CN101240018A - Peptides and related compounds having thrombopoietic activity - Google Patents

Abstract

The invention relates to peptides including thrombopoietic activity and relative compounds. More particularly, the invention relates to the compounds combining with mpl acceptor and physiologically acceptable salts thereof, the compounds comprises the following sequence: X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18. The invention also relates to a composite comprising the compounds, nucleotide which codes the composite and a carrier and a cell comprising the nucleotide. The compounds of the invention can be used for improving the yield of thrombocyte or thrombocyte precursor (e.g. megakaryocyte) in a mammal.

Description

Have the peptide of thrombopoietic activity and relevant compound

The application is to be on October 11st, 2002 applying date, and application number is 02824735.3, and denomination of invention is divided an application for the application for a patent for invention of " having the peptide of thrombopoietic activity and relevant compound ".

Invention field

Present invention relates in general to have the peptide of thrombopoietic activity and relevant compound.Compound of the present invention can be used for improving the generation of Mammals thrombocyte or platelet precursors (for example, megalokaryocyte).

Background technology

The present invention relates to have in stimulating platelet and their for example Megakaryocytic body of precursor cell and the compound, particularly peptide of external throughput.Be the known two kinds of protein that provide as a setting below: thrombopoietin (TPO) and megakaryocyte growth and the growth factor (MGDF) with thrombopoietic activity.

The clone of endogenous thrombopoietin (TPO) (Lok etc., nature, 369:568-571 (1994); Bartley etc., cell, 77:1117-1124 (1994); Kuter etc., institute of NAS periodical, 91:11104-11108 (1994); De Sauvage etc., nature, 369:533-538 (1994); Kato etc., journal of biological chemistry, 119:229-236 (1995); Chang etc., journal of biological chemistry, 270:511-514 (1995)) deepened us apace and megalokaryocyte has been generated the understanding of (megalokaryocyte production) and thrombocyte generation (thrombocyte production).

Endogenous people TPO, mainly be in liver and kidney, produce 60 to the glycosylated protein of 70kDa, form (Bartley etc., cell, 77:1117-1124 (1994) by 332 amino acid; Chang etc., journal of biological chemistry, 270:551-514 (1995)).This protein is high conservative between different plant species, (Bartley etc., cell, 77:1117-1124 (1994) and erythropoietin have 23% homology (Gurney etc. and at N-terminal (amino acid/11 to 172), blood, 85:981-988 (1995)).Endogenous TPO has shown to have all features that generate hematoblastic main biological instrumentality.Its external effect comprises mouse hematopoietic stem cell (Zeigler etc., blood, 84:4045-4052 (1994)) and the people CD34 from purifying ⁺Cell (Lok etc., nature, 369:568-571 (1994); Rasko etc., stem cell, 15:33-42 (1997)) induces the megalokaryocyte clone specifically, produce the megalokaryocyte (Broudy etc. that ploidy increases, blood, 85:402-413 (1995)) and induce terminal Megakaryocytic maturation and produce thrombocyte (Zeigler etc., blood, 84:40445-4052 (1994); Choi etc., blood, 85:402-413 (1995).On the contrary, synthetic TPO acceptor (c-mpl) oligomeric deoxynucleotide can obviously suppress megalokaryocyte ancestors' clonality (Methia etc., Blood, 82:1395-1401 (1993)).In addition, to pound out mouse be serious thrombopenia to c-mpl and lack Megakaryocytic (Alexander etc., blood, 87:2162-2170 (1996)).

(Thousand Oaks is that another thrombocyte relevant with TPO generates polypeptide CA) to the people MGDF of reorganization for rHuMGDF, Amgen Inc..It is that the intestinal bacteria that utilize plasmid to transform produce, and contains the proteinic cDNA of the brachymemma of encoding in this plasmid, comprises the N-terminal receptor binding domain (Ulich etc., blood, 86:971-976 (1995)) of people TPO in the protein of brachymemma.With this polypeptide extraction, folding again, and purifying, polyoxyethylene glycol (PEG) composition covalently is attached to N-terminal.The molecule that obtains is referred to herein as PEG-rHuMGDF or is called MGDF tout court.

Utilize various researchs (Ulich, T.R. etc., blood, the 86:971-976 (1995) of animal model; Hokom, M.M. etc., blood, 86:4486-4492 (1995)) clearly proved in bone marrow transplantation and in thrombocytopenic treatment, often be TPO in the treatment of the symptom that produces of chemotherapy or radiotherapy and the result of treatment of MGDF.Initial data in the people have been verified to improve in various device that MGDF has purposes (Basser etc., Lancet348:1279-81 (1996) in the platelet count; Kato etc., journal of biological chemistry, 119:229-236 (1995); Ulich etc., blood, 86:971-976 (1995)).MGDF can be used to strengthen the thrombocyte supply process, in the thrombocyte donor of health the round-robin platelet count has been improved about 3 times from originally value because use MGDF.

TPO and MGDF are by bringing into play their effect in conjunction with the c-mpl acceptor, and the c-mpl acceptor is at some hematopoietic cells such as Megakaryocytic surface, thrombocyte, CD34 at first ⁺(Debili, N. etc., blood, the 85:391-401 (1995) that express on cell and the initial ancester cell; De Sauvage, F.J.et al, nature, 369:533-538 (1994); Bartley, T.D. etc., cell, 77:1117-1124 (1994); Lok, S. etc., nature, 369:565-8 (1994)).As the acceptor of most of interleukin-and proteohormone, c-mpl belongs to I type cytokines receptor superfamily (Vigon, I. etc., institute of NAS periodical, 89:5640-5644 (1992)).The activation of this receptoroid comprises that part inductive acceptor homodimer turns usefulness into, and this effect has caused the signal transduction cascade of events successively.

Usually, the interaction of protein ligands and its acceptor often occurs in relative big interface.But, as with the situation of the human growth hormone of receptors bind in prove that having only a few the crucial residue on the interface is that most of bound energy are had real contribution (Clackson, T. etc., science, 267:383-386 (1995)).The effect of this and a large amount of residual protein part is showed in correct geometry just that the fact in conjunction with epi-position makes and might be found more small-sized active ligand.Therefore, have only the molecule of " peptide " length (for example, 2 to 80 amino acid) can be in conjunction with the receptor protein of given big protein ligands.Such peptide can be simulated the biological activity of big protein ligands, although competition is perhaps arranged in conjunction with the biological activity that also suppresses big protein ligands, and is commonly referred to " peptide mimics " or " simulating peptide ".

In identifying such peptide mimics, the phage display peptide library occurs as strong technology.Referring to for example, Scott, J.K. etc., science, 249:386 (1990); Devlin, J.J. etc., science, 249:404 (1990); On June 29th, 1993 laid-open U.S. Patents 5,223,409; On March 31st, 1998, laid-open U.S. Patents 5,733,731; March 12 disclosed 5,498,530 in 1996; July 11 nineteen ninety-five, laid-open U.S. Patents 5,432,018; On August 16th, 1994, laid-open U.S. Patents 5,338,665; On July 13rd, 1999, laid-open U.S. Patents 5,922,545; On December 19th, 1996 disclosed WO96/40987; With disclosed WO 98/15833 on April 16th, 1996 (above-mentioned each full patent texts is incorporated herein by reference).In such document, peptide sequence all is to merge by the dressing with filobactivirus to show arbitrarily.Usually, the peptide of displaying is that antibody fixed cell outskirt to acceptor comes affinity elution.The phage that keeps may be to accumulate by the circulation of successive affinity purification and breeding again.Can check order in conjunction with best peptide and to identify Key residues in the family of one or several structurally associated of peptide.Referring to for example, Cwirla etc. (1997), science, 276:1696-9.These peptide sequences have hinted that equally residue can be substituted by the L-Ala scanning (alanine scanning) or the mutagenesis of dna level safely.Can produce and screen the mutagenesis library, so that further make the sequence optimizing of best incorporated thing.Lowman(1997)，Ann.Rev.Biophys.Biomol.Struct.26：401-24。

The structural analysis of protein-protein interaction also can hint peptide simulated big protein ligands in conjunction with active.In a such analysis, crystalline texture can show, therefrom can design the identity and the relative orientation of Key residues of the big protein ligands of a peptide.Referring to for example, Takasaki etc. (1977), Nature Biotechnol, 15:1266-70.These analytical procedures also can be used to study the receptor protein of phage indicating meter selection and the interaction between the peptide, can show further that the modification of peptide has strengthened binding affinity.

In peptide research, additive method and phage display (phage display) technology competition are arranged.Peptide library can merge with the C-terminal of lac acceptor, and at expression in escherichia coli.Another, is showed on the adventitia of cell by merging with peptidoglycan bonded lipoprotein (PAL) based on colibacillary method.Hereinafter, these are generically and collectively referred to as " intestinal bacteria are showed (display) " with relevant method.In another method, before discharging, rrna stops the translation of any RNA, and cause the library of polypeptide still to adhere to their relevant RNA.Hereinafter, this is generically and collectively referred to as " ribosomal display " with relevant method.Additive method has utilized the peptide that is connected with RNA; For example, the PRO integration technology, Phylos company, referring to, Roberts﹠amp for example; Szostak (1997), institute of NAS periodical, 94:12297-303.Hereinafter, this is generically and collectively referred to as " RNA-peptide screening " with relevant method.The chemical origin peptide library has been developed, and wherein peptide is to be fixed on stable, the abiotic material, as polyethylene rod or solvent permeable resins.Another chemical origin peptide library has utilized fixed peptide on photolithography (photolithography) the scanning glass sheet.Hereinafter, these are generically and collectively referred to as " chemical peptide screening " with relevant method.The chemistry peptide screening may be favourable, because it allows to utilize D-amino acid and other non-natural analogues, and non-peptide element.The summary of biological and chemical method is at Wells﹠amp; Lowman (1992), Curr.Opin.Biotechnol is among the 3:355-62.In theory, can utilize display technique of bacteriophage, RNA-peptide screening and other methods above-mentioned to find any proteinic peptide mimics.

Utilize phage display peptide library technology, found little peptide molecule (Cwirla, S.E. etc., science, 276:1696-1699 (1977)) as the agonist of c-mpl acceptor.In such research, be demonstrated the small peptide sequence at random that merges with the dressing protein of filament phage and be at zone, the antibody fixed extracellular affinity elution of c-mpl, and the phage that keeps can accumulate and is used for second and takes turns affinity purification.This in conjunction with select and again reproductive process repeated many times, enrichment the aggregate of binding substances more closely.As a result of, two families of c-mpl binding peptide although uncorrelated mutually in their sequence, have at first obtained evaluation.Then, the mutagenesis library has produced, and further makes best binding substances optimizing, has caused the separation of active peptide very at last, IC ₅₀=2nM, EC ₅₀=400nM (Cwirla, S.E. etc., science, 276:1696-1699 (1997)).The TPO simulating peptide of these 14 residues and TPO or MGDF do not have the obvious sequence homology.The structure of TPO simulating peptide (TMP) compound that this is special is as follows:

Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala (SEQ IDNO:1) or utilize the single-letter amino acid abbreviations:

IEGPTLRQWLAARA

Past, in the same research of EPO simulating peptide, EPO simulating peptide (the EMP) (Wrighton that utilized identical scientific discovery, N.C. etc., and find to be to work as dimer in the combination of EPO acceptor (EPOR) science, 273:458-463 (1996)).According to X-ray crystal data, (part) of Xing Chenging like this ₂/ (acceptor) ₂Mixture has C2 symmetrical structure (Livnah, O. etc., science, 273:464-471 (1996)).According to this structural information, designed the covalently bound dimer of the crosslinked EMP of two monomeric C-terminal of EMP and flexible introns, discovery has biological activity (Wrighton in the huge combination of enhanced and the external/body, N.C., Deng, Nature Biotechnol, 15:1261-1265 (1997)).

The Dimerized scheme of C-terminal (Cwirla, S.E. etc., science, 276:1696-1699 (1997)) to TPO simulating peptide (TMP) applications similar.Have been found that in cell proliferation experiment the C-terminal of special TPO simulating peptide connects the binding affinity of dimer (C-C connection) and brings up to 0.5nM, and external activity improves (EC ₅₀=0.1nM) (Cwirla, S.E. etc., science, 276:1696-1699 (1997)).

In order to strengthen or improve the characteristic of such protein as pharmaceutical agent, the availability that is used for the treatment of the recombinant protein of purposes has caused further protein modification.Such modification can strengthen proteinic protection and reduce degraded by reducing or eliminating proteolysis.Additional advantage comprises, in some cases, strengthens treatment proteinic stability, cycling time and biological activity.A review article of narration protein modification is that Farncis " pays close attention to somatomedin (Focuson Growth Factor) 3:4-10 (in May, 1992) (Mediscript, London, Britain).

The modification of useful protein therapeutic reagent comprises connection polymer such as polyoxyethylene glycol (PEG) and dextran.Being modified in the patent application " as the modified peptides of treatment reagent " like this goes through, U.S. serial 09/428,082, and PCT applies for WO 00/24782, this full patent texts is incorporated herein by reference.

Another such modification is the Fc district that utilizes immunoglobulin molecules.Antibody comprises two function independent sectors; The variable region of known " Fab " as conjugated antigen, the constant region of known conduct " Fc " provides the constant region with " Fc " that be connected of effector functions, as complement or phage cell.The Fc of immunoglobulin (Ig) partly has long plasma half-life, and Fab is short-term life (Capon etc., nature, 337,525-531 (1989)).

Utilize the Fc district to make up the treatment protein, the longer transformation period is provided or has participated in receptors bind as Fc, the a-protein combination, the complementary fixing function that shifts with placenta, they all belong to the Fc protein of immunoglobulin (Ig).(Capon etc., nature, 337:525-531 (1989)).For example, the Fc district of an IgG1 antibody with CD30-L, be combined on Hodgkin ' the s disease tumour cell, anaplastic lymphocyte, the molecule of the CD30 acceptor of expressing on T chronic myeloid leukemia cell and other malignant cell types merges.Referring to, U.S. Patent number, 5,480,981.In order to strengthen the short-term circulating half-life of cytokine, anti-inflammatory and antireflection reagent merge (Zheng, X. etc., Journal of Immunology, 154:5590-5600 (1995)) with mouse Fc2a.Purposes (Fisher, C. etc., N.Engl.J.Med., the 334:1697-1702 (1996) of Tumor Necrosis Factor Receptors in the patient of treatment sepsis shock that is connected with human IgG1's Fc protein also estimated in many researchs; Van Zee, K. etc., Journal of Immunology, 156:2221-2230 (1996)).Fc also merges with the CD4 acceptor, produces the therapeutic protein of treatment AIDS.Referring to Capon etc., nature, 337:525-531 (1989).In addition, interleukin II with the Fc meromixis of IgG1 or IgG3 so that overcome the transformation period of the weak point of interleukin II and its system's toxin.Referring to, Harvill etc., immunological technique, 1:95-105 (1995).

Disclosed PCT application WO 00/24770 discloses special thrombocyte and has generated compound, and normally peptide has a series connection (tandem, be that N-is to the C-end) direction, with be attached to a carrier molecule at its N-terminal, as linear polymer, the placed in-line peptide dimer of polysaccharide or Fc group.

Provide super bioactive other compounds of other productions with stimulating platelet (thrombopoietic activity) and/or platelet precursors cell, particularly megalokaryocyte (megalokaryocyte generates active) to remain needs.Provide to have thrombopoietic activity and have super therapeutic quality simultaneously and also remain as the compound of long transformation period and need.Such compound separates having very favorable relevant production, purifying, biological activity, the characteristic of aspects such as stability and cycling time.The invention provides and have such active new compound and related aspect.

Summary of the invention

The present invention relates in conjunction with the c-mpl acceptor treatment compound of (hereinafter being called " mpl acceptor ").More particularly, the invention provides one group of compound, they are incorporated into and/or cause the ability raising of transmembrane signal by (promptly activating) mpl acceptor, and described acceptor is the active same receptor of the endogenous thrombopoietin of mediation (TPO).So the compound of invention has excellent thrombopoietic activity, can generate active ability with hematoblastic production of stimulated in vitro and/or megalokaryocyte in vivo, promptly in vivo with the ability of the generation of stimulated in vitro platelet precursors.In addition, some compound of the present invention also has excellent treatment characteristic, as plasma half-life, biological activity and the body-internal-circulation time of improving.

In one aspect, the invention provides the compound with the mpl receptors bind, comprise following sequence:

X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18

X1-X4 wherein, X9-X10 and X13-X18 are amino acid as defined herein independently of one another, wherein this compound has with the binding affinity of mpl acceptor and/or has biological activity greater than following sequence:

I-E-G-P-T-L-R-Q-W-L-A-A-R-A

And on the other hand, the invention provides compound in conjunction with the mpl acceptor with following sequence:

X1-X2-R-E-G-P-T-L-R-Q-W-L-X13-W-R-R-X17-X18

X1 wherein, X2, X13, X17 and X18 are amino acid independently of one another.

On the other hand, the invention provides the compound in conjunction with the mpl acceptor, this compound comprises and is selected from the sequence of SEQ ID NO:2 to SEQ ID NO:30.

On the other hand, the present invention is the dimer or the polymer of compound, and this compound comprises and is selected from the sequence of SEQ ID NO:2 to SEQ ID NO:30.

On the other hand, the invention provides the composition in conjunction with the mpl acceptor, it has following formula:

(LN1) _l--(TMP1) _a--(LN2) _m--(TMP2) _b--(LN3) _n--(TMP3) _c--(LN4) _o--(TMP4) _d

TMP1 wherein, TMP2, TMP3 and TMP4 are selected from TMP disclosed herein independently of one another; LN1, LN2, LN3 and LN4 are joint independently of one another; A, b, c and d are 0 to 10 integer independently of one another; L, m, n and o are 0 to 20 integer independently of one another.

On the other hand, the invention provides the composition in conjunction with the mpl acceptor, it has following formula:

(V1) _v--(LN1) _l--(TMP1) _a--(LN2) _m--(TMP2) _b--(LN3) _n--(TMP3) _c--(LN4) _o--(TMP4) _d--(V2) _w

Wherein V1 and V2 are carrier (vehicle) independently of one another, and v and w are 0 to 1 integer independently of one another.

Compound of the present invention can prepare by synthetic method, recombinant DNA technology or any other method for preparing peptide and fused protein of standard.Except that standard peptide chemical reaction (but when time spent), comprise that the compound of the present invention of non-peptide moiety can synthesize by the organic chemical reactions of standard.

Compound of the present invention can reach treatment or prevention purpose by preparing with suitable medicinal carrier substance and the people (or other Mammalss) of patient such as needs treatment being used with significant quantity.The peptide that carrier connects can have and peptide (being thrombopoietin here) mimic native ligand can compare or even bigger activity.

On the other hand, the invention provides the method for treatment thrombocytopenia.In other respects, the invention provides increases megalokaryocyte or hematoblastic method, and the method for producing compound as herein described.

On the other hand, the present invention also provides relevant pharmaceutical composition.

In other respects, the invention provides the polynucleotide of coding composition disclosed herein, comprise the expression vector and the host cell that comprises this expression vector of these polynucleotide.

The accompanying drawing summary

So, being described in detail below considering, with reference to the accompanying drawings, many other aspects of the present invention and advantage will be conspicuous.Wherein:

Fig. 1 shows the example of structure of peptide of the present invention and peptide linker compound.

Fig. 2 has shown the example of structure of peptide-carrier of the present invention and peptide-joint-carrier compound.

Fig. 3 has shown the nucleic acid and the aminoacid sequence (SEQ ID NOS:31 and 32) that can be used as the human IgG1 Fc of preferred carrier of the present invention.

Fig. 4 has shown can be from the example of IgG1 antibody deutero-Fc monomer of the present invention and dimer compound.Any Fc varient in the meaning in the Fc zone that " Fc " represents in the drawings at this paper." peptide " represents any peptide, joint-peptide, peptide-peptide combination, or as any combination disclosed herein.Special dimer is as follows:

Fig. 4 A and 4D have shown single disulfide linkage dimer.IgG1 antibody has two disulfide linkage in the twisting district of antibody usually.Fc district in Fig. 4 A and 4D can form by brachymemma between two disulfide linkage sites or by replacing cysteinyl residue with unreacted residue (for example, alanyl).In Fig. 4 A, the Fc district is connected with the N-terminal of peptide; The Fc district is at the C-terminal of peptide in 4D.

Fig. 4 B and 4E show the dimer of double disulfide-bonded.This Fc district can keep two cysteinyl residues in the Fc district chain or improve and comes expression from the construct of the sequence that comprises the Fc district that coding is such and form by the brachymemma parental antibody.In Fig. 4 B, the Fc district is connected with the N-terminal of peptide; It in 4E the C-terminal that is connected peptide.

Fig. 4 C and 4F have shown non-covalent dimer.This Fc zone can remove cysteinyl residue by brachymemma or replacement and form.People can expect to remove the impurity that other proteinic cysteinyl residues that cysteinyl residue avoids existing in cysteinyl residue and the host cell form.The non-covalent bonding in Fc district is to be enough to combine with dimer.Other dimers can utilize dissimilar antibody from antibody (for example, IgG2, IgM) deutero-Fc district forms.

Fig. 4 G and 4H have shown N-terminal (Fig. 4 G) at peptide and the strand Fc district of adhering at the C-terminal (Fig. 4 H) of peptide (Fig. 4 H).

Fig. 5 has shown the example of structure of the series connection multiple preferred compound of the present invention that is characterized as the pharmaceutically active peptides that is attached to the Fc zone.Fig. 5 A has shown to have the dimeric strand of the tandem polypeptide of being attached to (or Fc monomer) molecule, also can represent the DNA construct of this molecule simultaneously.Fig. 5 B has shown the Fc dimer, and its center tap-peptide moiety only appears on the dimeric chain of Fc.Fig. 5 C has shown at the dimer of the Fc dimer that has peptide moiety on two chains (in this case, being placed in-line peptide dimer) Fig. 5 C and will be have spontaneously formed in some host cells on the basis in the expression of the DNA construct of the strand shown in the code pattern 5A.In other host cells, cell can be positioned over and help forming dimer or can be under the dimeric condition of external formation.Fig. 5 D has represented other examples of strand (Fc monomer) and double-stranded (Fc dimer) preferred embodiment to 5I.

Fig. 6 has shown that being used to shown in this paper embodiment 3 makes up nucleotide sequence of the preferred carrier (20003180) of TMP-Fc syncretization compound (SEQ ID NO:33) and aminoacid sequence (SEQ ID NO:34).

Fig. 7 has shown the fragment of the example that the oligonucleotide that is used to produce the preferred peptide of the present invention shown in embodiment 3 is right.Each all provides nucleic acid and aminoacid sequence (SEQ ID NO:35-93).

Fig. 8 has shown the nucleotide sequence (SEQ IDNO:94) and the aminoacid sequence (SEQ ID NO:95) of the carrier as an example (20003182) that is used to make up C-terminal Fc syncretization compound (promptly being attached to the peptide of the N-terminal of Fc to C-terminal).

Fig. 9 has shown the ELISA dose response of selected phage clone.

Figure 10,11 and 12 have shown the biological activity of selected compound of the present invention.

Figure 13 and 14 has shown in mouse intravital platelet count behind the single injection selected compound of the present invention.

Detailed Description Of The Invention

I. the definition of term

Unless in a particular case especially restriction, the term definition that uses in the whole text in this manual is as follows.

Term " peptide " refers to about 2 to 80 amino acid whose molecules, and molecule is that 3 to 40 amino acid are preferred. The peptide that can be used as example can any means as herein described produce at random, and such as (for example, phage display library) of carrying in the peptide library, chemical synthesis produces, protein digestibility derive etc.

Term " randomization ", when being combined with peptide sequence, refer to that sequence (for example fully arbitrarily, select by phage display method or RNA peptide screening), and one or more residues of naturally occurring molecule are the sequences that is substituted by non-existent amino acid residue in that position of naturally occurring molecule. The example of the method for generation and evaluation randomization peptide sequence comprises phage display, Escherichia coli displaying, ribosomal display, RNA-peptide screening, Chemical Screening etc.

Term " dimer " refers to have and utilizes or do not utilize joint, the covalently or non-covalently molecule of two of combination peptide chains as being used for peptide. Peptide be the C end be connected to N terminal to the N end the peptide dimer also can be called " series connection repetition " or " series connection dimer ". Peptide is to be connected to the peptide dimer that C end or N be connected to the N end from C also can be called " parallel repetition " or " parallel dimer ".

