital (2) for lymphocyte function and stable state in the known this receptor family.The conservative property of Key residues is the feature of TNFR superfamily member among specificity homotype ECD contact and the PLAD, and described superfamily comprises by death domain and carries out the acceptor (p60, DR4 and Fas) of signal conduction and the acceptor (p80 and CD40) that does not carry out the signal conduction by death domain.On cell surface, chain presorted and to promote the validity and the specificity of replying for the homotype complex body." acceptor interference " should be avoided, and for example disturbs p80 chain (lacking death domain) to be raised by TNF α and p60 constitutes complex body at described acceptor, and causes the obvious inhibition to apoptosis.P80 has supported this viewpoint (15) by independently short actual this fact of p60-inductive apoptosis that strengthened of apoptotic signal is provided.The prerequisite that preformed tripolymer also can avoid conventional model to require is promptly raised receptor chain continuously with " foundation " complex body, therefore causes carrying out fast signal conduction (3) by TNFR sample acceptor.

Pre-assembled to other receptor family is on the books, particularly IL-1 and IL-2, and they constitute (16) by the different aggressiveness of homopolypeptide not.It should be noted that especially recently that about the pre-associating description of erythropoietin receptor dimer described dimer has obviously experienced " scissor " motion to admit part (17).In that case, then can contact by same amino acid and carry out the receptor chain self-association, combination is vital (17) for part in described amino acid contact.On the contrary, the TNFR superfamily has utilized the special-purpose self-association structural domain different with CRD2/3 part zone of action.The evaluation of PLAD can provide by specificity and suppress the preceding part assembling of TNFR sample acceptor and thereby stop the therapy of signal conduction to treat the new therapy of the disease that TNF α or associated ligands cause.

Example II

In human autoimmune lymphadenosis syndrome (ALPS), the heterozygous mutant of coding anomaly pattern Fas (CD95/APO-1) can dominant interference Fas inductive Lymphocyte Apoptosis.The present invention has proved that this situation derives from the wild-type of utilizing ectodomain and the pre-association of mutant Fas acceptor, and does not rely on part inductive acceptor oligomerization.Find that pre-associating Fas acceptor complex body is that signal transduction is necessary, and confirmed pre-associating Fas acceptor complex body in the viable cell by new being applied in of the FRET between green fluorescent protein (GFP) variant.These results provide the new molecular mechanism of a kind of human disease of being used for Fas signal conduction and dominant interference.

Fas (APO-1/CD95) is the cell surface receptor (19-21) of transduction for immune stable state and the vital apoptotic signal of tolerance.Fas has three 317 amino acid whose 1 type membrane glycoproteins that are rich in the ectodomain (CRD) of halfcystine, and described ectodomain is the feature of Tumor Necrosis Factor Receptors (TNFR) superfamily.

For the mankind, the lymphocyte that carries the patient who suffers from 1A type ALPS of heterozygosis Fas sudden change has the Fas inductive apoptosis of minimizing, and the transfection meeting of mutant allele causes the dominant interference (29-34) to Fas inductive apoptosis.This is considered to owing to ligand-mediated crosslinked wild-type and defective Fas chain have been cross-linked into the tripolymer complex body that blended can not be raised downstream signaling molecule.Yet, after deliberation cause the dominant interference sudden change of ectodomain (ECD) disappearance (Pt 2, disappearance a.a.52-96) of most of CRD2 by the RNA montage that changes.On the negative 293T cell of Fas-the expression of this sudden change show this mutant not with antagonism antibodies (Fig. 4 A) (33,35).This mutant can not combine with the FasL of trimerizing, and (for example Pt 6, A241D) do not influence FasL combination or APO-1 in conjunction with (Fig. 4 A) and the ALPS in the kytoplasm death domain suddenlys change.Even not with antagonism antibody or FasL bonded situation under, Pt 2 mutant also can be with almost the same with Pt 6 death domain mutant efficient dominant interference Fas inductive apoptosis (Fig. 4 B).The padding of cotransfection cell shows with those to be compared with WT Fas cells transfected separately, and Fas does not express and reduces, and gets rid of the possibility (36) that described mutant Fas molecules in inhibiting normal allele is expressed.Therefore, by routine can not explain dominant interference by the crosslinked signal of FasL inductive acceptor monomer conduction model because Pt 2 mutant Fas molecules can not become a part of mixing the acceptor complex body in this process.Therefore, the non-ligand dependent that uses construct to test between Pt 2 Fas and the wild-type Fas interacts, in described construct the cytoplasmic structure territory of wild-type Fas replace by green fluorescent protein (GFP) (HA-Fas 1-210:GFP), with the false appearance mutual effect of avoiding causing (24,37) by described death domain.Lacking under the situation of FasL, with Fas 1-210:GFP block polymer co-immunoprecipitation total length Fas acceptor and Pt 2 Fas acceptors (Fig. 4 C).This interaction is specific,--invades medium (HVEM Δ CD:GFP) with the simplexvirus that GFP merges--not with the Fas immunoprecipitation because another member of TNFR family.

For carrying out these immunoprecipitation research, according to the specification sheets of manufacturers Fugene 6 (BoehringerMannheim) transfection 293T cell.With 150mM NaCl, 20mM Tris.Cl[pH 7.5], 1mMEDTA, 5mM iodo-acid amide, 2mM dithiothreitol (DTT) (DTT), 10% glycerine, 1%TRITON X-100 and proteinase inhibitor (Boehringer Mannheim) lysing cell.After with Protein G agarose microbeads (Boehringer Mannheim) and normal mouse IgG preclearing, resist-GFP (RocheMolecular Biochemicals) and Protein G agarose microbeads immunoprecipitation albumen with 1mg.Wash immunocomplex three times with lysis buffer.With anti-AU1 of 2 μ l (Covance) and albumin A microballoon immunoprecipitation AU1.Albumen is gone up electrophoresis in Tris/ glycine gels (Novex), transfer on the nitrocellulose filter and and carry out trace with specified antibody.Band is developed with SuperSignal WestDura (Pierce).(Kodak) carries out photodensitometry with the 1D image analysis software.

In example I, described and be called as " preceding part assembling territory " conservative property N-end structure territory (PLAD), described other member's of structural domain mediation TNFR superfamily specificity self-association.Yet the N-end of Fas lacks several key amino acid conservative in other TNFR family receptors, has therefore produced the query whether Fas contains functional PLAD.

Made up brachymemma or removed the terminal Fas mutant of N-of a CRD, and tested part and associate and apoptosis function (Fig. 5) in conjunction with, Fas-Fas.Produce the Fas truncated mutant with suitable primer and Pwo high-fidelity polysaccharase (Roche Molecular Biochemicals) by PCR mutagenesis.For the acceptor of AU-1 mark, used a kind of template that is inserted into the AU-1 label in Fas CRD 1 upstream in advance that has.For carrying out the HA mark, in the EcoRI/XhoI site of the pcDNA3 carrier of transforming, described carrier contained the leader sequence of p80 before the HA sequence label with mutant clon.Point mutation is to use Quickchange technology (Stratagene) to produce, and replaces the Pfu polysaccharase with Pwo.Verify sudden change by restriction endonuclease mapping and automatic sequencing.

The above-mentioned disappearance that studies show that initial 43 amino acid (a.a.) of the one CRD subdomain of the formation in the ripe Fas albumen can significantly reduce part in conjunction with but can not stop the combination (39) of APO-1 agonistic antibody.Initial 66 amino acid of disappearance coding whole C RD1 can blocking-up and combine (Fig. 5 A) of FasL and APO-1.These two kinds of disappearances have shown the shortage of associated disadvantages (Fig. 5 C) in the apoptosis that is started by APO-1 antibody and described brachymemma chain and the proteic co-precipitation of Fas 1-210 with different labels, described Fas1-210 albumen contains complete ECD (Fig. 5 B, swimming lane 1-4).Therefore, although the FasL combination and the normal APO-1 combination of part are arranged, only remove from Fas N-end that 43 amino acid promptly can stop apoptosis induction, this associating disappearance with these truncation type acceptors and wild-type Fas is relevant.That predict all be present in this fact among CRD2 and the CRD3 with contacting of FasL based on most of, the FasL bonded disappearance that is caused by the disappearance of 66 amino acid (constituting CRD1) is unexpected (22,23).By relatively these results and those results who obtains with p60 and p80 in example I, it may be vital that the pre-installed pairing of non-ligand dependent of having supposed Fas acceptor complex body provides effective FasL combination and receptor signal to conduct.For further probing into part bonded prerequisite in the acceptor self-association, tested a kind of Fas point mutation-R86S (23), this R86S sudden change has removed in conjunction with the very important CRD2 contact residues of FasL, finds that it does not combine (Fig. 5 A, figure below) with FasL when cell surface expression.As utilize two kinds of different antagonism anti--coloration result of Fas antibody shown in, whole receptor structure is to preserve complete (Fig. 5 A and (18)); And shown in co-immunoprecipitation, the self-association (Fig. 5 B, swimming lane 5-7) of complete Fas can take place still.Even more meaningfully, when with wild-type (WT) acceptor coexpression, this mutant meeting dominant interference FasL inductive apoptosis himself does not combine with FasL (Fig. 5 D, solid bars) simultaneously.All not impaired in all transfections with APO-1 antibody inductive apoptosis in same cell, shown all functional expressions (Fig. 5 D, hollow strips) on cell surface of two kinds of acceptors.Therefore, dominant interference does not rely on the part combination of natural existence and genetic engineering modified Fas mutant.On the contrary, the Fas function is relevant with the self-association ability.

Being the Fas acceptor self-association in the quantitative assay viable cell, having developed based on having different spectrographic GFP mutant--the fluidic cell of FRET (fluorescence resonance energy transfer) (FRET) is measured and micro-mensuration between cyan fluorescent protein (CFP) and the yellow fluorescence protein (YFP).CFP and YFP have the spectral quality that helps FRET, because the maximum emission wavelength of CFP approaches the maximum absorption wavelength (40) of YFP.Since the FRET between these albumen distance greater than In time, can descend rapidly, so exist FRET to show that then their fluorescin structural domain is very approaching between CFP and the YFP fusion rotein.When the C-end being met the Fas acceptor cotransfection in the 293THEK cell time that has merged CFP and YFP (having replaced described death domain at the 210th) in frame ground, they are able to suitable expression (36) on cell surface.

Make Fas and other TNF family receptors and CFP and YFP produce in-frame fusion by Standard PC R clone technology, utilize western blotting and fluorescent microscopy to confirm correct protein expression.With described YFP fusion protein construct of 1 μ g and the described CFP construct of 2 μ g transfection 293T cell.After 24-36 hour, analyze, when being used for CFP Kr laser (Spectrophysics) is transferred to 413nm, when being used for YFP ILT air cooling laser apparatus is transferred to 514nm with the PBS collecting cell and with the FACSvantage cell instrument.Detect CFP with the 470nm/20nm bandpass filter.Detect YFP and FRET with the 546nm/10nm bandpass filter, the signal that uses 514nm and 413nm laser apparatus to produce respectively.Then with 514 and 413nm laser apparatus Continuous irradiation cell make and can from each cell, detect whole three kinds of signals.Adopt compensation correction so that in separately with CFP or YFP cells transfected, do not have visible FRET signal.From each sample, collect 50,000 incidents and utilize FlowJo software package (Treestar) analytical data.Be to measure the efficient of FRET,, measure the CFP emissive porwer of cotransfection cell with fluorescent microscope utilizing the 505-545nm bandpass filter with before by 5 minutes irradiation bleaching YFP and afterwards.Contrast shows that this hard intensity bleached YFP basically up hill and dale, the basic simultaneously CFP that directly do not bleach.Proofread and correct at this direct bleaching.Use following formula to calculate FRET efficient: E%=[1-(the CFP emission after the CFP emission/YFP bleaching before the YFP bleaching)] * 100%.

When utilizing Flow cytometry, because FRET, triggered the hyperfluorescence emission (Fig. 6 A, Fas1-210:CFP/Fas 1-210:YFP) at YFP wavelength place with the CFP emission of the cell of CFP and YFP Fas fusion rotein cotransfection, particularly when the YFP high level expression.Used and a kind ofly passed through construct that 9 amino acid whose peptide connectors merge (CFP-YFP) with CFP and YFP covalency as positive control (41).In these cells, also detected strong FRET signal with the expression level linear growth.Between the Fas fusion rotein that contains or do not contain described death domain, detect FRET, but between Fas and TNF family member TNFR1 or HVEM, do not detected FRET (Fig. 6 A and B).When with Fas 1-210 coexpression, brachymemma or removed the FRET signal that the Fas of the terminal clipped form of N-of PLAD produces and reduce (Fig. 6 B).Be the FRET efficient between these different acceptor mutants of quantitative measurment, the CFP behind the selective light bleaching YFP acceptor molecule taken off cancellation (dequenching) (another feature of FRET) carried out based on microscopical mensuration (40,24) (Fig. 6 C).It is 16% FRET that the self-association that lacks the Fas of death domain has produced observed efficiency.When all having death domain on two molecules, FRET efficient rises to 27%, and this has shown the oligomerization character (42) of death domain.Pt 2 Fas have produced the FRET efficient close with Fas 1-210, shown near normal self-association, but signal weakening when being to use Fas 43-210 do not have tangible FRET efficient when using Fas 67-210.These results show the specificity self-association on cell surface of Fas molecule, and this character depends on PLAD.

Whether can self-association on the T of untransfected lymphocytic cell surface for testing natural Fas acceptor, carried out chemically crosslinked research (Fig. 7).Adding can not penetration cell the linking agent 3 of cut sulfydryl, the two sulfosuccinimide propionic esters (DTSSP) of 3 '-two sulphur can become 140kD from 45 with the apparent molecular weight of Fas in the de-glycosylation cellular lysate, this is corresponding to forming Fas tripolymer (Fig. 7 A, swimming lane 2).Comparison shows that with the optical density(OD) of monomer band 60% Fas chain is cross-linked into tripolymer.Most of trimerization complex bodys can be reduced to monomer attitude (Fig. 7 A, swimming lane 5-8) with dithiothreitol (DTT) (DTT) cutting linking agent.DTSSP inductive complex body is similar to those complex bodys (Fig. 7 A, swimming lane 3-4) (25) that does not carry out existing after the chemically crosslinked using APO-1 antagonism antibody or FasL to excite.The complex body of agonist induction connects by intermolecular disulfide bond, and this can also prove (Fig. 7 A, swimming lane 7-8) originally by DTT.The detection that the Fas signal conduction complex body of the immunoprecipitation of these cells is carried out shows, antibody or part excite can trigger FADD and caspase-8 raising and cause caspase-8 by proteolysis become its 41 and the processing of 43kD after the caspase dependency cutting (Fig. 7 C) of form (Fig. 4 B) and poly-(ADP-ribose) polysaccharase.Yet the FADD that the signal conduction complex body in the cell of handling with DTSSP shows moderate associates, but do not have caspase-8 in conjunction with or processing and do not have the PARP cutting, show that the chemically crosslinked of pre-association acceptor complex body is not enough to trigger apoptotic signal.Pre-treatment that it should be noted that DTSSP can prevent the formation (Fig. 7 B) of the active signal conduction complex body handled in response to subsequently APO-1.These results show that the non-covalent pre-association of Fas acceptor did not rely on expression.Part is in conjunction with the variation that has triggered the acceptor complex structure, and described structural changes forms relevant with the conduction of intramolecularly signal with interchain disulfide bond.As if the chemically crosslinked of Fas acceptor can catch the pre-association complex body of non-signal conduction attitude.

The terminal PLAD of conservative property N-is that the suitable Fas function of receptors of performance is necessary, and therefore can play a key effect in the dominant interference of ALPS.Structure, dominant interference (DI) and the Fas-Fas self-association (SA) (29,33-35) of having compared a large amount of ALPS patients that NIH studies are (Fig. 8), it is found that in the dominant interference sudden change sample of each and disease-related all to have kept PLAD, comprise the sudden change that has influenced part in the outer or born of the same parents of Fas born of the same parents.In Pt 1 and 20, sudden change has produced the only premature termination polypeptide of encoding mature Fas proteic preceding 57 and 62a.a., shows that PLAD itself is enough to carry out dominant interference (Fig. 8 A).Utilize point mutation to remove all or part of death domain (Pt 5,30 or 33) or eliminate its FADD combined function (Pt 3,6,26,29 or 31) and can produce effective dominant interference Fas molecule (29).Therefore, whether the genetically engineered of having tested the Fas by the having removed death domain dominant interference that body (Fas 1-210) carries out that stops mutation needs PLAD.The result shows that the terminal brachymemma of N-has destroyed the dominant interference (Fig. 8 B) that is undertaken by Fas 1-210.(ALPS Pt 26, the dominance restraining effect (Fig. 8 B) of this natural mutation has been eliminated in the PLAD brachymemma (lacking to a.a.42 at most) in Fas death domain point mutation body D244V) from ALPS patient.

The mechanism of the normal Fas function of Fas sudden change dominant interference among the ALPS that these discoveries are clear and definite again together.Obviously dominant interference Fas sudden change has kept the terminal PLAD of N-, because this structural domain is responsible for wild-type and the intermolecular complex body of mutant Fas forms.The main molecules principle of heredity dominant interference be mutant protein must be fully with the specific function complex body in wild-type protein interact (43).Before, the dominant negative relevant with human disease sudden change had been proved to be and can have disturbed normal receptor signal conduction (44) by sheltering in part, the blocking-up cell signal conduction or stoping the WT chain to be transported to cell surface.For Fas, described data sheet palpability is interfered a kind of new mechanism that derives from, before described mechanism is included in the part combination, by the wild-type of PLAD mediation and the association between the mutant acceptor.Though why these discoveries have explained that ALPS Pt 2 intravital unusual Fas albumen and other Fas ECD mutant can not be in conjunction with FasL but also can produce the dominant interference that is enough to cause disease.Because the removing of PLAD can be eliminated the dominant negative function of the Fas molecule of the death domain that contains disappearance or sudden change, so the interaction of PLAD mediation also can cause carrying in a large number the ALPS patient's of the sudden change that can influence the Fas death domain dominant interference interaction.PLAD interacts and also may participate in the downward modulation of Fas inductive apoptosis, and described downward modulation is by containing (45) that whole Fas of the alternative splicing form of solubility of these structural domains carries out.Estimate that the natural receptor mutant of encoding function PLAD is not a dominant interference.The dominant interference of PLAD mediation also can work (20) in regulating by the signal conduction of inveigling acceptor to carry out, and works in the disease incidence that can cause unusually at the allogeneic heredity by other member of TNF family.

These results have also proposed a kind of new model that is used to understand the transmembrane signal conduction of being undertaken by Fas, and described model comprises by the part tripolymer that will associate in advance and changes into the oligomerization of signal conduction attitude rather than the single receptor chain of part inductive.FRET research makes and can be evaluated under the situation that lacks part the intermolecular distance of Fas of CFP and YFP mark on the cell surface.For the CFP and YFP of random orientation,

Radius-R ₀For

(23).Suppose that the CFP that merges with Fas and YFP are that equivalent is expressed, random orientation and be made into equilateral tripolymer at random mutually each other, so viewed FRET efficient (Fig. 3 C) shows: and between the CFP that merges of total length Fas molecule and the YFP chromophore apart from the upper limit be And between CFP that Fas 1-210 merges and the YFP chromophore be apart from the upper limit

Owing between Fas and other TNFR family receptors, do not observe FRET, so these distances are more much closer and be specific than observed distance between the molecule of stochastic distribution on the cell surface.FRET needs the YFP of threshold level to express that (Fig. 3 A, FAS1-210:CFP/FAS1-210:YFP) this fact has reflected that in fact statistics or the pre-association of the Fas of the Fas of blended CFP mark and YFP mark depend on Rd.Strengthened the conduction of Fas signal owing to associating in advance, therefore regulating and control acceptor association amount in advance by change Fas expression or other method is a kind of new mechanism that is used to regulate apoptosis signal transduction.

Resetting the signal conduction of carrying out by the acceptor complex body may be widely used mechanism, to guarantee the quick of part and specific cell response.Yet this signal transduction mechanism has also been given the susceptibility for the dominant interference that is caused by naturally occurring acceptor variant or pathogenicity bo allos sudden change among the ALPS.

EXAMPLE III

Method

Mouse and reagent

BALB/c, C57BL/6, DBA/1J, C3H/HeJ, C3H/HeN, TNFR1 and TNFR2 knock-out mice are available from Jackson Laboratory.TNF-α transgenic mice is available from Taconic.All use the male mice in age in 6-8 week in all experiments and it is raised in the Animal House of immunization experiment chamber (NIAID, NIH).Utilize CBER to synthesize CpG DNA.CpG DNA 1668 sequences are 5 '-TCCATGACGTTCCTGATGCT-3 ' (SEQ ID NO:61) (50).By using P60 PLAD and P80PLAD to prepare the mouse monoclonal antibody (MAb) of anti-P60 (1H11) or anti-P80PLAD (3H11).

The purifying of PLAD-GST fusion rotein

Carry out the clone and the purifying (78) of PLAD-GST fusion rotein according to the described method of document.Verify the PLAD-GST fusion rotein of purifying and be stored under-80 ℃ with 4-20%Tris-glycine gels and mass spectrum.Remove LPS with Detoxi-Gel AffinityPak post (Pierce).

P60 and the proteic vitro test of P80 PLAD

With collecting the L929 cell after the trypsin treatment.With the PLAD albumen pretreatment cell of various dose 30 minutes, add TNF-α (2ng) afterwards.Similarly, with PLAD albumen and GST pre-treatment 42.3 cells.After pre-treatment in 30 minutes, the antibody (11) that adds TNF-α (3ng), Fas (10ng) and albumin A (20ng) and 37 ℃ of cultivations down.Utilize hematimeter or flow cytometry to carry out quantitative assay in the death of specific time point pair cell.Specification sheets (R﹠amp according to manufacturers; D systems) measures TNF-α combination.