Term " polymer " is as using with peptide, refers to covalency, non-covalent or both also noncovalent interaction combinations of covalency, with or without 3 or the molecule of a plurality of peptide chains of joint.

Term " is derived " and " derivative " or " deriving " comprised the process of processing and produce compound, and wherein (1) compound has loop section; For example crosslinked between the cysteinyl residue in compound; (2) compound is crosslinked or has crosslink sites; For example, compound has cysteinyl residue, so in culture medium or form in vivo crosslinked dimer; (3) one or more peptide base keies are replaced by non-peptide base key; (4) the N end is-NRR¹，NRC(O)R ¹，- NRC(O)OR ¹，-NRS(O) ₂R ¹,-NHC (O) NHR, succinamide group, or replaced or unsubstituted phenyloxycarbonyl-NH-, wherein R and R¹With ring substituents as hereinafter the definition; (5) C-end is-C (O) R²Or-NR³R ⁴Substitute, wherein R²，R ³, and R⁴Such as hereinafter definition; (6) various compounds, wherein indivedual aminoacid ingredients can be modified with the reagent of the side chain of selecting or the reaction of terminal residue by using. Derivative is further as mentioned below.

Term " thrombopoietin mimic peptide ", " TPO simulating peptide " or " TMP " refer in conjunction with the mpl acceptor and/or have thrombopoietic activity, namely in the body or stimulated in vitro blood platelet or platelet precursors include but not limited to the peptide of the ability of Megakaryocytic production.

Term " mpl-land " refers in conjunction with the mpl acceptor and comprises the natural any amino acid sequence that has sequence or randomized sequence. Mpl land for example can be identified or derives by phage display or additive method mentioned in this article.

Term " mpl receptor stimulating agent " refers in conjunction with the mpl acceptor and such as endogenous TPO (eTPO), the molecule of one or more experiment parameters is equally strengthened or weakened to natural mpl receptors ligand.

Term " comprises " and refers to comprise other amino acid whose compounds at the N-of given sequence or one side or the both sides of C-end. Certainly, the obvious activity of interfering compound of these other amino acid.

In addition, the physiological acceptable salt of compound of the present invention also can be included in herein. Term " physiological acceptable salt " refers to the acceptable any salt of medicine known or that find afterwards. Some special examples are: acetic acid, trifluoroacetate; Hydrogen halides, example hydrochloric acid and hydrobromic acid; Sulfide; Citric acid; Tartaric acid; Hydroxyl acetic acid and oxalic acid.

Term " carrier (vehicle) " refers to the degraded of prophylactic treatment protein and/or increases the half-life, reduces toxicity, reduces immunogenicity and/or improves bioactive molecule. Carrier for example comprise Fc district (preferably) and linear polymer (for example, polyethylene glycol (PEG), polylysine, glucan, etc.); The polymer of side chain (referring to for example, on September 15th, 1981 disclosed patent 4,289,872 of authorizing Denkenwalter etc.; On July 20th, 1993 is disclosed authorize Tam 5,229,490,1993 on October 28, disclosed Frechet etc. WO 93/21259); Lipid; Cholesterol group (such as steroids); Carbohydrate or oligomerization polysaccharide (for example, glucan); In conjunction with any natural or synthetic protein, polypeptide or the peptide of remedying (salvage) acceptor; Albumin comprises human serum albumins (HSA), leucine zip district, and other such protein and protein fragments.

Term " natural Fc " refers to comprise from no matter be molecule or the sequence of the sequence of the non-Fab that obtains of the digestion of the whole antibody of monomer or polymer form. The originally immunoglobulin (Ig) source of natural Fc is people's origin preferably, and can be any immunoglobulin (Ig), although IgG1 and IgG2 are preferred. Natural Fcs is comprised of the monomer polypeptide, and these monomer polypeptide can connect into dimer or polymer form by covalency (being disulfide bond) and non-covalent combination. The scope of the number of the intermolecular disulfide bond between the monomer subunit of natural Fc molecule is 1 to 4 according to classification (for example, IgG, IgA, IgE) or subclass (for example, IgG1, IgG2, IgG3, IgA1, IgGA2). The example of natural Fc be the disulfide-bonded that obtains of the papain digestion from IgG dimer (referring to, Ellison etc. (1982), nucleic acids research, 10:4071-9). Term " natural Fc " monomer that belongs to as used herein, dimer and polymer form.

That term " Fc variant " refers to modify from natural Fc but still comprise and remedy acceptor, the molecule of the binding site of FcRn or sequence. International Application No. WO 97/34631 (on September 25th, 1997 open) and WO96/32478 have narrated the example of Fc variant, with and with the interaction of remedying acceptor, these patent applications are incorporated herein by reference in full. So term " Fc variant " comprises from molecule or the sequence of non-naive Fc peopleization. In addition, natural Fc comprises the site that can remove, because these sites provide the unwanted architectural feature of fusion molecule of the present invention or biologically active. So, term " Fc variant " comprises molecule or the sequence that lacks one or more natural Fc sites or residue, these sites or residue affect or have participated in the formation of (1) disulfide bond, the terminal heterogeneity of N on the basis of (2) in selected host cell, expressing with selected host cell incompatible (3), (4) glycosylation, (5) interact with complement, (6) except remedy acceptor also with the Fc receptors bind, or (7) rely on the cytotoxicity (ADCC) of antibody.

Term " Fc district " comprises natural Fc and Fc variant molecule and sequence as defined herein. Such as Fc variant and natural Fc, no matter term " Fc district " comprises whether from whole antibody digestion or the monomer of additive method generation or the molecule of polymer form.

" dimer " as with Fc zone or when comprising that the Fc zone is used, refers to have the molecule of two polymer chains of covalently or non-covalently combination to term.

Term " polymer " refers to have covalency as being applied to the Fc district or comprising minute period of the day from 11 p.m. to 1 a.m in Fc district, and is non-covalent, or the molecule of two or more polypeptide chains of covalency and the non-covalent combination that reacts to each other. The IgG molecule forms dimer usually; IgM, pentamer; IgD, dimer; IgA, monomer, dimer, trisome, or limbs. Polymer can be by development (exploit) this sequence, produces natural Ig source active of Fc or forms by derive (as defined herein) so natural Fc.

Term " peptide body " refers to comprise the molecule in the antibody Fc district that is attached at least at least one peptide. Such peptide body can be polymer or dimer or its fragment, and they can be derived.

II. the structure of compound

As a rule, the invention provides can in conjunction with and/or regulate the bioactive compound of mpl acceptor. More particularly, the invention provides can be by namely activating the mpl acceptor, namely mediate the active same receptor of endogenous TPO (TPO) come combination and/cause one group of compound of transmembrane signal. So the compound of invention has thrombopoietic activity, namely in vivo with the ability of the hematoblastic production of stimulated in vitro and/or have the megakaryocytopoiesis activity, namely in the body and the stimulated in vitro platelet precursors comprise the ability of Megakaryocytic production.

In brief, compound of the present invention comprises one or more peptides of the sequence with general formula I:

I：X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18；

X1-X4 wherein, X9-X10 and X13-X18 are amino acid independently of one another.

In other compositions prepared in accordance with the present invention, these compounds can comprise general formula I sequence-individual or a plurality of peptides, described peptide is attached to or interconnects, for example as dimer or polymer.

In other compositions of the present invention's preparation, these compounds can be included in the N-terminal of peptide or one or more peptides that C-terminal adheres to or be connected to the general formula I on the carrier.Any of these peptide can be with or without that joint is connected in series (that is, N is to C in order) or parallel (that is, N-is to the N-end, or C-is to the C-end).Peptide. compound of the present invention comprises separately or in conjunction with the TPO simulating peptide of another TMP, as dimer or polymer.TMP of the present invention comprises following sequences:

I：X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18；

X1-X4 wherein, X9-X10 and X13-X18 are amino acid independently separately.The preferred amino acids residue of top sequence is in addition as following table 1 definition.

Table 1-preferred amino acids residue

The position	Amino-acid residue
		X1	A，V，W，M，G，Y，C，Q，E，R，H
X2	A，V，L，I，G，S，C
		X3	L，I，P，W，G，S，D，K，R
X4	L，G，Q，D，E，H
		X9	K，R

X10	Q，E
		X13	A，V，L，S，Q，E，R，
X14	A，W，T，Y，C，Q
		X15	V，L，G，Y，R
X16	A，L，F，G，R
		X17	A，V，L，M，G，C，Q，N
X18	A，V，P，M，F，G，C，Q，K

Preferred TMP sequence of the present invention is to have those of following sequence:

I：X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18

X1-X4 wherein, X9-X10 and X13-X18 are amino acid independently of one another, wherein peptide has the binding affinity of mpl acceptor, and/or has the biological activity that is equal to or greater than following sequence:

I-E-G-P-T-L-R-Q-W-L-A-A-R-A(SEQ ID NO：1)

Binding affinity can be measured by the known any experimental technique that maybe can get of any those skilled in the art, includes but not limited to that BIAcore measures, ELISA experiment, competitive assay or the like.

Biological activity also can be by the known any experiment that maybe can get of those skilled in the art in vivo or in-vitro measurements.Experiment as an example includes but not limited to, based on the test of cell, i.e. megakaryocyte proliferation test, 32D cell tests (in WO 95/26746, having the IL-3 of the mouse 32D cell of the mpl acceptor transfection of choosing that more is described in detail to rely on the clone), the CD34+ test, CD61 cell tests etc.Biological activity also can be measured by various interior animal experiments.

Show in other preferred TMP sequences of the present invention table 2 below.

Table 2: preferred TMP sequence

TMP No.	Peptide sequence	SEQ ID NO：
			TMP2	GAREGPTLRQWLEWVRVG		2
TMP3	RDLDGPTLRQWLPLPSVQ	3
			TMP4	ALRDGPTLKQWLEYRRQA		4
TMP5	ARQEGPTLKEWLFWVRMG
			5
TMP6	EALLGPTLREWLAWRRAQ
			6
TMP7	MARDGPTLREWLRTYRMM			7
		TMP8	WMPEGPTLKQWLFHGRGQ
	8
		TMP9	HIREGPTLRQWLVALRMV	9
TMP10	QLGHGPTLRQWLSWYRGM					10
		TMP11	ELRQGPTLHEWLQHLASK				11
TMP12	VGIEGPTLRQWLAQKLNP					12
		TMP13	WSRDGPTLREWLAWRAVG	13
TMP14	AVPQGPTLKQWLLWRRCA					14
		TMP15	RIREGPTLKEWLAQRRGF	15
TMP16	RFAEGPTLREWLEQRKLV
			16
TMP17	DRFQGPTLREWLAAIRSV			17
		TMP18	AGREGPTLREWLNMRVWQ			18
TMP19	ALQEGPTLRQWLGWGQWG			19
		TMP20	YCDEGPTLKQWLVCLGLQ			20
TMP21	WCKEGPTLREWLRWGFLC			21
		TMP22	CSSGGPTLREWLQCRRMQ		22
TMP23	CSWGGPTLKQWLQCVRAK			23
		TMP24	CQLGGPTLREWLACRLGA		24
TMP25	CWEGGPTLKEWLQCLVER			25
		TMP26	CRGGGPTLHQWLSCFRWQ		26
TMP27	CRDGGPTLRQWLACLQQK			27
		TMP28	ELRSGPTLKEWLVWRLAQ		28
TMP29	GCRSGPTLREWLACREVQ			29
		TMP30	TCEQGPTLRQWLLCRQGR			30

The binding affinity of peptide TMP2-TMP30 and biologically active data are further as described in the embodiment.For simulation is better therefrom selected the phage environment of peptide and in order to protect the charged amino and the C-terminal of preferred 18 amino acid peptides, at each terminal two amino acid " cap (cap) " that add of each peptide.Particularly, the N-terminal of each all adds glutamine (Q) and halfcystine (C) among the TMP2-TMP30.Equally, add two amino acid " cap " at each C-terminal of peptide-Histidine (H) and Serine (S).It will be understood by those skilled in the art that cap only protects charged end, and do not plan the binding affinity of preferred peptide and/or biological activity are strengthened or lowered.

Because the known length along with peptide of peptide avidity increases, the length of benchmark biologically active peptides (SEQID NO:1) is brought up to 22 amino acid from 14 amino acid, is the length identical with test peptides TMP2-TMP30.Referring to embodiment 6-11.The biologically active zone that it will be appreciated by persons skilled in the art that comparison (comparator) peptide is 14 aminoacid sequences of core, is accredited as SEQ ID NO:1, is also referred to as TMP1.

Any peptide that contains cysteinyl residue can be crosslinked with another peptide that contains Cys, and each among both or both can be connected with a carrier.Any peptide with more than one Cys residue also can form disulfide linkage in the peptide.Any of these peptide can as mentioned belowly be derived.

Other useful peptide sequences can obtain from the conservative and/or non-conservative modification of the aminoacid sequence of TMP disclosed herein.The conservative modification has function and chemical feature similar in appearance to those peptides that carried out these modifications with generation.On the contrary, function and/or the basic modification in the chemical feature at peptide can be finished by selecting the replacement in the aminoacid sequence, being substituted in them and keeping (a) structure of molecular backbone in the zone that replaces in these aminoacid sequences, for example as lamella or helicoidal configuration, (b) in the electric charge or the hydrophobicity of target site molecule, or (c) effect on the bulk of molecule all is visibly different.

For example, " conserved amino acid replacement " can comprise with the non-natural residue and replace the natural amino acid residue, makes very little or do not have in the polarity of the amino-acid residue of that position or electric charge effect.In addition, any natural residue in polypeptide also can replace with L-Ala, as the front " in the alanine scanning mutagenesis " described (referring to, for example, MacLennan etc., Acta Physiol.Scand.Suppl.643:55-67; Sasaki etc., 1998, Adv.Biophys.35:1-24 has wherein discussed alanine scanning mutagenesis).

In the such replacement of needs, can determine the aminoacid replacement (no matter being conservative or nonconservative) that needs by those skilled in the art.For example, aminoacid replacement can be used to identify the important residue of peptide sequence, or improves or reduce the avidity of peptide as herein described or carrier-peptide molecule (referring to the general formula of front).Aminoacid replacement as an example is as described in Table 3.

Table 3-aminoacid replacement

Former residue	The typical case substitutes	Preferred substituting
			Ala(A)	Val，Leu，Ile	Val
Arg(R)	Lys，Gln，Asn	Lys
			Asn(N)	Gln	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser，Ala	Ser

Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro，Ala	Ala
			His(H)	Asn，Gln，Lys，Arg	Arg
Ile(I)	Leu, Val, Met, Ala, Phe, nor-leucine	Leu
			Leu(L)	Nor-leucine, Ile, Val, Met, Ala, Phe	Ile
Lys(K)	Arg, 1,4-DAB, Gln, Asn	Arg
			Met(M)	Leu，Phe，Ile	Leu
Phe(F)	Leu，Val，Ile，Ala，Tyr	Leu
			Pro(P)	Ala	Gly
Ser(S)	Thr，Ala，Cys	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr，Phe	Tyr
			Tyr(Y)	Trp，Phe，Thr，Ser	Phe
Val(V)	Ile, Met, Leu, Phe, Ala, nor-leucine	Leu

In some embodiments, conserved amino acid replaces the amino-acid residue comprise also that usually the synthetic non-natural that mixes in the synthetic rather than biosystem exists by chemical peptide.

Naturally occurring residue can be classified according to the side chain characteristic of the general modification that is used for sequence.For example, non-conservative replacement can comprise member's exchange of a member in these classifications and another classification.Such replacement residue can import the zone with inhuman homology evolution classification homologous peptide, or imports the non-homogeneous district of this molecule.In addition, people also can utilize P or G for the direction that influences chain and modify.

When carrying out such modification, hydrophilic index that can considered amino acid.On their basis of hydrophobic and charged feature, each amino acid has been specified hydrophilic index, and that is: Isoleucine (+4.5); Xie Ansuan (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Halfcystine/Gelucystine (+2.5); Methionine(Met) (+1.9); L-Ala (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophane (0.9); Tyrosine (1.3); Proline(Pro) (1.6); Histidine (3.2); L-glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); L-asparagine (3.5); Methionin (3.9); And arginine (4.5).

The importance of the hydrophilic amino acid index in the biological function that gives a protein interaction is that this area is understood.Kyte etc., J.Mol.Biol., 157:105-131 (1982).More known amino acid can replace other amino acid with similar hydrophilic index or score, and still keep similar biological activity.When changing on hydrophilic index, the aminoacid replacement of hydrophilic index in ± 2 is preferred, particularly preferred in ± 1, and in ± 0.5 is especially preferred.

This area is same accessible to be also can carry out similar amino acid whose replacement effectively according to hydrophobicity.The local average hydrophobicity of a proteinic maximum, controlling as its amino acid whose hydrophobicity of adjacency is immunogenicity and antigenicity with it, promptly relevant with proteinic biological nature.

Following hydrophobic value has been assigned to amino-acid residue: arginine (+3.0); Methionin (+3.0); Aspartic acid (+3.0 ± 1); L-glutamic acid (+3.0 ± 1); Serine (+0.3); L-asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline(Pro) (0.5 ± 1); L-Ala (0.5); Histidine (0.5); Halfcystine (1.0); Methionine(Met) (1.3); Xie Ansuan (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophane (3.4).When changing according to similar hydrophobic value, the aminoacid replacement of hydrophobic value in ± 2 is preferred, and those are particularly preferred in ± 1, and those are especially preferred in ± 0.5.On hydrophobic principle, can identify epi-position from initial aminoacid sequence equally.These zones also can be called " epi-position nucleus ".

Those skilled in the art can utilize known technology to determine suitable varient.In order to identify the appropriate area that can not destroy the activity change molecule, the technician in this area can not believe target is used as in the important zone of activity.For example, when having from same species or be known from the similar active similar polypeptide of other species, a technician of this area can be with the aminoacid sequence of a peptide and similar peptide relatively.In so relatively descending, can identify the residue and the part of molecule conservative in similar polypeptide.Should be understood that as if the variation in the zone of a peptide of not guarding with respect to so similar peptide can influence the biological activity and/or the structure of this peptide more sharply.A technician of this area also knows, even in quite conservative zone, also can use the chemically similar naturally occurring residue of aminoacid replacement (conservative amino acid residues replacement) under the situation of retentive activity.So, even may or also can carry out conserved amino acid to biological activity and replace, and not destroy biological activity or do not influence the structure of peptide unfriendly for the important zone of structure.

The amino acid and the TMPs of the present invention that may have the amino acid (except glycine, it is neither L neither D) of L or D stereochemical structure also can comprise stereochemical combination structure.But L type stereochemistry all is preferred to all amino acid in the TMP chain.The present invention also provides the TMP molecule that reverses, and wherein amino acid whose N-terminal reverses to the C-terminal sequence.For example, the X that has normal sequence ₁-X ₂-X ₃Molecule will be X after reversing ₃-X ₂-X ₁That the present invention also provides is back-reverse the TMP molecule, and wherein, as reversing TMP, amino acid whose N-terminal reverses to the C-terminal sequence, and normally " L " enantiomorph residue in TMP changes to " D " stereoisomeric forms in any ratio.

Should be noted that also TMP " derivative " can replace the TMP that narrates above.Such derivative TMP comprises the composition of one or several modification below having carried out:

● one or more peptidyls [C (O) NR-] connect (key) and are connected replacement by non-peptidyl, as-CH ₂Carbamate connects [CH ₂-OC (O) NR-]; Phosphoric acid connects;-CH ₂-sulphonamide [CH ₂-S (O) ₂NR-] connect; Urea [NHC (O) NH-] connects;-CH ₂Secondary amine connects; Or the alkylation peptidyl connects [C (O) NR ⁶-, R wherein ⁶Be low alkyl group];

● N-terminal is derivatized to-NRR ¹Group;-NRC (O) R group; NRC (O) OR group;-NRS (O) ₂The R group; The peptide of NHC (O) NHR group, wherein R and R ¹Be hydrogen or low alkyl group, if R and R ¹Not hydrogen; Be derivatized to the succinic diamide group so; Benzene oxygen carboxy-N H-(CBZ-NH-) group; Or benzene oxygen carboxy-N H-group, have 1 to 3 substituting group at phenyl ring, be selected from low alkyl group, lower alkoxy, groups such as chlorine and bromine.With

● the free C-terminal is derivatized to-C (O) R ²Peptide, R wherein ²Be selected from lower alkoxy and-NR ³R ⁴, R wherein ³And R ⁴Be selected from hydrogen and low alkyl group alone." rudimentary " is meant the group with 1 to 6 carbon atom.

In addition, indivedual amino acid whose modifications can be by importing the TMP molecule with the purpose amino-acid residue of peptide with can reacting with the organic derivatizing agent of selected side chain or terminal residue reaction.Example is as follows:

Methionin base and n terminal residue can react with succsinic acid or other carboxylic acid anhydride.Derive with these reagent and to have the effect of the electric charge that reverses lysine residue.Other the suitable reagent that contain alpha-amino residue of deriving comprise imido-ester, as the picoline methyl esters; Pyridoxal phosphate; Pyridoxal; Chlorine boron hydracid; Trinitro-benzene-sulfonic acid; The O-methyl-isourea; 2,4 diacetylmethanes; The reaction of and oxoethanoic acid catalytic with transaminase.

The arginyl residue can be by modifying with one or several conventional reagent react, phenyl glyoxal wherein, 2,3-dimethyl diketone, 1,2-cyclohexanedione, and triketohydrindene hydrate.Because the pKa height of guanidine functional group, the deriving need be reflected under the alkaline condition of arginine residues carries out.In addition, these reagent can react with Methionin group and arginine guanidino group.

The special modification of tyrosyl residue itself is research widely, special concern be by the spectrum mark being imported the tyrosyl residue with the reaction of aromatic diazo compound or tetranitromethane.Modal is that N-acetyl imidazole and tetranitromethane can be used to form O-ethanoyl tyrosyl thing class and 3-nitro-derivative.

Carboxyl side group (aspartyl or glutamyl) can be by (2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethyl amyl group) carbodiimide reacts optionally and modifies as 1-cyclohexyl-3-with carbodiimide (R '-N=C=N-R ').In addition, asparagyl and glutamyl residue can be by being transformed into asparagine residue and glutamine residue with the ammonium ion reaction.

The glutamy amido becomes corresponding glutamyl and asparagyl residue with the common deacylated tRNA amine of asparaginyl group.Perhaps, these residues can be under the acidic conditions of gentleness deacylated tRNA amine.The formation of these residues is within the scope of the invention.

Can be used for peptide and their the insoluble supported matrix of functional deriv or other macromolecular carriers crosslinked with deriving of bifunctional reagent with water.Normally used cross-linking reagent for example comprises, 1, and 1-two (diazonium ethanoyl)-2-phenylethane, glutaraldehyde, the N-hydroxy-succinamide ester for example has 4-nitrine sialic acid, and the difunctional imines ester of homotype comprises two-N-maleimide-1, the 8-octane.Derivative reagent such as methyl-3-[(p-azidophenyl) dithio] propionyl imines (propioimidate) can form the activable intermediate product of crosslinked light when being created in light and existing.Perhaps, United States Patent (USP) 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; With 4,330, the carbohydrate of insoluble matrix of 440 described water reactive such as cyanogen bromide-activated and reaction substrate can be used for proteinic fixing.

Other possible modifications comprise the hydroxylation of proline(Pro) and Methionin, the phosphorylation of the oh group of silk acyl group or Soviet Union's acyl residue, the oxidation of the sulphur atom in Cys, Methionin, methylate (Creighton, the T.E. of the alpha-amino group group of arginine and Histidine side chain, protein: structure and molecular characterization, W.H.Freeman﹠amp; Co., San Francisco, 79-86 (1983)), the ethylization of N-terminal amine and the amidation of C-terminal carboxylic group in some cases.

The composition of deriving has so preferably improved one or several feature, comprises the thrombopoietic activity of the compound of invention, and solubility absorbs biological half time etc.Perhaps, the deutero-composition can cause compound to have the feature and/or the characteristic of identical or essentially identical non-derived compounds.These compositions or can remove or weaken any unwanted side effect of these compounds etc.

Compound of the present invention can change at dna level etc.The dna sequence dna of any part of compound can change to the more compatible stage of host cell of codon and selection.Optimal codon is known in the art.Codon can replace, so that remove restriction site or comprise reticent restriction site, and the processing of the DNA in can the host cell of assisted Selection.Can modify carrier, the variation that joint and peptide dna sequence dna contain any foregoing sequence.So all modifications that this paper discusses replace, deriving etc. can be applicable to all aspects of the present invention equally, includes but not limited to peptide, peptide dimer and polymer, joint and carrier.