Proteic immunogenicity of PLAD and transformation period

Adopt ELISA to study immunogenicity and transformation period in the proteic body of PLAD.PLAD albumen can cause antibody response, and wherein half is at described GST part (accompanying drawing 5a) at least.The blood that utilization was taken a sample in each time adopts ELISA to measure the transformation period in P60 PLAD or the proteic body of P80 PLAD, and its transformation period was near 5 hours (accompanying drawing 5b, c).

Adopt the arthritic development by TNF-α, CpG DNA triggering of PLAD proteotherapy

Exist or do not exist under the proteic situation of 100 μ g PLAD, with TNF-α, CpG DNA or LPS intra-articular injection in the mouse knee joint.Inject and put to death mouse in back 3 days and take out the joint to carry out histopathological examination.

CIA induce and with PLAD protein for treatment CIA

Will be with isopyknic complete Freund's adjuvant (Sigma) emulsification II type chicken collagen (Sigma), the emulsion intradermal that 0.1ml is contained 100 μ gII Collagen Type VIs is injected to the tail base portion of male DBA/1J mouse.In the time of 21 days, append 100 μ g with isopyknic incomplete Freund's adjuvant (Sigma) emulsive II Collagen Type VI to mouse.After first immunisation 22 days begin to carry out preventative processing with PLAD albumen or PBS, by numbering (in coded fashion) weekly intraperitoneal be administered three times until the 52nd day.Three days begin to carry out therapeutic treatment with P60 PLAD albumen or PBS after mouse produces sacroiliitis.Intraperitoneal gives P60PLAD albumen (every other day giving 400 μ g) two weeks, conversion (switch) described processing between each group then.In " blind method " experiment, mouse is once analyzed and assesses arthritic degree by per three days of two independently surveyors that do not know described processing: paw swelling and clinical score.Measure arthroncus by measure sufficient pawl thickness with calipers.Use following scoring rank to assess clinical arthritis: 0 grade, no swelling; 1 grade, mild swelling and erythema; 2 grades, obviously swelling; 3 grades, joint stiffness.With 0-3 level and 12 pairs of each limb classifications of every animal of maximum possible scoring.

The histopathological examination in joint

Organizational routine is being fixed, behind decalcification and the paraffin embedding, made the joint section and dye with h and E.All sections are numbered and carry out the assessment of blind method by the researchist.According to judging that to the rank of 2 and 4 grades (degree of inflammatory cell infiltration of Zeng Jiaing or cartilage and osteoclasia gradually) synovitis, pannus form or the degree of bone/cartilage destruction from 0 grade (NIP sign), 1 grade (outgrowth mild inflammation of synovial membrane coating, bottom line is not for there being cartilage destruction).

External osteoclast forms to be measured

From the shin bone of BALB/c mouse, extract medullary cell, and in containing the α of M-CSF-MEM substratum, cultivate.After 3 days,, further induced bone marrow macrophage 4 days with TNF-α (20ng/ml) and M-CSF (50ng/ml) existing or not existing under the proteic situation of PLAD.Fixed cell and dye at osteoclast mark TRAP then according to the specification sheets (Sigma) of manufacturers.

Immunofluorescence (66)

Exist or do not exist under P60 and the proteic situation of P80 PLAD, at the spleen isolating monocyte of external use TNF-α processing from TNFR1 or TNFR2 knock-out mice.Rabbit antibody (C-20, Santa Cruz) with anti-NF-κ B p65 dyes to the cell through fixing and saturatingization.Washed cell is also with the anti-rabbit antibody incubation of FITC-link coupled donkey cell then.Analyze nuclear NF-κ B and marking with Laser Scanning Confocal Microscope in blind method mode.

Statistical study

Use the different of each sickness rate in organizing of Fisher check and Mann-Whitney U check analysis and severity respectively.It is significant that P≤0.05 is considered to.

The result

Recombinant chou PLAD-GST fusion rotein

Be the soluble proteins of the PLAD structural domain that obtains to contain people P60 or P80 TNFR, preparation glutathione-S-transferase (GST) fusion rotein.P60 and P80 PLAD-GST fusion rotein (Fig. 1 b and accompanying drawing 1) that the affinity purifying provides the full-length molecule amount to be respectively 34kD and 33kD (based on the expection size of aminoacid sequence).Carrying out western blotting and carrying out protein sequencing by mass spectrum all is P60 or P80 PLAD albumen (Fig. 1 c) with two bands on the conclusive evidence gel.

PLAD albumen suppresses the necrocytosis of TNF-α inductive

Carry out this experiment and whether can suppress the cytopathic effect (63) of TNF-α the L929 cell with the PLAD albumen of determining purifying.The P60 PLAD albumen of purifying relies on mode with dosage and has suppressed mouse and humanTNF-'s inductive necrocytosis (Fig. 2 a, b) significantly.This provide protection and infliximab and etanercept be (accompanying drawing 2) quite.On the contrary, not effect (Fig. 2 c) of contrast CD40 PLAD albumen.Also having tested need be from the signal of P60 and P80 with people 42.3 cells of experience by TNF-α (15) inductive death.It is found that P60 or P80 PLAD albumen can suppress TNF-α inductive necrocytosis but do not suppress anti--Fas inductive necrocytosis (Fig. 2 d).Only GST albumen can not suppress TNF-α inductive or anti--Fas inductive necrocytosis.In addition, the same TNF-α inductive caspase-8 that suppresses effectively in the L929 cell with etanercept and infliximab of P60 PLAD albumen activates (Fig. 2 e).Therefore, the people P60 of purifying or P80 PLAD albumen are blocked people or mouse TNF-α effectively.

P60 PLAD suppresses TNF-α or CpG DNA inductive sacroiliitis

Also test PLAD albumen and whether can suppress TNF-α inductive sacroiliitis in the body.Adding or not adding under the proteic situation of 100 μ g PLAD, induce sacroiliitis by intra-articular injection mouse TNF-α (45ng).P60 PLAD albumen brute force has suppressed the arthritic feature of TNF-α inductive, comprises synovitis, pannus and bone erosion (Fig. 3 a, b).On the contrary, independent P80 PLAD or GST albumen do not influence disease (Fig. 3 a, c) (accompanying drawing 3a) significantly.

TNF-α in the pathogenesis of SA, play an important role (49,50,61).The DNA of bacteria and the LPS that contain CpG can induce the bacterium composition (49,61) that can trigger arthritic TNF-α and discharge consumingly from monocyte and scavenger cell.Common injection 100 μ g P60 PLAD albumen can significantly alleviate pannus and the osteoclasia (P＜0.05) (Fig. 3 a, d) in the CpG DNA inductive sacroiliitis, but 100 μ gP80 PLAD then can not (accompanying drawing 3b).P60 PLAD albumen but not P80 PLAD albumen can alleviate LPS inductive sacroiliitis significantly.

The MMP that P60 PLAD suppresses in the collagen-induced sacroiliitis (CIA) expresses

Matrix metalloproteinase (MMP) is the important medium of cartilage and osteoclasia in the sacroiliitis process, and TNF-α can induce MMP to express.Therefore, this experiment has determined that the MMP whether P60-PLAD albumen suppresses among the CIA expresses.The result shows that P60 PLAD albumen has suppressed the MMP expression (Figure 25) of CIA significantly.Annotate: the dark color among the left figure (being brown in the original color section) coloured portions represents that MMP expresses.

The iNOS that P60 PLAD suppresses in the collagen-induced sacroiliitis (CIA) expresses

Nitrogen protoxide (NO) is fugitive hydroxyl radical gas.Excessive nitrogen protoxide is deleterious and can causes chronic inflammatory diseases.Importantly, proved that TNF α can stimulate monocytes/macrophages to produce nitrogen protoxide and raising NO level by activating derivable nitricoxide synthase (iNOS), causes local joint damage thus.Therefore determine that the iNOS whether P60 PLAD albumen can suppress among the CIA expresses.The result shows that P60 PLAD albumen significantly suppresses to be subjected to the iNOS in the joint of CIA invasion and attack to express (Figure 26).Annotate: the dark color among the left figure (being brown in the original color section) coloured portions represents that iNOS expresses.These results have supported the instruction of this paper, promptly by disturbing the TNFR assembling, TNF-α inductive tissue injury among the P60PLAD blocking-up CIA.

Conclusion

Result of the present invention has confirmed that the PLAD among the TNFR1 is important treatment target.It is found that CD95PLAD participates in the pathogenesis (11) of ALPS by the mediation dominant interference.P60 and P80 PLAD albumen TNF-α capable of blocking combines with TNFR1 but can directly not combine with TNF-α.The most important thing is that by whole body and intra-articular administration, in several arthritic mouse models, P60 PLAD shows significant improvement effect.In addition, can reduce the inflammatory cell infiltration in joint significantly by P60 PLAD treatment.In the example formerly, the homotype that it is found that P60 and P80 PLAD in conjunction be optionally and not to each other cross coupled or with CD95 PLAD cross coupled (11,53).But those experiment confirms P60 and P80 PLAD act preferentially on corresponding acceptor may not can be specific fully (Fig. 5 e, f).In addition, P60PLAD can suppress the critical aspects of described lysis in several arthritic mouse models.In a word, our data presentation PLAD is a kind of important arthritis drug target.

P60 PLAD albumen can suppress to be induced by the NF-κ B that TNF-α produces effectively.P60 and P80 PLAD albumen can the acceptor mode of priority suppress TNF-α inductive NF-κ B transposition.In addition, NF-κ B is that RANKL inductive osteoclast forms required (71).The bone of osteoclast mediation and cartilage erosion can cause the most of permanent damages (67,68,69) in the sacroiliitis.Result of the present invention demonstrated that osteoclast in the pannus activates and TNF-α transgenic mice in the site of osteoclasia.In the arthritis knuckle of TNF-α transgenic mice and collagen-induced sacroiliitis, exist a large amount of RANK and RANKL to express (69).The P60PLAD treatment has reduced RANK and the RANKL in coating and the pannus significantly.

Infliximab and etanercept can be directly in conjunction with and the effect of blocking-up TNF-α, and they have been widely used in RA and other treatment of diseases (48) now.Yet, observed side effect for example lupoid acne disease, mycobacterial infections and lymphoma sickness rate rising (72-75).Two kinds of medicines are all directly blocked combining of TNF-α and two kinds of TNFR.Therefore, their drug effects that can suppress TNFR2 effectively and mediated are blocked the disease inducing action of TNFR1 simultaneously.P60 PLAD is the small protein structural domain of preferentially target TNFR1.In our model, the P60 PLAD protein agent measurer of about 4mg/kg (intraarticular) or 16mg/kg (parenteral) has and infliximab (10mg/kg) (76) and etanercept (0.4mg/kg) ³³Dosage similarly acts on, and the dosage of described infliximab and etanercept has been used to improve sacroiliitis clinically.P60 PLAD albumen provides the described method of novel method of the pathogenic effects of a kind of TNF-of inhibition α can be used for the treatment of RA.

EXAMPLE IV

Direct and the TNF receptors bind of PLAD albumen

For determining whether PLAD albumen can directly combine with TNFR, P80-PLAD albumen is mixed with etanercept, by mixing the TNFR2 fusion rotein of testing the complete ectodomain that contains TNFR2 with P80 PLAD albumen.The result of immunoprecipitation (IP) and western blotting (WB) shows P80-PLAD-GST albumen but not simple GST albumen can associate with etanercept, has shown that PLAD albumen can directly combine with TNFR and has therefore suppressed TNF alpha active (Figure 24).

Table 1

Table 2

In this application, with reference to various publications.The whole modes by reference of the disclosure of these publications are included among the application, so that describe the prior art of the technical field of the invention more fully.

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Sequence table

SEQ?ID?NO：1

The 1-54 amino acids (TNFR1) of ripe p60

GenBank?Accession?No.M75866

GenBank?Accession?No.AAA61201

LVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRE

SEQ?ID?NO：2

The 10-54 amino acids (TNFR2) of ripe p80

GenBank registration number M32315

GenBank registration number AAA59929

YAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCD

SEQ?ID?NO：3

The 1-43 amino acids of Fas

GenBank registration number .M67454

GenBank registration number AAA63174

RLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFC

SEQ?ID?NO：4

The 1-66 amino acids of Fas

GenBank registration number M67454

GenBank registration number AAA63174

RLSS?KSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCH

KPCPPGERKARDCTVNGDEPDC

SEQ?ID?NO：5

The 13-50 amino acids of LtBR

GenBank registration number L04270

GenBank registration number AAA36757

CRDQEKEYYEPQHRICCSRCPPGTYVSAKCSRIRDTVC

SEQ?ID?NO：6

The 6-39 amino acids of CD40

GenBank registration number .X60592

GenBank registration number .CAA43045

CREKQYLlNSQCCSLCQPGQKLVSDCTEFTETEC

SEQ?ID?NO：7

The 11-51 amino acids of CD30

GenBank registration number .M83554

GenBank registration number .AAA51947

CHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQC

SEQ?ID?NO：8

The 7-42 amino acids of CD27

GenBank registration number .M63928

GenBank registration number AAA58411

CPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQC

SEQ?ID?NO：9

The 6-37 amino acids of HVEM

GenBank registration number U70321

GenBank registration number AAB58354

CKEDEYPVGSECCPKCSPGYRVKEACGELTGT

SEQ?ID?NO：10

The 3-36 amino acids of 0X40

GenBank registration number X75962

GenBank registration number CAA53576

CVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVC

SEQ?ID?NO：11

The 109-138 amino acids of DR4

GenBank registration number U90875

GenBank registration number AAC51226

CNRCTE?GVGYTNASNNLFACLPCTAC?KSDE

The full length sequence of TNFR1 (p60)

GenBank registration number M75866

455 amino acid

The full length sequence of TNFR2 (p80)

GenBank registration number M32315

461 amino acid

The Fas full length sequence

GenBank registration number M67454

335 amino acid

The LtBR full length sequence

GenBank registration number L04270

435 amino acid

The full length sequence of CD40 acceptor

GenBank registration number X60592

277 amino acid

The full length sequence of CD30 acceptor

GenBank registration number M83554

595 amino acid

The full length sequence of CD27 acceptor

GenBank registration number M63928

260 amino acid

The full length sequence of HVEM acceptor

GenBank registration number U70321

283 amino acid

The full length sequence of 0X40 acceptor

GenBank registration number X75962

283 amino acid

The full length sequence of DR4 acceptor

GenBank registration number U90875

283 amino acid

The CRD1 of CD40

SEQ?ID?NO：25

Cys?Arg?Glu?Lys?Gln?Tyr?Leu?Ile?Asn?Ser?Gln?Cys?Cys?Ser?Leu?Cys

Gln?Pro?Gly?Gln?Lys?Leu?Val?Ser?Asp?Cys?Thr?Glu?Phe?Thr?Glu?Thr

Glu?Cys

The CRD1 of HVEM

SEQ?ID?NO：26

Cys?Lys?Glu?Asp?Glu?Tyr?Pro?Val?Gly?Ser?Glu?Cys?Cys?Pro?Lys?Cys

Ser?Pro?Gly?Tyr?Arg?Val?Lys?Glu?Ala?Cys?Gly?Glu?Leu?Thr?Gly?Thr

Val?Cys

The CRD1 of CD3O

SEQ?ID?NO：27

Cys?His?Gly?Asn?Pro?Ser?His?Tyr?Tyr?Asp?Lys?Ala?Val?Arg?Arg?Cys

Cys?Tyr?Arg?Cys?Pro?Met?Gly?Leu?Phe?Pro?Thr?Gln?Gln?Pro?Gln

Arg?Pro?Thr?Asp?Cys?Arg?Lys?Gln?Cys

SEQ?ID?NO：28

The proteic 20-61 amino acids of T2 (myxoma virus)

GenBank registration number M95181

GenBank registration number AAA46632

PYGADRGKCRGNDYEKDGLCCTSCPPGSYASRLCGPGSDTVC

SEQ?ID?NO：29

The proteic 34-61 amino acids of s002R (rabbits fibromatosis virus)

GenBank registration number AF170722

GenBank registration number AAF18043

EKDGLCCASCHPGFYASRLCGPGSNTVC

SEQ?ID?NO：30

The 56-77 amino acids (HCMV) of UL144

GenBank registration number AF179208

GenBank registration number AF13375

CTPCPNGTYVSGLYNCTDCTEC

SEQ?ID?NO：31

The proteic 30-73 amino acids of CrmC (vaccinia virus)

GenBank registration number AF482758

Mark AAM13631 such as GenBank

HAPVNGSCDDGEYLDKTHNQCCNRCPPGEEAKIRCSGSDNTKCE

SEQ?ID?NO：32

The proteic 30-73 amino acids of CrmC (A53R) (vaccinia virus)

GenBank registration number AJ416893

GenBank registration number CAC95181

HAPVNGSCDEGEYLDKRHNQCCNRCPPGEFAKVRCNGNDNTKCE

SEQ?ID?NO：33

The proteic 31-67 amino acids of CrmD (vaccinia virus)

GenBank registration number U87581

GenBank registration number AAB94351

CNGTDYNSNSNNLCCKQCDPGMYMTHSCNTTSNTKCD

SEQ?ID?NO：34

The proteic 28-58 amino acids of CrmE (vaccinia virus)

GenBank registration number AJ272008

GenBank registration number CAC15562

YYNSQELKCCKLCKPGTYSDHRCDKYSDTIC

SEQ?ID?NO：35

The 80-117 amino acids (lymphocystis disease virus) of ORF167L putative protein

GenBank registration number AY380826

GenBank registration number AAU10940

CRQGYYYDPESEMCFPCSNCESSKVKVTTCNRTHDTVC

SEQ?ID?NO：36

The proteic 24-65 amino acids of EVM003 (infectious ectromelia virus)

GenBank registration number NC_004105

GenBank registration number NP_671521

YTPINGKCNGTDYNSNNLCCKQCNPGMYMTHSCNTTSNTKCD

SEQ?ID?NO：37

The proteic 25-65 amino acids of CrmB (camelpox virus)

GenBank registration number AF438165

GenBank registration number AAL73920

YAPSNGKCKDNEYKRHNLCCLSCPPGTYASRLCDSKTNTQC

SEQ?ID?NO：38

The proteic 25-65 amino acids of TNFR2 homologue (monkey pox virus)

GenBank registration number DQ011155

Genbank registration number AAY97399

HAPSNGKCKDNEYRSRNLCCLSCPPGTYASRLCDSKTNTQ

SEQ?ID?NO：39

The proteic 25-65 amino acids of G2R (alastrim virus)

GenBank registration number U88151

Genbank registration number AAB94376

YTPPNGKCKDTEYKRHNLCCLSCPPGTYASRLCDSKTNTQC

SEQ?ID?NO：40

The 1-124 amino acids of mature T NF1

VCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRH

CLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGT

VHLSCQEKQNTVC

SEQ?ID?NO：41

The 139-171 amino acids of putative protein (campylobacter jejuni RM1221)

GenBank registration number CP000025

Genbank registration number AW35633

DINSEGMEDLSFDDDAQDDNANKTLETQNLEHD

SEQ?ID?NO：42

The 438-454 amino acids (trypanosoma bocagei) of putative protein

GenBank registration number AC091702

Genbank registration number AAX79885

TEINELRQTITDVETNL

SEQ?ID?NO：43

The 488-517 amino acids of putative protein (golden yellow grape ball-bacterium-)

GenBank registration number BA000017

Genbank registration number BAB56791

DENSKELERLQKIAFNVETKTLESLVDEGQ

SEQ?ID?NO：44

The proteic full length sequence of T2 (myxoma virus)

GenBank registration number M95181

326 amino acid

MFRLTLLLAYVACVYGGGAPYGADRGKCRGNDYEKDGLCCTSCPPGSYASRLCGP

GSDTVCSPCKNETFTASTNHAPACVSCRGRCTGHLSESQSCDKTRDRVCDCSAGNY

CLLKGQEGCRICAPKTKCPAGYGVSGHTRTGDVLCTKCPRYTYSDAVSSTETCTSS

FNYISVEFNLYPVNDTSCTTTAGPNEVVKTSEFSVTLNHTDCDPVFHTEYYGTSGSE

GAGGFFTGMDRYQNTTKMCTLNIEIRCVEGDAVRTIPRTSDGVGVLSHSETITVIGG

CLSDVNVDIEYSDSNHPEEVDDFVEYHWGTRLRLFPSPKRCRLVS

SEQ?ID?NO：45

The proteic full length sequence of s002R (rabbits fibromatosis virus)

GenBank registration number AF170722

325 amino acid

MLRLIALLVCVYVYGDDVPYSSNQGKCGGHDYEKDGLCCASCHPGFYASRLCGP

GSNTVCSPCEDGTFTASTNHAPACVSCRGPCTGHLSESQPCDRTHDRVCNCSTGNY

CLLKGQNGCRICAPQTKCPAGYGVSGHTRAGDTLCEKCPPHTYSDSLSPTERCGTSF

NYISVGFNLYPVNETSCTTTAGHNEVIKTKEFTVTLNYTDCDPVFHTEYYATSGKEG

AGGFFTGTDIYQNTTKVCTLNVEIQCSEGDDIHTLQKTNGGSTMPHSETITVVGSCLS

DVNVDIMYSDTNHPGEVDDFVEYHWGTRLRFFPLPKRCTPVS

SEO?ID?NO：46

The proteic full length sequence of UL144 (HCMV)