In addition, this area technician can consult the structure-functional study of evaluation to the residue in the important similar peptide of activity or structure.From relatively such, be foreseeable corresponding to the importance of the amino-acid residue in the peptide of the important amino-acid residue of the activity of similar peptide or structure.A technician of this area can select the important amino-acid residue of some predictions of aminoacid replacement peptide like the chemofacies.

A technician of this area also can analyze to similar polypeptide in the three-dimensional structure and the aminoacid sequence of structurally associated.From this situation, a technician of this area can predict the arrangement of the amino-acid residue of the peptide relevant with three-dimensional structure.It is not to do basic change at proteinic lip-deep amino-acid residue to prediction that a technician of this area can select, because such residue can be included in the important interaction with other molecules.In addition, this area technician can generate at each amino-acid residue that needs and contain the test varient that single amino acids replaces.Then, this varient can utilize active testing well known by persons skilled in the art to screen.Such data can be used to collect the information about suitable varient.For example, if if find the variation of special amino-acid residue has been produced destructive, unwanted minimizing, or unsuitable activity have the varient of such variation to avoid.Talk about with another sentence, according to the information of collecting from such normal experiment, a technician of this area can be easy to determine to avoid separately or in conjunction with the amino acid of the further replacement of other sudden changes.

The prediction secondary structure has had many scientific publication things.Referring to Moult J., Curr.Op.inBiotech., 7 (4): 422-427 (1996), Chou etc., biological chemistry, 13 (2): 222-245 (1974); Chou etc., biological chemistry, 113, (2): 211-222 (1974); Chou etc., Adv.Enzymol.Relat.Areas Mol.Biol., 47:45-148 (1978); Chou etc., Ann.Rev.Biochem., 47:251-276 and Chou etc., Biophys.J., 26:367-384 (1979).In addition, the computer program that can get at present can be used for auxiliary prediction secondary structure.A method of prediction secondary structure is to carry out according to the homology model.For example, two polypeptide or protein have sequence identity greater than 30%, or similarity often has similar geometry greater than 40.The recent development of protein structure proximity database (pdb) proximity has made the predictability of secondary structure strengthen, and comprises the folding potential number in polypeptide or the protein structure.Referring to Holm etc., nucleic acids research, 27 (1): 244-247 (1999).Show that (7 (3): 369-376 (1997)) the folding number in given polypeptide is limited for Brenner etc., Curr.Op.Struct.Biol., and in case the structure of closing number of keys is differentiated, it is very accurate that structure prediction will reach.

The additive method of prediction secondary structure comprises " collimation method (threading) " (Jones, D., the comment of modern structure biology, 7 (3): 377-87 (1997); Sippl etc., structure, 4 (1): 15-9 (1996)), " distributional analysis (profile analysis) " (Bowie etc., science, 253:164-170 (1991); Gribskov etc., Enzymology method, 183:146-159 (1990); Gribskov etc., institute of NAS periodical, 84 (13): 4355-8 (1987)), with " evolve and be connected (evolutionarylinkage) " (referring to Home, ibid, and Brenner, and ibid).

The general formula of preferred peptide of the present invention and peptide linker molecule as shown in Figure 1.In addition, the physiological acceptable salt that has also comprised TMPs.

Peptide compounds

Except new peptide, the invention provides new peptide compounds, one or more peptides wherein of the present invention adhere to mutually or are connected on joint (LN) or the carrier (V).TMPs can connect (that is, in order, N-terminal is to C-terminal), or parallel (that is, N-to N-end or C-to the C-end).TMPs can be attached to other TMP or identical TMP being with or without under the situation of joint.TMP also can be attached to other TMP or identical TMP being with or without under joint and the situation that is with or without carrier.Peptide-joint of the present invention-carrier compound can be narrated by following general formula:

II

(V1) _v--(LN1) _l--(TMP) _a--(LN2) _m--(TMP2) _b--(LN3) _n--(TMP3) _c--(LN4) _o--(TMP4) _d--(V2) _w

Wherein:

V1 and V2 are carriers; LN1, LN2, LN3 and LN4 are joint independently of one another; TMP1, TMP2, TMP3 and TMP4 are the peptide sequence of general formula I independently of one another; A, b, c and d and l, m, n and o are 0 to 20 integer independently of one another, v and w are 0 to 1 integer independently of one another.

Compound as an example of the present invention is shown in following general formula:

TMP1-V1 TMP1-LN1-V1

TMP1-TMP2-V1

TMP1-LN1-TMP2-LN2-V1

With its other polymer, wherein V1 is carrier (preferably Fc district) and is being with or without the C-terminal that is attached to TMP under the situation of joint;

V1-TMP1 V1-LN1-TMP1

V1-TMP1-TMP2 V1-LN1-TMP1-LN2-TMP2

With its polymer, wherein V1 is carrier (preferably Fc district), and is being with or without the N-terminal that is attached to TMP under the situation of joint.The general formula of preferred peptide-carrier of the present invention and peptide-joint-carrier molecule as shown in Figure 2.

Many preferred compounds of the present invention are dimer or polymer, because they have two TMP compositions or polymer, because they have a plurality of TMP compositions.TMP1 can have identical or different structure to each of TMP4 etc.Preferably, compound of the present invention will have 2 to 5 TMP compositions, and particularly preferably 2-3, most preferably 2.

These compounds preferably directly adhere to or are connected (referring to as follows) with dimer by the joint group.Monomer TMP composition shows it is from left to right to read the N-of sign indicating number to the C-end on traditional direction.Therefore, can see that compound that can directed invention, the C-terminal that makes TMP1 are directly or be attached to the N-terminal (series connection dimer) of TMP2 by joint.Perhaps, compound that can directed invention directly is attached to the C-terminal of TMP1 or is attached to the C-terminal of TMP2 by joint, or the N-terminal of TMP1 directly or be attached to the N-terminal (parallel dimer) of TMP2 by joint.If even TMP1 and TMP2 be distinguishing on the structure, these compounds are also referred to as dimer.It is contemplated that it is homodimer and heterodimer.

Joint

In another embodiment, the invention provides by " joint " group (LN1, LN2, etc.) covalent bonding or connection or be attached to one or more TMP of another TMP peptide.The joint group can be selected arbitrarily.Owing to be as introns at first, when existing, its chemical structure is not crucial.Should select joint, so that can not disturb the biological activity of last compound, and the immunogenicity of last compound is not obviously increased.The amino acid that joint is preferably linked together by peptide bond is formed.So in preferred embodiments, 1 to 30 amino acid that joint is connected by peptide bond is formed, amino acid wherein is selected from 20 naturally occurring amino acid.Be readily appreciated that as those skilled in the art, these more amino acid whose can glycosylation.In a more preferred embodiment, from glycine, L-Ala, proline(Pro), l-asparagine has been selected 1 to 20 amino acid in glutamine and the Methionin.Even more preferably, joint by on the three-dimensional arrangement not many amino acid of twisting form, as glycine and L-Ala.So preferred joint is polyglycine (particularly (Gly) ₄, (Gly) ₅), poly-(Gly-Ala) and poly-L-Ala.The special example of other of joint is:

(Gly) ₃Lys(Gly) ₄ (SEQ ID NO：96)

(Gly) ₃AsnGlySer(Gly) ₂ (SEQ ID NO：97)

(Gly) ₃Cys (Gly) ₄(SEQ ID NO:98); With

GlyProAsnGlyGly (SEQ ID NO：99)

In order to explain top term, for example (Gly) ₃Lys (Gly) ₄Be meant Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly.The combination of Gly and Ala also is preferred.Joint shown in this article also is an example; Joint within the scope of the invention can be longer, and can comprise other residues.

Non-peptide linker also is fine.For example, the alkyl joint is as-NH-(CH ₂) _s-C (O)-, wherein s=2-20 is utilizable.These alkyl joints can be further with any non-three-dimensional twisting group such as low alkyl group (for example, C ₁-C ₆), lower acyl, and halogen (for example, Cl, Br), CN, NH ₂, phenyl or the like replaces.Non-peptide linker as an example is the PEG joint,

Wherein n makes joint have molecular weight 100 to 5000kD, preferably 100 arrives 500kD.Peptide linker can identical as mentioned above mode change the formation derivative.

Usually, have been found that the joint (for example, amino acid) for the about 0-14 of a thrombocyte generation compound length of the present invention subunit is preferred.Peptide linker can change the derivative that forms TMP with aforesaid identical method.In addition, the compound of this embodiment can also be linear or annular." annular " be meant at least two isolating, promptly the non-adjacent part of molecule is interconnective.For example, the amino of the end of molecule and C-terminal can covalently bound formation ring molecules.Perhaps, molecule can contain two or more Cys residues (for example, in joint), and this molecule also can be by the formation cyclisation of disulfide linkage.Merit attention in addition, a more than placed in-line peptide dimer can be connected to form a dimer in a plurality of dimers.So for example, the series connection dimer that contains the Cys residue can form intermolecular disulfide linkage with another so dimeric Cys.The example of peptide-linker compounds of the present invention is as follows:

CSSGGPTLREWLQCRRMQ--GGGGG--CSSGGPTLREWLQCRRMQ(SEQ ID NO 100)；

QLGHGPTLRQWLSWYRGM--(Gly) ₃Lys(Gly) ₄--ALRDGPTLKQWLEYRRQA(SEQ ID NO 101)；

RFAEGPTLREWLEQRKLV-GGG(PEG)GGG-RFAEGPTLREWLEQRKLV (SEQ IDNO 102).

So in preferred embodiments, joint comprises (LN1) _n, wherein LN1 is naturally occurring amino acid or its steric isomer, " n " is any one in 1 to 20.The general formula of preferred peptide-linkers as shown in Figure 1.Other preferred peptide-linkers comprise:

i)TMP1-LN1-TMP2-LN2

ii)LN1-TMP1-LN2-TMP2

iii)LN1-TMP1-LN2-TMP1

iv)TMP1-LN1-TMP1-LN1-TMP1-LN1

v)LN1-TMP1-LN2-TMP2-LN3-TMP3-LN4-TMP4

Wherein LN1-LN4 is a joint independently separately.

Carrier

Still in another embodiment, peptide of the present invention or peptide compounds can be connected or adhere to carrier (V).Carrier typically refers to the proteinic degraded of prophylactic treatment and/or increases the transformation period, reduces toxicity, reduces immunogenicity, or improves bioactive molecule.Carrier (V) can pass through N-terminal, C-terminal, and peptide backbone or side chain are attached on the peptide.

Carrier (V) can be a carrier molecule, as linear polymer (for example, polyoxyethylene glycol, polylysine, dextran or the like), the branched chain polymer (referring to, for example on September 15th, 1981 disclosed patent 4,289,872 of authorizing Denkenwalter etc.; On July 20th, 1993 disclosed 5,229,490 of the Tam that authorizes; October in 1993 disclosed Frechet on the 28th etc. WO93/21259); Lipid; Cholesterol group (as steroid); Or carbohydrate or oligomerization polysaccharide.Other possible carriers comprise one or more water soluble polymer dirt settlings, as polyoxyethylene glycol, or polypropylene glycol, as U.S. Patent number 4,640,835,4,496,689,4,301,144,4,670,417,4,791,192 and 4,179, described in 337.Remain other useful polymers known in the art and comprise single methoxy polyoxyethylene glycol, dextran, Mierocrystalline cellulose, or the polymer on other carbohydrate bases, poly-(N-V-Pyrol RC)-polyoxyethylene glycol, propylene glycol homopolymer, poly(propylene oxide)/ethylene oxide copolymer, polyoxy ethylization polyvalent alcohol (for example, glycerine) and polyvinyl alcohol, and the mixture of these polymers.The example of carrier also comprises:

● the Fc district;

● can be in conjunction with other protein of remedying acceptor, polypeptide, or peptide;

● human serum albumin (HSA);

● leucine zipper (LZ) district;

● polyoxyethylene glycol (PEG) comprises 5kD, 20kD and 30kD PEG, and other polymers;

● dextran

With other molecules known in the art, the transformation period that prolongs is provided, and/or the degraded of protected protein matter or removing.

Carrier as an example can be polyoxyethylene glycol (PEG).The PEG group can be the molecular weight of any routine and can be straight chain or branch.The molecular-weight average of PEG preferably scope is to arrive about 100kDa at about 2kDa, more preferably is that about 5kDa arrives about 50kDa, most preferably is that about 5kDa is to about 10kDa.

The PEG group passes through acidylate usually, standard reductive alkylation, and Michael is additional; mercaptoalkylization or other chemistry are selected combination/method of attachment, by (for example, the aldehyde of the reactive group on the PEG composition; amino, ester, sulfydryl; the halo ethanoyl, maleimide or diazanyl group) (for example, aldehyde to the reactive group of target compound; amino, ester, sulfydryl; the halo ethanoyl, maleinamide, or diazanyl group) be attached on the compound of the present invention.

Carbohydrate (oligomerization polysaccharide) group can be attached to the site of proteinic glycosylation site easily.Usually, when they are the part of sequence A sh-X-Ser/Thr, wherein X can be any amino acid beyond the proline(Pro), and the oligomerization polysaccharide that O-connects is to be attached to Serine (Ser) or Threonine (Thr) residue, and the oligomerization polysaccharide that N-connects is attached to l-asparagine (Asn) residue.X does not preferably comprise in 19 naturally occurring amino acid of proline(Pro).It is different that the N that finds in each type connects the structure that is connected oligomerization polysaccharide and saccharide residue with O.The sugar that both common discoveries are types is N-ethyl neuraminic acid (being called sialic acid).Sialic acid can be that N connects the terminal residue that is connected the oligomerization polysaccharide with O by its negative charge usually, can give glycosyl compound acid characteristic.Such site can be mixed in the joint of compound of the present invention, and preferably in the recombinant production process of polypeptide compound by cell (for example, at mammalian cell such as CHO, BHK, COS) glycosylation.But such site can be further by synthetic or semi-synthetic process glycosylation known in the art.

In a more preferred embodiment, carrier (V) can comprise one or more antibody Fc district.So aforesaid peptide compounds can directly or by joint merge with one or more Fc district in addition.The Fc carrier can be from human normal immunoglobulin IgG-1 heavy chain, referring to J.W. etc., nucleic acids research, 10:4071-4079 (1982), or any other Fc sequence of this area (for example, other IgG classifications, include but not limited to IgG-2, IgG-3 and IgG4, or other immunoglobulin (Ig)s) select.

Known, the monomer polypeptide section of dimer or polymer form forms by connecting into by disulfide linkage or by non-covalent combination in the Fc district of antibody.According to the classification of the antibody that comprises (for example, IgG, IgA, IgE) or subclass (for example, IgG1, IgG2, IgG3, IgA1, IgGA2), the scope of the number of the intermolecular disulfide bond between the monomer subunit of natural Fc molecule is 1 to 4.Term " Fc " monomer that normally belongs to the Fc molecule as used herein, dimer and polymer form.Should be noted that prevent Dimerized special conditions of contract unless exist by disulfide linkage formation, when having suitable Cys residue, the Fc monomer will be spontaneously Dimerized.If even remove or be substituted in the Fc dimer the normal Cys residue that forms disulfide linkage by other residues, the monomer chain usually will be Dimerized by noncovalent interaction.The term of this paper " Fc " is to mean any these following forms: natural monomer, natural dimer (disulfide linkage connection), the monomer (that is derivative) of modifying dimer (two sulphur and/or non-covalent connection) and modifying.

The varient of Fc part, analogue or derivative can make up by the replacement of for example carrying out various residues or sequence.

Varient (or analogue) polypeptide comprises the insertion varient, and wherein one or more amino-acid residues replenish the Fc aminoacid sequence.Insertion can be positioned at this proteinic one or two end, maybe can be positioned at the interior region of Fc aminoacid sequence.The insertion varient that has other residues at one or two end comprises for example fused protein and the protein that comprises amino-terminal end or mark.For example, particularly when molecule is recombinant expressed in bacterial cell such as intestinal bacteria, Fc molecule or can contain N-terminal Met.

In Fc deletion mutation body, the one or more amino-acid residues in the Fc polypeptide have been removed.Disappearance can be removed one or more residues one or two terminal generation of Fc polypeptide in the Fc aminoacid sequence.So the deletion mutation body comprises the fragment of all Fc peptide sequences.

In Fc replaces varient, removed one or several amino-acid residue of Fc polypeptide, and with or the residue replacement of choosing.In one aspect, be substituted in and guard in natural, still, the present invention also comprises nonconservative replacement.

For example, cysteine residues can be deleted or replace with other amino acid, so that prevent some or all of two sulfur-crosslinked formation of Fc sequence.Can remove each in these cysteine residues, perhaps replace cysteine residues so one or more as Ala or Ser with other amino acid.In another example, modify and also can import aminoacid replacement to (1) disengaging (ablate) Fc receptor binding site; (2) break away from complement (Clq) binding site; And/or (3) break away from cytotoxicity (ADCC) site of antibody-dependant cell mediation.Such site is known in the art, and any known replacement all as used herein be in the scope of Fc.For example, the ADCC site among the relevant IgG1 can be referring to molecular immunology, 29 volumes, No.5,633-639 (1992).

Equally, one or more tyrosine residues also can replace with phenylalanine residue.In addition, the aminoacid insertion of other variations, disappearance (for example, from 1-25 amino acid) and/or replacement also receive publicity, and are within the scope of the invention.Conserved amino acid replaces normally preferred.In addition, change can be the amino acid whose form that has changed, as peptide mimics or D amino acid.

Fc sequence of the present invention also can be a deutero-, promptly contains the insertion of amino-acid residue, disappearance, or the modification beyond replacing.Preferably, these are modified on natural can be covalency, for example comprise, and with polymer, fat, other chemical bondings organic and inorganic components.Can prepare derivative of the present invention increases circulating half-life, maybe can design and improve the cell of polypeptide to needs, the target capability of tissue or organ.

As WO 96/32478, exercise question " polypeptide of the change that the transformation period increases " is described, and utilizing the receptor binding domain of remedying of complete Fc molecule also is possible as the Fc part of the compound of invention.Other members of the classification of the molecule of called after Fc are that exercise question is those described in the WO 97/34631 of " transformation period increase immunoglobulin like protein zone " herein.Two disclosed PCT applications of quoting as proof in this chapter are incorporated herein by reference.

Fc merges can be at TMP ₁Or TMP ₂N-or C-end, or at TMP ₁Or TMP ₂N-and C-terminal.Equally, the Fc fusion can be at the N-or the C-end in Fc zone.

Preferred compound of the present invention comprises dimer or the polymeric IgG1 Fc fused dimer that connects or be attached to TMP disclosed herein.In such a case, each Fc district will be with or without on the dimer or polymer that is connected to the TMP peptide under the situation of joint.The illustrated example of such compound as shown in Figure 2.

Also can utilize a plurality of carriers; For example, at each terminal Fc, or at the Fc of an end with at the PEG of other ends or side chain group.

Peptide-carrier compound as an example is provided at following table 4.

Table 4-peptide-carrier compound as an example

Aminoacid sequence	SEQ ID NO：
		HIREGPTLRQWLVALRMV-GGG(PEG)GGG-HIREGPTLRQWLVALRMV	103
Fc-TCEQGPTLRQWLLCRQGR-GGGKGGG-TCEQGPTLRQWLLCRqGR-Fc	104
		Fc-QLGHGPTLRQWLSWYRGM-GPNG-ELRSGPTLKEWLVWRLAq	105
CSWGGPTLKQWLQCVRAK-Fc | SWGGPTLKQWLQCVRAK	106
		Fc-GGGKGGG-AVPQGPTLKQWLLWRRCA	107
PEG-CSSGGPTLREWLQCRRMQ | CSSGGPTLREWLQCRRMQ	108
		Fc-GGGGG-YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ	109
CSWGGPTLKQWLQCVRAK-GGGAGGG-CSWGGPTLKQWLQCVRAK-GGGAGGG- CSWGGPTLKQWLQCVRAK-GGGAGGG-Fc	110
		VGIEGPTLRQWLAQRLNP-GGGCGGG-VGIEGPTLRQWLAQRLNP-PEG	111
Fc-ELRSGPTLKEWLVWRLAq-GGGG-ELRSGPTLKEWLVWRLAQ	112
		Fc-ALRDGPTLKQWLEYRRQA-GGGKGGG-ALRDGPTLKQWLEYRRQA-Fc	113

In addition, the preferred embodiment of the invention is listed in the table 5.

The specific embodiment preferred of table 5-

Aminoacid sequence	SEQ ID NO：
		ALRDGPTLKQWLEYRRQA-ALRDGPTLKQWLEYRRQA	114
EALLGPTLRRWLAWRRAQ-EALLGPTLREWLAWRRAQ	115
		AVPQGPTLKQWLLWRRCA-AVPQGPTLKQWLLWRRCA	116
YCDEGPTLKQWLVCLGLQ-YCDEGPTLKQWLVCLGLQ	117
		CSSGGPTLREWLQCRRMQ-CSSGGPTLREWLQCRRMQ	118
CSWGGPTLKQWLQCVRAK-CSWGGPTLKQWLQCVRAK	119
		ALRDGPTLKQWLEYRRQA-GGGGG-ALRDGPTLKQWLEYRRQA	120
EALLGPTLREWLAWRRAQ-GGGGG-EALLGPTLREWLAWRRAQ	121
		AVPQGPTLKQWLLWRRCA-GGGGG-AVPQGPTLKQWLLWRRCA	122
YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ	123
		CSSGGPTLREWLQCRRMQ-GGGGG-CSSGGPTLREWLQCRRMQ	124
CSWGGPTLKQWLQCVRAK-GGGGG-CSWGGPTLKQWLQCVRAK	125
		Fc-GGGGG-ALRDGPTLKQWLEYRRQA	126
Fc-GGGGG-EALLGPTLREWLAWRRAQ	127
		Fc-GGGGG-AVPQGPTLKQWLLWPRCA	128
Fc-GGGGG-YCDEGPTLKQWLVCLGLQ	129
		Fc-GGGGG-CSSGGPTLREWLQCRRMQ	130
Fc-GGGGG-CSWGGPTLKQWLQCVRAK	131
		Fc-GGGGG-ALRDGPTLKQWLEYRRQA-GGGGG-ALRDGPTLKQWLEYRRQA	132
Fc-GGGGG-EALLGPTLREWLAWRRAQ-GGGGG-EALLGPTLREWLAWRRAQ	133
		Fc-GGGGG-AVPQGPTLKQWLLWRRCA-GGGGG-AVPQGPTLKQWLLWRRCA	134
Fc-GGGGG-YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ	135
		Fc-GGGGG-CSSGGPTLREWLQCRRMQ-GGGGG-CSSGGPTLREWLQCRRMQ	136
Fc-GGGGG-CSWGGPTLKQWLQCVRAK-GGGGG-CSWGGPTLKQWLQCVRAK	137
		ALRDGPTLKQWLRYRRQA-GGGGG-ALRDGPTLKQWLEYRRQA-GGGGG-Fc	138
EALLGPTLREWLAWRRAQ-GGGGG-RALLGPTLREWLAWRRAQ-GGGGG-Fc	139
		AVPQGPTLKQWLLWRRCA-GGGGG-AVPQGPTLKQWLLWRRCA-GGGGG-Fc	140
YCDEGPTLKQWLVCLGLQ-GGGGG-YCDEGPTLKQWLVCLGLQ-GGGGG-Fc	141
		CSSGGPTLREWLQCRRMQ-GGGGG-CSSGGPTLREWLQCRRMQ-GGGGG-Fc	142
CSWGGPTLKQWLQCVRAK-GGGGG-CSWGGPTLKQWLQCVRAK-GGGGG-Fc	143
		ALRDGPTLKQWLEYRRQA-GGGGG-Fc	144
EALLGPTLREWLAWRRAQ-GGGGG-Fc	145
		AVPQGPTLKQWLLWRRCA-GGGGG-Fc	146
YCDEGPTLKQWLVCLGLQ-GGGGG-Fc	147
		CS SGGPTLREWLQCRRMQ-GGGGG-Fc	148
CSWGGPTLKQWLQCVRAK-GGGGG-Fc	149

III. the method for Sheng Chaning

Compound of the present invention can produced in several ways.Because chemical compound lot is a peptide, or comprise peptide, the method for synthetic peptide is relevant especially in this article.Solid phase synthesis technique also can utilize.Suitable technology is known in the art, and comprises Merrifield, chemical polypeptide, 335-61 (Katsoyannis and Panayotis eds.1973); Merrifield, J.Am.Chem.Soc.85:2149 (1963); Davis etc., Biochem.Intl.10:394-414 (1985); Stewart and Young, solid-phase peptide is synthesized (1969); United States Patent (USP) 3,941,763; Finn etc., protein, the 3rd edition, 2 volumes, 105-253 (1976); With Erickson etc., protein, the 3rd edition, 2 volumes, 257-527 (1976) described those.Solid phase synthesis is a preferred technology of making indivedual peptides, because it is the effective means of consumption valency of producing little peptide.

Peptide also can utilize recombinant DNA technology to produce in transformed host cell.For this reason, the encode recombinant DNA molecules of this peptide is preferred.The method for preparing such DNA and/or RNA molecule is known in the art.For example, the encode sequence of this peptide can utilize suitable restriction enzyme to cut out from DNA.The polymerase chain reaction (PCR) that utilization comprises clone's subsequently useful restriction site can produce relevant sequence.Perhaps, utilize chemical synthesising technology, can synthetic DNA/RNA molecule as the phosphoramidite method.Simultaneously, the combination of these or other technologies also can utilize.

The present invention has also comprised a carrier of encoded peptide in suitable host.This carrier comprises the dna molecular of the peptide that coding is operably connected with suitable expression control sequenc.Before the dna molecular of encoded peptide inserts carrier or after, it is known influencing this method that is operatively connected.Expression control sequenc comprises promotor, activator, and enhanser, operon, ribosome bind site, start signal, termination signal adds the cap signal, polyadenylation signal and other signal under the control of transcribing or translating.

The carrier that obtains that comprises the dna molecular of encoded peptide is used to transform suitable host.This conversion can utilize methods known in the art to carry out.

In a large amount of available and known host cells any one may be used in the practice of the present invention.Select special host to depend on many factors known in the art.These factors for example comprise the compatibility with the expression vector of selecting, with toxicity, the speed of conversion, the easness of the discovery of peptide, expression characteristic, biological safety and the consumption valency of the host cell of the peptide of dna molecule encode.These factors of balance must deeply be understood not every host can equality express special dna sequence dna effectively.

Under these general guidance rules, useful microorganism host comprises the bacterium (as intestinal bacteria) in the substratum, yeast (as cereuisiae fermentum and pichia pastoris phaff) and other fungies, insect, plant, Mammals (comprising the people) cell, or other hosts known in the art.Host transformed is to cultivate under the fermentation condition of routine, makes the peptide that needs obtain expressing.Such fermentation condition is known in the art.Then, can purified peptide from the host cell of fermention medium or their expression.These purification process also are known in the art.

The compound that contains derived peptide or contain non-peptide group can synthesize by known technique of organic chemistry.For example, can utilize solid phase synthesis technique.Suitable technology is known in the art, and comprises Merrifield (1973), chemiluminescent polypeptide, 335-61 (Katsoyannis and Panayotiseds.); Merrifield (1963), J.Am.Chem.Soc.85:2149; Davis etc. (1985), Biochem.Intl.10:394-414; Stewart and Young (1969), solid-phase peptide is synthetic; United States Patent (USP) 3,941,763; Finn etc. (1976), protein (the 3rd edition), 2:105-253; With (1976) such as Erickson, protein (the 3rd edition), those described in the 2:257-527.Solid phase synthesis technique is the preferred technology of producing single peptide, because it is the effective means of consumption valency of producing little peptide.

IV. the purposes of compound

Compound of the present invention has combination and/or activates the ability of mpl acceptor, and/or has the ability of the production of (in vivo with external) stimulating platelet (" thrombopoietic activity ") and platelet precursors (" megalokaryocyte generates active ").In order to measure the activity of these compounds, can utilize the testing method of standard, as exercise question be the WO 95/26746 of " composition of stimulating megakaryocyte growth and differentiation and method " described those.In the embodiment of this paper part, further narrated in the body and tested.

The symptom of method and composition of the present invention treatment is normally comprising in the future those of the megalokaryocyte/platelet defect of existence or the megalokaryocyte/platelet defect of expectation or expection (for example, because surgical operation of having planned or thrombocyte donor).Such symptom can be the result of the defective (temporary transient or permanent) of intravital active mpl part.Platelet defect be commonly referred to as thrombopenia, so method and composition of the present invention normally can get in thrombocytopenic prevention of treatment and the treatment in the patient of needs.

The World Health Organization is to the thrombocytopenic degree of the hematoblastic number of round-robin in the individuality classify (Miller etc., cancer, 47:210-21 (1981)).For example, show do not have the individuality (0 grade) of thrombopenia signal will have 100,000 thrombocyte/mm at least usually ³Gentle thrombopenia (1 grade) shows, hematoblastic cyclical level is 79,000 and 99,000/mm ³Medium thrombopenia (2 grades) is presented at 50,000 to 74,000 thrombocytes/mm ³, serious thrombocytopenic feature is 25,000 and 49,000 thrombocyte/mm ³Life-threatening or weak thrombocytopenic feature are that hematoblastic circulation composition is less than 25,000/mm ³

Can there be a variety of causes in thrombopenia (platelet defect), comprises the treatment of chemotherapy and other many medicines, radiation treatment, surgical operation, accidental losing blood and disease symptoms that other are special.The example of the special disease symptoms that comprises thrombopenia and can treat according to the present invention has: aplastic anemia; Spontaneous or immune thrombopenia (ITP) comprises the purple scar of the spontaneous thrombopenia relevant with the chest cancer; The ITP that HIV is relevant, the purple scar of the thrombus thrombopenia relevant with HIV; Cause thrombocytopenic metastatic tumo(u)r; Systemic lupus erythematous; Comprise the comprehensive splenomegaly of neonatal lupus; The Fanconi syndromes; The vitamin B12 defective, the folic acid defective; May-Hegglin is unusual; The Wiskott-Aldrich syndromes; Chronic hepatopathy; The infull syndromes of the marrow plasticity-relevant with thrombopenia; The hemoglobinuria of outbreak at night; Acute degree of depth thrombopenia after C7E3Fab (Abciximab) treatment; The alloimmunization thrombopenia comprises maternal alloimmunization thrombopenia; The thrombopenia relevant with anti-phospholipid antibody and thrombosis; The autoimmunization thrombopenia; Drug-induced immune thrombopenia comprises the dull and stereotyped inductive thrombopenia of carbon, heparin-induced thrombopenia; Newborn infant's thrombopenia; The pregnant thrombopenia that causes seized with terror; The Hughes syndromes; The lupoid thrombopenia; Accidental and/or a large amount of loses blood; The bone marrow proliferation disorder; Thrombopenia among the patient of malignant tumour is arranged; The purple scar of thrombotic thrombopenia comprises that thrombosis microangiography shadow demonstrate,proves the haemolysis uraemic syndrome among thrombotic thrombocytopenic purple scar/cancer patients; Autoimmunization haemolysis anaemia; Hide the jejunum blind pipe perforation of property; Pure red cell underdevelopment; The autoimmunization thrombopenia; The ephrosis prevailing disease; The acute renal failure that Rifampin is relevant; Paris Te Suolu image thrombopenia; Newborn infant's isoimmunization thrombopenia; Paroxysm formula nocturnal hemoglobinuria; Blood in the cancer of the stomach changes; Child's haemolysis uraemic syndrome; The blood phenomenon relevant with virus infection comprises the thrombopenia that hepatitis A virus is relevant with CMV.Equally, the treatment of some AIDS also causes thrombopenia (for example, AZT).Some wound healing disorders also can have benefited from the platelet count purpose to be increased.

The hematoblastic defective of relevant expection, for example because surgery in the future, compound of the present invention will the needs thrombocyte a few days ago by several hours in use.In acute situations, for example accidental and a large amount of losing blood, compound of the present invention can be used with blood or pure blood platelet.

If these cells find it is to express the mpl acceptor, compound of the present invention also can be used to stimulate some cell types except megalokaryocyte.With the relevant condition of these cells of expressing the mpl acceptor, be the stimulation of replying the mpl part, also be within the scope of the invention.

Compound of the present invention can be used for any situation, the production that needs thrombocyte and platelet precursors cell is wherein arranged, or needs the stimulation of mpl acceptor.So for example, compound of the present invention can be used for the treatment of needs thrombocyte, megalokaryocyte, any symptom in the Mammals of waiting.Such symptom is described in detail in the source of giving an example below: WO 95/26746; WO95/21919; WO95/18858; WO 95/21920, and these all are incorporated herein.

Compound of the present invention also can be used to keep the viability of thrombocyte and/or megalokaryocyte and relevant cell or store the lifetime.Therefore, it can be used for comprising at the composition that contains such cell one or several such compound of significant quantity.

" Mammals " is meant any Mammals, comprises the people, and domestic animal is as dog and cat; The animal in external and/or zoological park comprises, monkey, and laboratory animal comprises mouse, rat, and cavy; Farm-animals comprises horse, ox, sheep, goat and pig etc.Preferred Mammals is the people.

V. pharmaceutical composition

The present invention also provides pharmaceutical composition and has used the method for the pharmaceutical composition of The compounds of this invention.Such pharmaceutical composition can be injected, or oral, nasal feeding, use through skin or other forms, for example comprise intravenously, intracutaneous, intramuscular, intramammary, endoperitoneal, in the sheath, intraocular, retrobulbar, (for example, aerosol medication) or subcutaneous injection in the lung (comprising that the long-term storage that discharges is used); By the hypogloeeis, anus, vagina, or by the surgery implantation, for example at the spleen capsule, brain or embedding under cornea.Treatment can one period single dose or multiple doses treatment.Usually, the present invention is appreciated that compound of the present invention and medicine acceptable diluent, sanitas, solvent, emulsifying agent, adjuvant and/or the carrier that comprises significant quantity.Such composition comprises the thinner (for example, Tris-HCl, acetate, phosphoric acid) of various damping fluid content, pH, and ionic strength; Additive such as washing agent, and solvating agent (for example, tween 80, polysorbate80), antioxidant (for example, xitix, sodium metabisulfite), sanitas (for example, Thiomersalate, phenyl alcohol) and a large amount of materials (for example, lactose, sorbyl alcohol); In polymer compound such as poly(lactic acid), in the special preparation of polyglycolic acid etc., or in liposome, mix this material.Hyaluronic acid also can utilize, and this can have in circulation and to promote to keep persistent effect.Pharmaceutical composition or can still comprise the acceptable liquid of other drug, semisolid, or as pharmaceutical carrier, vehicle, or the solid diluent of medium include but not limited to, polyoxyethylene sorbitan mono laurate salt, Magnesium Stearate, methyl-propyl hydroxy benzoate, starch, sucrose, dextran, gum arabic mouth balosam, calcium phosphate, mineral oil, theobroma oil, and theobroma oil.Such composition can influence physical condition, stability, the speed of removing in the speed that discharges in the body and the body of this protein and derivative.Referring to, for example, Remington pharmaceutical science handbook, the 18th edition, (1990, Mack publishing company, Easton, PA 18042), the 1435-1712 page or leaf, the document is incorporated herein by reference.Composition can be prepared into liquid form, can be dry powder maybe, as lyophilized form.Delivery formulations is kept in the implantation that receives publicity equally, as the preparation through skin.

This paper also utilizes oral administration solid dosage form, usually at Remington pharmaceutical science handbook, and the 18th edition, in 1990 (Mack publishing company, Easton PA 18042), 89 chapters narration is arranged, the document is incorporated herein by reference.The solid dosage form comprises tablet, capsule, pill, lozenge or lozenge, cachet or piller.Simultaneously, liposome or proteinoid packing also can be used to prepare composition of the present invention (for example, United States Patent (USP) 4,925, the proteinoid microsphere of report in 673).Can utilize liposomes enclose, liposome can derive with various polymers (for example, United States Patent (USP) 5,013,556).The possible solid dosage form that is used for the treatment of be described in Marshall, K., modern medicines, G.S.Banker and C.T.Rhodes the 10th chapter is stated in 1979, the document is incorporated herein by reference.Usually, said preparation comprises the compound and the opposing gastric environment of invention, the inert fraction of release of bioactive substances in intestines.

What receive publicity equally especially is the oral dosage form of top invention compound.If desired, this compound can be made oral delivery effective by chemically modified.Usually, the chemically modified that receives publicity is that at least one composition is attached to this compound itself, and described composition allows the hydrolysis of (a) arrestin matter; And (b) advance blood by stomach or intestinal absorption.What need equally is the whole stability of strengthening compound, increases intravital cycling time.The example of such composition comprises, polyoxyethylene glycol, the multipolymer of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline (Abuchowski and Davis, soluble polymer-enzyme adducts is as the enzyme of medicine, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, (1981), 367-383; Newmark etc., applied biochemistry magazine, 4:185-189 (1982)).Utilizable other polymers are poly--1,3-dioxolane and poly--1,3,6-tioxocane.Having preferred pharmaceutical use, is the polyoxyethylene glycol composition as mentioned above.

For the dosage form of oral delivery, may utilize the salt of the aliphatic amino acid of modification equally, as N-(8-[2-(2-hydroxybenzoyl)] amino) Sodium octoate (SNAC), can be used as the carrier of the absorption that strengthens treatment compound of the present invention.Utilize the clinical efficiency of the heparin preparations of SNAC in the II chapter of " Emisphere technical manual ", to obtain proof, referring to United States Patent (USP) 5,792,451, " oral pharmaceutical are sent and are formed and method ".

Therapeutical agent can be included in the preparation as the trickle microparticle of the particle of the granular size of about 1mm or piller form.The preparation of the material that capsule is used also can be a powder, the suppository of light compression, or even use as tablet.Therapeutical agent can prepare by compressing.

Toner and seasonings can be included.For example, can prepare protein (or derivative) (as liposome or microsphere packing), further be included in the edible product then, as contain the frozen beverage of toner or seasonings.

People can dilute or improve the volume of the therapeutical agent with inert substance.These thinners can comprise carbohydrate, particularly, and N.F,USP MANNITOL, lactose, lactose hydrous, Mierocrystalline cellulose, sucrose, the dextran of modification, and starch.Some inorganic salt also can be used as weighting agent, comprise calcium triphosphate, magnesiumcarbonate and sodium-chlor.The thinner that some commerce can get is Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell.

Disintegrating agent can be included in the preparation of therapeutical agent of solid dosage form.Material as disintegrating agent includes but not limited to starch, based on starch, and the commercial disintegrating agent of dispersive tablet.Primojel, Amberlite, Xylo-Mucine, over-expense chain starch, alginate sodium, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite can utilize.The other form of disintegrating agent is insoluble Zeo-karb.The glue of powdered can be as washing agent with as wedding agent, and these can comprise plastic powder, as agar, and Karaya or tragakanta.Alginic acid and its sodium salt also can be used as disintegrating agent.

Binding substances can be combined together to form therapeutical agent hard tablet, and comprises from natural product such as gum arabic, tragakanta, the material of starch and gelatin.Other comprise methylcellulose gum (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinylpyrrolidone (PVP) and HYDROXY PROPYL METHYLCELLULOSE (HPMC) may be used to ethanolic soln with the therapeutical agent granulating.

The anti-friction liniment can comprise in the preparation to prevent the adhesion in process for preparation.Lubricant can be as the aspect between therapeutical agent and the die wall, and these can include but not limited to, stearic acid comprises its magnesium and calcium salt, polytetrafluoroethylene (PTFE), whiteruss, vegetables oil, and paraffin.Soluble lubricant also can utilize the Sodium Lauryl Sulphate BP/USP of various molecular weight, lauryl magnesium sulfate, polyoxyethylene glycol, Carbowax 4000 and 6000.

Can improve the flow characteristics of process for preparation Chinese traditional medicine and the antiseize paste of the rearrangement in the compression process also can add.Antiseize paste can comprise starch, and talcum powder is forged system silica and hydrated aluminosilicate.

For auxiliary therapeutical agent dissolves, can add tensio-active agent as wetting agent in water surrounding.Tensio-active agent can comprise, teepol such as Sulfuric acid,monododecyl ester, sodium salt, dioctyl sulfuric acid sodium succinate and dioctyl sodium sulfonate.Cationic detergent also can use and can comprise Benzalkonii Chloridum or benzyl rope chloramines.The series as the potential nonionic washing agent of tensio-active agent that can be included in the preparation is the bay poly(oxyethylene glycol) 400, polyoxy 40 stearate, polyoxyethylene hydrogenated castor oil 10,50 and 60, the glycerine Monostearate, polysorbate40,60,65 and 80, sucrose fatty ester, methylcellulose gum and carboxymethyl cellulose.These tensio-active agents can protein or derivative exist separately or as the preparation of the mixture under the different ratios.

The additive that can strengthen to potentiality the absorption of compound for example is a lipid acid oleic acid, linoleic acid plus linolenic acid.

The preparation that discharges control may also need.Such medicine may mix the inert base that permission discharges by diffusion or leaching mechanism, for example glue.Chronic matrix degradation also can mix in the preparation, as alginic acid, and poly-polysaccharide.Another form of the sustained release of this therapeutical agent is by the method (Alza company) based on the Oros therapy system, and promptly such medicine is included in because osmotic effect allows water to enter and promotes medicine from the semipermeable partition that an osculum is gone out.Some enteric coatings also have postpones the effect that discharges.

Other dressings also can be used for preparation.These comprise the various sugar that can be used for coating pan.Treatment reagent can be the film coating tablet that provides also, and the material that is used for this situation can be divided into 2 groups.First group is the material of non-intestines and comprises methylcellulose gum, ethyl cellulose, Natvosol, methyl hydroxyl ethyl cellulose, hydroxy propyl cellulose, HYDROXY PROPYL METHYLCELLULOSE, sodium carboxy methyl cellulose, pyrrolidone and polyoxyethylene glycol.Second group comprises intestines material, normally phthalic ester.

The mixture of these materials can be used to provide optimal film coating.Film coating can be undertaken in dish dressing device or in the liquid bed or by the compression dressing.

What receive publicity equally herein is that protein of the present invention (or derivatives thereof) is delivered to lung.When protein of the present invention (or derivative) when being delivered to mammiferous lung, just suck and transmit endodermis by lung in blood.(other reports comprise Adjei etc., drug research, 7:565-569 (1990); Adjei etc., pharmacology international magazine, 63:135-144 (1990) (leuprolide acetate); Braquet etc., cardiovascular agent is learned magazine, 13 (the 5th volumes): s.143-146 (1989) (endotheliocyte element-1); Hubbard etc., international medical science annual, 3:206-212 (1989) (1-antitrypsin); Smith etc., Journal of Clinical Investigation, 84:1145-1146 (1989) (1-proteolytic enzyme); Oswein etc., " proteinic aerosol thinner ", respiratory medications is sent symposium summary II, Keystone, Colorado, March nineteen ninety (recombinant human somatropin); Deb etc., Journal of Immunology, 140:3482-3488 (1988) (Interferon, rabbit-and tumour necrosis factor) and Platz etc., United States Patent (USP) 5,284,656 (granulocyte colony-stimulating factors).

What receive publicity in the utilization of practice of the present invention is the many mechanisms of treatment product that design treatment lung is sent, and includes but not limited to atomizer, metered-dose inhaler, and powder inhalator, and all these is that those skilled in the art are familiar with.

Being suitable for the example that some special commerce of practice of the present invention can install is the Ultravent aerosolizer, is made St. Louis, the Missouri State by Mallinckrodt company; Acorn II aerosolizer is made by Marquest medical product company, at Englewood, and the Colorado; The Ventolin metered dose inhaler is made by Glaxo company, Research Triangle park, the North Carolina that; Suck powder inhalator with rotation, make Bedford, Massachusetts by Fisons company.

All these devices need to utilize the preparation of the compound that is suitable for distributing invention.Usually, each preparation is the type that is specific to the device of utilization, and can comprise and utilize suitable propelling agent material, and thinner, adjuvant, and/or the carrier in being used for the treatment of.

Compound of the present invention the most advantageously should be prepared into mean particle size less than 10 μ m (or micron), 0.5 to 5 μ m most preferably, the far-end of the lung that can be delivered to most effectively.

Carrier comprises carbohydrate, as trehalose, and N.F,USP MANNITOL, Xylitol, sucrose, lactose and sorbyl alcohol.Other compositions that are used for preparing can comprise DPPC, DOPE, DSPC and DOPC.Can utilize natural or the synthetic tensio-active agent.Can utilize polyoxyethylene glycol (even separate with its purposes when derived protein or the analogue).Dextran also can be utilized as the ring dextran.The toughener that cholate is relevant with other also can utilize.Mierocrystalline cellulose and derivatived cellulose also can utilize.Amino acid also can utilize, as is used for buffer formulation.

Simultaneously, liposome, microcapsule or microsphere, the purposes of carrier that includes the other types of mixture or carrier also receives publicity.

Be applicable to and utilize aerosolizer, promptly spray or hyperacoustic preparation will comprise the compound of the present invention that the concentration with about 0.1 to the 25mg biological activity protein of every ml solution is dissolved in the water usually.Said preparation also comprises damping fluid and simple the sugar adjusting of protein stabilization and osmotic pressure (for example, for).Aerosol formulation also can contain tensio-active agent, so that the proteinic gathering that the atomize of the solution of the formation aerosol of minimizing or prevention spatial induction causes.

Utilize the preparation of note amount inhaler device will comprise the powder of dispersive fine that contains the auxiliary low suspension compound of the present invention in propelling agent of tensio-active agent usually.Propelling agent can be any conventional substances that is used for this purpose, as Chlorofluorocarbons, and Chlorofluorocarbons (CFCs), fluorohydrocarbon, or hydrocarbon comprise trichlorofluoromethane, Refrigerant 12, dichloro-tetrafluoro ethanol and 1,1,1,2-Tetrafluoroethane, or their combination.Suitable tensio-active agent comprises sorbitanic trioleate and soybean lecithin.Oleic acid also can be used as tensio-active agent.

The dry powder that will comprise the fine dispersion that contains compound of the present invention from powder inhalator device dispersive preparation, and also can comprise swelling agent, as lactose, sorbyl alcohol, sucrose, N.F,USP MANNITOL, trehalose or Xylitol, their amount can be simplified powder diffusion from device, for example 50 of weight of formulation to 90%.

The nasal delivery of compound of the present invention also should be paid close attention to.Nasal delivery allows directly protein to be delivered to blood after product is administered to nose will treating, and can be in lung sedimentation products.The preparation of nasal delivery comprise with dextran or the ring dextran those.Delivering method by other mucous membrane transmission receives publicity too.

Dosage

The dosage that comprises will be by the doctor who participates in the method for treatment above-mentioned symptom, consider to change the various factors of the effect of medicine, patient's age for example, symptom, body weight, sex, and diet, the seriousness of any infection, the time of using and other clinical factors are determined.

Compound of the present invention can make medication group at first, and the treatment cyclical level of keeping medicament production of then infusing is continuously used.As another example, compound of the present invention can be used as disposable dosage and uses.Those of ordinary skill in the art makes the clinical symptom of effective dose and application program such as outstanding medical approaches and individual patient determine easily and reaches optimized.The frequency of dosage will be according to the pharmacokinetic parameter of reagent of using and approach.Best pharmaceutical preparation will be determined according to the dosage of approach of using and needs by those skilled in the art.Referring to for example, Remington pharmaceutical science handbook, the 18th edition, (1990, Mack publishing company, Easton, PA 18042 (1435-1712) page or leaf, the disclosure thing is incorporated herein by reference.Such preparation can influence the physical condition of the reagent of using, stability, the speed of removing in speed that discharges in the body and the body.According to the approach of using, can be according to body weight, body surface area or organ size are calculated suitable dosage.Determine to comprise the needed meticulous calculating of suitable dosage that each preparation above-mentioned is used for the treatment of normally those of ordinary skills just do not need to experimentize can routine operation, dosage information disclosed herein and experiment have particularly been arranged, and easier carrying out during observed pharmacokinetic data in the people's who discusses in the above the clinical experiment.Suitable dosage can determine that blood levels dosage and suitable dose response data determine by the experiment of having set up.Last dosage will be the doctor by participating in, consider the various factors of the effect of change medicine, for example, the specific activity of medicine, the seriousness of damage and patient reply, patient's age, symptom, body weight, sex and diet, the seriousness of any infection, the time of using and other clinical factors are determined.When studying, about suitable dosage level, news will appear in the time span of the treatment of various diseases and symptom.