GenBank registration number AF179208

175 amino acid

MKPLVMLICFAVILLQLGVTKVCQHNEVQLGNECCPPCGSGQRVTKVCTDYTSVTC

TPCPNGTYVSGLYNCTDCTECNDTEVTIRNCTSTNNTVCASKNYTSFSISGVQHHKQ

RQNHTAHVTVKQGKSGRHTLARLSLFIFLVGIILLILYLIAAYRSEKCQQCCSIGKIFY

RTL

SEQ?ID?NO：47

The proteic full length sequence of CrmC (vaccinia virus)

GenBank registration number AF482758

186 amino acid

MDIKNLLTVCTILYISTLVTADIPTSSLPHAPVNGSCDDGEYLDKTHNQCCNRCPPGE

FAKIRCSGSDNTKCERCPPHTYTTVPNYSNGCHQCRKCPTGSFDKVKCTGTQNSKC

SCLPGWFCATDSSKTEDCRDCIPKRKCPCGYFGGIDELGNPLCKSCCVGEYCDDIRN

HRVGPFPPCKLSKCN

SEQ?ID?NO：48

The proteic full length sequence of CrmC (A53R) (vaccinia virus)

GenBank registration number AJ416893

186 amino acid

MDIKNLLTACTIFYITTLATADIPTSSLPHAPVNGSCDEGEYLDKRHNQCCNRCPPGE

FAKVRCNGNDNTKCERCPPHTYTAIPNYSNGCHQCRKCPTGSFDKVKCTGTQNSK

CSCLPGWYCATDSSQTEDCRDCIPKRRCPCGYFGGIDEQGNPICKSCCVGEYCDYL

RNYRLDPFPPCKLSKCN

SEQ?ID?NO：49

The proteic full length sequence of CrmD (vaccinia virus)

GenBank registration number U87581

148 amino acid

MMKMTPSYILLVYMFVVVSGDVPYEHINGKCNGTDYNSNSNNLCCKQCDPGMYM

THSCNTTSNTKCDKCPDGTFTSIPNHIPTCLSCRGKCSSNQVETKSCSNTQAEYVSV

HPDTTANLKDQMVAGYVYHKQSVILVTAYMATHLKEM

SEQ?ID?NO：50

The proteic full length sequence of CrmE (vaccinia virus)

GenBank registration number AJ272008

167 amino acid

MTKVIIILGFLIINTNSLSMKCEQGVSYYNSQELKCCKLCKPGTYSDHRCDKYSDTIC

GHCPSDTFTSIYNRSPWCHSCRGSCGTNRVEVTPCTPTTNRICHCDSNSYCLLKASD

GNCVTCAPKTKCGRGYGKKGEDEMGNTICKKCRKGTYSDIVSDSDQCKPMTR

SEQ?ID?NO：51

The full length sequence of ORF167L putative protein (lymphocystis disease virus)

GenBank registration number AY380826

289 amino acid

MMLFILFLLPITVHTATDCPPGYYISKYYPAGTPMCSPCSPGTYTGLQNSLRKCLRCS

TCSHNEEPKVACSTTSDVQCQCRQGYYYDPESEMCFPCSNCESSKVKVTTCNRTHD

TVCKCKEGYYDKNGVCVKCGNCYLGEGVKSKCANNTDVTCELCKNGTFSDKVSS

SNICYLYTMCTLGLTQLNFNVTWFDTICINCSMSTDLLDLETFFTINFVTQRRFPEDD

LKNMFRLTYNKTRNDIDSANRDDVEGSFTYDPNLPYTMKQLDYLIASKFLMTAYK

KISQICQL

SEQ?ID?NO：52

The proteic full length sequence of EVM003 (infectious ectromelia virus)

GenBank registration number NC_004105

320 amino acid

MMKMTPSYILLVYMFVVVSGDVPYTPINGKCNGTDYNSNNLCCKQCNPGMYMTH

SCNTTSNTKCDKCPDDTFTSIPNHSPACLSCRGKCSSNQVETKSCSNTQDRVCVCAS

GYYCEFEGSNGCRLCVPQTKCGSGYGVYGYSSKGDVICKKCPGNIDKCDLSFNSID

VEINMYPVNKTSCNSSIGSSSTISTSELTITLTHEDCTPVFIGDYYSVVDKLATSGFFT

NDKVHQDLTTQCKINLEIKCNSGRESRQLTPTTKVYFMPHSETVTVVGDCLSNLDV

YIVYANTDAIYSDMDVVAYHTSYILNVDHIPPNDCERD

SEQ?ID?NO：53

The proteic full length sequence of CrmB (camelpox virus)

GenBank registration number AF438165

349 amino acid

MKSVLYSYILFLSCIIINGRDVTPYAPSNGKCKDNEYKRHNLCCLSCPPGTYASRLCD

SKTNTQCTPCGSGTFTSRNNHLPACLSCNGRCDSNQVETRSCNTTHNRICECSPGYY

CILKGSSGCKACVSQTKCGIGYGVSGHTSAGDVICSPCGLGTYSRTVSSADKCEPVP

SNTFNYIDVEINLYPVNDTSCTRTTTTGISESISTSELTITMNHKDCDPVFREEYFSVL

NKVATSGFFTGENRYQNISKVCTLNFEIKCNNKGSSSKQLTKAKNDDGIMPHSETVT

LVGDCLSSVDIYILYSNTNTQDYETDTISYHAGNVLDVDSHMPGSCDIHKLITNSKPT

HFL

SEQ?ID?NO：54

The proteic full length sequence of TNFR2 homologue (monkey pox virus)

GenBank registration number DQ011155

348 amino acid

MRSVLYSYILFLSCIIINGRDLAPHAPSNGKCKDNEYRSRNLCCLSCPPGTYASRLCD

SKTNTQCTPCGSDTFTSHNNHLQACLSCNGRCDSNQVETRSCNTTHNRICECSPGY

YCLLKGSSGCRTCISKTKCGIGYGVSGYTSTGDVICSPCGPGTYSHTVSSTDKCEPVT

SNTFNYIDVEINLYPVNDTSCTRTTTTGLSESISTSELTITMNHKDCDPVFRAEYFSVL

NNVATSGFFTGENRYQNTSKICTLNFEIKCNNKDSSSKQLTKTKNDTIMPHSETVTL

VGDCLSSVDIYILYSNTNTQDYETDTISYHMGNVLDVNSHMPASCDIHKLITNSQNP

THL

SEQ?ID?NO：55

The proteic full length sequence of G2R (alastrim virus)

GenBank registration number U88151

349 amino acid

MKSVLYLYILFLSCIIINGRDAAPYTPPNGKCKDTEYKRHNLCCLSCPPGTYASRLCD

SKTNTQCTPCGSGTFTSRNNHLPACLSCNGRCNSNQVETRSCNTTHNRICECSPGYY

CLLKGSSGCKACVSQTKCGIGYGVSGHTSVGDVICSPCGFGTYSYTVSSTDKCEPVP

NNTFNYIDVEITLYPVNDTSCTRTTTTGLSESILTSELTITMNHTDCNPVFREEYFSVL

NKVATSGFFTGENRYQNISKVCTLNFEIKCNNKGSSFKQLTKAKNDDGMMSHSETV

TLAGDCLSSVDIYILYSNTNAQDYETDTISYRVGNVLDDDSHMPGSCDIHKLITNSKP

TRFL

SEQ?ID?NO：56

The full length sequence of putative protein (campylobacter jejuni RM1221)

GenBank registration number CP000025

547 amino acid

MKILLLNENPVVSRLVSLSAKKMSYDFEELNAYSENLGNYDVIVVDSDTPAPLKILK

EKCDRLIFLAPRNQNVEDIDAQILQKPFLPTDFLNLLNNKDANKHTSIDLPMLSNDEN

PYADISLDLDNLNLDDLPDENSLDINSEGMEDLSFDDDAQDDNANKTLETQNLEHD

NLEQETIKEQTQEDTQTDLDLTLEDSESEKEDLSQEHTALDTEPSLDELDDKNDEDL

EIKEDDKNEEIEKQELLDDSKTNTLEMQEELSESQDDNANKTLETQNLEHDNLEQET

IKEQTQEDTQTDLDLTLEDGESEKEDLSQEHTALDTEPSLDELDDKNDEDLEDNKEL

QANISDFDDLPEVEEQEKEMDFDDLPEDAEFLGQAKYNEESEENLEEFAPVVEEDV

QDEIDDFASNLSTQDQIKEELAQLDELDYGIDSDNSSKVLEDFKDEPILDDKELGTNE

EEVVVPNLNISDFDTLKESDIQEALGEEILEKNEEPIVSDVTKDDNSEEIVNELSQSIA

GAITSSIKDDTLKAAKGMNMNININISFKED

SEQ?ID?NO：57

The full length sequence of putative protein (trypanosoma bocagei)

GenBank registration number AC091702

496 amino acid

MLRCRVADVVSSDQMCEMTNFPPVQATVGYVQLLRRLSFKREFFVPFVPTKHVIFT

FQTRNPLEEGKVKDEVTDLVSHYFNWIRERGVYSFQILQLYVCILSGCTVVVIE

CPDTLTTLDVHSFSQEPEAPTVGKRRVLVFPCDNDEISDSELESNYYIVPTCTLCAER

LEPTLTGYSSPTCSCVDGRECRCLLEQSSCVVCQTSITMQHESQKVQCEQCSRTGDP

WICLVCGYVGCSRYQAKHAREHYLQHKHLFSMSLLTQQIWDYDSDAFVHRVVVLL

DNATGAVNRVQYPDRDNIPSSLADEYVDAAAEKVSKKHINAKFDSKVETSNEQLA

LMIISELNTRRVEYETEMHGGSHHLNDELMGDPSLCSVIVAERACAASRERWWQLH

NANKSIQEELMQRRREEEAHQKTIDELQQELRSVVQHYATREYSLLTEINELRQTIT

DVETNLRTFAKLSRGLGNDTLEHVRIVGGTKEPKPRRRGGNNTRDGRA

SEQ?ID?NO：58

The full length sequence of putative protein (streptococcus aureus)

GenBank registration number BA000017

680 amino acid

MEIFETILIFIAVVILSSFVHTFIPKVPLAFIQIFLGMLLFITPIPVQFNFDSELFMVTMIAP

LLFVEGVNVSRVHLRKYIKPVMMMALGLVITTVIGVGLFIHWIWPDLPIGAAFAIAAI

LCPTDAVAVQAITKGKVLPKGAMTILEGESLLNDAAGIISFKIAVGVLVTGAFSLVD

AVQLFLIASIGGAVVGLLIGMALVRFRLTLMRRGYENINMFTIIQLLTPFVTYLIAELF

HASGIIAAVVAGLVHGFERDRIMQVRTQLQMSYNHTWNILGYVLNGFVFSILGFLVP

EVIIKIIKTEPHNLIFLIGITIVVALAVYLFRFVWVYVLYPYFYLAISPFQKMMTKNDD

DNPTTEKPPKRSLYALIMTLCGVHGTISLAIALTLPYFLAGHHAFTYRNDLLFIASGM

VIISLVVAQVLLPLLTKPAPKTVIGNMSFKVARIYILEQVIDYLNQKSTFETSFKYGNV

IKEYHDKLAFLKTVEKDDENSKELERLQKLAFNVETKTLESLVDEGQITNSVLENYM

RYAERTQVYRQASLIRRMIVLLRGALLKRRVQTRVNSASSLSVTDNLMELNKINKL

VHYNVVSRLSKETTKDNTLEIGMVCDGYLMRIENLTPSNFFNSASEDTITKIKLNAL

REQRRILRELIDTDEVSEGTALKLREAINYDEMVIVDSMT

SEQ?ID?NO.59

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEF

PNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG

VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD

ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQAT

FGGGDHPPKSDLVPRGSPEFLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKC

HKGTYLYNDCPGPGQDTDCREFPGRLERPHRD

SEQ?ID?NO：60

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEF

PNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG

VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD

ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQAT

FGGGDHPPKSDLVPRGSPEFYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHA

KVFCTKTSDTVCDEFPGRLERPHRD

SEQ?ID?NO.61

TCCATGACGTTCCTGATGCT

Sequence table

<110>The?Government?of?the?United?States?of?America，as?represented?by?theSecretary，Department?of?Health?and?Human?Services

Lenardo，Michael?J.

Chang，Francis?Ka-Ming

Siegel，Richard?M.

＜120〉come amelioration of inflammatory arthritis by the preceding part assembling territory (PLAD) of target tumor mecrosis factor receptors

<130>14014/0365P2

<140>PCT/US2006/024909

<141>2006-06-26

<150>60/694,015

<151>2005-06-24

<150>60/717,589

<151>2005-09-16

<160>61

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>54

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>1

Leu?Val?Pro?His?Leu?Gly?Asp?Arg?Glu?Lys?Arg?Asp?Ser?Val?Cys?Pro

1 5 10 15

Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys

20 25 30

Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro?Gly?Gln

35 40 45

Asp?Thr?Asp?Cys?Arg?Glu

50

<210>2

<211>45

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>2

Tyr?Ala?Pro?Glu?Pro?Gly?Ser?Thr?Cys?Arg?Leu?Arg?Glu?Tyr?Tyr?Asp

1 5 10 15

Gln?Thr?Ala?Gln?Met?Cys?Cys?Ser?Lys?Cys?Ser?Pro?Gly?Gln?His?Ala

20 25 30

Lys?Val?Phe?Cys?Thr?Lys?Thr?Ser?Asp?Thr?Val?Cys?Asp

35 40 45

<210>3

<211>43

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>3

Arg?Leu?Ser?Ser?Lys?Ser?Val?Asn?Ala?Gln?Val?Thr?Asp?Ile?Asn?Ser

1 5 10 15

Lys?Gly?Leu?Glu?Leu?Arg?Lys?Thr?Val?Thr?Thr?Val?Glu?Thr?Gln?Asn

20 25 30

Leu?Glu?Gly?Leu?His?His?Asp?Gly?Gln?Phe?Cys

35 40

<210>4

<211>66

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>4

Arg?Leu?Ser?Ser?Lys?Ser?Val?Asn?Ala?Gln?Val?Thr?Asp?Ile?Asn?Ser

1 5 10 15

Lys?Gly?Leu?Glu?Leu?Arg?Lys?Thr?Val?Thr?Thr?Val?Glu?Thr?Gln?Asn

20 25 30

Leu?Glu?Gly?Leu?His?His?Asp?Gly?Gln?Phe?Cys?His?Lys?Pro?Cys?Pro

35 40 45

Pro?Gly?Glu?Arg?Lys?Ala?Arg?Asp?Cys?Thr?Val?Asn?Gly?Asp?Glu?Pro

50 55 60

Asp?Cys

65

<210>5

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>5

Cys?Arg?Asp?Gln?Glu?Lys?Glu?Tyr?Tyr?Glu?Pro?Gln?His?Arg?Ile?Cys

1 5 10 15

Cys?Ser?Arg?Cys?Pro?Pro?Gly?Thr?Tyr?Val?Ser?Ala?Lys?Cys?Ser?Arg

20 25 30

Ile?Arg?Asp?Thr?Val?Cys

35

<210>6

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>6

Cys?Arg?Glu?Lys?Gln?Tyr?Leu?Ile?Asn?Ser?Gln?Cys?Cys?Ser?Leu?Cys

1 5 10 15

Gln?Pro?Gly?Gln?Lys?Leu?Val?Ser?Asp?Cys?Thr?Glu?Phe?Thr?GIu?Thr

20 25 30

Glu?Cys

<210>7

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>7

Cys?His?Gly?Asn?Pro?Ser?His?Tyr?Tyr?Asp?Lys?Ala?Val?Arg?Arg?Cys

1 5 10 15

Cys?Tyr?Arg?Cys?Pro?Met?Gly?Leu?Phe?Pro?Thr?Gln?Gln?Cys?Pro?Gln

20 25 30

Arg?Pro?Thr?Asp?Cys?Arg?Lys?Gln?Cys

35 40

<210>8

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>8

Cys?Pro?Glu?Arg?His?Tyr?Trp?Ala?Gln?Gly?Lys?Leu?Cys?Cys?Gln?Met

1 5 10 15

Cys?Glu?Pro?Gly?Thr?Phe?Leu?Val?Lys?Asp?Cys?Asp?Gln?His?Arg?Lys

20 25 30

Ala?Ala?Gln?Cys

35

<210>9

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>9

Cys?Lys?Glu?Asp?Glu?Tyr?Pro?Val?Gly?Ser?Glu?Cys?Cys?Pro?Lys?Cys

1 5 10 15

Ser?Pro?Gly?Tyr?Arg?Val?Lys?Glu?Ala?Cys?Gly?Glu?Leu?Thr?Gly?Thr

20 25 30

<210>10

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>10

Cys?Val?Gly?Asp?Thr?Tyr?Pro?Ser?Asn?Asp?Arg?Cys?Cys?His?Glu?Cys

1 5 10 15

Arg?Pro?Gly?Asn?Gly?Met?Val?Ser?Arg?Cys?Ser?Arg?Ser?Gln?Asn?Thr

20 25 30

Val?Cys

<210>11

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>11

Cys?Asn?Arg?Cys?Thr?Glu?Gly?Val?Gly?Tyr?Thr?Asn?Ala?Ser?Asn?Asn

1 5 10 15

Leu?Phe?Ala?Cys?Leu?Pro?Cys?Thr?Ala?Cys?Lys?Ser?Asp?Glu

20 25 30

<210>12

<211>455

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>12

Met?Gly?Leu?Ser?Thr?Val?Pro?Asp?Leu?Leu?Leu?Pro?Leu?Val?Leu?Leu

1 5 10 15

Glu?Leu?Leu?Val?Gly?Ile?Tyr?Pro?Ser?Gly?Val?Ile?Gly?Leu?Val?Pro

20 25 30

His?Leu?Gly?Asp?Arg?Glu?Lys?Arg?Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys

35 40 45

Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys

50 55 60

Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp

65 70 75 80

Cys?Arg?Glu?Cys?Glu?Ser?Gly?Ser?Phe?Thr?Ala?Ser?Glu?Asn?His?Leu

85 90 95

Arg?His?Cys?Leu?Ser?Cys?Ser?Lys?Cys?Arg?Lys?Glu?Met?Gly?Gln?Val

100 105 110

Glu?Tle?Ser?Ser?Cys?Thr?Val?Asp?Arg?Asp?Thr?Val?Cys?Gly?Cys?Arg

115 120 125

Lys?Asn?Gln?Tyr?Arg?His?Tyr?Trp?Ser?Glu?Asn?Leu?Phe?Gln?Cys?Phe

130 135 140

Asn?Cys?Ser?Leu?Cys?Leu?Asn?Gly?Thr?Val?His?Leu?Ser?Cys?Gln?Glu

145 150 155 160

Lys?Gln?Asn?Thr?Val?Cys?Thr?Cys?His?Ala?Gly?Phe?Phe?Leu?Arg?Glu

165 170 175

Asn?Glu?Cys?Val?Ser?Cys?Ser?Asn?Cys?Lys?Lys?Ser?Leu?Glu?Cys?Thr

180 185 190

Lys?Leu?Cys?Leu?Pro?Gln?Ile?Glu?Asn?Val?Lys?Gly?Thr?Glu?Asp?Ser

195 200 205

Gly?Thr?Thr?Val?Leu?Leu?Pro?Leu?Val?Ile?Phe?Phe?Gly?Leu?Cys?Leu

210 215 220

Leu?Ser?Leu?Leu?Phe?Ile?Gly?Leu?Met?Tyr?Arg?Tyr?Gln?Arg?Trp?Lys

225 230 235 240

Ser?Lys?Leu?Tyr?Ser?Ile?Val?Cys?Gly?Lys?Ser?Thr?Pro?Glu?Lys?Glu

245 250 255

Gly?Glu?Leu?Glu?Gly?Thr?Thr?Thr?Lys?Pro?Leu?Ala?Pro?Asn?Pro?Ser

260 265 270

Phe?Ser?Pro?Thr?Pro?Gly?Phe?Thr?Pro?Thr?Leu?Gly?Phe?Ser?Pro?Val

275 280 285

Pro?Ser?Ser?Thr?Phe?Thr?Ser?Ser?Ser?Thr?Tyr?Thr?Pro?Gly?Asp?Cys

290 295 300

Pro?Asn?Phe?Ala?Ala?Pro?Arg?Arg?Glu?Val?Ala?Pro?Pro?Tyr?Gln?Gly

305 310 315 320

Ala?Asp?Pro?Ile?Leu?Ala?Thr?Ala?Leu?Ala?Ser?Asp?Pro?Ile?Pro?Asn

325 330 335

Pro?Leu?Gln?Lys?Trp?Glu?Asp?Ser?Ala?His?Lys?Pro?Gln?Ser?Leu?Asp

340 345 350

Thr?Asp?Asp?Pro?Ala?Thr?Leu?Tyr?Ala?Val?Val?Glu?Asn?Val?Pro?Pro

355 360 365

Leu?Arg?Trp?Lys?Glu?Phe?Val?Arg?Arg?Leu?Gly?Leu?Ser?Asp?His?Glu

370 375 380

Ile?Asp?Arg?Leu?Glu?Leu?Gln?Asn?Gly?Arg?Cys?Leu?Arg?Glu?Ala?Gln

385 390 395 400

Tyr?Ser?Met?Leu?Ala?Thr?Trp?Arg?Arg?Arg?Thr?Pro?Arg?Arg?Glu?Ala

405 410 415

Thr?Leu?Glu?Leu?Leu?Gly?Arg?Val?Leu?Arg?Asp?Met?Asp?Leu?Leu?Gly

420 425 430

Cys?Leu?Glu?Asp?Ile?Glu?Glu?Ala?Leu?Cys?Gly?Pro?Ala?Ala?Leu?Pro

435 440 445

Pro?Ala?Pro?Ser?Leu?Leu?Arg

450 455

<210>13

<211>461

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>13

Met?Ala?Pro?Val?Ala?Val?Trp?Ala?Ala?Leu?Ala?Val?Gly?Leu?Glu?Leu

1 5 10 15

Trp?Ala?Ala?Ala?His?Ala?Leu?Pro?Ala?Gln?Val?Ala?Phe?Thr?Pro?Tyr

20 25 30

Ala?Pro?Glu?Pro?Gly?Ser?Thr?Cys?Arg?Leu?Arg?Glu?Tyr?Tyr?Asp?Gln

35 40 45

Thr?Ala?Gln?Met?Cys?Cys?Ser?Lys?Cys?Ser?Pro?Gly?Gln?His?Ala?Lys

50 55 60

Val?Phe?Cys?Thr?Lys?Thr?Ser?Asp?Thr?Val?Cys?Asp?Ser?Cys?Glu?Asp

65 70 75 80

Ser?Thr?Tyr?Thr?Gln?Leu?Trp?Asn?Trp?Val?Pro?Glu?Cys?Leu?Ser?Cys

85 90 95

Gly?Ser?Arg?Cys?Ser?Ser?Asp?Gln?Val?Glu?Thr?Gln?Ala?Cys?Thr?Arg

100 105 110

Glu?Gln?Asn?Arg?Ile?Cys?Thr?Cys?Arg?Pro?Gly?Trp?Tyr?Cys?Ala?Leu

115 120 125

Ser?Lys?Gln?Glu?Gly?Cys?Arg?Leu?Cys?Ala?Pro?Leu?Arg?Lys?Cys?Arg

130 135 140

Pro?Gly?Phe?Gly?Val?Ala?Arg?Pro?Gly?Thr?Glu?Thr?Ser?Asp?Val?Val

145 150 155 160

Cys?Lys?Pro?Cys?Ala?Pro?Gly?Thr?Phe?Ser?Asn?Thr?Thr?Ser?Ser?Thr

165 170 175

Asp?Ile?Cys?Arg?Pro?His?Gln?Ile?Cys?Asn?Val?Val?Ala?Ile?Pro?Gly

180 185 190

Asn?Ala?Ser?Met?Asp?Ala?Val?Cys?Thr?Ser?Thr?Ser?Pro?Thr?Arg?Ser

195 200 205

Met?Ala?Pro?Gly?Ala?Val?His?Leu?Pro?Gln?Pro?Val?Ser?Thr?Arg?Ser

210 215 220

Gln?His?Thr?Gln?Pro?Thr?Pro?Glu?Pro?Ser?Thr?Ala?Pro?Ser?Thr?Ser

225 230 235 240

Phe?Leu?Leu?Pro?Met?Gly?Pro?Ser?Pro?Pro?Ala?Glu?Gly?Ser?Thr?Gly

245 250 255

Asp?Phe?Ala?Leu?Pro?Val?Gly?Leu?Ile?Val?Gly?Val?Thr?Ala?Leu?Gly

260 265 270

Leu?Leu?Ile?Ile?Gly?Val?Val?Asn?Cys?Val?Ile?Met?Thr?Gln?Val?Lys

275 280 285

Lys?Lys?Pro?Leu?Cys?Leu?Gln?Arg?Glu?Ala?Lys?Val?Pro?His?Leu?Pro

290 295 300

Ala?Asp?Lys?Ala?Arg?Gly?Thr?Gln?Gly?Pro?Glu?Gln?Gln?His?Leu?Leu

305 310 315 320

Ile?Thr?Ala?Pro?Ser?Ser?Ser?Ser?Ser?Ser?Leu?Glu?Ser?Ser?Ala?Ser

325 330 335

Ala?Leu?Asp?Arg?Arg?Ala?Pro?Thr?Arg?Asn?Gln?Pro?Gln?Ala?Pro?Gly

340 345 350

Val?Glu?Ala?Ser?Gly?Ala?Gly?Glu?Ala?Arg?Ala?Ser?Thr?Gly?Ser?Ser

355 360 365

Asp?Ser?Ser?Pro?Gly?Gly?His?Gly?Thr?Gln?Val?Asn?Val?Thr?Cys?Ile

370 375 380

Val?Asn?Val?Cys?Ser?Ser?Ser?Asp?His?Ser?Ser?Gln?Cys?Ser?Ser?Gln

385 390 395 400

Ala?Ser?Ser?Thr?Met?Gly?Asp?Thr?Asp?Ser?Ser?Pro?Ser?Glu?Ser?Pro

405 410 415

Lys?Asp?Glu?Gln?Val?Pro?Phe?Ser?Lys?Glu?Glu?Cys?Ala?Phe?Arg?Ser

420 425 430

Gln?Leu?Glu?Thr?Pro?Glu?Thr?Leu?Leu?Gly?Ser?Thr?Glu?Glu?Lys?Pro

435 440 445

Leu?Pro?Leu?Gly?Val?Pro?Asp?Ala?Gly?Met?Lys?Pro?Ser

450 455 460

<210>14

<211>335

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>14

Met?Leu?Gly?Ile?Trp?Thr?Leu?Leu?Pro?Leu?Val?Leu?Thr?Ser?Val?Ala

1 5 10 15

Arg?Leu?Ser?Ser?Lys?Ser?Val?Asn?Ala?Gln?Val?Thr?Asp?Ile?Asn?Ser

20 25 30

Lys?Gly?Leu?Glu?Leu?Arg?Lys?Thr?Val?Thr?Thr?Val?Glu?Thr?Gln?Asn

35 40 45

Leu?Glu?Gly?Leu?His?His?Asp?Gly?Gln?Phe?Cys?His?Lys?Pro?Cys?Pro

50 55 60

Pro?Gly?Glu?Arg?Lys?Ala?Arg?Asp?Cys?Thr?Val?Asn?Gly?Asp?Glu?Pro

65 70 75 80

Asp?Cys?Val?Pro?Cys?Gln?Glu?Gly?Lys?Glu?Tyr?Thr?Asp?Lys?Ala?His

85 90 95

Phe?Ser?Ser?Lys?Cys?Arg?Arg?Cys?Arg?Leu?Cys?Asp?Glu?Gly?His?Gly

100 105 110

Leu?Glu?Val?Glu?Ile?Asn?Cys?Thr?Arg?Thr?Gln?Asn?Thr?Lys?Cys?Arg

115 120 125

Cys?Lys?Pro?Asn?Phe?Phe?Cys?Asn?Ser?Thr?Val?Cys?Glu?His?Cys?Asp

130 135 140

Pro?Cys?Thr?Lys?Cys?Glu?His?Gly?Ile?Ile?Lys?Glu?Cys?Thr?Leu?Thr

145 150 155 160

Ser?Asn?Thr?Lys?Cys?Lys?Glu?Glu?Gly?Ser?Arg?Ser?Asn?Leu?Gly?Trp

165 170 175

Leu?Cys?Leu?Leu?Leu?Leu?Pro?Ile?Pro?Leu?Ile?Val?Trp?Val?Lys?Arg

180 185 190

Lys?Glu?Val?Gln?Lys?Thr?Cys?Arg?Lys?His?Arg?Lys?Glu?Asn?Gln?Gly

195 200 205

Ser?His?Glu?Ser?Pro?Thr?Leu?Asn?Pro?Glu?Thr?Val?Ala?Ile?Asn?Leu

210 215 220

Ser?Asp?Val?Asp?Leu?Ser?Lys?Tyr?Ile?Thr?Thr?Ile?Ala?Gly?Val?Met

225 230 235 240

Thr?Leu?Ser?Gln?Val?Lys?Gly?Phe?Val?Arg?Lys?Asn?Gly?Val?Asn?Glu

245 250 255

Ala?Lys?Ile?Asp?Glu?Ile?Lys?Asn?Asp?Asn?Val?Gln?Asp?Thr?Ala?Glu

260 265 270

Gln?Lys?Val?Gln?Leu?Leu?Arg?Asn?Trp?His?Gln?Leu?His?Gly?Lys?Lys

275 280 285

Glu?Ala?Tyr?Asp?Thr?Leu?Ile?Lys?Asp?Leu?Lys?Lys?Ala?Asn?Leu?Cys

290 295 300

Thr?Leu?Ala?Glu?Lys?Ile?Gln?Thr?Ile?Ile?Leu?Lys?Asp?Ile?Thr?Ser

305 310 315 320

Asp?Ser?Glu?Asn?Ser?Asn?Phe?Arg?Asn?Glu?Ile?Gln?Ser?Leu?Val

325 330 335

<210>15

<211>435

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>15

Met?Leu?Leu?Pro?Trp?Ala?Thr?Ser?Ala?Pro?Gly?Leu?Ala?Trp?Gly?Pro

1 5 10 15

Leu?Val?Leu?Gly?Leu?Phe?Gly?Leu?Leu?Ala?Ala?Ser?Gln?Pro?Gln?Ala

20 25 30

Val?Pro?Pro?Tyr?Ala?Ser?Glu?Asn?Gln?Thr?Cys?Arg?Asp?Gln?Glu?Lys

35 40 45

Glu?Tyr?Tyr?Glu?Pro?Gln?His?Arg?Ile?Cys?Cys?Ser?Arg?Cys?Pro?Pro

50 55 60

Gly?Thr?Tyr?Val?Ser?Ala?Lys?Cys?Ser?Arg?Ile?Arg?Asp?Thr?Val?Cys

65 70 75 80

Ala?Thr?Cys?Ala?Glu?Asn?Ser?Tyr?Asn?Glu?His?Trp?Asn?Tyr?Leu?Thr

85 90 95

Ile?Cys?Gln?Leu?Cys?Arg?Pro?Cys?Asp?Pro?Val?Met?Gly?Leu?Glu?Glu

100 105 110

Ile?Ala?Pro?Cys?Thr?Ser?Lys?Arg?Lys?Thr?Gln?Cys?Arg?Cys?Gln?Pro

115 120 125

Gly?Met?Phe?Cys?Ala?Ala?Trp?Ala?Leu?Glu?Cys?Thr?His?Cys?Glu?Leu

130 135 140

Leu?Ser?Asp?Cys?Pro?Pro?Gly?Thr?Glu?Ala?Glu?Leu?Lys?Asp?Glu?Val

145 150 155 160

Gly?Lys?Gly?Asn?Asn?His?Cys?Val?Pro?Cys?Lys?Ala?Gly?His?Phe?Gln

165 170 175

Asn?Thr?Ser?Ser?Pro?Ser?Ala?Arg?Cys?Gln?Pro?His?Thr?Arg?Cys?Glu

180 185 190

Asn?Gln?Gly?Leu?Val?Glu?Ala?Ala?Pro?Gly?Thr?Ala?Gln?Ser?Asp?Thr

195 200 205

Thr?Cys?Lys?Asn?Pro?Leu?Glu?Pro?Leu?Pro?Pro?Glu?Met?Ser?Gly?Thr

210 215 220

Met?Leu?Met?Leu?Ala?Val?Leu?Leu?Pro?Leu?Ala?Phe?Phe?Leu?Leu?Leu

225 230 235 240

Ala?Thr?Val?Phe?Ser?Cys?Ile?Trp?Lys?Ser?His?Pro?Ser?Leu?Cys?Arg

245 250 255

Lys?Leu?Gly?Ser?Leu?Leu?Lys?Arg?Arg?Pro?Gln?Gly?Glu?Gly?Pro?Asn

260 265 270

Pro?Val?Ala?Gly?Ser?Trp?Glu?Pro?Pro?Lys?Ala?His?Pro?Tyr?Phe?Pro

275 280 285

Asp?Leu?Val?Gln?Pro?Leu?Leu?Pro?Ile?Ser?Gly?Asp?Val?Ser?Pro?Val

290 295 300

Ser?Thr?Gly?Leu?Pro?Ala?Ala?Pro?Val?Leu?Glu?Ala?Gly?Val?Pro?Gln

305 310 315 320

Gln?Gln?Ser?Pro?Leu?Asp?Leu?Thr?Arg?Glu?Pro?Gln?Leu?Glu?Pro?Gly

325 330 335

Glu?Gln?Ser?Gln?Val?Ala?His?Gly?Thr?Asn?Gly?Ile?His?Val?Thr?Gly

340 345 350

Gly?Ser?Met?Thr?Ile?Thr?Gly?Asn?Ile?Tyr?Ile?Tyr?Asn?Gly?Pro?Val

355 360 365

Leu?Gly?Gly?Pro?Pro?Gly?Pro?Gly?Asp?Leu?Pro?Ala?Thr?Pro?Glu?Pro

370 375 380

Pro?Tyr?Pro?Ile?Pro?Glu?Glu?Gly?Asp?Pro?Gly?Pro?Pro?Gly?Leu?Ser

385 390 395 400

Thr?Pro?His?Gln?Glu?Asp?Gly?Lys?Ala?Trp?His?Leu?Ala?Glu?Thr?Glu

405 410 415

His?Cys?Gly?Ala?Thr?Pro?Ser?Asn?Arg?Gly?Pro?Arg?Asn?Gln?Phe?Ile

420 425 430

Thr?His?Asp

435

<210>16

<211>277

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>16

Met?Val?Arg?Leu?Pro?Leu?Gln?Cys?Val?Leu?Trp?Gly?Cys?Leu?Leu?Thr

1 5 10 15

Ala?Val?His?Pro?Glu?Pro?Pro?Thr?Ala?Cys?Arg?Glu?Lys?Gln?Tyr?Leu

20 25 30

Ile?Asn?Ser?Gln?Cys?Cys?Ser?Leu?Cys?Gln?Pro?Gly?Gln?Lys?Leu?Val

35 40 45

Ser?Asp?Cys?Thr?Glu?Phe?Thr?Glu?Thr?Glu?Cys?Leu?Pro?Cys?Gly?Glu

50 55 60

Ser?Glu?Phe?Leu?Asp?Thr?Trp?Asn?Arg?Glu?Thr?His?Cys?His?Gln?His

65 70 75 80

Lys?Tyr?Cys?Asp?Pro?Asn?Leu?Gly?Leu?Arg?Val?Gln?Gln?Lys?Gly?Thr

85 90 95

Ser?Glu?Thr?Asp?Thr?Ile?Cys?Thr?Cys?Glu?Glu?Gly?Trp?His?Cys?Thr

100 105 110

Ser?Glu?Ala?Cys?Glu?Ser?Cys?Val?Leu?His?Arg?Ser?Cys?Ser?Pro?Gly

115 120 125

Phe?Gly?Val?Lys?Gln?Ile?Ala?Thr?Gly?Val?Ser?Asp?Thr?Ile?Cys?Glu

130 135 140

Pro?Cys?Pro?Val?Gly?Phe?Phe?Ser?Asn?Val?Ser?Ser?Ala?Phe?Glu?Lys

145 150 155 160

Cys?His?Pro?Trp?Thr?Ser?Cys?Glu?Thr?Lys?Asp?Leu?Val?Val?Gln?Gln

165 170 175

Ala?Gly?Thr?Asn?Lys?Thr?Asp?Val?Val?Cys?Gly?Pro?Gln?Asp?Arg?Leu

180 185 190

Arg?Ala?Leu?Val?Val?Ile?Pro?Ile?Ile?Phe?Gly?Ile?Leu?Phe?Ala?Ile

195 200 205

Leu?Leu?Val?Leu?Val?Phe?Ile?Lys?Lys?Val?Ala?Lys?Lys?Pro?Thr?Asn

210 215 220

Lys?Ala?Pro?His?Pro?Lys?Gln?Glu?Pro?Gln?Glu?Ile?Asn?Phe?Pro?Asp

225 230 235 240

Asp?Leu?Pro?Gly?Ser?Asn?Thr?Ala?Ala?Pro?Val?Gln?Glu?Thr?Leu?His

245 250 255

Gly?Cys?Gln?Pro?Val?Thr?Gln?Glu?Asp?Gly?Lys?Glu?Ser?Arg?Ile?Ser

260 265 270

Val?Gln?Glu?Arg?Gln

275

<210>17

<211>595

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>17

Met?Arg?Val?Leu?Leu?Ala?Ala?Leu?Gly?Leu?Leu?Phe?Leu?Gly?Ala?Leu

1 5 10 15

Arg?Ala?Phe?Pro?Gln?Asp?Arg?Pro?Phe?Glu?Asp?Thr?Cys?His?Gly?Asn

20 25 30

Pro?Ser?His?Tyr?Tyr?Asp?Lys?Ala?Val?Arg?Arg?Cys?Cys?Tyr?Arg?Cys

35 40 45

Pro?Met?Gly?Leu?Phe?Pro?Thr?Gln?Gln?Cys?Pro?Gln?Arg?Pro?Thr?Asp

50 55 60

Cys?Arg?Lys?Gln?Cys?Glu?Pro?Asp?Tyr?Tyr?Leu?Asp?Glu?Ala?Asp?Arg

65 70 75 80

Cys?Thr?Ala?Cys?Val?Thr?Cys?Ser?Arg?Asp?Asp?Leu?Val?Glu?Lys?Thr

85 90 95

Pro?Cys?Ala?Trp?Asn?Ser?Ser?Arg?Val?Cys?Glu?Cys?Arg?Pro?Gly?Met

100 105 110

Phe?Cys?Ser?Thr?Ser?Ala?Val?Asn?Ser?Cys?Ala?Arg?Cys?Phe?Phe?His

115 120 125

Ser?Val?Cys?Pro?Ala?Gly?Met?Ile?Val?Lys?Phe?Pro?Gly?Thr?Ala?Gln

130 135 140

Lys?Asn?Thr?Val?Cys?Glu?Pro?Ala?Ser?Pro?Gly?Val?Ser?Pro?Ala?Cys

145 150 155 160

Ala?Ser?Pro?Glu?Asn?Cys?Lys?Glu?Pro?Ser?Ser?Gly?Thr?Ile?Pro?Gln

165 170 175

Ala?Lys?Pro?Thr?Pro?Val?Ser?Pro?Ala?Thr?Ser?Ser?Ala?Ser?Thr?Met

180 185 190

Pro?Val?Arg?Gly?Gly?Thr?Arg?Leu?Ala?Gln?Glu?Ala?Ala?Ser?Lys?Leu

195 200 205

Thr?Arg?Ala?Pro?Asp?Ser?Pro?Ser?Ser?Val?Gly?Arg?Pro?Ser?Ser?Asp

210 215 220

Pro?Gly?Leu?Ser?Pro?Thr?Gln?Pro?Cys?Pro?Glu?Gly?Ser?Gly?Asp?Cys

225 230 235 240

Arg?Lys?Gln?Cys?Glu?Pro?Asp?Tyr?Tyr?Leu?Asp?Glu?Ala?Gly?Arg?Cys

245 250 255

Thr?Ala?Cys?Val?Ser?Cys?Ser?Arg?Asp?Asp?Leu?Val?Glu?Lys?Thr?Pro

260 265 270

Cys?Ala?Trp?Asn?Ser?Ser?Arg?Thr?Cys?Glu?Cys?Arg?Pro?Gly?Met?Ile

275 280 285

Cys?Ala?Thr?Ser?Ala?Thr?Asn?Ser?Cys?Ala?Arg?Cys?Val?Pro?Tyr?Pro

290 295 300

Ile?Cys?Ala?Ala?Glu?Thr?Val?Thr?Lys?Pro?Gln?Asp?Met?Ala?Glu?Lys

305 310 315 320

Asp?Thr?Thr?Phe?Glu?Ala?Pro?Pro?Leu?Gly?Thr?Gln?Pro?Asp?Cys?Asn

325 330 335

Pro?Thr?Pro?Glu?Asn?Gly?Glu?Ala?Pro?Ala?Ser?Thr?Ser?Pro?Thr?Gln

340 345 350

Ser?Leu?Leu?Val?Asp?Ser?Gln?Ala?Ser?Lys?Thr?Leu?Pro?Ile?Pro?Thr

355 360 365

Ser?Ala?Pro?Val?Ala?Leu?Ser?Ser?Thr?Gly?Lys?Pro?Val?Leu?Asp?Ala

370 375 380

Gly?Pro?Val?Leu?Phe?Trp?Val?Ile?Leu?Val?Leu?Val?Val?Val?Val?Gly

385 390 395 400

Ser?Ser?Ala?Phe?Leu?Leu?Cys?His?Arg?Arg?Ala?Cys?Arg?Lys?Arg?Ile

405 410 415

Arg?Gln?Lys?Leu?His?Leu?Cys?Tyr?Pro?Val?Gln?Thr?Ser?Gln?Pro?Lys

420 425 430

Leu?Glu?Leu?Val?Asp?Ser?Arg?Pro?Arg?Arg?Ser?Ser?Thr?Gln?Leu?Arg

435 440 445

Ser?Gly?Ala?Ser?Val?Thr?Glu?Pro?Val?Ala?Glu?Glu?Arg?Gly?Leu?Met

450 455 460