In treatment other symptoms and platelet defect, methods of treatment of the present invention, composition and compound can use separately, or with other cytokines, soluble mpl acceptor, Hemopoietic factor, LeIF, somatomedin or antibodies are used.Can expect that compound of the present invention can combine with general hemopoiesis stimulant (as IL-3 or GM-CSF), useful when the thrombopenia of some forms of treatment.Other megakaryocyte stimulating factors, i.e. meg-CSF, STEM CELL FACTOR (SCF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megalokaryocyte stimulating activity also can use with the mpl part.The collaborative cytokine of using like this or other examples of Hemopoietic factor comprise IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony-stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony-stimulating factor (G-CSF), EPO, interferon-' alpha ' (IFN-α), the synonym Interferon, rabbit, IFN-β, IFN-γ, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, thrombopoietin (TPO), angiogenin, for example, Ang-1, Ang-2, Ang-4, Ang-Y, the human angiogenin similar polypeptide, vascular endothelial growth factor (VEGF), angiogenin, bone morphogenetic protein 1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein acceptor IA, bone morphogenetic protein acceptor IB, the close neural factor of brain origin, ciliary parent neural factor, ciliary parent neural factor acceptor, the neutrophilia CF 1 of cytokine induction, the neutrophilia CF 2 of cytokine induction, endothelial cell growth factor (ECGF), endothelin 1, Urogastron, the neutrophilia attractant of epidermis origin, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acid, fibroblast growth factor alkali, the close neural factor acceptor 1 of glial cell-line origin, the close neural factor acceptor 2 of glial cell-line origin, growth associated protein matter, heparin associative list skin growth factor, pHGF, hepatocyte growth factor receptor, insulin analogous growth factor I, insulin-like growth factor acceptor, insulin-like growth factor receptor II, the insulin-like growth factor conjugated protein, keratinocyte growth factor, leukaemia inhibitory factor, leukaemia inhibitory factor acceptor, nerve growth factor, trk C, close neural factor-3, close neural factor-4, placenta growth factor, placenta growth factor 2, thrombocyte origin endothelial cell growth factor (ECGF), thrombocyte origin somatomedin, thrombocyte origin somatomedin A chain, thrombocyte origin somatomedin AA, thrombocyte origin somatomedin AB, thrombocyte origin growth factor B chain, thrombocyte origin growth factor B B, thrombocyte origin growth factor receptors, thrombocyte origin growth factor receptors, pre-B cell growth-stimulating factor, the STEM CELL FACTOR acceptor, TNF comprises TNF0, TNF1, TNF2, transforming growth factor, transforming growth factor, transforming growth factor 1, transforming growth factor 1,2, transforming growth factor 2, transforming growth factor 3, transforming growth factor 5, potential transforming growth factor 1, the growth factor binding proteins I of conversion, the growth factor binding proteins II of conversion, the growth factor binding proteins III that transforms, I type Tumor Necrosis Factor Receptors, II type Tumor Necrosis Factor Receptors, urokinase-type plasminogen activator acceptor, vascular endothelial growth factor and chimeric protein and its biology or immunocompetence fragment.Simultaneously or the solvable Mammals mpl acceptor of using significant quantity in order may be useful, in case megalokaryocyte reaches mature form, as if described acceptor has the megalokaryocyte of causing becomes hematoblastic effect to fragment.So, use compound of the present invention (strengthening sophisticated Megakaryocytic number), then use soluble mpl acceptor (inactivation part, and allow sophisticated megalokaryocyte to produce thrombocyte) and can wish it is the especially effective stimulating platelet method of producing.The dosage of quoting as proof more above will be adjusted to and replenish other such compositions in therapeutic composition.The patient's who has treated state can detect by the method for routine.

In compound of the present invention has joined situation in thrombocyte and/or Megakaryocytic composition and the relevant cell, the amount that comprises will be by technology known in the art and experiment usually, determine in experiment.The scope of amount as an example is per 10 ⁶Individual cell 0.1 μ g-1mg compound of the present invention.

Should be appreciated that content application of the present invention will hold in this paper comprises in special problem or situation, in the limit of power of the those of ordinary skill in this area.The exemplary process of the separating of the example of product of the present invention and they, purposes and manufacturing is described below.

Embodiment

Stated the example methodology of making and identify some compounds disclosed herein below.

Embodiment 1

Make up the secondary peptide library

A. prepare electroreception attitude Bacillus coli cells:

At 37 ℃, at the 2xYT of 10ml substratum (1.6%Bacto Tryptones, 1% yeast extract, 85.5mMNaCl) middle culture of Escherichia coli (the TG1 bacterial strain that spends the night for preparing; Amersham Pharma Biotech, Piscataway, New Jersey).This overnight culture of 1 microlitre is used to inoculate 1 liter contains 0.4% glucose and 1mM MgCl ₂The 2xYT substratum, at 37 ℃, in a jolting device growth this rise culture up to OD ₆₀₀=0.8.Frozen cultures on ice 15 minutes, at 4 ℃, 4000rpm centrifugal (Beckman JA-10 whizzer) 20 minutes.Resuspending bacterial precipitation in the glycerine solution of the ice refrigerated 10% of 500ml is at 4 ℃, in the centrifugal mixture that obtains of 4000rpm 20 minutes.Once more in ice refrigerated 10% glycerine solution of 500ml at the suspension bacterial precipitation, at 4 ℃, the mixture that obtains at the 4000rpm recentrifuge 20 minutes.Resuspending bacterial precipitation once more in the 10% freezing glycerine solution of 500ml, at 4 ℃, at 4000rpm, the mixture that recentrifuge obtains 20 minutes.Then, resuspending cell precipitation in the glycerine solution of the ice refrigerated 10% of 25ml.This spissated bacteria samples is transferred in the garden Taper Pipe of ice refrigerated 50ml, 4 ℃ in desk centrifuge (Beckman CS-6R), centrifugal at 3500rpm.Freeze resuspending cell precipitation in the glycerine solution at small volume ice-cold,, and under-80 ℃, keep in cold storage immediately at ethanol/the dry ice bath freezing 100 or 300 μ l bacterium reserves.

The modification of B.pCES1 carrier

Utilize the long template PCR system (Roche Diagnostics company, Indianapolis, Indiana State) of expansion, as template, carry out the PCR reaction with 1 μ gpCES, 1 carrier (TargetQuest company).The volume of PCR mixture is 100 μ l, wherein contain 1X PCR damping fluid, each two primer of 200nM, 5 '-CAAACGAATGGATCCTCATTAAAGCCAGA-3 ' and 5 '-GGTGGTGCGGCCGCACTCGAGACTGTTGAAAGTTGTTTAGCA-3 ', 200nM dNTP, 3U Tag archaeal dna polymerase.TRIO-Thermoblock (Biometra) PCR system is used to carry out following procedure: 94 ℃ 5 minutes, 30 circulations [94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 45 seconds]; 72 ℃ 10 minutes, be cooled to 4 ℃.At the electrophoresis of the enterprising performing PCR product of 1% agar gel, carry out purifying with QIAGEN Spin Column (QIAGEN company, Valencia, Gary Fu Niya) according to the scheme of manufacturers.With 5 μ l PCR products, each two primer of 200nM, 5 '-CAAACGAATGGATCCTCATTAAAGCCAGA-3 ' and 5 '-AACACAAAAGTGCACAGGGTGGAGGTGGTGGTGCGGCCGCACT-3 ' utilizes identical as mentioned above PCR condition to carry out second PCR reaction.

At 37 ℃, containing the 1xNEB2 damping fluid, separating digesting PCR product and pCES11 originally hour in the reaction of the 100 μ l of the ApaLI of 60U (New England's biology laboratory, Beverly, Massachusetts).With the DNA of QIAGEN Spin column purification digestion, and contain in the reaction mixture that 1x connects damping fluid and 40U T4 dna ligase (New England's biology laboratory) at 40 μ l, at room temperature spending the night links together.

Carrier is transferred in the intestinal bacteria, and at 37 ℃ of incubations that spend the night.Select isolating single bacterium colony, with QIAGEN Spin column purification.Confirm to insert accurately with dna sequencing.

C. prepare carrier DNA

Utilize gene pulse producer II (BIO-RAD, Hercules, Gary Fu Niya), be set in 2500V, 25 , the p CES1 carrier DNA (part 1B) that 200ohms modifies 1 microgram transforms 100 μ l electroreception attitude TG1 intestinal bacteria (part 1A).Then, the bacteria samples that transforms is transformed into contain 900 μ l SOC (2% peptone, 0.5% yeast extract, 10mMNaCl, 2.5mM KCl, 20mM glucose, 10mMMgSO immediately ₄, 10mM MgCl ₂) test tube in, and allow this culture at 37 ℃, jolting 1 hour is 37 ℃ of growths.Then, at 2xYTAG (2xYT) with 100 μ g/ml penbritins and 2% glucose, agar plate upper berth cell, and at 37 ℃ of incubations that spend the night.At 37 ℃, utilize the 2xYTAG of 1 liter of single colony inoculation, jolting is spent the night.With qiagen plasmid Maxi test kit, come the plasmid purification carrier DNA according to the scheme of manufacturers.

D. digested vector DNA

Containing 1xNEB damping fluid 2, digestion 50 microgram carrier DNAs (part 1C) spend the night at 37 ℃ in the reaction mixture of the 400 μ l of the ApaLI of 200U and 200U XhoI.At 37 ℃ of incubation restriction digestion reaction mixtures that spend the night, in the agar gel (Embi Tec, San Diego, Gary Fu Niya) of ready-formed 1%, analyze.According to the guidance of manufacturers, from gel, cut out linear carrier DNA, extract (QIAGEN company) with QIAquick gel extraction test kit.

E. prepare the library oligonucleotide

Design two library oligonucleotide (fixed and prediction).Synthesized fixed library oligonucleotide 5 '-

CACAGTGCACAGGGTNNKNNKNNKNNKGGTCCTACTCTGMRKSARTGGCTGNNKNNKNNKNNKNNKNNKCATTCTCTCGAGATCG-3′

And prediction (doped) library oligonucleotide 5 '-

5′-CACAGTGCAC-AGGGTNNKNNKNNKNNKggKcc-KacKctKNNKNNKtgKNNKNNKNNKNNKNNKNNKNNKCATTCTCTCGAGATCG-3′

(lowercase is represented the mixture of 70% base that indicates and each other three kinds of Nucleotide of 10%).In these oligonucleotide each can be as the template in the polymerase chain reaction.

Utilize the accurate PCR of unfolded height system test kit (Roche diagnostic companies) to carry out the PCR reaction.It is 100 μ l that each PCR is reflected on the volume, contains the library oligonucleotide of 10nM, 1XPCR damping fluid, each primer 5 ' of 300nM-CACAGTGCACAGGGT-3 ' and 5 '-TGATCTCGAGAATG-3 ', 200nM dNTP, 2mMCaCl ₂And the polysaccharase of 5U expansion.Utilize thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, Inc.) to carry out following procedure: 94 ℃ 5 minutes, 30 round-robin [94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds]; 72 ℃ 7 minutes, be cooled to 4 ℃.Utilize QIAquick Nucleotide to remove test kit (QIAGEN company) and remove free Nucleotide according to the scheme of manufacturers.

F. the digestion of library oligonucleotide

Containing 1xNEB damping fluid 2, each PCR product (part IE) of digestion 5 micrograms in the reaction mixture of the 400 μ l of 200U ApaLI and 200U XhoI, 37 ℃ are spent the night.The DNA of separating digesting on 3% agar gel (Embi Tec).DNA band from the needs of each reaction cuts out from gel, with the extraction of QIAquick gel extraction test kit.

G. carrier is connected with the library oligonucleotide

Containing the 400 μ l reaction mixtures that 1xNEB connects the T4 dna ligase of damping fluid and 80U, spending the night at 16 ℃ connects the PCR product (1F part, 5 μ g) of linearizing carrier (1D part, 25 μ g) and each digestion.At 65 ℃, the product that incubation connects 20 minutes, the inactivated dna ligase enzyme, further 37 ℃ with 8U NotI incubation 2 hours, carrier self is connected minimizes.Then, the product (molecular cloning, Maniatis et al, the 3rd edition) that the phenol by standard/chloroform extraction purifying connects, resuspending is in the H of 30 μ l ₂Among the O.

H. electroporation transforms

For each library, carry out 10 electroporation reactions.For each conversion, the carrier DNA (1G part) of the connection of (BIO-RAD) mixing 3 μ l and the TG1 cell (1A part) of 300 μ l in the tubule of 0.2cm.By gene pulse device II, be set in 2500V, the mixture that 25uF and 200ohms pulse obtain.To merge from the transform bacteria sample of 10 electroporations reaction, and shift to advance to contain in the SOC flask of 27ml, 37 ℃ of incubations 1 hour.Then, in 170ml 2xYTAG, add cell, 37 ℃ of growths, jolting 3 hours.At the 5000rpm eccentric cell, 4 ℃, 10 minutes.Then, resuspending cell precipitation in glycerine/2xYT of 15% of 10ml is stored in-80 ℃.This is the reserve in initial library.Titration shows that the library is respectively that size is 1.0 * 10 in fixed and the prediction library ⁹Individual independently transformant and 2.4 * 10 ⁹Individual independently transformant.

2. amplification library

A. make the secondary reserves thing (stock) in library

Utilize elementary library cell reserve (1H part) inoculation 1300ml (for the fixed library) and 2600ml (for the prediction library) 2xYTAG substratum, make initial OD ₆₀₀=0.1.Allow culture 37 ℃ of growths, jolting several hours is up to OD ₆₀₀=0.5.Take out the aliquots containig in the prediction library of the aliquots containig in fixed library of 120ml and 240ml, in other flask 37 ℃ of regrowths 2 hours.Under 4 ℃, centrifugal these subculture things of 5000rpm (Beckman JA-14 whizzer) 10 minutes, resuspending bacterial precipitation in glycerine/2xYT of 10ml (for each library) 10% was-80 ℃ of storages.

B. phage induction

(Amersham Pharma Biotech) joins OD with M13KO7 helper phage aliquots containig ₆₀₀The remaining bacterial cultures of=0.5 (2A part), to the last concentration is 3 * 10 ⁹Pfu/ml.Allow helper phage 37 ℃ of not jolting bacterial infections 30 minutes, slow jolting was infected 30 minutes.At 4 ℃, in the cell of the centrifugal infection of 5000rpm 10 minutes.With 2xYTAK (2YT with 100 μ g/ml penbritins and the 40 μ g/ml kantlex) resuspending of cell precipitation with 1300ml (fixedly library) and 2600ml (prediction library).Allow to carry out phagemid production 37 ℃ of joltings of spending the night.

C. gather in the crops phage

At 4 ℃, the centrifugal bacterial cultures of 5000rpm (2B part) 10 minutes.Supernatant liquor is transferred in the new bottle, added the 20%PEG/2.5M NaCl of 0.2 volume, incubation is 1 hour on ice bath, the precipitation phagemid.At 4 ℃, the phagemid of 8000rpm centrifugation 20 minutes.Careful with the cold PBS resuspending of 100ml.By at 4 ℃, removed remaining cell in centrifugal 10 minutes with 8000rpm, and be further purified phagemid solution by the 20%PEG/2.5M NaCl precipitation phagemid that adds 0.2 volume.At 4 ℃, the centrifugal phagemid of 8000rpm 20 minutes, with the cold PBS resuspending of 12ml phagemid precipitation.In phagemid solution, add the glycerine solution of 4 microlitres 60%, be stored in-80 ℃.Determine phagemid tire (molecular cloning, Maniatis et al, the 3rd edition) by the method for standard.

3. people MPL is in conjunction with the selection of phage

A. the biotinylation of people MPL

The Sulfo-NHS-LC-biotinylation test kit that utilizes EZ-to connect, according to the guidance of manufacturers with 1 microgram recombinant human MPL biotinylation.

B. fixing MPL on magnetic bead

Since from the concentration of per 100 μ l globule reserves, 1 μ g MPL of manufacturers biotinylated MPL (3A part) is fixed on Dyn globule M-280 streptavidin (DYNAL, Lake Success, New York).Globule is attracted to a side of test tube under magnetic attraction, inhales and remove liquid, with phosphate-buffered salt (PBS) washing globule twice, resuspending is in PBS.With top concentration biotinylated MPL protein is added on the globule of washing, at room temperature rotated incubation 1 hour.Then, the globule of the final concn sealing MPL dressing by adding BSA to 2% is incubated overnight 4 ℃ of rotations.Then, the globule of the MPL dressing that before carrying out chosen process, obtains with PBST (having 0.05% tween 20) washed twice.

C. utilize the globule of MPL dressing to select

(the 2C part is for fixing library 1 * 10 when phagemid to seal about 100 times library etc. with the PBS that contains 2% BSA of 1ml ¹¹Cfu is for prediction library 2.4 * 10 ¹¹Cfu).By it being joined blank globule (identical globule as the 3B part, but be not the MPL dressing), the phagemid sample of sealing bear the selection step, at room temperature rotated this mixture of incubation 1 hour.Utilize magnetic that the phagemid that contains in the supernatant liquor is attracted out, transfer to the new test tube (3B part) of the globule that contains the MPL dressing, at room temperature rotated this mixture of incubation 1 hour.After abandoning supernatant liquor, wash phagemid bonded globule 10 times with PBST, with PBS washing 10 times.Then, allow phagemid on whizzer, wash-out is 10 minutes in 1ml 100mM triethylamine solution (Sigma, St. Louis, the Missouri State).The pH that contains the solution of phagemid by 1MTris-HCl (pH7.5) neutralization that adds 0.5ml.At 37 ℃, the phagemid that not jolting utilization obtains infects the TG1 bacterium (OD of the fresh growth of 5ml ₆₀₀About 0.5) 30 minutes and slow jolting were infected 30 minutes.At the TG1 cell of dull and stereotyped all infection of upper berth of big 2xYTAG, at 30 ℃ of incubations that spend the night.

D. induce and gather in the crops phage

Go up adding 10ml 2xYTAG substratum aliquots containig, resuspending TG1 cell at dull and stereotyped (part 3C).In test tube, collect all TG1 cells, in the 2xYTAG of 25ml, add the aliquots containig of 250 these cells of μ l, and 37 ℃ of growths up to OD ₆₀₀=0.5.Adding M13K07 helper phage to the last concentration is 3 * 10 ⁹Cfu/ml, and at 37 ℃ of incubations, not jolting 30 minutes and slow jolting 30 minutes.At 4 ℃, 5000rpm eccentric cell 10 minutes, with the 2xYTAK resuspending of 25ml.When allowing jolting on 30 ℃ of these bacteriums of overnight growth.Results inductive phagemid, and as purifying in the 2C part.

E. second take turns selection

Except following step, partly point out to carry out second as 3B to 3C and take turns selection.The aliquots containig of about 0.5ml of the phagemid solution that will obtain from the 3D section is as the phagemid of input.Biotinylated MPL (3A part) dressing that has only 0.1 μ g is to Dyna globule M-280 streptavidin.In conjunction with globule 16 times, last washing is included among the PBST with PBST washing phage, and at room temperature incubation is 30 minutes.Wash globule more than 10 time with PBS.

4. clonal analysis

A. preparation is main dull and stereotyped

Select the second single bacterium colony of taking turns selection, contain incubation in the 96 hole flat boards of 2xYTAG of 120 μ l in each hole.Incubation 96 hole flat boards spend the night in 30 ℃ of jolting devices.60% of adding 50 microlitres glycerine is used for-80 ℃ of storages in each hole.

B. phagemid ELISA

In the future autonomous dull and stereotyped about 3 μ l aliquots containig cell inoculations contain to every hole in the 96 fresh hole flat boards of 2xYTAG of 120 μ l, at 37 ℃ of these new flat boards of growth up to about OD ₆₀₀=0.5.The 2xYTAG (1.2 * 10 that contains M1 3K07 helper phage that in each hole, adds 50 microlitres ¹⁰Cfu/ml), not jolting, dull and stereotyped 30 minutes, other 30 minutes of slow jolting incubation in 37 ℃ of incubation 96 holes.At 4 ℃, centrifugal dull and stereotyped 10 minutes of 2000rpm (Beckman CS-6R desk centrifuge).From the hole, remove supernatant liquor, every hole 160 each cell precipitations of μ l 2xYTAK resuspending.Being used for phagemid at 30 ℃ of incubation flat boards that spend the night expresses.

At 4 ℃, with 5 μ g/ml in the 0.1M carbonic acid buffer, pH9.6 spends the night the people MPL dressing (NUNC) to the 96 hole Maxisorp flat boards of reorganization.In contrast, with 5 μ g/ml with BSA (Sigma) dressing to isolated M axisorp flat board.

At second day, at 4 ℃, the cell culture that spends the night centrifugal 10 minutes at 2000rpm.The supernatant liquor of 20 microlitres in each hole is transferred in the 96 new hole flat boards of 2% the BSA/PBS solution that contains 180 μ l in each hole.At room temperature the mixture that obtains of jolting incubation is 1 hour, the sealing phagemid.Simultaneously, in jolting, at room temperature with the flat board of 2% the BSA/PBS solution sealing MPL dressing of every hole 200 μ l 1 hour.Abandon BSA solution, use each hole of PB ST solution washing 3 times.Behind last washing step, in dull and stereotyped each hole of the dull and stereotyped of MPL dressing and contrast, add the sealing phagemid solution of 50 μ l, and when jolting incubation 1 hour at room temperature.Abandon liquid once more, use each hole of PBST solution washing 3 times.With 1: 15,000 extent of dilution added the anti-M13mAb of HRP bonded (Amersham Pharmacia biotech company) of 50 microlitres in dull and stereotyped each hole of MPL dressing and contrast, with these flat boards room temperature jolting incubation 1 hour.Abandon liquid once more, use each hole of PBST solution washing 3 times.In the hole, add 50 microlitre LumiGLO chemical luminous substrate (Kirkegaard﹠amp; Perry laboratory, Gaithersburg, Myelosan state), by Luminoskan AscentDLRearly machine with each hole reading (laboratory system company, Franklin, Massachusetts).

C. the order-checking of phage clone

The 1 μ l bacterium (4A part) that is used to each hole of autonomous flat board carries out the PCR reaction as template.The volume of each PCR mixture is 20 μ l, wherein contains the 1xPCR damping fluid, each two primer of 300nM, 5 '-GTTAGCTCACTCATTAGGCAC-3 ' and 5 '-GTACCGTAACACTGAGTTTCG-3 ', 200nM dNTP, 2mM CaCl ₂And 5Utaq archaeal dna polymerase (Roche molecular biochemistry company).Utilize GeneAmp PCR system 9700 (Applied Biosystems, Inc.) to carry out following procedure: 94 ℃ 5 minutes; 40 circulations [94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 90 seconds]; 72 ℃ 10 minutes; Be cooled to 4 ℃.With QIAquick 96 PCR purification kits (QIAGEN company), according to the purified pcr product that instructs of manufacturers.With primer 5 '-CGGATAACAATTTCACACAGG-3 ', utilize ABI 3770 sequence instrument, according to the explanation of manufacturers PCR product order-checking with all purifying.

5. sequence classification

Relevant from the peptide sequence of top nucleotide sequence translation with the ELISA data.In the hole of MPL dressing, show high OD reading and show that in the hole of BSA dressing the clone of low OD reading may be thought of as the material standed for of further research.Repeatedly sequence takes place also think the material standed for that can be used as further research.Can further in the ELISA titration experiments, identify according to the phage clone that these principles are selected.Referring to Fig. 9 (the ELISA dose response of the phage clone of selection).

Embodiment 2

The preparation of peptide

Prepare all peptides by fixed solid phase synthesis process progressively.Merrifield(1963)，J.Am.Chem.Soc.85：2149。Steward and Young (1969), solid-phase peptide is synthetic.The amino acid of Fmoc protection is used as the structure piece, utilizes ABI or Symphony peptide synthesizer to make up peptide chain.Usually, peptide is synthetic to be that Wang resin with preload begins, and being created in C-terminal has free carboxy acid's peptide (perhaps, can utilize the Rink production of resins to have the functional peptide of C-terminal acid amides).Removing of Fmoc is to carry out in the piperidines scheme of standard.Utilize uronium (as HBTU) or carbodiimide (as DCC/HOBt) chemistry to carry out coupling.The side chain protected group is: Glu (O-t-Bu), Asp (O-t-Bu), Ser (t-Bu), Thr (t-Bu), Arg (Pbf), Asn (Trt), Gln (Trt), His (Trt), Lys (t-Boc), Trp (t-Boc), and Cys (Trt).Use RT, 4 hours, utilize and contain 2.5%H ₂O, 5% phenol, the trifluoroacetic acid (TFA) of 2.5% triisopropyl siloxanes and 2.5% sulphur phenylmethylether or mercaptoethanol carry out last the going of all peptide-based resins and protect and cracking.After removing TFA, with cold anhydrous ether precipitation cracked peptide.For those peptides that contain disulfide linkage, utilize the thick material of the DMSO (pH7.5) of 15% in the water directly to form cyclisation product.By all former peptides of anti-phase HPLC purifying, prove the structure of the peptide of purifying by ESI-MS and amino acid analysis.

Embodiment 3

The preparation of TMP-Fc peptide body compound

Select several peptides to be used for merging and express (that is the Fc that, is attached to the C-terminal of peptide) (C-terminal fusion) as peptide-Fc.Place coding and the dna sequence dna of each TPO simulating peptide under the control of the luxPR promotor in following plasmid expression vector pAMG21 in the human IgG1's of framework endomixis Fc district.

Change the plasmid (Amgen bacterial strain #3788) of coding TMP1-Fc,, allow easily from annealed oligonucleotide clone small peptide so that contain ApaLI site and XhoI site.Primer 2 396-69 is used to add ApaLI and XhoI restriction enzyme sites.Utilize the long polysaccharase of expansion of 2396-69 and ubiquity 3 ' the primer 191-24 on 3788 dna profilings to carry out PCR.Primer sequence is as follows:

2396-69 ACAAACAAACATATGGGTGCACAGAAAGCGGCCGCAAAAAAACTCGAGGGTGGAGGCGGTGGGGACA

191-24 GGTCATTACTGGACCGGATC

With NdeI and BsrGI, gel-purified digests the PCR fragment that obtains, and as inserting fragment.Plasmid from bacterial strain #3788 also digests with NdeI and BsrGI, and gel-purified is as carrier.Carrier and insertion fragment are linked together, the connection mixture electroporation that obtains is advanced GM221 cell (referring to as follows).Select single bacterium colony, the preparation plasmid DNA is with dna sequencing.A plasmid that obtains, 200003180 show to have dna sequence dna accurately, and as making up the carrier that TMP-Fc merges.Carrier is presented among Fig. 6.

With ApaLI and XhoI digested plasmid 200003180, and as carrier.With every pair of oligonucleotide (referring to Fig. 7) annealing, form amphiploid with ApaLI and XhoI sticky end.These molecules are connected the into fused protein of carrier generation needs.The ApaLI to reply of each oligonucleotide is provided at Fig. 7 to the XhoI fragment.

TMP 1-23,25,26,28 express as the C-terminal syzygy.

Embodiment 4

The preparation of Fc-TMP peptide body compound

Some peptides are expressed as Fc peptide syzygy (that is, Fc is attached to the N-terminal of peptide) (N-terminal fusion).Change the plasmid (Amgen bacterial strain #3728) of coding Fc-TMP1,, allow easily from annealed oligonucleotide clone small peptide so that contain ApaLI site and XhoI site.Design primer 2 396-70 adds ApaLI and XhoI restriction enzyme sites.Utilize the long polysaccharase of expansion of 2396-70 and 5 ' the general primer 1209-85 on 3728 dna profilings to carry out PCR.Primer sequence is as follows:

1209-85 CGTACAGGTTTACGCAAGAAAATGG

2396-70 TTTGTTGGATCCATTACTCGAGTTTTTTTGCGGCC

GCTTTCTGTGCACCACCACCTCCACCTTTAC

With the PCR fragment that BsrGI and BamHI digestion obtain, gel-purified is as inserting fragment.Digest plasmid from bacterial strain #3728 simultaneously with BsrGI and BamHI, gel-purified is as carrier.Carrier and insertion fragment are linked together, the connection mixture electroporation that obtains is advanced the GM221 cell.Select single bacterium colony, the preparation plasmid DNA is with dna sequencing.A plasmid that obtains, 200003182 (Fig. 8) show to have dna sequence dna accurately, and merge as vector construction Fc-TMP.

Digest 200003182 plasmids with ApaLI and XhoI, and as carrier.The annealed oligomer that will have ApaLI and XhoI sticky end is connected the into fusion of carrier generation needs.

TMP20, TMP24, TMP27, TMP29 and TMP30 are as the N-terminal fusions that produces by this way.

Transform

As described below, by electroporation the connector above each is transformed into host strain GM221.To clone's production recombinant protein product, and the ability with gene fusion of accurate nucleotide sequence is screened.

pAMG21

Expression plasmid pAMG21 can obtain with preserving number 98113 (preservation on July 24 in 1996) from ATCC.

GM221 (Amgen host strain #2596)

Amgen host strain #2596 is modified e. coli k-12 bacterial strain, contains temperature sensitive λ acceptor cI857s7 in the ebg district and contains lacI in ebg district, back in morning ^QAcceptor (68 minutes).The existence of these two repressor genes allows this host to utilize various expression systems, still, these two repressors be with from luxP _RExpression incoherent.Unconverted host does not have antibiotics resistance.

The ribosome bind site of CI857s7 gene has been modified and has been comprised enhanced RBS.At gene pool registration number M64441Gb Ba, in numbering, between nucleotide position 1170 and 1411, inserted the ebg operon, lacked interferential ebg sequence.

The recombinant phage enhanced RBS#4 that utilization is called MMebg-cI857s7 is delivered to karyomit(e) with construct and enters F ' tet/393.After reorganization and dissolving, have only aforesaid karyomit(e) to insert fragment still in cell.Its name is called F ' tet/GM101.

Then, in the gene pool registration number M64441Gb Ba of disappearance interference ebg sequence, be numbered between Nucleotide 2493 and 2937 positions, by sending lacI ^QConstruct enters the ebg operon and modifies F ' tet/GM101.The recombinant phage that utilization is called Agebg-LacIQ#5 enters F ' tet/GM101 construct is delivered in the karyomit(e).After reorganization and decomposing, have only aforesaid karyomit(e) to insert fragment and be retained in the cell.It is called after F ' tet/GM221 still.Utilize bifurcation pyridine dried tangerine peel preservation F ' tet episome from bacterial strain of 25 μ g/ml concentration among the LB.The identification of strains of preservation is a sensitive tetracycline, saves as GM221.

Express

In the Luria broth culture, express the GM221 culture of each fused protein 37 ℃ of growths.In substratum, add synthetic auto-induction thing N-(3-oxygen hexanol)-DL-homoserine lactone concentration 20ng/ml to the end, and incubation just can be from the expression of luxPR promotor induced gene product after 3 hours again at 37 ℃.After 3 hours, by the microscopic examination bacterial cultures, detect the existence of inclusion body, then, by centrifugal collection.In the inductive substratum, observe the refraction inclusion body, show that fused protein most possibly is to produce in the insoluble part in intestinal bacteria, by the direct dissolved cell precipitation of resuspending in containing the Laemmli sample buffer of 10% mercaptoethanol, and analyze by SDS-PAGE.Each protein can both be observed the intensive coomassie colored zone of suitable size (about 30kDa).

Embodiment 5

The purifying of peptide body compound

By high pressure homogenization somatoblast (14,000PSI has 2 to be passed through) in water (1/10), by centrifugal results inclusion body (at 1 hour, 4200RPM in J-6B).At the 6M guanidine, 50mMTris, 8mMDTT, the dissolving inclusion body is 1 hour among the pH8.7, and ratio is 1/10.At the 2M urea, 50mMTris, the 160mM arginine, the 3mM halfcystine, among the pH8.5 with 20 times of dissolved mixture diluted.In cold, stir the mixture and spend the night.Then, by 10 times of ultra-filtration enriched mixtures.Then, use 10mM Tris, 1.5M urea, 3 times of pH9 dilutions.Then, with acetate the pH of mixture is adjusted to pH5.By the centrifugal precipitation of removing, at 20mM NaAc, 100mMNaCl loads supernatant liquor on the mobile fast post of equilibrated SP-Sepharose among the pH5 (10mg/ml protein loads, under the room temperature).In identical damping fluid, scope 10mM NaCl elute protein in 20 column volume gradients of 500mMNaCl.Amalgamation liquid from post has diluted 3 times, is loaded into 20mM NaAc, 150mM NaCl, pH5) (10mg/ml protein loads, room temperature) on the SP-Sepharose HP post in.Utilize the 20 column volume gradient elution protein of scope 150mM NaCl in the same buffer of 400mM NaCl scope.Liquid when merging peak value filters.

Embodiment 6

Peptide is affine in conjunction with research

At room temperature utilize BIACORE 3000 to experimentize, determine the binding affinity (TMP1-TMP23) of several TMP peptides.Utilize the amine coupling process, the fixing fixing Hu-mpl of Hu-mpl (sealing) by the activation of NHS/EDC with by thanomin on sensor bar (CM5) surface.At the TMP peptide of injection 0.78nM in hu-mpl surface to 100nM.The BIACORE damping fluid is the PBS with 0.005% tensio-active agent P20.Blank surface while injected sample for a contrast.Utilize BIAEVALUATION 3.1 software packages to analyze experimental data.

As discussed earlier, in order to simulate the phage environment better, therefrom select peptide, with charged amino and the C-terminal of hiding 18 preferred peptides of amino acid (TMP2-TMP30) from acceptor, C-terminal and N-terminal at each peptide add two amino acid " cap ": add glutamine-halfcystine (QC) at N-terminal, add Histidine-Serine (HS) at C-terminal, make the length of each peptide reach 22 amino acid.Because the avidity of known peptide is the length that increases peptide, 14 amino acid peptide sequences of the biological activity of benchmark (SEQ ID NO:1) also are increased to 22 amino acid altogether.But the biologically active zone of each peptide is still identical, and represents with following black matrix (bold).

TMP No.	Peptide sequence	K D(nM)	The avidity relevant with TMP1
				TMP1	SAQGIEGPTLRQWLAARALETV	5.40	-
TMP2	QGGAREGPTLRQWLEWVRVGHS	1.60	3.38
				TMP3	QGRDLDGPTLRQWLPLPSVQHS	45.00	0.12
TMP4	QGALRDGPTLKQWLEYRRQAHS	0.86	6.28
				TMP5	QGARQEGPTLKEWLFWVRMGHS	6.66	0.81
TMP6	QGEALLGPTLREWLAWRRAQHS	0.37	14.59
				TMP7	QGMARDGPTLREWLRTYRMMHS	1.20	4.50
TMP8	QGWMPEGPTLKQWLFHGRGQHS	23.20	0.23
				TMP9	QGHIREGPTLRQWLVALRMVHS	1.67	3.23
TMP10	QGQLGHGPTLRQWLSWYRGMHS	1.22	4.43
				TMP11	QGELRQGPTLHEWLQHLASKHS	35.90	0.15
TMP12	QGVGIEGPTLRQWLAQRLNPHS	5.20	1.04
				TMP13	QGWSRDGPTLREWLAWRAVGHS	4.44	1.22
TMP14	QGAVPQGPTLKQWLLWRRCAHS	0.88	6.14
				TMP15	QGRIREGPTLKEWLAQRRGFHS	1.03	5.24
TMP16	QGRFAEGPTLPEWLEQRKLVHS	6.58	0.82
				TMP17	QGDRFQGPTLREWLAAIRSVHS	12.90	0.42
TMP18	QGAGREGPTLREWLNMRVWQHS	12.80	0.42
				TMP19	QGALQEGPTLRQWLGWGQWGHS	78.50	0.07
TMP20	QGYCDEGPTLKQWLVCLGLQHS	0.56	9.64
				TMP21	QGWCKEGPTLREWLRWGFLCHS	1.53	3.53
TMP22	QGCSSGGPTLREWLQCRRMQHS	＜0.1	＞54
				TMP23	QGCSWGGPTLKQWLQCVRAKHS	＜0.1	＞54

Embodiment 7

Peptide biological activity research

Utilization is based on the biological activity of determining peptide TMP1-TMP23 of cell.

Mouse 32D cell proliferation experiment comprises the chosen mouse 32D cell of mpl acceptor transfection of utilization.Following result's report is relevant with TMP1.

The CD61 cell experiment comprises and utilizes initial people CD34+ cell that they were cultivated several days when having peptide TMP1-TMP23.Then, with these cell sortings, determine the percentage ratio of the cell of the lip-deep megalokaryocyte specific mark of express cell (CD16).When active compound stimulated the appearance of these platelet precursors cells in the mode of dependent dose, the mark of cell born of the same parents class precursor (CD36+) and neutrophilia precursor (CD15+) was still on baseline.Represent the quantitative results of CD61 cell experiment of the mean value of three different concns to be expressed as follows:

Peptide	Mouse 32D cell proliferation experiment (with respect to TMP1)	CD61 cell experiment (with respect to TMP)
			TMP01	100％	-/+
TMP02	290％	+
			TMP03	39％	++
TMP04	42％	-
			TMP05	85％	++
TMP06	569％	++
			TMP07	289％	++
TMP08	39％	+
			TMP09	2％	-
TMP10	12％	-
			TMP11	21％	-
TMP12	10％	-
			TMP13	328％	++
TMP14	635％	+++
			TMP15	35％	-
TMP16	32％	+
			TMP17	21％	-
TMP18	337％	++
			TMP19	27％	+
TMP20	Can not detect	-/+
			TMP21	312％	-/+
TMP22	Can not detect	-
			TMP23	Can not detect	+++

Embodiment 8

The combination research of peptide body

On BIAcore, in direct binding analysis, it is active with combining of hu-MPL to test several TMP peptide bodies.At 25 ℃, utilize BIAcore 2000 (BIACORE company) to experimentize.The damping fluid that utilizes is the PBS with 0.005% tensio-active agent P20.According to the amine coupling process of standard (NHS/EDC and seal with thanomin), the protein G (Pierce 21193ZZ) of fixing reorganization on the CM5 bar is caught TMP peptide body to about 400RU.With 50 μ l/min before the peptide surface of catching injection 3 minutes, the hu-MPL (Lot 27315-53) that in sample buffer (PBS that 0.005% tensio-active agent P20 and 100 μ g/ml BSA are arranged), will recombinate from the 1uM serial dilution to 0.15nM.Also injected the rhu-MPL sample on barren protein G surface, so that deduct any non-specific in conjunction with background.(Pierce 21009zz, pH2) with 100 μ l 8mM glycine pH1.5,1M NaCl is with the injection of 50 μ l/min series, the surface of regenerated protein G with 100 μ l ImmunoPure IgG elution buffers between two circulations.Utilize BIAevaluation3.1 (BIACORE company), determine the binding affinity (K of peptide body and rhu-MPL by the nonlinear regression analysis of data _D).The result is summarized as follows:

Peptide body (TMP-Fc)	k a(1/Ms)	k d(1/s)	K D(M)
				TMP20-Fc	5.06×10 4	7.34×10 -3	1.45×10 -7
Fc-TMP24	4.01×10 4	8.75×10 -3	2.18×10 -7
				TMP25-Fc	2.35×10 4	1.40×10 -3	5.97×10 -8
TMP26-Fc	2.58×10 4	5.72×10 -3	2.22×10 -7
				Fc-TMP27	1.3×10 5	8.42×10 -3	6.49×10 -8
TMP28-Fc	6.78×10 4	2.52×10 -2	3.71×10 -7

Embodiment 9

Peptide body active testing

When having several TMP-Fc fused protein, cultivated primary human CD34+ cell several days.Then, sort these cells and determine percentage ratio at the cell of cell surface expression megalokaryocyte specific mark (CD61).When active compound stimulated the appearance of these platelet precursors cells in the mode of dependent dose, the mark of cell born of the same parents class precursor (CD36+) (not having to show) and neutrophilia precursor (CD15+) (not having demonstration) was still on baseline.Referring to Figure 10,11 and 12 (CD61 cell experiments).

Embodiment 10

Activity in vivo

Normal female BDF1 mouse at the age in about 10-12 week, is used for activity in vivo research.

Carrying out pill with the subcutaneous injection mouse handles.The volume that subcutaneous injection is sent is 0.2ml.BSA with 0.1% diluted compounds in PBS.All experiments only comprise a control group with this diluent treatment, mark " carrier ".

The 0th day every group of 10 mouse, two groups of beginnings in the 4th day, every group of 20 mouse altogether.Get blood at each time point 5 mouse, mouse is got blood minimum one time.Use the isoflurane anesthesia mouse, obtain the blood of volume 140-160 μ l altogether by acupuncture socket of the eye axle.On TechniconHlE blood analyser electrophoresis software, mouse blood is counted.The parameter that detects is white blood cell, red blood cell, hematocrit, oxyphorase, thrombocyte, neutrophilic granulocyte.Referring to Figure 13 and 14.

The present invention has so far all narrated, and those of ordinary skills should understand, and wherein can carry out many changes and modification, and not break away from the spirit and scope of the invention as herein described.

Sequence table

<110>Amgen Inc.

＜120〉have the peptide of thrombopoietic activity and relevant compound

<130>A-750A PCT

<140>PCT/US 02/32552

<141>2002-10-11

<150>10/269,806

<151>2002-10-10

<150>60/328,666

<151>2001-10-11

<160>199

<170>PatentIn version 3.2

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＜223〉synthetic peptide sequence

<400>24

Cys Gln Leu Gly Gly Pro Thr Leu Arg Glu Trp Leu Ala Cys Arg Leu

1 5 10 15

Gly Ala

<210>25

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>25

Cys Trp Glu Gly Gly Pro Thr Leu Lys Glu Trp Leu Gln Cys Leu Val

1 5 10 15

Glu Arg

<210>26

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>26

Cys Arg Gly Gly Gly Pro Thr Leu His Gln Trp Leu Ser Cys Phe Arg

1 5 10 15

Trp Gln

<210>27

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>27

Cys Arg Asp Gly Gly Pro Thr Leu Arg Gln Trp Leu Ala Cys Leu Gln

1 5 10 15

Gln Lys

<210>28

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>28

Glu Leu Arg Ser Gly Pro Thr Leu Lys Glu Trp Leu Val Trp Arg Leu

1 5 10 15

Ala Gln

<210>29

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>29

Gly Cys Arg Ser Gly Pro Thr Leu Arg Glu Trp Leu Ala Cys Arg Glu

1 5 10 15

Val Gln

<210>30

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>30

Thr Cys Glu Gln Gly Pro Thr Leu Arg Gln Trp Leu Leu Cys Arg Gln

1 5 10 15

Gly Arg

<210>31

<211>684

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(1)..(684)

<400>31

atg gac aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc ctg 48

Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

1 5 10 15

ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 96

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 144

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 192

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 240

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

65 70 75 80

tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 288

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 336

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

100 105 110

atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 384

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 432

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

130 135 140

agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 480

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 528

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 576

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 624

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 672

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

tct ccg ggt aaa 684

Ser Pro Gly Lys

225

<210>32

<211>228

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>32

Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

1 5 10 15

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Pro Gly Lys

225

<210>33

<211>835

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(60)..(791)

<400>33

tagtcgatta atcgatttga ttctagattt gttttaacta attaaaggag gaataacat 59

atg ggt gca cag aaa gcg gcc gca aaa aaa ctc gag ggt gga ggc ggt 107

Met Gly Ala Gln Lys Ala Ala Ala Lys Lys Leu Glu Gly Gly Gly Gly

1 5 10 15

ggg gac aaa act cac aca tgt cca cct tgc cca gca cct gaa ctc ctg 155

Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

20 25 30

ggg gga ccg tca gtt ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 203

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

35 40 45

atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 251

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

50 55 60

cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 299

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

65 70 75 80

gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 347

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

85 90 95

tac cgt gtg gtc agc gtc crc acc gtc ctg cac cag gac tgg ctg aat 395

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

100 105 110

ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 443

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

115 120 125

atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 491

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

130 135 140

gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 539

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

145 150 155 160

agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 587

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

165 170 175

gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 635

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

180 185 190

ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 683

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

195 200 205

gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 731

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

210 215 220

atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 779

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

225 230 235 240

tct ccg ggt aaa taatggatcc gcggaaagaa gaagaagaag aagaaagccc gaaa 835

Ser Pro Gly Lys

<210>34

<211>244

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>34

Met Gly Ala Gln Lys Ala Ala Ala Lys Lys Leu Glu Gly Gly Gly Gly

1 5 10 15

Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

20 25 30

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

35 40 45

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

50 55 60

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

65 70 75 80

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

85 90 95

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

100 105 110

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

115 120 125

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

130 135 140

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

145 150 155 160

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

165 170 175

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

180 185 190

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

195 200 205

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

210 215 220

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

225 230 235 240

Ser Pro Gly Lys

<210>35

<211>66

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(4)..(66)

<400>35

cat atg ggt gca cag ggt atc gaa ggt ccg act ctg cgt cag tgg ctg 48

Met Gly Ala Gln Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

gct gct cgt gct ctc gag 66

Ala Ala Arg Ala Leu Glu

20

<210>36

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>36

Met Gly Ala Gln Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala

1 5 10 15

Ala Arg Ala Leu Glu

20

<210>37

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>37

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Gly Gly Ala Gly Cys Ala

1 5 10 15

Cys Gly Thr Gly Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Gly Thr Cys Ala Ala Thr Gly Gly Cys Thr Thr Gly Ala

35 40 45

Ala Thr Gly Gly Gly Thr Thr Cys Gly Thr Gly Thr Thr Gly Gly Thr

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>38

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>38

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Ala Cys Cys Ala Ala

1 5 10 15

Cys Ala Cys Gly Ala Ala Cys Cys Cys Ala Thr Thr Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Ala Cys Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Cys Ala Cys Gly Thr Gly Cys Thr Cys

50 55 60

Cys Ala Cys Cys Thr Thr Gly

65 70

<210>39

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>39

gt gca caa ggt gga gca cgt gaa gga cca act ctt cgt caa tgg ctt 47

Ala Gln Gly Gly Ala Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

gaa tgg gtt cgt gtt ggt cat tct ctc gag 77

Glu Trp Val Arg Val Gly His Ser Leu Glu

20 25

<210>40

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>40

Ala Gln Gly Gly Ala Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Glu

1 5 10 15

Trp Val Arg Val Gly His Ser Leu Glu

20 25

<210>41

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>41

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Cys Gly Thr Gly Ala Thr

1 5 10 15

Cys Thr Thr Gly Ala Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Gly Thr Cys Ala Ala Thr Gly Gly Cys Thr Thr Cys Cys

35 40 45

Ala Cys Thr Thr Gys Cys Ala Thr Cys Thr Gly Thr Thr Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>42

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>42

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly Ala Ala

1 5 10 15

Cys Ala Gly Ala Thr Gly Gly Ala Ala Gly Thr Gly Gly Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Ala Cys Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Thr Cys Ala Ala Gly Ala Thr Cys Ala Cys

50 55 60

Gly Thr Cys Cys Thr Thr Gly

65 70

<210>43

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>43

gt gca caa gga cgt gat ctt gat ggt cca act ctt cgt caa tgg ctt 47

Ala Gln Gly Arg Asp Leu Asp Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

cca ctt cca tct gtt caa cat tct ctc gag 77

Pro Leu Pro Ser Val Gln His Ser Leu Glu

20 25

<210>44

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>44

Ala Gln Gly Arg Asp Leu Asp Gly Pro Thr Leu Arg Gln Trp Leu Pro

1 5 10 15

Leu Pro Ser Val Gln His Ser Leu Glu

20 25

<210>45

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>45

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Cys Thr Thr Thr Ala

1 5 10 15

Cys Gly Thr Gly Ala Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Ala Ala Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Ala

35 40 45

Ala Thr Ala Thr Cys Gly Thr Cys Gly Thr Cys Ala Ala Gly Cys Thr

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>46

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>46

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Gly Cys Thr Thr

1 5 10 15

Gly Ala Cys Gly Ala Cys Gly Ala Thr Ala Thr Thr Cys Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Gly Thr Thr Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Thr Cys Ala Cys Gly Thr Ala Ala Ala Gly

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>47

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>47

gt gca caa gga gct tta cgt gat ggt cca act ctt aaa caa tgg tta 47

Ala Gln Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu

1 5 10 15

gaa tat cgt cgt caa gct cat tca ctc gag 77

Glu Tyr Arg Arg Gln Ala His Ser Leu Glu

20 25

<210>48

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>48

Ala Gln Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu

1 5 10 15

Tyr Arg Arg Gln Ala His Ser Leu Glu

20 25

<210>49

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>49

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Cys Ala Cys Gly Thr

1 5 10 15

Cys Ala Ala Gly Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Ala Ala Gly Ala Ala Thr Gly Gly Thr Thr Ala Thr Thr

35 40 45

Thr Thr Gly Gly Gly Thr Thr Cys Gly Thr Ala Thr Gly Gly Gly Thr

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>50

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>50

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Cys Cys Cys Ala

1 5 10 15

Thr Ala Cys Gly Ala Ala Cys Cys Cys Ala Ala Ala Ala Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Cys Thr Thr Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Cys Thr Thr Gly Ala Cys Gly Thr Gly