Ser?Gln?Pro?Leu?Met?Glu?Thr?Cys?His?Ser?Val?Gly?Ala?Ala?Tyr?Leu

465 470 475 480

Glu?Ser?Leu?Pro?Leu?Gln?Asp?Ala?Ser?Pro?Ala?Gly?Gly?Pro?Ser?Ser

485 490 495

Pro?Arg?Asp?Leu?Pro?Glu?Pro?Arg?Val?Ser?Thr?Glu?His?Thr?Asn?Asn

500 505 510

Lys?Ile?Glu?Lys?Ile?Tyr?Ile?Met?Lys?Ala?Asp?Thr?Val?Ile?Val?Gly

515 520 525

Thr?Val?Lys?Ala?Glu?Leu?Pro?Glu?Gly?Arg?Gly?Leu?Ala?Gly?Pro?Ala

530 535 540

Glu?Pro?Glu?Leu?Glu?Glu?Glu?Leu?Glu?Ala?Asp?His?Thr?Pro?His?Tyr

545 550 555 560

Pro?Glu?Gln?Glu?Thr?Glu?Pro?Pro?Leu?Gly?Ser?Cys?Ser?Asp?Val?Met

565 570 575

Leu?Ser?Val?Glu?Glu?Glu?Gly?Lys?Glu?Asp?Pro?Leu?Pro?Thr?Ala?Ala

580 585 590

Ser?Gly?Lys

595

<210>18

<211>260

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>18

Met?Ala?Arg?Pro?His?Pro?Trp?Trp?Leu?Cys?Val?Leu?Gly?Thr?Leu?Val

1 5 10 15

Gly?Leu?Ser?Ala?Thr?Pro?Ala?Pro?Lys?Ser?Cys?Pro?Glu?Arg?His?Tyr

20 25 30

Trp?Ala?Gln?Gly?Lys?Leu?Cys?Cys?Gln?Met?Cys?Glu?Pro?Gly?Thr?Phe

35 40 45

Leu?Val?Lys?Asp?Cys?Asp?Gln?His?Arg?Lys?Ala?Ala?Gln?Cys?Asp?Pro

50 55 60

Cys?Ile?Pro?Gly?Val?Ser?Phe?Ser?Pro?Asp?His?His?Thr?Arg?Pro?His

65 70 75 80

Cys?Glu?Ser?Cys?Arg?His?Cys?Asn?Ser?Gly?Leu?Leu?Val?Arg?Asn?Cys

85 90 95

Thr?Ile?Thr?Ala?Asn?Ala?Glu?Cys?Ala?Cys?Arg?Asn?Gly?Trp?Gln?Cys

100 105 110

Arg?Asp?Lys?Glu?Cys?Thr?Glu?Cys?Asp?Pro?Leu?Pro?Asn?Pro?Ser?Leu

115 120 125

Thr?Ala?Arg?Ser?Ser?Gln?Ala?Leu?Ser?Pro?His?Pro?Gln?Pro?Thr?His

130 135 140

Leu?Pro?Tyr?Val?Ser?Glu?Met?Leu?Glu?Ala?Arg?Thr?Ala?Gly?His?Met

145 150 155 160

Gln?Thr?Leu?Ala?Asp?Phe?Arg?Gln?Leu?Pro?Ala?Arg?Thr?Leu?Ser?Thr

165 170 175

His?Trp?Pro?Pro?Gln?Arg?Ser?Leu?Cys?Ser?Ser?Asp?Phe?Ile?Arg?Ile

180 185 190

Leu?Val?Ile?Phe?Ser?Gly?Met?Phe?Leu?Val?Phe?Thr?Leu?Ala?Gly?Ala

195 200 205

Leu?Phe?Leu?His?Gln?Arg?Arg?Lys?Tyr?Arg?Ser?Asn?Lys?Gly?Glu?Ser

210 215 220

Pro?Val?Glu?Pro?Ala?Glu?Pro?Cys?Arg?Tyr?Ser?Cys?Pro?Arg?Glu?Glu

225 230 235 240

Glu?Gly?Ser?Thr?Ile?Pro?Ile?Gln?Glu?Asp?Tyr?Arg?Lys?Pro?Glu?Pro

245 250 255

Ala?Cys?Ser?Pro

260

<210>19

<211>283

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>19

Met?Glu?Pro?Pro?Gly?Asp?Trp?Gly?Pro?Pro?Pro?Trp?Arg?Ser?Thr?Pro

1 5 10 15

Arg?Thr?Asp?Val?Leu?Arg?Leu?Val?Leu?Tyr?Leu?Thr?Phe?Leu?Gly?Ala

20 25 30

Pro?Cys?Tyr?Ala?Pro?Ala?Leu?Pro?Ser?Cys?Lys?Glu?Asp?Glu?Tyr?Pro

35 40 45

Val?Gly?Ser?Glu?Cys?Cys?Pro?Lys?Cys?Ser?Pro?Gly?Tyr?Arg?Val?Lys

50 55 60

Glu?Ala?Cys?Gly?Glu?Leu?Thr?Gly?Thr?Val?Cys?Glu?Pro?Cys?Pro?Pro

65 70 75 80

Gly?Thr?Tyr?Ile?Ala?His?Leu?Asn?Gly?Leu?Ser?Lys?Cys?Leu?Gln?Cys

85 90 95

Gln?Met?Cys?Asp?Pro?Ala?Met?Gly?Leu?Arg?Ala?Ser?Arg?Asn?Cys?Ser

100 105 110

Arg?Thr?Glu?Asn?Ala?Val?Cys?Gly?Cys?Ser?Pro?Gly?His?Phe?Cys?Ile

115 120 125

Val?Gln?Asp?Gly?Asp?His?Cys?Ala?Ala?Cys?Arg?Ala?Tyr?Ala?Thr?Ser

130 135 140

Ser?Pro?Gly?Gln?Arg?Val?Gln?Lys?Gly?Gly?Thr?Glu?Ser?Gln?Asp?Thr

145 150 155 160

Leu?Cys?Gln?Asn?Cys?Pro?Pro?Gly?Thr?Phe?Ser?Pro?Asn?Gly?Thr?Leu

165 170 175

Glu?Glu?Cys?Gln?His?Gln?Thr?Lys?Cys?Ser?Trp?Leu?Val?Thr?Lys?Ala

180 185 190

Gly?Ala?Gly?Thr?Ser?Ser?Ser?His?Trp?Val?Trp?Trp?Phe?Leu?Ser?Gly

195 200 205

Ser?Leu?Val?Ile?Val?Ile?Val?Cys?Ser?Thr?Val?Gly?Leu?Ile?Ile?Cys

210 215 220

Val?Lys?Arg?Arg?Lys?Pro?Arg?Gly?Asp?Val?Val?Lys?Val?Ile?Val?Ser

225 230 235 240

Val?Gln?Arg?Lys?Arg?Gln?Glu?Ala?Glu?Gly?Glu?Ala?Thr?Val?Ile?Glu

245 250 255

Ala?Leu?Gln?Ala?Pro?Pro?Asp?Val?Thr?Thr?Val?Ala?Val?Glu?Glu?Thr

260 265 270

Ile?Pro?Ser?Phe?Thr?Gly?Arg?Ser?Pro?Asn?His

275 280

<210>20

<211>277

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>20

Met?Cys?Val?Gly?Ala?Arg?Arg?Leu?Gly?Arg?Gly?Pro?Cys?Ala?Ala?Leu

1 5 10 15

Leu?Leu?Leu?Gly?Leu?Gly?Leu?Ser?Thr?Val?Thr?Gly?Leu?His?Cys?Val

20 25 30

Gly?Asp?Thr?Tyr?Pro?Ser?Asn?Asp?Arg?Cys?Cys?His?Glu?Cys?Arg?Pro

35 40 45

Gly?Asn?Gly?Met?Val?Ser?Arg?Cys?Ser?Arg?Ser?Gln?Asn?Thr?Val?Cys

50 55 60

Arg?Pro?Cys?Gly?Pro?Gly?Phe?Tyr?Asn?Asp?Val?Val?Ser?Ser?Lys?Pro

65 70 75 80

Cys?Lys?Pro?Cys?Thr?Trp?Cys?Asn?Leu?Arg?Ser?Gly?Ser?Glu?Arg?Lys

85 90 95

Gln?Leu?Cys?Thr?Ala?Thr?Gln?Asp?Thr?Val?Cys?Arg?Cys?Arg?Ala?Gly

100 105 110

Thr?Gln?Pro?Leu?Asp?Ser?Tyr?Lys?Pro?Gly?Val?Asp?Cys?Ala?Pro?Cys

115 120 125

Pro?Pro?Gly?His?Phe?Ser?Pro?Gly?Asp?Asn?Gln?Ala?Cys?Lys?Pro?Trp

130 135 140

Thr?Asn?Cys?Thr?Leu?Ala?Gly?Lys?His?Thr?Leu?Gln?Pro?Ala?Ser?Asn

145 150 155 160

Ser?Ser?Asp?Ala?Ile?Cys?Glu?Asp?Arg?Asp?Pro?Pro?Ala?Thr?Gln?Pro

165 170 175

Gln?Glu?Thr?Gln?Gly?Pro?Pro?Ala?Arg?Pro?Ile?Thr?Val?Gln?Pro?Thr

180 185 190

Glu?Ala?Trp?Pro?Arg?Thr?Ser?Gln?Gly?Pro?Ser?Thr?Arg?Pro?Val?Glu

195 200 205

Val?Pro?Gly?Gly?Arg?Ala?Val?Ala?Ala?Ile?Leu?Gly?Leu?Gly?Leu?Val

210 215 220

Leu?Gly?Leu?Leu?Gly?Pro?Leu?Ala?Ile?Leu?Leu?Ala?Leu?Tyr?Leu?Leu

225 230 235 240

Arg?Arg?Asp?Gln?Arg?Leu?Pro?Pro?Asp?Ala?His?Lys?Pro?Pro?Gly?Gly

245 250 255

Gly?Ser?Phe?Arg?Thr?Pro?Ile?Gln?Glu?Glu?Gln?Ala?Asp?Ala?His?Ser

260 265 270

Thr?Leu?Ala?Lys?Ile

275

<210>21

<211>468

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>21

Met?Ala?Pro?Pro?Pro?Ala?Arg?Val?His?Leu?Gly?Ala?Phe?Leu?Ala?Val

1 5 10 15

Thr?Pro?Asn?Pro?Gly?Ser?Ala?Ala?Ser?Gly?Thr?Glu?Ala?Ala?Ala?Ala

20 25 30

Thr?Pro?Ser?Lys?Val?Trp?Gly?Ser?Ser?Ala?Gly?Arg?Ile?Glu?Pro?Arg

35 40 45

Gly?Gly?Gly?Arg?Gly?Ala?Leu?Pro?Thr?Ser?Met?Gly?Gln?His?Gly?Pro

50 55 60

Ser?Ala?Arg?Ala?Arg?Ala?Gly?Arg?Ala?Pro?Gly?Pro?Arg?Pro?Ala?Arg

65 70 75 80

Glu?Ala?Ser?Pro?Arg?Leu?Arg?Val?His?Lys?Thr?Phe?Lys?Phe?Val?Val

85 90 95

Val?Gly?Val?Leu?Leu?Gln?Val?Val?Pro?Ser?Ser?Ala?Ala?Thr?Ile?Lys

100 105 110

Leu?His?Asp?Gln?Ser?Ile?Gly?Thr?Gln?Gln?Trp?Glu?His?Ser?Pro?Leu

115 120 125

Gly?Glu?Leu?Cys?Pro?Pro?Gly?Ser?His?Arg?Ser?Glu?Arg?Pro?Gly?Ala

130 135 140

Cys?Asn?Arg?Cys?Thr?Glu?Gly?Val?Gly?Tyr?Thr?Asn?Ala?Ser?Asn?Asn

145 150 155 160

Leu?Phe?Ala?Cys?Leu?Pro?Cys?Thr?Ala?Cys?Lys?Ser?Asp?Glu?Glu?Glu

165 170 175

Arg?Ser?Pro?Cys?Thr?Thr?Thr?Arg?Asn?Thr?Ala?Cys?Gln?Cys?Lys?Pro

180 185 190

Gly?Thr?Phe?Arg?Asn?Asp?Asn?Ser?Ala?Glu?Met?Cys?Arg?Lys?Cys?Ser

195 200 205

Thr?Gly?Cys?Pro?Arg?Gly?Met?Val?Lys?Val?Lys?Asp?Cys?Thr?Pro?Trp

210 215 220

Ser?Asp?Ile?Glu?Cys?Val?His?Lys?Glu?Ser?Gly?Asn?Gly?His?Asn?Ile

225 230 235 240

Trp?Val?Ile?Leu?Val?Val?Thr?Leu?Val?Val?Pro?Leu?Leu?Leu?Val?Ala

245 250 255

Val?Leu?Ile?Val?Cys?Cys?Cys?Ile?Gly?Ser?Gly?Cys?Gly?Gly?Asp?Pro

260 265 270

Lys?Cys?Met?Asp?Arg?Val?Cys?Phe?Trp?Arg?Leu?Gly?Leu?Leu?Arg?Gly

275 280 285

Pro?Gly?Ala?Glu?Asp?Asn?Ala?His?Asn?Glu?Ile?Leu?Ser?Asn?Ala?Asp

290 295 300

Ser?Leu?Ser?Thr?Phe?Val?Ser?Glu?Gln?Gln?Met?Glu?Ser?Gln?Glu?Pro

305 310 315 320

Ala?Asp?Leu?Thr?Gly?Val?Thr?Val?Gln?Ser?Pro?Gly?Glu?Ala?Gln?Cys

325 330 335

Leu?Leu?Gly?Pro?Ala?Glu?Ala?Glu?Gly?Ser?Gln?Arg?Arg?Arg?Leu?Leu

340 345 350

Val?Pro?Ala?Asn?Gly?Ala?Asp?Pro?Thr?Glu?Thr?Leu?Met?Leu?Phe?Phe

355 360 365

Asp?Lys?Phe?Ala?Asn?Ile?Val?Pro?Phe?Asp?Ser?Trp?Asp?Gln?Leu?Met

370 375 380

Arg?Gln?Leu?Asp?Leu?Thr?Lys?Asn?Glu?Ile?Asp?Val?Val?Arg?Ala?Gly

385 390 395 400

Thr?Ala?Gly?Pro?Gly?Asp?Ala?Leu?Tyr?Ala?Met?Leu?Met?Lys?Trp?Val

405 410 415

Asn?Lys?Thr?Gly?Arg?Asn?Ala?Ser?Ile?His?Thr?Leu?Leu?Asp?Ala?Leu

420 425 430

Glu?Arg?Met?Glu?Glu?Arg?His?Ala?Lys?Glu?Lys?Ile?Gln?Asp?Leu?Leu

435 440 445

Val?Asp?Ser?Gly?Lys?Phe?Ile?Tyr?Leu?Glu?Asp?Gly?Thr?Gly?Ser?Ala

450 455 460

Val?Ser?Leu?Glu

465

<210>22

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>22

Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys

1 5 10 15

Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly?Pro

20 25 30

Gly?Gln?Asp?Thr?Asp?Cys

35

<210>23

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>23

Cys?Arg?Leu?Arg?Glu?Tyr?Tyr?Asp?Gln?Thr?Ala?Gln?Met?Cys?Cys?Ser

1 5 10 15

Lys?Cys?Ser?Pro?Gly?Gln?His?Ala?Lys?Val?Phe?Cys?Thr?Lys?Thr?Ser

20 25 30

Asp?Thr?Val?Cys

35

<210>24

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>24

Cys?Arg?Asp?Gln?Glu?Lys?Glu?Tyr?Tyr?Glu?Pro?Gln?His?Arg?Ile?Cys

1 5 10 15

Cys?Ser?Arg?Cys?Pro?Pro?Gly?Thr?Tyr?Val?Ser?Ala?Lys?Cys?Ser?Arg

20 25 30

Ile?Arg?Asp?Thr?Val?Cys

35

<210>25

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>25

Cys?Arg?Glu?Lys?Gln?Tyr?Leu?Ile?Asn?Ser?Gln?Cys?Cys?Ser?Leu?Cys

1 5 10 15

Gln?Pro?Gly?Gln?Lys?Leu?Val?Ser?Asp?Cys?Thr?Glu?Phe?Thr?Glu?Thr

20 25 30

Glu?Cys

<210>26

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>26

Cys?Lys?Glu?Asp?Glu?Tyr?Pro?Val?Gly?Ser?Glu?Cys?Cys?Pro?Lys?Cys

1 5 10 15

Ser?Pro?Gly?Tyr?Arg?Val?Lys?Glu?Ala?Cys?Gly?Glu?Leu?Thr?Gly?Thr

20 25 30

Val?Cys

<210>27

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>27

Cys?His?Gly?Asn?Pro?Ser?His?Tyr?Tyr?Asp?Lys?Ala?Val?Arg?Arg?Cys

1 5 10 15

Cys?Tyr?Arg?Cys?Pro?Met?Gly?Leu?Phe?Pro?Thr?Gln?Gln?Cys?Pro?Gln

20 25 30

Arg?Pro?Thr?Asp?Cys?Arg?Lys?Gln?Cys

35 40

<210>28

<211>42

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>28

Pro?Tyr?Gly?Ala?Asp?Arg?Gly?Lys?Cys?Arg?Gly?Asn?Asp?Tyr?Glu?Lys

1 5 10 15

Asp?Gly?Leu?Cys?Cys?Thr?Ser?Cys?Pro?Pro?Gly?Ser?Tyr?Ala?Ser?Arg

20 25 30

Leu?Cys?Gly?Pro?Gly?Ser?Asp?Thr?Val?Cys

35 40

<210>29

<211>28

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>29

Glu?Lys?Asp?Gly?Leu?Cys?Cys?Ala?Ser?Cys?His?Pro?Gly?Phe?Tyr?Ala

1 5 10 15

Ser?Arg?Leu?Cys?Gly?Pro?Gly?Ser?Asn?Thr?Val?Cys

20 25

<210>30

<211>22

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>30

Cys?Thr?Pro?Cys?Pro?Asn?Gly?Thr?Tyr?Val?Ser?Gly?Leu?Tyr?Asn?Cys

1 5 10 15

Thr?Asp?Cys?Thr?Glu?Cys

20

<210>31

<211>44

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>31

His?Ala?Pro?Val?Asn?Gly?Ser?Cys?Asp?Asp?Gly?Glu?Tyr?Leu?Asp?Lys

1 5 10 15

Thr?His?Asn?Gln?Cys?Cys?Asn?Arg?Cys?Pro?Pro?Gly?Glu?Phe?Ala?Lys

20 25 30

Ile?Arg?Cys?Ser?Gly?Ser?Asp?Asn?Thr?Lys?Cys?Glu

35 40

<210>32

<211>44

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>32

His?Ala?Pro?Val?Asn?Gly?Ser?Cys?Asp?Glu?Gly?Glu?Tyr?Leu?Asp?Lys

1 5 10 15

Arg?His?Asn?Gln?Cys?Cys?Asn?Arg?Cys?Pro?Pro?Gly?Glu?Phe?Ala?Lys

20 25 30

Val?Arg?Cys?Asn?Gly?Asn?Asp?Asn?Thr?Lys?Cys?Glu

35 40

<210>33

<211>37

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>33

Cys?Asn?Gly?Thr?Asp?Tyr?Asn?Ser?Ash?Ser?Asn?Asn?Leu?Cys?Cys?Lys

1 5 10 15

Gln?Cys?Asp?Pro?Gly?Met?Tyr?Met?Thr?His?Ser?Cys?Ash?Thr?Thr?Ser

20 25 30

Asn?Thr?Lys?Cys?Asp

35

<210>34

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>34

Tyr?Tyr?Asn?Ser?Gln?Glu?Leu?Lys?Cys?Cys?Lys?Leu?Cys?Lys?Pro?Gly

1 5 10 15

Thr?Tyr?Ser?Asp?His?Arg?Cys?Asp?Lys?Tyr?Ser?Asp?Thr?Ile?Cys

20 25 30

<210>35

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>35

Cys?Arg?Gln?Gly?Tyr?Tyr?Tyr?Asp?Pro?Glu?Ser?Glu?Met?Cys?Phe?Pro

1 5 10 15

Cys?Ser?Asn?Cys?Glu?Ser?Ser?Lys?Val?Lys?Val?Thr?Thr?Cys?Asn?Arg

20 25 30

Thr?His?Asp?Thr?Val?Cys

35

<210>36

<211>42

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>36

Tyr?Thr?Pro?Ile?Asn?Gly?Lys?Cys?Asn?Gly?Thr?Asp?Tyr?Asn?Ser?Asn

1 5 10 15

Asn?Leu?Cys?Cys?Lys?Gln?Cys?Asn?Pro?Gly?Met?Tyr?Met?Thr?His?Ser

20 25 30

Cys?Asn?Thr?Thr?Ser?Asn?Thr?Lys?Cys?Asp

35 40

<210>37

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>37

Tyr?Ala?Pro?Ser?Asn?Gly?Lys?Cys?Lys?Asp?Asn?Glu?Tyr?Lys?Arg?His

1 5 10 15

Asn?Leu?Cys?Cys?Leu?Ser?Cys?Pro?Pro?Gly?Thr?Tyr?Ala?Ser?Arg?Leu

20 25 30

Cys?Asp?Ser?Lys?Thr?Asn?Thr?Gln?Cys

35 40

<210>38

<211>40

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>38

His?Ala?Pro?Ser?Asn?Gly?Lys?Cys?Lys?Asp?Asn?Glu?Tyr?Arg?Ser?Arg

1 5 10 15

Asn?Leu?Cys?Cys?Leu?Ser?Cys?Pro?Pro?Gly?Thr?Tyr?Ala?Ser?Arg?Leu

20 25 30

Cys?Asp?Ser?Lys?Thr?Asn?Thr?Gln

35 40

<210>39

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>39

Tyr?Thr?Pro?Pro?Asn?Gly?Lys?Cys?Lys?Asp?Thr?Glu?Tyr?Lys?Arg?His

1 5 10 15

Asn?Leu?Cys?Cys?Leu?Ser?Cys?Pro?Pro?Gly?Thr?Tyr?Ala?Ser?Arg?Leu

20 25 30

Cys?Asp?Ser?Lys?Thr?Asn?Thr?Gln?Cys

35 40

<210>40

<211>124

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>40

Val?Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys

1 5 10 15

Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys?Pro?Gly

20 25 30

Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Glu?Cys?Glu?Ser?Gly?Ser?Phe?Thr

35 40 45

Ala?Ser?Glu?Asn?His?Leu?Arg?His?Cys?Leu?Ser?Cys?Ser?Lys?Cys?Arg

50 55 60

Lys?Glu?Met?Gly?Gln?Val?Glu?Ile?Ser?Ser?Cys?Thr?Val?Asp?Arg?Asp

65 70 75 80

Thr?Val?Cys?Gly?Cys?Arg?Lys?Asn?Gln?Tyr?Arg?His?Tyr?Trp?Ser?Glu

85 90 95

Asn?Leu?Phe?Gln?Cys?Phe?Asn?Cys?Ser?Leu?Cys?Leu?Asn?Gly?Thr?Val

100 105 110

His?Leu?Ser?Cys?Gln?Glu?Lys?Gln?Asn?Thr?Val?Cys

115 120

<210>41

<211>33

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>41

Asp?Ile?Asn?Ser?Glu?Gly?Met?Glu?Asp?Leu?Ser?Phe?Asp?Asp?Asp?Ala

1 5 10 15

Gln?Asp?Asp?Asn?Ala?Asn?Lys?Thr?Leu?Glu?Thr?Gln?Asn?Leu?Glu?His

20 25 30

Asp

<210>42

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>42

Thr?Glu?Ile?Asn?Glu?Leu?Arg?Gln?Thr?Ile?Thr?Asp?Val?Glu?Thr?Asn

1 5 10 15

Leu

<210>43

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>43

Asp?Glu?Asn?Ser?Lys?Glu?Leu?Glu?Arg?Leu?Gln?Lys?Ile?Ala?Phe?Asn

1 5 10 15

Val?Glu?Thr?Lys?Thr?Leu?Glu?Ser?Leu?Val?Asp?Glu?Gly?Gln

20 25 30

<210>44

<211>326

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>44

Met?Phe?Arg?Leu?Thr?Leu?Leu?Leu?Ala?Tyr?Val?Ala?Cys?Val?Tyr?Gly

1 5 10 15

Gly?Gly?Ala?Pro?Tyr?Gly?Ala?Asp?Arg?Gly?Lys?Cys?Arg?Gly?Asn?Asp

20 25 30

Tyr?Glu?Lys?Asp?Gly?Leu?Cys?Cys?Thr?Ser?Cys?Pro?Pro?Gly?Ser?Tyr

35 40 45

Ala?Ser?Arg?Leu?Cys?Gly?Pro?Gly?Ser?Asp?Thr?Val?Cys?Ser?Pro?Cys

50 55 60

Lys?Asn?Glu?Thr?Phe?Thr?Ala?Ser?Thr?Asn?His?Ala?Pro?Ala?Cys?Val

65 70 75 80

Ser?Cys?Arg?Gly?Arg?Cys?Thr?Gly?His?Leu?Ser?Glu?Ser?Gln?Ser?Cys

85 90 95

Asp?Lys?Thr?Arg?Asp?Arg?Val?Cys?Asp?Cys?Ser?Ala?Gly?Asn?Tyr?Cys

100 105 110

Leu?Leu?Lys?Gly?Gln?Glu?Gly?Cys?Arg?Ile?Cys?Ala?Pro?Lys?Thr?Lys

115 120 125

Cys?Pro?Ala?Gly?Tyr?Gly?Val?Ser?Gly?His?Thr?Arg?Thr?Gly?Asp?Val

130 135 140

Leu?Cys?Thr?Lys?Cys?Pro?Arg?Tyr?Thr?Tyr?Ser?Asp?Ala?Val?Ser?Ser

145 150 155 160

Thr?Glu?Thr?Cys?Thr?Ser?Ser?Phe?Asn?Tyr?Ile?Ser?Val?Glu?Phe?Asn

165 170 175

Leu?Tyr?Pro?Val?Asn?Asp?Thr?Ser?Cys?Thr?Thr?Thr?Ala?Gly?Pro?Asn

180 185 190

Glu?Val?Val?Lys?Thr?Ser?Glu?Phe?Ser?Val?Thr?Leu?Asn?His?Thr?Asp

195 200 205

Cys?Asp?Pro?Val?Phe?His?Thr?Glu?Tyr?Tyr?Gly?Thr?Ser?Gly?Ser?Glu

210 215 220

Gly?Ala?Gly?Gly?Phe?Phe?Thr?Gly?Met?Asp?Arg?Tyr?Gln?Asn?Thr?Thr

225 230 235 240

Lys?Met?Cys?Thr?Leu?Asn?Ile?Glu?Ile?Arg?Cys?Val?Glu?Gly?Asp?Ala

245 250 255

Val?Arg?Thr?Ile?Pro?Arg?Thr?Ser?Asp?Gly?Val?Gly?Val?Leu?Ser?His

260 265 270

Ser?Glu?Thr?Ile?Thr?Val?Ile?Gly?Gly?Cys?Leu?Ser?Asp?Val?Asn?Val

275 280 285

Asp?Ile?Glu?Tyr?Ser?Asp?Ser?Asn?His?Pro?Glu?Glu?Val?Asp?Asp?Phe

290 295 300

Val?Glu?Tyr?His?Trp?Gly?Thr?Arg?Leu?Arg?Leu?Phe?Pro?Ser?Pro?Lys

305 310 315 320

Arg?Cys?Arg?Leu?Val?Ser

325

<210>45

<211>325

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>45

Met?Leu?Arg?Leu?Ile?Ala?Leu?Leu?Val?Cys?Val?Val?Tyr?Val?Tyr?Gly

1 5 10 15

Asp?Asp?Val?Pro?Tyr?Ser?Ser?Asn?Gln?Gly?Lys?Cys?Gly?Gly?His?Asp

20 25 30

Tyr?Glu?Lys?Asp?Gly?Leu?Cys?Cys?Ala?Ser?Cys?His?Pro?Gly?Phe?Tyr

35 40 45

Ala?Ser?Arg?Leu?Cys?Gly?Pro?Gly?Ser?Asn?Thr?Val?Cys?Ser?Pro?Cys

50 55 60

Glu?Asp?Gly?Thr?Phe?Thr?Ala?Ser?Thr?Asn?His?Ala?Pro?Ala?Cys?Val

65 70 75 80

Ser?Cys?Arg?Gly?Pro?Cys?Thr?Gly?His?Leu?Ser?Glu?Ser?Gln?Pro?Cys

85 90 95

Asp?Arg?Thr?His?Asp?Arg?Val?Cys?Asn?Cys?Ser?Thr?Gly?Asn?Tyr?Cys

100 105 110

Leu?Leu?Lys?Gly?Gln?Asn?Gly?Cys?Arg?Ile?Cys?Ala?Pro?Gln?Thr?Lys

115 120 125

Cys?Pro?Ala?Gly?Tyr?Gly?Val?Ser?Gly?His?Thr?Arg?Ala?Gly?Asp?Thr

130 135 140

Leu?Cys?Glu?Lys?Cys?Pro?Pro?His?Thr?Tyr?Ser?Asp?Ser?Leu?Ser?Pro

145 150 155 160

Thr?Glu?Arg?Cys?Gly?Thr?Ser?Phe?Asn?Tyr?Ile?Ser?Val?Gly?Phe?Asn

165 170 175

Leu?Tyr?Pro?Val?Asn?Glu?Thr?Ser?Cys?Thr?Thr?Thr?Ala?Gly?His?Asn

180 185 190

Glu?Val?Ile?Lys?Thr?Lys?Glu?Phe?Thr?Val?Thr?Leu?Asn?Tyr?Thr?Asp

195 200 205

Cys?Asp?Pro?Val?Phe?His?Thr?Glu?Tyr?Tyr?Ala?Thr?Ser?Gly?Lys?Glu

210 215 220

Gly?Ala?Gly?Gly?Phe?Phe?Thr?Gly?Thr?Asp?Ile?Tyr?Gln?Asn?Thr?Thr

225 230 235 240

Lys?Val?Cys?Thr?Leu?Asn?Val?Glu?Ile?Gln?Cys?Ser?Glu?Gly?Asp?Asp

245 250 255

Ile?His?Thr?Leu?Gln?Lys?Thr?Asn?Gly?Gly?Ser?Thr?Met?Pro?His?Ser

260 265 270

Glu?Thr?Ile?Thr?Val?Val?Gly?Ser?Cys?Leu?Ser?Asp?Val?Asn?Val?Asp

275 280 285

Ile?Met?Tyr?Ser?Asp?Thr?Asn?His?Pro?Gly?Glu?Val?Asp?Asp?Phe?Val

290 295 300

Glu?Tyr?His?Trp?Gly?Thr?Arg?Leu?Arg?Phe?Phe?Pro?Leu?Pro?Lys?Arg

305 310 315 320

Cys?Thr?Pro?Val?Ser

325

<210>46

<211>175

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>46

Met?Lys?Pro?Leu?Val?Met?Leu?Ile?Cys?Phe?Ala?Val?Ile?Leu?Leu?Gln

1 5 10 15

Leu?Gly?Val?Thr?Lys?Val?Cys?Gln?His?Asn?Glu?Val?Gln?Leu?Gly?Asn

20 25 30

Glu?Cys?Cys?Pro?Pro?Cys?Gly?Ser?Gly?Gln?Arg?Val?Thr?Lys?Val?Cys

35 40 45

Thr?Asp?Tyr?Thr?Ser?Val?Thr?Cys?Thr?Pro?Cys?Pro?Asn?Gly?Thr?Tyr

50 55 60

Val?Ser?Gly?Leu?Tyr?Asn?Cys?Thr?Asp?Cys?Thr?Glu?Cys?Ash?Asp?Thr

65 70 75 80

Glu?Val?Thr?Ile?Arg?Asn?Cys?Thr?Ser?Thr?Asn?Asn?Thr?Val?Cys?Ala

85 90 95

Ser?Lys?Asn?Tyr?Thr?Ser?Phe?Ser?Ile?Ser?Gly?Val?Gln?His?His?Lys

100 105 110

Gln?Arg?Gln?Asn?His?Thr?Ala?His?Val?Thr?Val?Lys?Gln?Gly?Lys?Ser

115 120 125

Gly?Arg?His?Thr?Leu?Ala?Arg?Leu?Ser?Leu?Phe?Ile?Phe?Leu?Val?Gly

130 135 140

Ile?Ile?Leu?Leu?Ile?Leu?Tyr?Leu?Ile?Ala?Ala?Tyr?Arg?Ser?Glu?Lys

145 150 155 160

Cys?Gln?Gln?Cys?Cys?Ser?Ile?Gly?Lys?Ile?Phe?Tyr?Arg?Thr?Leu

165 170 175

<210>47

<211>186

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>47

Met?Asp?Ile?Lys?Asn?Leu?Leu?Thr?Val?Cys?Thr?Ile?Leu?Tyr?Ile?Ser

1 5 10 15

Thr?Leu?Val?Thr?Ala?Asp?Ile?Pro?Thr?Ser?Ser?Leu?Pro?His?Ala?Pro

20 25 30

Val?Asn?Gly?Ser?Cys?Asp?Asp?Gly?Glu?Tyr?Leu?Asp?Lys?Thr?His?Asn

35 40 45

Gln?Cys?Cys?Asn?Arg?Cys?Pro?Pro?Gly?Glu?Phe?Ala?Lys?Ile?Arg?Cys

50 55 60

Ser?Gly?Ser?Asp?Asn?Thr?Lys?Cys?Glu?Arg?Cys?Pro?Pro?His?Thr?Tyr

65 70 75 80

Thr?Thr?Val?Pro?Asn?Tyr?Ser?Asn?Gly?Cys?His?Gln?Cys?Arg?Lys?Cys

85 90 95

Pro?Thr?Gly?Ser?Phe?Asp?Lys?Val?Lys?Cys?Thr?Gly?Thr?Gln?Asn?Ser

100 105 110

Lys?Cys?Ser?Cys?Leu?Pro?Gly?Trp?Phe?Cys?Ala?Thr?Asp?Ser?Ser?Lys

115 120 125

Thr?Glu?Asp?Cys?Arg?Asp?Cys?Ile?Pro?Lys?Arg?Lys?Cys?Pro?Cys?Gly

130 135 140

Tyr?Phe?Gly?Gly?Ile?Asp?Glu?Leu?Gly?Asn?Pro?Leu?Cys?Lys?Ser?Cys

145 150 155 160

Cys?Val?Gly?Glu?Tyr?Cys?Asp?Asp?Ile?Arg?Asn?His?Arg?Val?Gly?Pro

165 170 175

Phe?Pro?Pro?Cys?Lys?Leu?Ser?Lys?Cys?Asn

180 185

<210>48

<211>186

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>48

Met?Asp?Ile?Lys?Asn?Leu?Leu?Thr?Ala?Cys?Thr?Ile?Phe?Tyr?Ile?Thr

1 5 10 15

Thr?Leu?Ala?Thr?Ala?Asp?Ile?Pro?Thr?Ser?Ser?Leu?Pro?His?Ala?Pro

20 25 30

Val?Asn?Gly?Ser?Cys?Asp?Glu?Gly?Glu?Tyr?Leu?Asp?Lys?Arg?His?Asn

35 40 45

Gln?Cys?Cys?Asn?Arg?Cys?Pro?Pro?Gly?Glu?Phe?Ala?Lys?Val?Arg?Cys

50 55 60

Asn?Gly?Asn?Asp?Asn?Thr?Lys?Cys?Glu?Arg?Cys?Pro?Pro?His?Thr?Tyr

65 70 75 80

Thr?Ala?Ile?Pro?Asn?Tyr?Ser?Asn?Gly?Cys?His?Gln?Cys?Arg?Lys?Cys

85 90 95

Pro?Thr?Gly?Ser?Phe?Asp?Lys?Val?Lys?Cys?Thr?Gly?Thr?Gln?Asn?Ser

100 105 110

Lys?Cys?Ser?Cys?Leu?Pro?Gly?Trp?Tyr?Cys?Ala?Thr?Asp?Ser?Ser?Gln

115 120 125

Thr?Glu?Asp?Cys?Arg?Asp?Cys?Ile?Pro?Lys?Arg?Arg?Cys?Pro?Cys?Gly

130 135 140

Tyr?Phe?Gly?Gly?Ile?Asp?Glu?Gln?Gly?Asn?Pro?Ile?Cys?Lys?Ser?Cys

145 150 155 160

Cys?Val?Gly?Glu?Tyr?Cys?Asp?Tyr?Leu?Arg?Asn?Tyr?Arg?Leu?Asp?Pro

165 170 175

Phe?Pro?Pro?Cys?Lys?Leu?Ser?Lys?Cys?Asn

180 185

<210>49

<211>148

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>49

Met?Met?Lys?Met?Thr?Pro?Ser?Tyr?Ile?Leu?Leu?Val?Tyr?Met?Phe?Val

1 5 10 15

Val?Val?Ser?Gly?Asp?Val?Pro?Tyr?Glu?His?Ile?Asn?Gly?Lys?Cys?Asn

20 25 30

Gly?Thr?Asp?Tyr?Asn?Ser?Asn?Ser?Asn?Asn?Leu?Cys?Cys?Lys?Gln?Cys

35 40 45

Asp?Pro?Gly?Met?Tyr?Met?Thr?His?Ser?Cys?Asn?Thr?Thr?Ser?Asn?Thr

50 55 60

Lys?Cys?Asp?Lys?Cys?Pro?Asp?Gly?Thr?Phe?Thr?Ser?Ile?Pro?Asn?His

65 70 75 80

Ile?Pro?Thr?Cys?Leu?Ser?Cys?Arg?Gly?Lys?Cys?Ser?Ser?Asn?Gln?Val

85 90 95

Glu?Thr?Lys?Ser?Cys?Ser?Asn?Thr?Gln?Ala?Glu?Tyr?Val?Ser?Val?His

100 105 110

Pro?Asp?Thr?Thr?Ala?Asn?Leu?Lys?Asp?Gln?Met?Val?Ala?Gly?Tyr?Val

115 120 125

Tyr?His?Lys?Gln?Ser?Val?Ile?Leu?Val?Thr?Ala?Tyr?Met?Ala?Thr?His

130 135 140

Leu?Lys?Glu?Met

145

<210>50

<211>167

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>50

Met?Thr?Lys?Val?Ile?Ile?Ile?Leu?Gly?Phe?Leu?Ile?Ile?Asn?Thr?Asn

1 5 10 15

Ser?Leu?Ser?Met?Lys?Cys?Glu?Gln?Gly?Val?Ser?Tyr?Tyr?Asn?Ser?Gln

20 25 30

Glu?Leu?Lys?Cys?Cys?Lys?Leu?Cys?Lys?Pro?Gly?Thr?Tyr?Ser?Asp?His

35 40 45

Arg?Cys?Asp?Lys?Tyr?Ser?Asp?Thr?Ile?Cys?Gly?His?Cys?Pro?Ser?Asp

50 55 60

Thr?Phe?Thr?Ser?Ile?Tyr?Asn?Arg?Ser?Pro?Trp?Cys?His?Ser?Cys?Arg

65 70 75 80

Gly?Ser?Cys?Gly?Thr?Asn?Arg?Val?Glu?Val?Thr?Pro?Cys?Thr?Pro?Thr

85 90 95

Thr?Asn?Arg?Ile?Cys?His?Cys?Asp?Ser?Asn?Ser?Tyr?Cys?Leu?Leu?Lys

100 105 110

Ala?Ser?Asp?Gly?Asn?Cys?Val?Thr?Cys?Ala?Pro?Lys?Thr?Lys?Cys?Gly

115 120 125

Arg?Gly?Tyr?Gly?Lys?Lys?Gly?Glu?Asp?Glu?Met?Gly?Asn?Thr?Ile?Cys

130 135 140

Lys?Lys?Cys?Arg?Lys?Gly?Thr?Tyr?Ser?Asp?Ile?Val?Ser?Asp?Ser?Asp

145 150 155 160

Gln?Cys?Lys?Pro?Met?Thr?Arg

165

<210>51

<211>289

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>51

Met?Met?Leu?Phe?Ile?Leu?Phe?Leu?Leu?Pro?Ile?Thr?Val?His?Thr?Ala

1 5 10 15

Thr?Asp?Cys?Pro?Pro?Gly?Tyr?Tyr?Ile?Ser?Lys?Val?Tyr?Pro?Ala?Gly

20 25 30

Thr?Pro?Met?Cys?Ser?Pro?Cys?Ser?Pro?Gly?Thr?Tyr?Thr?Gly?Leu?Gln

35 40 45

Asn?Ser?Leu?Arg?Lys?Cys?Leu?Arg?Cys?Ser?Thr?Cys?Ser?His?Asn?Glu

50 55 60

Glu?Pro?Lys?Val?Ala?Cys?Ser?Thr?Thr?Ser?Asp?Val?Gln?Cys?Gln?Cys

65 70 75 80

Arg?Gln?Gly?Tyr?Tyr?Tyr?Asp?Pro?Glu?Ser?Glu?Met?Cys?Phe?Pro?Cys

85 90 95

Ser?Asn?Cys?Glu?Ser?Ser?Lys?Val?Lys?Val?Thr?Thr?Cys?Asn?Arg?Thr

100 105 110

His?Asp?Thr?Val?Cys?Lys?Cys?Lys?Glu?Gly?Tyr?Tyr?Asp?Lys?Asn?Gly

115 120 125

Val?Cys?Val?Lys?Cys?Gly?Asn?Cys?Tyr?Leu?Gly?Glu?Gly?Val?Lys?Ser

130 135 140

Lys?Cys?Ala?Asn?Asn?Thr?Asp?Val?Thr?Cys?Glu?Leu?Cys?Lys?Asn?Gly

145 150 155 160

Thr?Phe?Ser?Asp?Lys?Val?Ser?Ser?Ser?Asn?Ile?Cys?Tyr?Leu?Tyr?Thr

165 170 175

Met?Cys?Thr?Leu?Gly?Leu?Thr?Gln?Leu?Asn?Phe?Asn?Val?Thr?Trp?Phe

180 185 190

Asp?Thr?Ile?Cys?Ile?Asn?Cys?Ser?Met?Ser?Thr?Asp?Leu?Leu?Asp?Leu

195 200 205

Glu?Thr?Phe?Phe?Thr?Ile?Asn?Phe?Val?Thr?Gln?Arg?Arg?Phe?Pro?Glu

210 215 220

Asp?Asp?Leu?Lys?Asn?Met?Phe?Arg?Leu?Thr?Tyr?Asn?Lys?Thr?Arg?Asn

225 230 235 240

Asp?Ile?Asp?Ser?Ala?Asn?Arg?Asp?Asp?Val?Glu?Gly?Ser?Phe?Thr?Tyr

245 250 255

Asp?Pro?Asn?Leu?Pro?Tyr?Thr?Met?Lys?Gln?Leu?Asp?Tyr?Leu?Ile?Ala

260 265 270

Ser?Lys?Phe?Leu?Met?Thr?Ala?Tyr?Lys?Lys?Ile?Ser?Gln?Ile?Cys?Gln

275 280 285

Leu

<210>52

<211>320

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>52

Met?Met?Lys?Met?Thr?Pro?Ser?Tyr?Ile?Leu?Leu?Val?Tyr?Met?Phe?Val

1 5 10 15

Val?Val?Ser?Gly?Asp?Val?Pro?Tyr?Thr?Pro?Ile?Asn?Gly?Lys?Cys?Asn

20 25 30

Gly?Thr?Asp?Tyr?Asn?Ser?Asn?Asn?Leu?Cys?Cys?Lys?Gln?Cys?Asn?Pro

35 40 45

Gly?Met?Tyr?Met?Thr?His?Ser?Cys?Asn?Thr?Thr?Ser?Asn?Thr?Lys?Cys

50 55 60

Asp?Lys?Cys?Pro?Asp?Asp?Thr?Phe?Thr?Ser?Ile?Pro?Asn?His?Ser?Pro

65 70 75 80

Ala?Cys?Leu?Ser?Cys?Arg?Gly?Lys?Cys?Ser?Ser?Asn?Gln?Val?Glu?Thr

85 90 95

Lys?Ser?Cys?Ser?Asn?Thr?Gln?Asp?Arg?Val?Cys?Val?Cys?Ala?Ser?Gly

100 105 110

Tyr?Tyr?Cys?Glu?Phe?Glu?Gly?Ser?Asn?Gly?Cys?Arg?Leu?Cys?Val?Pro

115 120 125

Gln?Thr?Lys?Cys?Gly?Ser?Gly?Tyr?Gly?Val?Tyr?Gly?Tyr?Ser?Ser?Lys

130 135 140

Gly?Asp?Val?Ile?Cys?Lys?Lys?Cys?Pro?Gly?Asn?Ile?Asp?Lys?Cys?Asp

145 150 155 160

Leu?Ser?Phe?Asn?Ser?Ile?Asp?Val?Glu?Ile?Asn?Met?Tyr?Pro?Val?Asn

165 170 175

Lys?Thr?Ser?Cys?Asn?Ser?Ser?Ile?Gly?Ser?Ser?Ser?Thr?Ile?Ser?Thr

180 185 190

Ser?Glu?Leu?Thr?Ile?Thr?Leu?Thr?His?Glu?Asp?Cys?Thr?Pro?Val?Phe

195 200 205

Ile?Gly?Asp?Tyr?Tyr?Ser?Val?Val?Asp?Lys?Leu?Ala?Thr?Ser?Gly?Phe

210 215 220

Phe?Thr?Asn?Asp?Lys?Val?His?Gln?Asp?Leu?Thr?Thr?Gln?Cys?Lys?Ile

225 230 235 240

Asn?Leu?Glu?Ile?Lys?Cys?Asn?Ser?Gly?Arg?Glu?Ser?Arg?Gln?Leu?Thr

245 250 255

Pro?Thr?Thr?Lys?Val?Tyr?Phe?Met?Pro?His?Ser?Glu?Thr?Val?Thr?Val

260 265 270

Val?Gly?Asp?Cys?Leu?Ser?Asn?Leu?Asp?Val?Tyr?Ile?Val?Tyr?Ala?Asn

275 280 285