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>51

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>51

gt gca caa gga gca cgt caa gaa gga cca act ctt aaa gaa tgg tta 47

Ala Gln Gly Ala Arg Gln Glu Gly Pro Thr Leu Lys Glu Trp Leu

1 5 10 15

ttt tgg gtt cgt atg ggt cat tca ctc gag 77

Phe Trp Val Arg Met Gly His Ser Leu Glu

20 25

<210>52

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>52

Ala Gln Gly Ala Arg Gln Glu Gly Pro Thr Leu Lys Glu Trp Leu Phe

1 5 10 15

Trp Val Arg Met Gly His Ser Leu Glu

20 25

<210>53

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>53

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Ala Ala Gly Cys Thr

1 5 10 15

Thr Thr Ala Thr Thr Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Thr

20 25 30

Thr Ala Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys

35 40 45

Thr Thr Gly Gly Cys Gly Thr Cys Gly Thr Gly Cys Ala Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>54

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>54

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly Thr Gly

1 5 10 15

Cys Ala Cys Gly Ala Cys Gly Cys Cys Ala Ala Gly Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Thr Ala Ala Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Ala Ala Thr Ala Ala Ala Gly Cys Thr Thr

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>55

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>55

gt gca caa gga gaa gct tta tta ggt cca act tta cgt gaa tgg ctt 47

Ala Gln Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

gct tgg cgt cgt gca caa cat tct ctc gag 77

Ala Trp Arg Arg Ala Gln His Ser Leu Glu

20 25

<210>56

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>56

Ala Gln Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala

1 5 10 15

Trp Arg Arg Ala Gln His Ser Leu Glu

20 25

<210>57

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>57

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Ala Thr Gly Gly Cys Ala

1 5 10 15

Cys Gly Thr Gly Ala Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Cys Gly

35 40 45

Thr Ala Cys Thr Thr Ala Thr Cys Gly Thr Ala Thr Gly Ala Thr Gly

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>58

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>58

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Cys Ala Thr Cys Ala

1 5 10 15

Thr Ala Cys Gly Ala Thr Ala Ala Gly Thr Ala Cys Gly Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Thr Cys Ala Cys Gly Thr Gly Cys Cys Ala

50 55 60

Thr Ala Cys Cys Thr Thr Gly

65 70

<210>59

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>59

gt gca caa ggt atg gca cgt gat ggt cca act ctt cgt gaa tgg ctt 47

Ala Gln Gly Met Ala Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

cgt act tat cgt atg atg cat tct ctc gag 77

Arg Thr Tyr Arg Met Met His Ser Leu Glu

20 25

<210>60

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>60

Ala Gln Gly Met Ala Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu Arg

1 5 10 15

Thr Tyr Arg Met Met His Ser Leu Glu

20 25

<210>61

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>61

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Gly Ala Thr Gly

1 5 10 15

Cys Cys Ala Gly Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Ala Thr

20 25 30

Thr Ala Ala Ala Ala Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Thr

35 40 45

Thr Cys Ala Thr Gly Gly Thr Cys Gly Thr Gly Gly Thr Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>62

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>62

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly Ala Cys

1 5 10 15

Cys Ala Cys Gly Ala Cys Cys Ala Thr Gly Ala Ala Ala Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Thr Thr Thr Thr Ala Ala Thr Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Cys Thr Gly Gly Cys Ala Thr Cys Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>63

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>63

gt gca caa gga tgg atg cca gaa gga cca aca tta aaa caa tgg ctt 47

Ala Gln Gly Trp Met Pro Glu Gly Pro Thr Leu Lys Gln Trp Leu

1 5 10 15

ttt cat ggt cgt ggt caa cat tct ctc gag 77

Phe His Gly Arg Gly Gln His Ser Leu Glu

20 25

<210>64

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400> 64

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

1 5 10 15

His Gly Arg Gly Gln His Ser Leu Glu

20 25

<210>65

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>65

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Cys Ala Thr Ala Thr Thr

1 5 10 15

Cys Gly Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Ala Thr

20 25 30

Thr Ala Cys Gly Thr Cys Ala Ala Thr Gly Gly Cys Thr Thr Gly Thr

35 40 45

Thr Gly Cys Thr Cys Thr Thr Cys Gly Thr Ala Thr Gly Gly Thr Thr

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>66

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>66

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Ala Ala Cys Cys Ala

1 5 10 15

Thr Ala Cys Gly Ala Ala Gly Ala Gly Cys Ala Ala Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Ala Cys Gly Thr Ala Ala Thr Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Cys Ala Cys Gly Ala Ala Thr Ala Thr

50 55 60

Gly Thr Cys Cys Thr Thr Gly

65 70

<210>67

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>67

gt gca caa gga cat att cgt gaa ggt cca aca tta cgt caa tgg ctt 47

Ala Gln Gly His Ile Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

gtt gct ctt cgt atg gtt cat tct ctc gag 77

Val Ala Leu Arg Met Val His Ser Leu Glu

20 25

<210>68

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>68

Ala Gln Gly His Ile Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Val

1 5 10 15

Ala Leu Arg Met Val His Ser Leu Glu

20 25

<210>69

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>69

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Cys Ala Ala Thr Thr Ala

1 5 10 15

Gly Gly Ala Cys Ala Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Gly Thr Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Cys

35 40 45

Thr Thr Gly Gly Thr Ala Thr Cys Gly Thr Gly Gly Thr Ala Thr Gly

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>70

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>70

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Cys Ala Thr Ala Cys

1 5 10 15

Cys Ala Cys Gly Ala Thr Ala Cys Cys Ala Ala Gly Ala Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Ala Cys Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Thr Gly Thr Cys Cys Thr Ala Ala Thr Thr

50 55 60

Gly Ala Cys Cys Thr Thr Gly

65 70

<210>71

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>71

gt gca caa ggt caa tta gga cat ggt cca act ctt cgt caa tgg ctt 47

Ala Gln Gly Gln Leu Gly His Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

tct tgg tat cgt ggt atg cat tct ctc gag 77

Ser Trp Tyr Arg Gly Met His Ser Leu Glu

20 25

<210>72

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>72

Ala Gln Gly Gln Leu Gly His Gly Pro Thr Leu Arg Gln Trp Leu Ser

1 5 10 15

Trp Tyr Arg Gly Met His Ser Leu Glu

20 25

<210>73

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>73

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Ala Ala Thr Thr Ala

1 5 10 15

Cys Gly Thr Cys Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Ala Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Cys Ala

35 40 45

Ala Cys Ala Thr Thr Thr Ala Gly Cys Ala Ala Gly Cys Ala Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>74

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>74

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Thr Gly Cys

1 5 10 15

Thr Thr Gly Cys Thr Ala Ala Ala Thr Gly Thr Thr Gly Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Thr Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Gly Ala Cys Gly Thr Ala Ala Thr Thr

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>75

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>75

gt gca caa gga gaa tta cgt caa gga cca act ctt cat gaa tgg ctt 47

Ala Gln Gly Glu Leu Arg Gln Gly Pro Thr Leu His Glu Trp Leu

1 5 10 15

caa cat tta gca agc aaa cat tct ctc gag 77

Gln His Leu Ala Ser Lys His Ser Leu Glu

20 25

<210>76

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>76

Ala Gln Gly Glu Leu Arg Gln Gly Pro Thr Leu His Glu Trp Leu Gln

1 5 10 15

His Leu Ala Ser Lys His Ser Leu Glu

20 25

<210>77

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>77

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Thr Ala Gly Gly Thr

1 5 10 15

Ala Thr Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Ala Thr

20 25 30

Thr Ala Cys Gly Thr Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Cys

35 40 45

Thr Cys Ala Ala Cys Gly Thr Cys Thr Thr Ala Ala Thr Cys Cys Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>78

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>78

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Gly Gly Ala Thr

1 5 10 15

Thr Ala Ala Gly Ala Cys Gly Thr Thr Gly Ala Gly Cys Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Gly Ala Cys Gly Thr Ala Ala Thr Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Cys Ala Ala Thr Ala Cys Cys Thr Ala

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>79

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>79

gt gca caa gga gta ggt att gaa ggt cca aca tta cgt caa tgg tta 47

Ala Gln Gly Val Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

gct caa cgt ctt aat cca cat tct ctc gag 77

Ala Gln Arg Leu Asn Pro His Ser Leu Glu

20 25

<210>80

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>80

Ala Gln Gly Val Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala

1 5 10 15

Gln Arg Leu Asn Pro His Ser Leu Glu

20 25

<210>81

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>81

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Gly Thr Cys Ala

1 5 10 15

Cys Gly Thr Gly Ala Thr Gly Gly Thr Cys Cys Ala Ala Cys Ala Cys

20 25 30

Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys

35 40 45

Thr Thr Gly Gly Cys Gly Thr Gly Cys Thr Gly Thr Thr Gly Gly Ala

50 55 60

Cys Ala Thr Ala Gly Thr Cys

65 70

<210>82

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>82

Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Thr Cys Cys Ala Ala

1 5 10 15

Cys Ala Gly Cys Ala Cys Gly Cys Cys Ala Ala Gly Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Thr Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Thr Cys Ala Cys Gly Thr Gly Ala Cys Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>83

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>83

gt gca caa gga tgg tca cgt gat ggt cca aca ctt cgt gaa tgg ctt 47

Ala Gln Gly Trp Ser Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

gct tgg cgt gct gtt gga cat agt ctc gag 77

Ala Trp Arg Ala Val Gly His Ser Leu Glu

20 25

<210>84

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>84

Ala Gln Gly Trp Ser Arg Asp Gly Pro Thr Leu Arg Glu Trp Leu Ala

1 5 10 15

Trp Arg Ala Val Gly His Ser Leu Glu

20 25

<210>85

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>85

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Cys Ala Gly Thr Thr

1 5 10 15

Cys Cys Ala Cys Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Ala Ala Cys Ala Gly Thr Gly Gly Thr Thr Ala Thr Thr

35 40 45

Ala Thr Gly Gly Cys Gly Thr Cys Gly Thr Thr Gly Thr Gly Cys Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>86

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>86

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Gly Cys Ala Cys

1 5 10 15

Ala Ala Cys Gly Ala Cys Gly Cys Cys Ala Thr Ala Ala Thr Ala Ala

20 25 30

Cys Cys Ala Cys Thr Gly Thr Thr Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Gly Thr Gly Gly Ala Ala Cys Thr Gly

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>87

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>87

gt gca caa gga gca gtt cca caa gga cca act ctt aaa cag tgg tta 47

Ala Gln Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu

1 5 10 15

tta tgg cgt cgt tgt gca cat tct ctc gag 77

Leu Trp Arg Arg Cys Ala His Ser Leu Glu

20 25

<210>88

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>88

Ala Gln Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu

1 5 10 15

Trp Arg Arg Cys Ala His Ser Leu Glu

20 25

<210>89

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>89

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Cys Gly Thr Ala Thr Thr

1 5 10 15

Cys Gly Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Ala Ala Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys

35 40 45

Thr Cys Ala Ala Cys Gly Thr Cys Gly Thr Gly Gly Thr Thr Thr Thr

50 55 60

Cys Ala Thr Ala Gly Thr Cys

65 70

<210>90

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>90

Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Ala Ala Ala Ala Cys

1 5 10 15

Cys Ala Cys Gly Ala Cys Gly Thr Thr Gly Ala Gly Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Thr Thr Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Cys Ala Cys Gly Ala Ala Thr Ala Cys

50 55 60

Gly Ala Cys Cys Thr Thr Gly

65 70

<210>91

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>91

gt gca caa ggt cgt att cgt gaa ggt cca act ctt aaa gaa tgg ctt 47

Ala Gln Gly Arg Ile Arg Glu Gly Pro Thr Leu Lys Glu Trp Leu

1 5 10 15

gct caa cgt cgt ggt ttt cat agt ctc gag 77

Ala Gln Arg Arg Gly Phe His Ser Leu Glu

20 25

<210>92

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>92

Ala Gln Gly Arg Ile Arg Glu Gly Pro Thr Leu Lys Glu Trp Leu Ala

1 5 10 15

Gln Arg Arg Gly Phe His Ser Leu Glu

20 25

<210>93

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>93

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Cys Gly Thr Thr Thr Cys

1 5 10 15

Gly Cys Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Ala Cys

20 25 30

Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Thr Thr Ala Gly Ala

35 40 45

Ala Cys Ala Ala Cys Gly Thr Ala Ala Ala Cys Thr Thr Gly Thr Thr

50 55 60

Cys Ala Thr Ala Gly Thr Cys

65 70

<210>94

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>94

Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Ala Ala Cys Ala Ala

1 5 10 15

Gly Thr Thr Thr Ala Cys Gly Thr Thr Gly Thr Thr Cys Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Thr Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Cys Ala Gly Cys Gly Ala Ala Ala Cys

50 55 60

Gly Ala Cys Cys Thr Thr Gly

65 70

<210>95

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>95

gt gca caa ggt cgt ttc gct gaa ggt cca aca ctt cgt gaa tgg tta 47

Ala Gln Gly Arg Phe Ala Glu Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

gaa caa cgt aaa ctt gtt cat agt ctc gag 77

Glu Gln Arg Lys Leu Val His Ser Leu Glu

20 25

<210>96

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>96

Ala Gln Gly Arg Phe Ala Glu Gly Pro Thr Leu Arg Glu Trp Leu Glu

1 5 10 15

Gln Arg Lys Leu Val His Ser Leu Glu

20 25

<210>97

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>97

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Gly Ala Thr Cys Gly Thr

1 5 10 15

Thr Thr Cys Cys Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys

35 40 45

Thr Gly Cys Ala Ala Thr Cys Cys Gly Thr Ala Gly Cys Gly Thr Ala

50 55 60

Cys Ala Thr Ala Gly Thr Cys

65 70

<210>98

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>98

Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Thr Ala Cys Gly Cys

1 5 10 15

Thr Ala Cys Gly Gly Ala Thr Thr Gly Cys Ala Gly Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Gly Gly Ala Ala Ala Cys Gly Ala Thr

50 55 60

Cys Ala Cys Cys Thr Thr Gly

65 70

<210>99

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>99

gt gca caa ggt gat cgt ttc caa ggt cca act ctt cgt gaa tgg ctt 47

Ala Gln Gly Asp Arg Phe Gln Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

gct gca atc cgt agc gta cat agt ctc gag 77

Ala Ala Ile Arg Ser Val His Ser Leu Glu

20 25

<210>100

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>100

Ala Gln Gly Asp Arg Phe Gln Gly Pro Thr Leu Arg Glu Trp Leu Ala

1 5 10 15

Ala Ile Arg Ser Val His Ser Leu Glu

20 25

<210>101

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>101

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Gly Cys Thr Gly Gly Thr

1 5 10 15

Cys Gly Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Ala Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Ala Ala

35 40 45

Thr Ala Thr Gly Cys Gly Thr Gly Thr Thr Thr Gly Gly Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>102

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>102

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly Cys Cys

1 5 10 15

Ala Ala Ala Cys Ala Cys Gly Cys Ala Thr Ala Thr Thr Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Thr Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Cys Ala Cys Gly Ala Cys Cys Ala Gly

50 55 60

Cys Ala Cys Cys Thr Thr Gly

65 70

<210>103

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>103

gt gca caa ggt gct ggt cgt gaa ggt cca act cta cgt gaa tgg ctt 47

Ala Gln Gly Ala Gly Arg Glu Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

aat atg cgt gtt tgg caa cat tct ctc gag 77

Asn Met Arg Val Trp Gln His Ser Leu Glu

20 25

<210>104

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>104

Ala Gln Gly Ala Gly Arg Glu Gly Pro Thr Leu Arg Glu Trp Leu Asn

1 5 10 15

Met Arg Val Trp Gln His Ser Leu Glu

20 25

<210>105

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>105

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Cys Thr Thr Thr Ala

1 5 10 15

Cys Ala Ala Gly Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Ala Thr

20 25 30

Thr Ala Cys Gly Thr Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Gly

35 40 45

Ala Thr Gly Gly Gly Gly Thr Cys Ala Ala Thr Gly Gly Gly Gly Ala

50 55 60

Cys Ala Cys Thr Cys Thr Cys

65 70

<210>106

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>106

Thr Cys Gly Ala Gly Ala Gly Ala Gly Thr Gly Thr Cys Cys Cys Cys

1 5 10 15

Ala Thr Thr Gly Ala Cys Cys Cys Cys Ala Thr Cys Cys Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Gly Ala Cys Gly Thr Ala Ala Thr Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Cys Thr Thr Gly Thr Ala Ala Ala Gly

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>107

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>107

gt gca caa gga gct tta caa gaa gga cca aca tta cgt caa tgg tta 47

Ala Gln Gly Ala Leu Gln Glu Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

gga tgg ggt caa tgg gga cac tct ctc gag 77

Gly Trp Gly Gln Trp Gly His Ser Leu Glu

20 25

<210>108

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>108

Ala Gln Gly Ala Leu Gln Glu Gly Pro Thr Leu Arg Gln Trp Leu Gly

1 5 10 15

Trp Gly Gln Trp Gly His Ser Leu Glu

20 25

<210>109

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>109

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Ala Cys Thr Gly Thr

1 5 10 15

Gly Ala Thr Gly Ala Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Ala Ala Cys Ala Ala Thr Gly Gly Thr Thr Ala Gly Thr

35 40 45

Ala Thr Gly Thr Cys Thr Thr Gly Gly Thr Thr Thr Ala Cys Ala Ala

50 55 60

Cys Ala Thr Ala Gly Thr Cys

65 70

<210>110

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>110

Thr Cys Gly Ala Gly Ala Cys Thr Ala Thr Gly Thr Thr Gly Thr Ala

1 5 10 15

Ala Ala Cys Cys Ala Ala Gly Ala Cys Ala Thr Ala Cys Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Gly Thr Thr Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Thr Cys Ala Thr Cys Ala Cys Ala Gly Thr

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>111

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>111

gt gca caa gga tac tgt gat gaa ggt cca act ctt aaa caa tgg tta 47

Ala Gln Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu

1 5 10 15

gta tgt ctt ggt tta caa cat agt ctc gag 77

Val Cys Leu Gly Leu Gln His Ser Leu Glu

20 25

<210>112

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>112

Ala Gln Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val

1 5 10 15

Cys Leu Gly Leu Gln His Ser Leu Glu

20 25

<210>113

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>113

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Ala Gly Thr

1 5 10 15

Thr Cys Ala Gly Gly Ala Gly Gly Thr Cys Cys Ala Ala Cys Thr Thr

20 25 30

Thr Ala Cys Gly Thr Gly Ala Ala Thr Gly Gly Thr Thr Ala Cys Ala

35 40 45

Ala Thr Gly Thr Cys Gly Thr Cys Gly Thr Ala Thr Gly Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>114

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>114

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Gly Cys Ala

1 5 10 15

Thr Ala Cys Gly Ala Cys Gly Ala Cys Ala Thr Thr Gly Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Thr Ala Ala Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Ala Ala Cys Thr Ala Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>115

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>115

gt gca caa gga tgt agt tca gga ggt cca act tta cgt gaa tgg tta 47

Ala Gln Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

caa tgt cgt cgt atg caa cat tct ctc gag 77

Gln Cys Arg Arg Met Gln His Ser Leu Glu

20 25

<210>116

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>116

Ala Gln Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln

1 5 10 15

Cys Arg Arg Met Gln His Ser Leu Glu

20 25

<210>117

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>117

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Thr Cys Ala

1 5 10 15

Thr Gly Gly Gly Gly Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Ala Ala Cys Ala Ala Thr Gly Gly Thr Thr Ala Cys Ala

35 40 45

Ala Thr Gly Thr Gly Thr Thr Cys Gly Thr Gly Cys Thr Ala Ala Ala

50 55 60

Cys Ala Thr Thr Cys Thr Cys

65 70

<210>118

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>118

Thr Cys Gly Ala Gly Ala Gly Ala Ala Thr Gly Thr Thr Thr Ala Gly

1 5 10 15

Cys Ala Cys Gly Ala Ala Cys Ala Cys Ala Thr Thr Gly Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Gly Thr Thr Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Cys Cys Cys Cys Ala Thr Gly Ala Ala Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>119

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>119

gt gca caa gga tgt tca tgg ggt ggt cca act ctt aaa caa tgg tta 47

Ala Gln Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu

1 5 10 15

caa tgt gtt cgt gct aaa cat tct ctc gag 77

Gln Cys Val Arg Ala Lys His Ser Leu Glu

20 25

<210>120

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>120

Ala Gln Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln

1 5 10 15

Cys Val Arg Ala Lys His Ser Leu Glu

20 25

<210>121

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>121

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Cys Ala Ala

1 5 10 15

Thr Thr Ala Gly Gly Thr Gly Gly Thr Cys Cys Gly Ala Cys Thr Cys

20 25 30

Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys

35 40 45

Thr Thr Gly Thr Cys Gly Thr Cys Thr Thr Gly Gly Thr Gly Cys Thr

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>122

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>122

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Gly Cys Ala Cys

1 5 10 15

Cys Ala Ala Gly Ala Cys Gly Ala Cys Ala Ala Gly Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Ala Gly Thr Cys

35 40 45

Gly Gly Ala Cys Cys Ala Cys Cys Thr Ala Ala Thr Thr Gly Ala Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>123

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>123

gt gca caa gga tgt caa tta ggt ggt ccg act ctt cgt gaa tgg ctt 47

Ala Gln Gly Cys Gln Leu Gly Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

gct tgt cgt ctt ggt gct cat tca ctc gag 77

Ala Cys Arg Leu Gly Ala His Ser Leu Glu

20 25

<210>124

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>124

Ala Gln Gly Cys Gln Leu Gly Gly Pro Thr Leu Arg Glu Trp Leu Ala

1 5 10 15

Cys Arg Leu Gly Ala His Ser Leu Glu

20 25

<210>125

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>125

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Thr Gly Gly

1 5 10 15

Gly Ala Ala Gly Gly Thr Gly Gly Thr Cys Cys Thr Ala Cys Ala Cys

20 25 30

Thr Thr Ala Ala Ala Gly Ala Ala Thr Gly Gly Cys Thr Thr Cys Ala

35 40 45

Ala Thr Gly Thr Cys Thr Thr Gly Thr Ala Gly Ala Ala Cys Gly Thr

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>126

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>126

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Ala Cys Gly Thr Thr

1 5 10 15

Cys Thr Ala Cys Ala Ala Gly Ala Cys Ala Thr Thr Gly Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Thr Thr Thr Ala Ala Gly Thr Gly Thr Ala

35 40 45

Gly Gly Ala Cys Cys Ala Cys Cys Thr Thr Cys Cys Cys Ala Ala Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>127

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>127

gt gca caa gga tgt tgg gaa ggt ggt cct aca ctt aaa gaa tgg ctt 47

Ala Gln Gly Cys Trp Glu Gly Gly Pro Thr Leu Lys Glu Trp Leu

1 5 10 15

caa tgt ctt gta gaa cgt cat tca ctc gag 77

Gln Cys Leu Val Glu Arg His Ser Leu Glu

20 25

<210>128

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>128

Ala Gln Gly Cys Trp Glu Gly Gly Pro Thr Leu Lys Glu Trp Leu Gln

1 5 10 15

Cys Leu Val Glu Arg His Ser Leu Glu

20 25

<210>129

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>129

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Thr Gly Thr Cys Gly Thr

1 5 10 15

Gly Gly Thr Gly Gly Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Cys Ala Thr Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Cys

35 40 45

Thr Thr Gly Thr Thr Thr Thr Cys Gly Thr Thr Gly Gly Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>130

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>130

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Gly Cys Cys

1 5 10 15

Ala Ala Cys Gly Ala Ala Ala Ala Cys Ala Ala Gly Ala Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Ala Thr Gly Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Cys Cys Ala Cys Cys Ala Cys Gly Ala Cys

50 55 60

Ala Ala Cys Cys Thr Thr Gly

65 70

<210>131

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>131

gt gca caa ggt tgt cgt ggt ggt ggt cca act ctt cat caa tgg ctt 47

Ala Gln Gly Cys Arg Gly Gly Gly Pro Thr Leu His Gln Trp Leu

1 5 10 15

tct tgt ttt cgt tgg caa cat tca ctc gag 77

Ser Cys Phe Arg Trp Gln His Ser Leu Glu

20 25

<210>132

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>132

Ala Gln Gly Cys Arg Gly Gly Gly Pro Thr Leu His Gln Trp Leu Ser

1 5 10 15

Cys Phe Arg Trp Gln His Ser Leu Glu

20 25

<210>133

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>133

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Thr Gly Thr Cys Gly Thr

1 5 10 15

Gly Ala Thr Gly Gly Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Thr Ala Gly Ala Cys Ala Ala Thr Gly Gly Cys Thr Thr Gly Cys