Thr?Asp?Ala?Ile?Tyr?Ser?Asp?Met?Asp?Val?Val?Ala?Tyr?His?Thr?Ser

290 295 300

Tyr?Ile?Leu?Asn?Val?Asp?His?Ile?Pro?Pro?Asn?Asp?Cys?Glu?Arg?Asp

305 310 315 320

<210>53

<211>349

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>53

Met?Lys?Ser?Val?Leu?Tyr?Ser?Tyr?Ile?Leu?Phe?Leu?Ser?Cys?Ile?Ile

1 5 10 15

Ile?Asn?Gly?Arg?Asp?Val?Thr?Pro?Tyr?Ala?Pro?Ser?Asn?Gly?Lys?Cys

20 25 30

Lys?Asp?Asn?Glu?Tyr?Lys?Arg?His?Asn?Leu?Cys?Cys?Leu?Ser?Cys?Pro

35 40 45

Pro?Gly?Thr?Tyr?Ala?Ser?Arg?Leu?Cys?Asp?Ser?Lys?Thr?Asn?Thr?Gln

50 55 60

Cys?Thr?Pro?Cys?Gly?Ser?Gly?Thr?Phe?Thr?Ser?Arg?Asn?Asn?His?Leu

65 70 75 80

Pro?Ala?Cys?Leu?Ser?Cys?Asn?Gly?Arg?Cys?Asp?Ser?Asn?Gln?Val?Glu

85 90 95

Thr?Arg?Ser?Cys?Asn?Thr?Thr?His?Asn?Arg?Ile?Cys?Glu?Cys?Ser?Pro

100 105 110

Gly?Tyr?Tyr?Cys?Ile?Leu?Lys?Gly?Ser?Ser?Gly?Cys?Lys?Ala?Cys?Val

115 120 125

Ser?Gln?Thr?Lys?Cys?Gly?Ile?Gly?Tyr?Gly?Val?Ser?Gly?His?Thr?Ser

130 135 140

Ala?Gly?Asp?Val?Ile?Cys?Ser?Pro?Cys?Gly?Leu?Gly?Thr?Tyr?Ser?Arg

145 150 155 160

Thr?Val?Ser?Ser?Ala?Asp?Lys?Cys?Glu?Pro?Val?Pro?Ser?Asn?Thr?Phe

165 170 175

Asn?Tyr?Tle?Asp?Val?Glu?Ile?Asn?Leu?Tyr?Pro?Val?Asn?Asp?Thr?Ser

180 185 190

Cys?Thr?Arg?Thr?Thr?Thr?Thr?Gly?Ile?Ser?Glu?Ser?Ile?Ser?Thr?Ser

195 200 205

Glu?Leu?Thr?Ile?Thr?Met?Asn?His?Lys?Asp?Cys?Asp?Pro?Val?Phe?Arg

210 215 220

Glu?Glu?Tyr?Phe?Ser?Val?Leu?Asn?Lys?Val?Ala?Thr?Ser?Gly?Phe?Phe

225 230 235 240

Thr?Gly?Glu?Asn?Arg?Tyr?Gln?Asn?Ile?Ser?Lys?Val?Cys?Thr?Leu?Asn

245 250 255

Phe?Glu?Ile?Lys?Cys?Asn?Asn?Lys?Gly?Ser?Ser?Ser?Lys?Gln?Leu?Thr

260 265 270

Lys?Ala?Lys?Asn?Asp?Asp?Gly?Ile?Met?Pro?His?Ser?Glu?Thr?Val?Thr

275 280 285

Leu?Val?Gly?Asp?Cys?Leu?Ser?Ser?Val?Asp?Ile?Tyr?Ile?Leu?Tyr?Ser

290 295 300

Asn?Thr?Asn?Thr?Gln?Asp?Tyr?Glu?Thr?Asp?Thr?Ile?Ser?Tyr?His?Ala

305 310 315 320

Gly?Asn?Val?Leu?Asp?Val?Asp?Ser?His?Met?Pro?Gly?Ser?Cys?Asp?Ile

325 330 335

His?Lys?Leu?Ile?Thr?Asn?Ser?Lys?Pro?Thr?His?Phe?Leu

340 345

<210>54

<211>348

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>54

Met?Arg?Ser?Val?Leu?Tyr?Ser?Tyr?Ile?Leu?Phe?Leu?Ser?Cys?Ile?Ile

1 5 10 15

Ile?Asn?Gly?Arg?Asp?Leu?Ala?Pro?His?Ala?Pro?Ser?Asn?Gly?Lys?Cys

20 25 30

Lys?Asp?Asn?Glu?Tyr?Arg?Ser?Arg?Asn?Leu?Cys?Cys?Leu?Ser?Cys?Pro

35 40 45

Pro?Gly?Thr?Tyr?Ala?Ser?Arg?Leu?Cys?Asp?Ser?Lys?Thr?Asn?Thr?Gln

50 55 60

Cys?Thr?Pro?Cys?Gly?Ser?Asp?Thr?Phe?Thr?Ser?His?Asn?Asn?His?Leu

65 70 75 80

Gln?Ala?Cys?Leu?Ser?Cys?Asn?Gly?Arg?Cys?Asp?Ser?Asn?Gln?Val?Glu

85 90 95

Thr?Arg?Ser?Cys?Asn?Thr?Thr?His?Asn?Arg?Ile?Cys?Glu?Cys?Ser?Pro

100 105 110

Gly?Tyr?Tyr?Cys?Leu?Leu?Lys?Gly?Ser?Ser?Gly?Cys?Arg?Thr?Cys?Ile

115 120 125

Ser?Lys?Thr?Lys?Cys?Gly?Ile?Gly?Tyr?Gly?Val?Ser?Gly?Tyr?Thr?Ser

130 135 140

Thr?Gly?Asp?Val?Ile?Cys?Ser?Pro?Cys?Gly?Pro?Gly?Thr?Tyr?Ser?His

145 150 155 160

Thr?Val?Ser?Ser?Thr?Asp?Lys?Cys?Glu?Pro?Val?Thr?Ser?Asn?Thr?Phe

165 170 175

Asn?Tyr?Ile?Asp?Val?Glu?Ile?Asn?Leu?Tyr?Pro?Val?Asn?Asp?Thr?Ser

180 185 190

Cys?Thr?Arg?Thr?Thr?Thr?Thr?Gly?Leu?Ser?Glu?Ser?Ile?Ser?Thr?Ser

195 200 205

Glu?Leu?Thr?Ile?Thr?Met?Asn?His?Lys?Asp?Cys?Asp?Pro?Val?Phe?Arg

210 215 220

Ala?Glu?Tyr?Phe?Ser?Val?Leu?Asn?Asn?Val?Ala?Thr?Ser?Gly?Phe?Phe

225 230 235 240

Thr?Gly?Glu?Asn?Arg?Tyr?Gln?Asn?Thr?Ser?Lys?Ile?Cys?Thr?Leu?Asn

245 250 255

Phe?Glu?Ile?Lys?Cys?Asn?Asn?Lys?Asp?Ser?Ser?Ser?Lys?Gln?Leu?Thr

260 265 270

Lys?Thr?Lys?Asn?Asp?Thr?Ile?Met?Pro?His?Ser?Glu?Thr?Val?Thr?Leu

275 280 285

Val?Gly?Asp?Cys?Leu?Ser?Ser?Val?Asp?Ile?Tyr?Ile?Leu?Tyr?Ser?Asn

290 295 300

Thr?Asn?Thr?Gln?Asp?Tyr?Glu?Thr?Asp?Thr?Ile?Ser?Tyr?His?Met?Gly

305 310 315 320

Asn?Val?Leu?Asp?Val?Asn?Ser?His?Met?Pro?Ala?Ser?Cys?Asp?Ile?His

325 330 335

Lys?Leu?Ile?Thr?Asn?Ser?Gln?Asn?Pro?Thr?His?Leu

340 345

<210>55

<211>349

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>55

Met?Lys?Ser?Val?Leu?Tyr?Leu?Tyr?Ile?Leu?Phe?Leu?Ser?Cys?Ile?Ile

1 5 10 15

Ile?Asn?Gly?Arg?Asp?Ala?Ala?Pro?Tyr?Thr?Pro?Pro?Asn?Gly?Lys?Cys

20 25 30

Lys?Asp?Thr?Glu?Tyr?Lys?Arg?His?Asn?Leu?Cys?Cys?Leu?Ser?Cys?Pro

35 40 45

Pro?Gly?Thr?Tyr?Ala?Ser?Arg?Leu?Cys?Asp?Ser?Lys?Thr?Asn?Thr?Gln

50 55 60

Cys?Thr?Pro?Cys?Gly?Ser?Gly?Thr?Phe?Thr?Ser?Arg?Asn?Asn?His?Leu

65 70 75 80

Pro?Ala?Cys?Leu?Ser?Cys?Asn?Gly?Arg?Cys?Asn?Ser?Asn?Gln?Val?Glu

85 90 95

Thr?Arg?Ser?Cys?Asn?Thr?Thr?His?Asn?Arg?Ile?Cys?Glu?Cys?Ser?Pro

100 105 110

Gly?Tyr?Tyr?Cys?Leu?Leu?Lys?Gly?Ser?Ser?Gly?Cys?Lys?Ala?Cys?Val

115 120 125

Ser?Gln?Thr?Lys?Cys?Gly?Ile?Gly?Tyr?Gly?Val?Ser?Gly?His?Thr?Ser

130 135 140

Val?Gly?Asp?Val?Ile?Cys?Ser?Pro?Cys?Gly?Phe?Gly?Thr?Tyr?Ser?Tyr

145 150 155 160

Thr?Val?Ser?Ser?Thr?Asp?Lys?Cys?Glu?Pro?Val?Pro?Asn?Asn?Thr?Phe

165 170 175

Asn?Tyr?Ile?Asp?Val?Glu?Ile?Thr?Leu?Tyr?Pro?Val?Asn?Asp?Thr?Ser

180 185 190

Cys?Thr?Arg?Thr?Thr?Thr?Thr?Gly?Leu?Ser?Glu?Ser?Ile?Leu?Thr?Ser

195 200 205

Glu?Leu?Thr?Ile?Thr?Met?Asn?His?Thr?Asp?Cys?Asn?Pro?Val?Phe?Arg

210 215 220

Glu?Glu?Tyr?Phe?Ser?Val?Leu?Asn?Lys?Val?Ala?Thr?Ser?Gly?Phe?Phe

225 230 235 240

Thr?Gly?Glu?Asn?Arg?Tyr?Gln?Asn?Ile?Ser?Lys?Val?Cys?Thr?Leu?Asn

245 250 255

Phe?Glu?Ile?Lys?Cys?Asn?Asn?Lys?Gly?Ser?Ser?Phe?Lys?Gln?Leu?Thr

260 265 270

Lys?Ala?Lys?Asn?Asp?Asp?Gly?Met?Met?Ser?His?Ser?Glu?Thr?Val?Thr

275 280 285

Leu?Ala?Gly?Asp?Cys?Leu?Ser?Ser?Val?Asp?Ile?Tyr?Ile?Leu?Tyr?Ser

290 295 300

Asn?Thr?Asn?Ala?Gln?Asp?Tyr?Glu?Thr?Asp?Thr?Ile?Ser?Tyr?Arg?Val

305 310 315 320

Gly?Asn?Val?Leu?Asp?Asp?Asp?Ser?His?Met?Pro?Gly?Ser?Cys?Asp?Ile

325 330 335

His?Lys?Leu?Ile?Thr?Asn?Ser?Lys?Pro?Thr?Arg?Phe?Leu

340 345

<210>56

<211>547

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>56

Met?Lys?Ile?Leu?Leu?Leu?Asn?Glu?Asn?Pro?Val?Val?Ser?Arg?Leu?Val

1 5 10 15

Ser?Leu?Ser?Ala?Lys?Lys?Met?Ser?Tyr?Asp?Phe?Glu?Glu?Leu?Asn?Ala

20 25 30

Tyr?Ser?Glu?Asn?Leu?Gly?Asn?Tyr?Asp?Val?Ile?Val?Val?Asp?Ser?Asp

35 40 45

Thr?Pro?Ala?Pro?Leu?Lys?Ile?Leu?Lys?Glu?Lys?Cys?Asp?Arg?Leu?Ile

50 55 60

Phe?Leu?Ala?Pro?Arg?Asn?Gln?Asn?Val?Glu?Asp?Ile?Asp?Ala?Gln?Ile

65 70 75 80

Leu?Gln?Lys?Pro?Phe?Leu?Pro?Thr?Asp?Phe?Leu?Asn?Leu?Leu?Asn?Asn

85 90 95

Lys?Asp?Ala?Asn?Lys?His?Thr?Ser?Ile?Asp?Leu?Pro?Met?Leu?Ser?Asn

100 105 110

Asp?Glu?Asn?Pro?Tyr?Ala?Asp?Ile?Ser?Leu?Asp?Leu?Asp?Asn?Leu?Asn

115 120 125

Leu?Asp?Asp?Leu?Pro?Asp?Glu?Asn?Ser?Leu?Asp?Ile?Asn?Ser?Glu?Gly

130 135 140

Met?Glu?Asp?Leu?Ser?Phe?Asp?Asp?Asp?Ala?Gln?Asp?Asp?Asn?Ala?Asn

145 150 155 160

Lys?Thr?Leu?Glu?Thr?Gln?Asn?Leu?Glu?His?Asp?Asn?Leu?Glu?Gln?Glu

165 170 175

Thr?Ile?Lys?Glu?Gln?Thr?Gln?Glu?Asp?Thr?Gln?Thr?Asp?Leu?Asp?Leu

180 185 190

Thr?Leu?Glu?Asp?Ser?Glu?Ser?Glu?Lys?Glu?Asp?Leu?Ser?Gln?Glu?His

195 200 205

Thr?Ala?Leu?Asp?Thr?Glu?Pro?Ser?Leu?Asp?Glu?Leu?Asp?Asp?Lys?Asn

210 215 220

Asp?Glu?Asp?Leu?Glu?Ile?Lys?Glu?Asp?Asp?Lys?Asn?Glu?Glu?Ile?Glu

225 230 235 240

Lys?Gln?Glu?Leu?Leu?Asp?Asp?Ser?Lys?Thr?Asn?Thr?Leu?Glu?Met?Gln

245 250 255

Glu?Glu?Leu?Ser?Glu?Ser?Gln?Asp?Asp?Asn?Ala?Asn?Lys?Thr?Leu?Glu

260 265 270

Thr?Gln?Asn?Leu?Glu?His?Asp?Asn?Leu?Glu?Gln?Glu?Thr?Ile?Lys?Glu

275 280 285

Gln?Thr?Gln?Glu?Asp?Thr?Gln?Thr?Asp?Leu?Asp?Leu?Thr?Leu?Glu?Asp

290 295 300

Gly?Glu?Ser?Glu?Lys?Glu?Asp?Leu?Ser?Gln?Glu?His?Thr?Ala?Leu?Asp

305 310 315 320

Thr?Glu?Pro?Ser?Leu?Asp?Glu?Leu?Asp?Asp?Lys?Asn?Asp?Glu?Asp?Leu

325 330 335

Glu?Asp?Asn?Lys?Glu?Leu?Gln?Ala?Asn?Ile?Ser?Asp?Phe?Asp?Asp?Leu

340 345 350

Pro?Glu?Val?Glu?Glu?Gln?Glu?Lys?Glu?Met?Asp?Phe?Asp?Asp?Leu?Pro

355 360 365

Glu?Asp?Ala?Glu?Phe?Leu?Gly?Gln?Ala?Lys?Tyr?Asn?Glu?Glu?Ser?Glu

370 375 380

Glu?Asn?Leu?Glu?Glu?Phe?Ala?Pro?Val?Val?Glu?Glu?Asp?Val?Gln?Asp

385 390 395 400

Glu?Ile?Asp?Asp?Phe?Ala?Ser?Asn?Leu?Ser?Thr?Gln?Asp?Gln?Ile?Lys

405 410 415

Glu?Glu?Leu?Ala?Gln?Leu?Asp?Glu?Leu?Asp?Tyr?Gly?Ile?Asp?Ser?Asp

420 425 430

Asn?Ser?Ser?Lys?Val?Leu?Glu?Asp?Phe?Lys?Asp?Glu?Pro?Ile?Leu?Asp

435 440 445

Asp?Lys?Glu?Leu?Gly?Thr?Asn?Glu?Glu?Glu?Val?Val?Val?Pro?Asn?Leu

450 455 460

Asn?Ile?Ser?Asp?Phe?Asp?Thr?Leu?Lys?Glu?Ser?Asp?Ile?Gln?Glu?Ala

465 470 475 480

Leu?Gly?Glu?Glu?Ile?Leu?Glu?Lys?Asn?Glu?Glu?Pro?Ile?Val?Ser?Asp

485 490 495

Val?Thr?Lys?Asp?Asp?Asn?Ser?Glu?Glu?Ile?Val?Asn?Glu?Leu?Ser?Gln

500 505 510

Ser?Ile?Ala?Gly?Ala?Ile?Thr?Ser?Ser?Ile?Lys?Asp?Asp?Thr?Leu?Lys

515 520 525

Ala?Ala?Leu?Lys?Gly?Met?Asn?Met?Asn?Ile?Asn?Ile?Asn?Ile?Ser?Phe

530 535 540

Lys?Glu?Asp

545

<210>57

<211>496

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>57

Met?Leu?Arg?Cys?Arg?Val?Ala?Asp?Val?Val?Ser?Ser?Asp?Gln?Met?Cys

1 5 10 15

Glu?Met?Thr?Asn?Phe?Pro?Pro?Val?Gln?Ala?Thr?Val?Gly?Tyr?Val?Gln

20 25 30

Leu?Leu?Arg?Arg?Leu?Ser?Phe?Lys?Arg?Glu?Phe?Phe?Val?Pro?Phe?Val

35 40 45

Pro?Thr?Lys?His?Val?Ile?Phe?Thr?Phe?Gln?Thr?Arg?Asn?Pro?Leu?Glu

50 55 60

Glu?Gly?Lys?Val?Lys?Asp?Glu?Val?Thr?Asp?Leu?Val?Ser?His?Tyr?Phe

65 70 75 80

Asn?Trp?Ile?Arg?Glu?Arg?Gly?Val?Tyr?Ser?Phe?Gln?Ile?Leu?Gln?Leu

85 90 95

Tyr?Val?Cys?Ile?Leu?Ser?Gly?Cys?Thr?Val?Val?Val?Ile?Glu?Cys?Pro

100 105 110

Asp?Thr?Leu?Thr?Thr?Leu?Asp?Val?His?Ser?Phe?Ser?Gln?Glu?Pro?Glu

115 120 125

Ala?Pro?Thr?Val?Gly?Lys?Arg?Arg?Val?Leu?Val?Phe?Pro?Cys?Asp?Asn

130 135 140

Asp?Glu?Ile?Ser?Asp?Ser?Glu?Leu?Glu?Ser?Asn?Tyr?Tyr?Ile?Val?Pro

145 150 155 160

Thr?Cys?Thr?Leu?Cys?Ala?Glu?Arg?Leu?Glu?Pro?Thr?Leu?Thr?Gly?Tyr

165 170 175

Ser?Ser?Pro?Thr?Cys?Ser?Cys?Val?Asp?Gly?Arg?Glu?Cys?Arg?Cys?Leu

180 185 190

Leu?Glu?Gln?Ser?Ser?Cys?Val?Val?Cys?Gln?Thr?Ser?Ile?Thr?Met?Gln

195 200 205

His?Glu?Ser?Gln?Lys?Val?Gln?Cys?Glu?Gln?Cys?Ser?Arg?Thr?Gly?Asp

210 215 220

Pro?Trp?Ile?Cys?Leu?Val?Cys?Gly?Tyr?Val?Gly?Cys?Ser?Arg?Tyr?Gln

225 230 235 240

Ala?Lys?His?Ala?Arg?Glu?His?Tyr?Leu?Gln?His?Lys?His?Leu?Phe?Ser

245 250 255

Met?Ser?Leu?Leu?Thr?Gln?Gln?Ile?Trp?Asp?Tyr?Asp?Ser?Asp?Ala?Phe

260 265 270

Val?His?Arg?Val?Val?Val?Leu?Leu?Asp?Asn?Ala?Thr?Gly?Ala?Val?Asn

275 280 285

Arg?Val?Gln?Tyr?Pro?Asp?Arg?Asp?Asn?Ile?Pro?Ser?Ser?Leu?Ala?Asp

290 295 300

Glu?Tyr?Val?Asp?Ala?Ala?Ala?Glu?Lys?Val?Ser?Lys?Lys?His?Ile?Asn

305 310 315 320

Ala?Lys?Phe?Asp?Ser?Lys?Val?Glu?Thr?Ser?Asn?Glu?Gln?Leu?Ala?Leu

325 330 335

Met?Ile?Ile?Ser?Glu?Leu?Asn?Thr?Arg?Arg?Val?Glu?Tyr?Glu?Thr?Glu

340 345 350

Met?His?Gly?Gly?Ser?His?His?Leu?Asn?Asp?Glu?Leu?Met?Gly?Asp?Pro

355 360 365

Ser?Leu?Cys?Ser?Val?Ile?Val?Ala?Glu?Arg?Ala?Cys?Ala?Ala?Ser?Arg

370 375 380

Glu?Arg?Trp?Trp?Gln?Leu?His?Asn?Ala?Asn?Lys?Ser?Ile?Gln?Glu?Glu

385 390 395 400

Leu?Met?Gln?Arg?Arg?Arg?Glu?Glu?Glu?Ala?His?Gln?Lys?Thr?Ile?Asp

405 410 415

Glu?Leu?Gln?Gln?Glu?Leu?Arg?Ser?Val?Val?Gln?His?Tyr?Ala?Thr?Arg

420 425 430

Glu?Tyr?Ser?Leu?Leu?Thr?Glu?Ile?Asn?Glu?Leu?Arg?Gln?Thr?Ile?Thr

435 440 445

Asp?Val?Glu?Thr?Asn?Leu?Arg?Thr?Phe?Ala?Lys?Leu?Ser?Arg?Gly?Leu

450 455 460

Gly?Asn?Asp?Thr?Leu?Glu?His?Val?Arg?Ile?Val?Gly?Gly?Thr?Lys?Glu

465 470 475 480

Pro?Lys?Pro?Arg?Arg?Arg?Gly?Gly?Asn?Asn?Thr?Arg?Asp?Gly?Arg?Ala

485 490 495

<210>58

<211>680

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>58

Met?Glu?Ile?Phe?Glu?Thr?Ile?Leu?Ile?Phe?Ile?Ala?Val?Val?Ile?Leu

1 5 10 15

Ser?Ser?Phe?Val?His?Thr?Phe?Ile?Pro?Lys?Val?Pro?Leu?Ala?Phe?Ile

20 25 30

Gln?Ile?Phe?Leu?Gly?Met?Leu?Leu?Phe?Ile?Thr?Pro?Ile?Pro?Val?Gln

35 40 45

Phe?Asn?Phe?Asp?Ser?Glu?Leu?Phe?Met?Val?Thr?Met?Ile?Ala?Pro?Leu

50 55 60

Leu?Phe?Val?Glu?Gly?Val?Asn?Val?Ser?Arg?Val?His?Leu?Arg?Lys?Tyr

65 70 75 80

Ile?Lys?Pro?Val?Met?Met?Met?Ala?Leu?Gly?Leu?Val?Ile?Thr?Thr?Val

85 90 95

Ile?Gly?Val?Gly?Leu?Phe?Ile?His?Trp?Ile?Trp?Pro?Asp?Leu?Pro?Ile

100 105 110

Gly?Ala?Ala?Phe?Ala?Ile?Ala?Ala?Ile?Leu?Cys?Pro?Thr?Asp?Ala?Val

115 120 125

Ala?Val?Gln?Ala?Ile?Thr?Lys?Gly?Lys?Val?Leu?Pro?Lys?Gly?Ala?Met

130 135 140

Thr?Ile?Leu?Glu?Gly?Glu?Ser?Leu?Leu?Asn?Asp?Ala?Ala?Gly?Ile?Ile

145 150 155 160

Ser?Phe?Lys?Ile?Ala?Val?Gly?Val?Leu?Val?Thr?Gly?Ala?Phe?Ser?Leu

165 170 175

Val?Asp?Ala?Val?Gln?Leu?Phe?Leu?Ile?Ala?Ser?Ile?Gly?Gly?Ala?Val

180 185 190

Val?Gly?Leu?Leu?Ile?Gly?Met?Ala?Leu?Val?Arg?Phe?Arg?Leu?Thr?Leu

195 200 205

Met?Arg?Arg?Gly?Tyr?Glu?Asn?Ile?Asn?Met?Phe?Thr?Ile?Ile?Gln?Leu

210 215 220

Leu?Thr?Pro?Phe?Val?Thr?Tyr?Leu?Ile?Ala?Glu?Leu?Phe?His?Ala?Ser

225 230 235 240

Gly?Ile?Ile?Ala?Ala?Val?Val?Ala?Gly?Leu?Val?His?Gly?Phe?Glu?Arg

245 250 255

Asp?Arg?Ile?Met?Gln?Val?Arg?Thr?Gln?Leu?Gln?Met?Ser?Tyr?Asn?His

260 265 270

Thr?Trp?Asn?Ile?Leu?Gly?Tyr?Val?Leu?Asn?Gly?Phe?Val?Phe?Ser?Ile

275 280 285

Leu?Gly?Phe?Leu?Val?Pro?Glu?Val?Ile?Ile?Lys?Ile?Ile?Lys?Thr?Glu

290 295 300

Pro?His?Asn?Leu?Ile?Phe?Leu?Ile?GIy?Ile?Thr?Ile?Val?Val?Ala?Leu

305 310 315 320

Ala?Val?Tyr?Leu?Phe?Arg?Phe?Val?Trp?Val?Tyr?Val?Leu?Tyr?Pro?Tyr

325 330 335

Phe?Tyr?Leu?Ala?Ile?Ser?Pro?Phe?Gln?Lys?Met?Met?Thr?Lys?Asn?Asp

340 345 350

Asp?Asp?Asn?Pro?Thr?Thr?Glu?Lys?Pro?Pro?Lys?Arg?Ser?Leu?Tyr?Ala

355 360 365

Leu?Ile?Met?Thr?Leu?Cys?Gly?Val?His?Gly?Thr?Ile?Ser?Leu?Ala?Ile

370 375 380

Ala?Leu?Thr?Leu?Pro?Tyr?Phe?Leu?Ala?Gly?His?His?Ala?Phe?Thr?Tyr

385 390 395 400

Arg?Asn?Asp?Leu?Leu?Phe?Ile?Ala?Ser?Gly?Met?Val?Ile?Ile?Ser?Leu

405 410 415

Val?Val?Ala?Gln?Val?Leu?Leu?Pro?Leu?Leu?Thr?Lys?Pro?Ala?Pro?Lys

420 425 430

Thr?Val?Ile?Gly?Asn?Met?Ser?Phe?Lys?Val?Ala?Arg?Ile?Tyr?Ile?Leu

435 440 445

Glu?Gln?Val?Ile?Asp?Tyr?Leu?Asn?Gln?Lys?Ser?Thr?Phe?Glu?Thr?Ser

450 455 460

Phe?Lys?Tyr?Gly?Asn?Val?Ile?Lys?Glu?Tyr?His?Asp?Lys?Leu?Ala?Phe

465 470 475 480

Leu?Lys?Thr?Val?Glu?Lys?Asp?Asp?Glu?Asn?Ser?Lys?Glu?Leu?Glu?Arg

485 490 495

Leu?Gln?Lys?Ile?Ala?Phe?Asn?Val?Glu?Thr?Lys?Thr?Leu?Glu?Ser?Leu

500 505 510

Val?Asp?Glu?Gly?Gln?Ile?Thr?Asn?Ser?Val?Leu?Glu?Asn?Tyr?Met?Arg

515 520 525

Tyr?Ala?Glu?Arg?Thr?Gln?Val?Tyr?Arg?Gln?Ala?Ser?Leu?Ile?Arg?Arg

530 535 540

Met?Ile?Val?Leu?Leu?Arg?Gly?Ala?Leu?Leu?Lys?Arg?Arg?Val?Gln?Thr

545 550 555 560

Arg?Val?Asn?Ser?Ala?Ser?Ser?Leu?Ser?Val?Thr?Asp?Asn?Leu?Met?Glu

565 570 575

Leu?Asn?Lys?Ile?Asn?Lys?Leu?Val?His?Tyr?Asn?Val?Val?Ser?Arg?Leu

580 585 590

Ser?Lys?Glu?Thr?Thr?Lys?Asp?Asn?Thr?Leu?Glu?Ile?Gly?Met?Val?Cys

595 600 605

Asp?Gly?Tyr?Leu?Met?Arg?Ile?Glu?Asn?Leu?Thr?Pro?Ser?Asn?Phe?Phe

610 615 620

Asn?Ser?Ala?Ser?Glu?Asp?Thr?Ile?Thr?Lys?Ile?Lys?Leu?Asn?Ala?Leu

625 630 635 640

Arg?Glu?Gln?Arg?Arg?Ile?Leu?Arg?Glu?Leu?Ile?Asp?Thr?Asp?Glu?Val

645 650 655

Ser?Glu?Gly?Thr?Ala?Leu?Lys?Leu?Arg?Glu?Ala?Ile?Asn?Tyr?Asp?Glu

660 665 670

Met?Val?Ile?Val?Asp?Ser?Met?Thr

675 680

<210>59

<211>294

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>59

Met?Ser?Pro?Ile?Leu?Gly?Tyr?Trp?Lys?Ile?Lys?Gly?Leu?Val?Gln?Pro

1 5 10 15

Thr?Arg?Leu?Leu?Leu?Glu?Tyr?Leu?Glu?Glu?Lys?Tyr?Glu?Glu?His?Leu

20 25 30

Tyr?Glu?Arg?Asp?Glu?Gly?Asp?Lys?Trp?Arg?Asn?Lys?Lys?Phe?Glu?Leu

35 40 45

Gly?Leu?Glu?Phe?Pro?Asn?Leu?Pro?Tyr?Tyr?Ile?Asp?Gly?Asp?Val?Lys

50 55 60

Leu?Thr?Gln?Ser?Met?Ala?Ile?Ile?Arg?Tyr?Ile?Ala?Asp?Lys?His?Asn

65 70 75 80

Met?Leu?Gly?Gly?Cys?Pro?Lys?Glu?Arg?Ala?Glu?Ile?Ser?Met?Leu?Glu

85 90 95

Gly?Ala?Val?Leu?Asp?Ile?Arg?Tyr?Gly?Val?Ser?Arg?Ile?Ala?Tyr?Ser

100 105 110

Lys?Asp?Phe?Glu?Thr?Leu?Lys?Val?Asp?Phe?Leu?Ser?Lys?Leu?Pro?Glu

115 120 125

Met?Leu?Lys?Met?Phe?Glu?Asp?Arg?Leu?Cys?His?Lys?Thr?Tyr?Leu?Asn

130 135 140

Gly?Asp?His?Val?Thr?His?Pro?Asp?Phe?Met?Leu?Tyr?Asp?Ala?Leu?Asp

145 150 155 160

Val?Val?Leu?Tyr?Met?Asp?Pro?Met?Cys?Leu?Asp?Ala?Phe?Pro?Lys?Leu

165 170 175

Val?Cys?Phe?Lys?Lys?Arg?Ile?Glu?Ala?Ile?Pro?Gln?Ile?Asp?Lys?Tyr

l80 185 190

Leu?Lys?Ser?Ser?Lys?Tyr?Ile?Ala?Trp?Pro?Leu?Gln?Gly?Trp?Gln?Ala

195 200 205

Thr?Phe?Gly?Gly?Gly?Asp?His?Pro?Pro?Lys?Ser?Asp?Leu?Val?Pro?Arg

210 215 220

Gly?Ser?Pro?Glu?Phe?Leu?Val?Pro?His?Leu?Gly?Asp?Arg?Glu?Lys?Arg

225 230 235 240

Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?Tyr?Ile?His?Pro?Gln?Asn?Asn?Ser

245 250 255

Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?Asn?Asp?Cys

260 265 270

Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Glu?Phe?Pro?Gly?Arg?Leu

275 280 285

Glu?Arg?Pro?His?Arg?Asp

290

<210>60

<211>286

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>60

Met?Ser?Pro?Ile?Leu?Gly?Tyr?Trp?Lys?Ile?Lys?Gly?Leu?Val?Gln?Pro

1 5 10 15

Thr?Arg?Leu?Leu?Leu?Glu?Tyr?Leu?Glu?Glu?Lys?Tyr?Glu?Glu?His?Leu

20 25 30

Tyr?Glu?Arg?Asp?Glu?Gly?Asp?Lys?Trp?Arg?Asn?Lys?Lys?Phe?Glu?Leu

35 40 45

Gly?Leu?Glu?Phe?Pro?Asn?Leu?Pro?Tyr?Tyr?Ile?Asp?Gly?Asp?Val?Lys

50 55 60

Leu?Thr?Gln?Ser?Met?Ala?Ile?Ile?Arg?Tyr?Ile?Ala?Asp?Lys?His?Asn

65 70 75 80

Met?Leu?Gly?Gly?Cys?Pro?Lys?Glu?Arg?Ala?Glu?Ile?Ser?Met?Leu?Glu

85 90 95

Gly?Ala?Val?Leu?Asp?Ile?Arg?Tyr?Gly?Val?Ser?Arg?Ile?Ala?Tyr?Ser

100 105 110

Lys?Asp?Phe?Glu?Thr?Leu?Lys?Val?Asp?Phe?Leu?Ser?Lys?Leu?Pro?Glu

115 120 125

Met?Leu?Lys?Met?Phe?Glu?Asp?Arg?Leu?Cys?His?Lys?Thr?Tyr?Leu?Asn

130 135 140

Gly?Asp?His?Val?Thr?His?Pro?Asp?Phe?Met?Leu?Tyr?Asp?Ala?Leu?Asp

145 150 155 160

Val?Val?Leu?Tyr?Met?Asp?Pro?Met?Cys?Leu?Asp?Ala?Phe?Pro?Lys?Leu

165 170 175

Val?Cys?Phe?Lys?Lys?Arg?Ile?Glu?Ala?Ile?Pro?Gln?Ile?Asp?Lys?Tyr

180 185 190

Leu?Lys?Ser?Ser?Lys?Tyr?Ile?Ala?Trp?Pro?Leu?Gln?Gly?Trp?Gln?Ala

195 200 205

Thr?Phe?Gly?Gly?Gly?Asp?His?Pro?Pro?Lys?Ser?Asp?Leu?Val?Pro?Arg

210 215 220

Gly?Ser?Pro?Glu?Phe?Tyr?Ala?Pro?Glu?Pro?Gly?Ser?Thr?Cys?Arg?Leu

225 230 235 240

Arg?Glu?Tyr?Tyr?Asp?Gln?Thr?Ala?Gln?Met?Cys?Cys?Ser?Lys?Cys?Ser

245 250 255

Pro?Gly?Gln?His?Ala?Lys?Val?Phe?Cys?Thr?Lys?Thr?Ser?Asp?Thr?Val

260 265 270

Cys?Asp?Glu?Phe?Pro?Gly?Arg?Leu?Glu?Arg?Pro?His?Arg?Asp

275 280 285

<210>61

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: mark=synthetic construct

<400>61

tccatgacgt?tcctgatgct 20

Claims

1. 38 to 125 amino acid whose polypeptide comprise that the preceding part of TNF acceptor sample acceptor assembles the isolating aminoacid sequence in territory (PLAD).

2. the polypeptide of claim 1, wherein said PLAD is selected from: the PLAD of TNF-R, the PLAD of p60, the PLAD of p80, the PLAD of Fas (CD95/APO-1), the PLAD of TRAIL, the PLAD of LT β R, the PLAD of CD40, the PLAD of CD30, the PLAD of CD27, the PLAD of HVEM, the PLAD of OX40, the PLAD of DR4, the PLAD of NGFR, the PLAD of Troy, the PLAD of EDAR, the PLAD of XEDAR, the PLAD of DcR3, the PLAD of AITR, the PLAD of 4-1BB, the PLAD of DR3, the PLAD of RANK, the PLAD of TACI, the PLAD of BCMA, the PLAD of DR6, the PLAD of OPG, the PLAD of DRS, the PLAD of DcR1 and the PLAD of DcR2.

3. polypeptide of forming by the aminoacid sequence in the preceding part assembling territory of TNF acceptor sample acceptor.

4. one kind comprises that the preceding part of TNF acceptor sample acceptor assembles the polypeptide of the isolating aminoacid sequence in territory (PLAD), and wherein said polypeptide is R ₁-TNF acceptor sample acceptor PLAD-R ₂, R wherein ₁Or R ₂Be included in the aminoacid sequence that is not positioned at TNF acceptor sample acceptor PLAD flank in the naturally occurring TNF acceptor sample acceptor.

5. the polypeptide of claim 1, wherein said polypeptide is R ₁-TNF acceptor sample acceptor PLAD-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

6. the polypeptide of claim 1, wherein said polypeptide is R ₁The amino acid/11 of-p60-54 (SEQ IDNO:1)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

7. the polypeptide of claim 1, wherein said polypeptide is R ₁The amino acid/11 0-54 of-p80 (SEQ IDNO:2)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

8. the polypeptide of claim 1, wherein said polypeptide is R ₁The amino acid/11 of-Fas-43 (SEQ IDNO:3)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

9. the polypeptide of claim 1, wherein said polypeptide is R ₁The amino acid/11 of-Fas-66 (SEQ IDNO:4)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

10. the polypeptide of claim 1, wherein said polypeptide is R ₁The amino acid/11 3-50 of-LT β R (SEQ IDNO:5)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

11. the polypeptide of claim 1, wherein said polypeptide are R ₁The amino acid 6-39 of-CD40 (SEQ IDNO:6)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

12. the polypeptide of claim 1, wherein said polypeptide are R ₁The amino acid/11 1-51 of-CD30 (SEQ IDNO:7)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

13. the polypeptide of claim 1, wherein said polypeptide are R ₁The amino acid 7-42 of-CD27 (SEQ IDNO:8)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

14. the polypeptide of claim 1, wherein said polypeptide are R ₁The amino acid 6-37 of-HVEM (SEQ IDNO:9)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

15. the polypeptide of claim 1, wherein said polypeptide are R ₁The amino acid 3-36 of-OX40 (SEQ IDNO:10)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

16. the polypeptide of claim 1, wherein said polypeptide are R ₁The amino acid/11 09-138 of-DR4 (SEQ IDNO:11)-R ₂, R wherein ₁Be H, acyl group, NH ₂, amino acid or peptide, R ₂Be H, acyl group, NH ₂, amino acid or peptide.

17. the polypeptide of claim 3, wherein said PLAD is selected from: the PLAD of the PLAD of TNF-R, Fas (CD95/APO-1) and the PLAD of TRAIL.

18. the isolating nucleic acid of the polypeptide of the claim 1 of encoding.

19. the isolating nucleic acid of claim 18, described nucleic acid is in carrier.

20. carrier that is suitable in the host, expressing the described nucleic acid of claim 19.

21. claim 1 or 3 polypeptide method of suppressing TNF acceptor oligomerization in the cell by giving significant quantity.

22. claim 1 or 3 polypeptide method of suppressing Fas oligomerization in the cell by giving significant quantity.

23. one kind by giving significant quantity claim 1 or 3 polypeptide suppress TNF acceptor sample acceptor and part bonded method.

24. one kind by giving significant quantity claim 1 or 3 polypeptide suppress Fas and part bonded method.

25. claim 1 or 3 polypeptide method for the treatment of inflammation in the subject by giving significant quantity.

26. the method for claim 25, wherein said inflammation is relevant with autoimmune disorder.

27. the method for claim 25, wherein said inflammation is relevant with rheumatoid arthritis, osteoarthritis or septic arthritis.

28. composition that comprises PLAD association inhibitor.

29. the method for claim 25, wherein said inhibitor are the PLAD specificity bonded antibody with TNF acceptor sample acceptor.

30. the method for claim 25, wherein said inhibitor are the PLAD of TNF acceptor sample acceptor.

31. the method for claim 30, wherein said solubility PLAD is the PLAD of p60.

32. a method of screening PLAD association inhibitor comprises:

A) with following two kinds of same cells of plasmid transfection, the nucleic acid that wherein a kind of plasmid contains comprises coding and the nucleotide sequence of the functional isolating PLAD that is connected of fluorescence donor, and another kind of plasmid contains contains the nucleotide sequence of encoding with the functional isolating PLAD that is connected of fluorescent receptor;

B) described cell is contacted with the inhibitor of supposition; And

C) measure FRET, wherein with not with cell that described supposition inhibitor contact in the FRET measuring result compare, FRET decline shows and has PLAD association inhibitor.

33. a method of screening PLAD association inhibitor comprises:

A) with following two kinds of same cells of plasmid transfection, wherein a kind of plasmid contains the nucleic acid of the nucleotide sequence that comprises a kind of isolating PLAD that encodes, and another kind of plasmid contains the nucleotide sequence of the another kind of isolating PLAD that encodes;

B) described cell is contacted with the inhibitor of supposition;

C) measure the self-association of PLAD, wherein with not with cell that the inhibitor of described supposition contact in the PLAD association compare, the associating minimizing of PLAD has shown and has had PLAD association inhibitor in the step b) cell.