35 40 45

Thr Thr Gly Thr Cys Thr Thr Cys Ala Ala Cys Ala Ala Ala Ala Ala

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>134

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>134

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Thr Thr Thr

1 5 10 15

Gly Thr Thr Gly Ala Ala Gly Ala Cys Ala Ala Gly Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Thr Cys Thr Ala Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala Cys Gly Ala Cys

50 55 60

Ala Thr Cys Cys Thr Thr Gly

65 70

<210>135

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>135

gt gca caa gga tgt cgt gat ggt ggt cca act ctt aga caa tgg ctt 47

Ala Gln Gly Cys Arg Asp Gly Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

gct tgt ctt caa caa aaa cat tca ctc gag 77

Ala Cys Leu Gln Gln Lys His Ser Leu Glu

20 25

<210>136

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>136

Ala Gln Gly Cys Arg Asp Gly Gly Pro Thr Leu Arg Gln Trp Leu Ala

1 5 10 15

Cys Leu Gln Gln Lys His Ser Leu Glu

20 25

<210>137

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>137

Thr Cys Gly Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Gly Ala Gly

1 5 10 15

Cys Ala Ala Gly Ala Cys Gly Cys Cys Ala Ala Ala Cys Ala Ala Gly

20 25 30

Cys Cys Ala Thr Thr Cys Thr Thr Thr Thr Ala Ala Ala Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Gly Ala Thr Cys Thr Thr Ala Ala Thr Thr

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>138

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>138

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Ala Ala Thr Thr Ala

1 5 10 15

Ala Gly Ala Thr Cys Thr Gly Gly Thr Cys Cys Ala Ala Cys Thr Thr

20 25 30

Thr Ala Ala Ala Ala Gly Ala Ala Thr Gly Gly Cys Thr Thr Gly Thr

35 40 45

Thr Thr Gly Gly Cys Gly Thr Cys Thr Thr Gly Cys Thr Cys Ala Ala

50 55 60

Cys Ala Thr Thr Cys Ala Cys

65 70

<210>139

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>139

gt gca caa gga gaa tta aga tct ggt cca act tta aaa gaa tgg ctt 47

Ala Gln Gly Glu Leu Arg Ser Gly Pro Thr Leu Lys Glu Trp Leu

1 5 10 15

gtt tgg cgt ctt gct caa cat tca ctc gag 77

Val Trp Arg Leu Ala Gln His Ser Leu Glu

20 25

<210>140

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>140

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

1 5 10 15

Trp Arg Leu Ala Gln His Ser Leu Glu

20 25

<210>141

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>141

Thr Gly Cys Ala Cys Ala Ala Gly Gly Ala Gly Gly Ala Thr Gly Thr

1 5 10 15

Ala Gly Ala Thr Cys Thr Gly Gly Thr Cys Cys Ala Ala Cys Ala Cys

20 25 30

Thr Thr Cys Gly Thr Gly Ala Ala Thr Gly Gly Thr Thr Ala Gly Cys

35 40 45

Thr Thr Gly Thr Ala Gly Ala Gly Ala Gly Gly Thr Thr Cys Ala Ala

50 55 60

Cys Ala Cys Thr Cys Thr Cys

65 70

<210>142

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>142

Thr Cys Gly Ala Gly Ala Gly Ala Gly Thr Gly Thr Thr Gly Ala Ala

1 5 10 15

Cys Cys Thr Cys Thr Cys Thr Ala Cys Ala Ala Gly Cys Thr Ala Ala

20 25 30

Cys Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Gly Thr Gly Thr Thr

35 40 45

Gly Gly Ala Cys Cys Ala Gly Ala Thr Cys Thr Ala Cys Ala Thr Cys

50 55 60

Cys Thr Cys Cys Thr Thr Gly

65 70

<210>143

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>143

gt gca caa gga gga tgt aga tct ggt cca aca ctt cgt gaa tgg tta 47

Ala Gln Gly Gly Cys Arg Ser Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

gct tgt aga gag gtt caa cac tct ctc gag 77

Ala Cys Arg Glu Val Gln His Ser Leu Glu

20 25

<210>144

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>144

Ala Gln Gly Gly Cys Arg Ser Gly Pro Thr Leu Arg Glu Trp Leu Ala

1 5 10 15

Cys Arg Glu Val Gln His Ser Leu Glu

20 25

<210>145

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>145

Thr Gly Cys Ala Cys Ala Ala Gly Gly Thr Ala Cys Ala Thr Gly Cys

1 5 10 15

Gly Ala Ala Cys Ala Ala Gly Gly Ala Cys Cys Ala Ala Cys Thr Cys

20 25 30

Thr Ala Ala Gly Ala Cys Ala Ala Thr Gly Gly Cys Thr Ala Cys Thr

35 40 45

Ala Thr Gly Thr Ala Gly Ala Cys Ala Ala Gly Gly Ala Ala Gly Ala

50 55 60

Cys Ala Cys Thr Cys Ala Cys

65 70

<210>146

<211>71

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>146

Thr Cys Gly Ala Gly Thr Gly Ala Gly Thr Gly Thr Cys Thr Thr Cys

1 5 10 15

Cys Thr Thr Gly Thr Cys Thr Ala Cys Ala Thr Ala Gly Thr Ala Gly

20 25 30

Cys Cys Ala Thr Thr Gly Thr Cys Thr Thr Ala Gly Ala Gly Thr Thr

35 40 45

Gly Gly Thr Cys Cys Thr Thr Gly Thr Thr Cys Gly Cys Ala Thr Gly

50 55 60

Thr Ala Cys Cys Thr Thr Gly

65 70

<210>147

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>147

gt gca caa ggt aca tgc gaa caa gga cca act cta aga caa tgg cta 47

Ala Gln Gly Thr Cys Glu Gln Gly Pro Thr Leu Arg Gln Trp Leu

1 5 10 15

cta tgt aga caa gga aga cac tca ctc gag 77

Leu Cys Arg Gln Gly Arg His Ser Leu Glu

20 25

<210>148

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>148

Ala Gln Gly Thr Cys Glu Gln Gly Pro Thr Leu Arg Gln Trp Leu Leu

1 5 10 15

Cys Arg Gln Gly Arg His Ser Leu Glu

20 25

<210>149

<211>77

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(3)..(77)

<400>149

gt gca cag ggt tgg tgt aag gag ggt cct act ctg cgt gag tgg ctg 47

Ala Gln Gly Trp Cys Lys Glu Gly Pro Thr Leu Arg Glu Trp Leu

1 5 10 15

cgg tgg ggt ttt ctg tgt cat tct ctc gag 77

Arg Trp Gly Phe Leu Cys His Ser Leu Glu

20 25

<210>150

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>150

Ala Gln Gly Trp Cys Lys Glu Gly Pro Thr Leu Arg Glu Trp Leu Arg

1 5 10 15

Trp Gly Phe Leu Cys His Ser Leu Glu

20 25

<210>151

<211>837

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

<221>CDS

<222>(57)..(785)

<400>151

tcgattaatc gatttgattc tagatttgtt ttaactaatt aaaggaggaa taacat atg 59

Met

1

gac aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc ctg ggg 107

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

5 10 15

gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 155

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 203

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 251

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60 65

cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 299

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

70 75 80

cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 347

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

85 90 95

aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 395

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 443

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 491

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140 145

ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 539

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

150 155 160

tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 587

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 635

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 683

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 731

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220 225

ccg ggt aaa ggt gga ggt ggt ggt gca cag aaa gcg gcc gca aaa aaa 779

Pro Gly Lys Gly Gly Gly Gly Gly Ala Gln Lys Ala Ala Ala Lys Lys

230 235 240

ctc gag taatggatcc gcggaaagaa gaagaagaag aagaaagccc gaaaggaagc tg 837

Leu Glu

<210>152

<211>243

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>152

Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

1 5 10 15

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Pro Gly Lys Gly Gly Gly Gly Gly Ala Gln Lys Ala Ala Ala Lys

225 230 235 240

Lys Leu Glu

<210>153

<211>45

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(22)..(22)

＜223〉position 22, the PEG joint

<400>153

His Ile Arg Glu Gly Pro Thr Leu Arg Gln Trp Leu Val Ala Leu Arg

1 5 10 15

Met Val Gly Gly Gly Pro Glu Gly Gly Gly Gly His Ile Arg Glu Gly

20 25 30

Pro Thr Leu Arg Gln Trp Leu Val Ala Leu Arg Met Val

35 40 45

<210>154

<211>43

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<220>

＜221〉misc_ characteristic

<222>(43)..(43)

＜223〉position 43, C-terminal Fc

<400>154

Thr Cys Glu Gln Gly Pro Thr Leu Arg Gln Trp Leu Leu Cys Arg Gln

1 5 10 15

Gly Arg Gly Gly Gly Lys Gly Gly Gly Thr Cys Glu Gln Gly Pro Thr

20 25 30

Leu Arg Gln Trp Leu Leu Cys Arg Gln Gly Arg

35 40

<210>155

<211>40

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>155

Gln Leu Gly His Gly Pro Thr Leu Arg Gln Trp Leu Ser Trp Tyr Arg

1 5 10 15

Gly Met Gly Pro Asn Gly Glu Leu Arg Ser Gly Pro Thr Leu Lys Glu

20 25 30

Trp Leu Val Trp Arg Leu Ala Gln

35 40

<210>156

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(19)..(19)

＜223〉position 19, C-terminal Fc

<400>156

Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg

1 5 10 15

Ala Lys

<210>157

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>157

Gly Gly Gly Lys Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys

1 5 10 15

Gln Trp Leu Leu Trp Arg Arg Cys Ala

20 25

<210>158

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, the PEG group of connection

<400>158

Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg

1 5 10 15

Met Gln

<210>159

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>159

Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Val Cys Leu Gly Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu

20 25 30

Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly Leu Gln

35 40 45

<210>160

<211>75

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(76)..(76)

＜223〉position 76, C-terminal Fc

<400>160

Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg

1 5 10 15

Ala Lys Gly Gly Gly Ala Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr

20 25 30

Leu Lys Gln Trp Leu Gln Cys Val Arg Ala Lys Gly Gly Gly Ala Gly

35 40 45

Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys

50 55 60

Val Arg Ala Lys Gly Gly Gly Ala Gly Gly Gly

65 70 75

<210>161

<211>43

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(44)..(44)

＜223〉position 44, C-terminal PEG group

<400>161

Val Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Gln Arg Leu

1 5 10 15

Asn Pro Gly Gly Gly Cys Gly Gly Gly Val Gly Ile Glu Gly Pro Thr

20 25 30

Leu Arg Gln Trp Leu Ala Gln Arg Leu Asn Pro

35 40

<210>162

<211>40

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>162

Glu Leu Arg Ser Gly Pro Thr Leu Lys Glu Trp Leu Val Trp Arg Leu

1 5 10 15

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

20 25 30

Trp Leu Val Trp Arg Leu Ala Gln

35 40

<210>163

<211>43

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<220>

＜221〉misc_ characteristic

<222>(44)..(44)

＜223〉position 44, C-terminal Fc

<400>163

Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg

1 5 10 15

Gln Ala Gly Gly Gly Lys Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr

20 25 30

Leu Lys Gln Trp Leu Glu Tyr Arg Arg Gln Ala

35 40

<210>164

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>164

Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg

1 5 10 15

Gln Ala Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr

20 25 30

Arg Arg Gln Ala

35

<210>165

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>165

Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg

1 5 10 15

Ala Gln Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp

20 25 30

Arg Arg Ala Gln

35

<210>166

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>166

Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg

1 5 10 15

Cys Ala Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp

20 25 30

Arg Arg Cys Ala

35

<210>167

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>167

Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly

1 5 10 15

Leu Gln Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys

20 25 30

Leu Gly Leu Gln

35

<210>168

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>168

Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg

1 5 10 15

Met Gln Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys

20 25 30

Arg Arg Met Gln

35

<210>169

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>169

Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg

1 5 10 15

Ala Lys Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys

20 25 30

Val Arg Ala Lys

35

<210>170

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>170

Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg

1 5 10 15

Gln Ala Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Glu Tyr Arg Arg Gln Ala

35 40

<210>171

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>171

Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg

1 5 10 15

Ala Gln Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg

20 25 30

Glu Trp Leu Ala Trp Arg Arg Ala Gln

35 40

<210>172

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>172

Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg

1 5 10 15

Cys Ala Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Leu Trp Arg Arg Cys Ala

35 40

<210>173

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>173

Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly

1 5 10 15

Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Val Cys Leu Gly Leu Gln

35 40

<210>174

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>174

Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg

1 5 10 15

Met Gln Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg

20 25 30

Glu Trp Leu Gln Cys Arg Arg Met Gln

35 40

<210>175

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>175

Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg

1 5 10 15

Ala Lys Gly Gly Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Gln Cys Val Arg Ala Lys

35 40

<210>176

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>176

Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Glu Tyr Arg Arg Gln Ala

20

<210>177

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>177

Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp

1 5 10 15

Leu Ala Trp Arg Arg Ala Gln

20

<210>178

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>178

Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Leu Trp Arg Arg Cys Ala

20

<210>179

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>179

Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Val Cys Leu Gly Leu Gln

20

<210>180

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>180

Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp

1 5 10 15

Leu Gln Cys Arg Arg Met Gln

20

<210>181

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>181

Gly Gly Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Gln Cys Val Arg Ala Lys

20

<210>182

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>182

Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Glu Tyr Arg Arg Gln Ala Gly Gly Gly Gly Gly Ala Leu Arg Asp

20 25 30

Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg Gln Ala

35 40 45

<210>183

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>183

Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp

1 5 10 15

Leu Ala Trp Arg Arg Ala Gln Gly Gly Gly Gly Gly Glu Ala Leu Leu

20 25 30

Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg Ala Gln

35 40 45

<210>184

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>184

Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Leu Trp Arg Arg Cys Ala Gly Gly Gly Gly Gly Ala Val Pro Gln

20 25 30

Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg Cys Ala

35 40 45

<210>185

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>185

Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Val Cys Leu Gly Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu

20 25 30

Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly Leu Gln

35 40 45

<210>186

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>186

Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp

1 5 10 15

Leu Gln Cys Arg Arg Met Gln Gly Gly Gly Gly Gly Cys Ser Ser Gly

20 25 30

Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg Met Gln

35 40 45

<210>187

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(1)..(1)

＜223〉position 1, N-terminal Fc

<400>187

Gly Gly Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp

1 5 10 15

Leu Gln Cys Val Arg Ala Lys Gly Gly Gly Gly Gly Cys Ser Trp Gly

20 25 30

Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg Ala Lys

35 40 45

<210>188

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(47)..(47)

＜223〉position 47, C-terminal Fc

<400>188

Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg

1 5 10 15

Gln Ala Gly Gly Gly Gly Gly Ala Leu Arg Asp Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Glu Tyr Arg Arg Gln Ala Gly Gly Gly Gly Gly

35 40 45

<210>189

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(47)..(47)

＜223〉position 47, C-terminal Fc

<400>189

Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg

1 5 10 15

Ala Gln Gly Gly Gly Gly Gly Glu Ala Leu Leu Gly Pro Thr Leu Arg

20 25 30

Glu Trp Leu Ala Trp Arg Arg Ala Gln Gly Gly Gly Gly Gly

35 40 45

<210>190

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(47)..(47)

＜223〉position 47, C-terminal Fc

<400>190

Ala Val Pro Gln Gly Pro Thr Leu Lys Gln Trp Leu Leu Trp Arg Arg

1 5 10 15

Cys Ala Gly Gly Gly Gly Gly Ala Val Pro Gln Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Leu Trp Arg Arg Cys Ala Gly Gly Gly Gly Gly

35 40 45

<210>191

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(47)..(47)

＜223〉position 47, C-terminal Fc

<400>191

Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly

1 5 10 15

Leu Gln Gly Gly Gly Gly Gly Tyr Cys Asp Glu Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Val Cys Leu Gly Leu Gln Gly Gly Gly Gly Gly

35 40 45

<210>192

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(47)..(47)

＜223〉position 47, C-terminal Fc

<400>192

Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg

1 5 10 15

Met Gln Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Pro Thr Leu Arg

20 25 30

Glu Trp Leu Gln Cys Arg Arg Met Gln Gly Gly Gly Gly Gly

35 40 45

<210>193

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(47)..(47)

＜223〉position 47, C-terminal Fc

<400>193

Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg

1 5 10 15

Ala Lys Gly Gly Gly Gly Gly Cys Ser Trp Gly Gly Pro Thr Leu Lys

20 25 30

Gln Trp Leu Gln Cys Val Arg Ala Lys Gly Gly Gly Gly Gly

35 40 45

<210>194

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(24)..(24)

＜223〉position 24, C-terminal Fc

<400>194

Ala Leu Arg Asp Gly Pro Thr Leu Lys Gln Trp Leu Glu Tyr Arg Arg

1 5 10 15

Gln Ala Gly Gly Gly Gly Gly

20

<210>195

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(24)..(24)

＜223〉position 24, C-terminal Fc

<400>195

Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg

1 5 10 15

Ala Gln Gly Gly Gly Gly Gly

20

<210>196

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(24)..(24)

＜223〉position 24, C-terminal

<400>196

Glu Ala Leu Leu Gly Pro Thr Leu Arg Glu Trp Leu Ala Trp Arg Arg

1 5 10 15

Ala Gln Gly Gly Gly Gly Gly

20

<210>197

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(24)..(24)

＜223〉position 24, C-terminal Fc

<400>197

Tyr Cys Asp Glu Gly Pro Thr Leu Lys Gln Trp Leu Val Cys Leu Gly

1 5 10 15

Leu Gln Gly Gly Gly Gly Gly

20

<210>198

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(24)..(24)

＜223〉position 24, C-terminal Fc

<400>198

Cys Ser Ser Gly Gly Pro Thr Leu Arg Glu Trp Leu Gln Cys Arg Arg

1 5 10 15

Met Gln Gly Gly Gly Gly Gly

20

<210>199

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<220>

＜221〉misc_ characteristic

<222>(24)..(24)

＜223〉position 24, C-terminal Fc

<400>199

Cys Ser Trp Gly Gly Pro Thr Leu Lys Gln Trp Leu Gln Cys Val Arg

1 5 10 15

Ala Lys Gly Gly Gly Gly Gly

20

Claims (25)

1. in conjunction with compound and its physiological acceptable salt of mpl acceptor, it comprises following sequence:

X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18，

X1-X4 wherein, X9-X10 and X13-X18 are amino acid independently of one another, described compound to the binding affinity of mpl acceptor greater than following sequences:

X19-X20-I-E-G-P-T-L-R-Q-W-L-A-A-R-A-X21-X22，

Wherein X19-X20 and X21-X22 are amino acid independently of one another.

2. in conjunction with compound and its physiological acceptable salt of mpl acceptor, it comprises following sequence:

X1-X2-X3-X4-G-P-T-L-X9-X10-W-L-X13-X14-X15-X16-X17-X18，

X1-X4 wherein, X9-X10 and X13-X18 are amino acid independently of one another, the biological activity that described compound has is greater than following sequences:

X19-X20-I-E-G-P-T-L-R-Q-W-L-A-A-R-A-X21-X22，

Wherein X19-X20 and X21-X22 are amino acid independently of one another.

3. the compound of claim 1, wherein:

X1 is selected from A, V, W, M, G, Y, C, Q, E, R and H;

X2 is selected from A, V, L, I, G, S and C;

X3 is selected from L, I, P, W, G, S, D, K and R;

X4 is selected from L, G, Q, D, E and H;

X9 is selected from K and R;

X10 is selected from Q and E;

X13 is selected from A, V, and L, S, Q, E and R,

X14 is selected from A, W, T, Y, C and Q;

X15 is selected from V, L, G, Y and R;

X16 is selected from A, L, F, G and R;

X17 is selected from A, V, L, M, G, C, Q and N;

X18 is selected from A, V, P, M, F, G, C, Q and K.

4. the compound of claim 2, wherein:

X1 is selected from A, V, W, M, G, C, E and R;

X2 is selected from A, V, L, M, F, G, S, C, D and R;

X3 is selected from A, L, I, P, W, Q, K and R;

X4 is selected from L, G, Q, D and E;

X9 is selected from K, R and H;

X10 is selected from Q and E;

X13 is selected from A, L, P, F, G, Q, N, E and R;

X14 is selected from L, W, M, C, Q and H;

X15 is selected from V, L, P, G, Y and R;

X16 is selected from A, V, L, F, S, Q, K and R;

X17 is selected from A, V, L, W, M, G, S, C and N;

X18 is selected from A, V, P, M, G, C, Q and K.

5. in conjunction with the compound of mpl acceptor, it comprises following sequence:

X1-X2-R-E-G-P-T-L-R-Q-W-L-X13-W-R-R-X17-X18, X1 wherein, X2, X13, X17 and X18 are amino acid independently of one another.

6. in conjunction with the compound of mpl acceptor, it comprises and is selected from the sequence of SEQ ID NO:2 to SEQ IDNO:30:

Peptide sequence SEQ ID NO： GAREGPTLRQWLEWVRVG 2 RDLDGPTLRQWLPLPSVQ 3 ALRDGPTLKQWLEYRRQA 4 ARQEGPTLKEWLFWVRMG 5 EALLGPTLREWLAWRRAQ 6 MARDGPTLREWLRTYRMM 7 WMPEGPTLKQWLFHGRGQ 8 HIREGPTLRQWLVALRMV 9 QLGHGPTLRQWLSWYRGM 10 ELRQGPTLHEWLQHLASK 11 VGIEGPTLRQWLAQRLNP 12 WSRDGPTLREWLAWRAVG 13 AVPQGPTLKQWLLWRRCA 14 RIREGPTLKEWLAQRRGF 15 RFAEGPTLREWLEQRKLV 16 DRFQGPTLREWLAAIRSV 17 AGREGPTLREWLNMRVWQ 18 ALQEGPTLRQWLGWGQWG 19 YCDEGPTLKQWLVCLGLQ 20 WCKEGPTLREWLRWGFLC 21 CSSGGPTLREWLQCRRMQ 22 CSWGGPTLKQWLQCVRAK 23 CQLGGPTLREWLACRLGA 24 CWEGGPTLKEWLQCLVER 25 CRGGGPTLHQWLSCFRWQ 26 CRDGGPTLRQWLACLQQK 27 ELRSGPTLKEWLVWRLAQ 28 GCRSGPTLREWLACREVQ 29 TCEQGPTLRQWLLCRQGR 30

7. the compound of claim 1,2 or 6, it is a cyclic.

8. the compound of claim 1,2 or 6, wherein at least one amino-acid residue has the D configuration.

9. the compound of claim 1,2 or 6, wherein all amino-acid residues have the D configuration.

10. a kind of dimer or the polymer of the compound of claim 1,2 or 6.

11. in conjunction with the composition of mpl acceptor, it comprises following formula:

(LN1) _l--(TMP1) _a--(LN2) _m--(TMP2) _b--(LN3) _n--(TMP3) _c--(LN4) _o--(TMP4) _d

TMP1 wherein, TMP2, TMP3, TMP4 are selected from the compound of claim 1,2 and 6 independently of one another; LN1, LN2, LN3 and LN4 are joint independently of one another; A, b, c and d are 0 to 20 integer independently of one another; L, m, n and o are 0 to 20 integer independently of one another.

12. the composition of claim 11, it further comprises carrier, and has following formula: (V1) _v--(LN1) _l--(TMP1) _a--(LN2) _m--(TMP2) _b--(LN3) _n--(TMP3) _c--(LN4) _o--(TMP4) _d--(V2) _w

Wherein V1 and V2 are carrier independently of one another, and v and w are 0 to 1 integer independently of one another.

13. the compound of claim 12, LN1 wherein, LN2, LN3 and LN4 contain peptide.

14. the composition of claim 12, wherein V1 and/or V2 contain the Fc district.

15. the composition of claim 12, wherein V1 and/or V2 contain the IgG1Fc district.

16. the polynucleotide of code set compound, described composition is selected from the composition of claim 12.

17. comprise the expression vector of the polynucleotide of claim 12.

18. contain the host cell of the expression vector of claim 12.

19. the host cell of claim 12, wherein cell is a Bacillus coli cells.

20. the host cell of claim 12, wherein cell is a prokaryotic cell prokaryocyte.

21. the host cell of claim 12, wherein cell is an eukaryotic cell.

22. a pharmaceutical composition, it comprises significant quantity and composition pharmaceutical carrier blended claim 12.

23. in Mammals, treat the method for thrombocytopenia, comprise the composition of the claim 12 of administering therapeutic significant quantity.

24. increase megalokaryocyte or hematoblastic method among the patient, comprise the compound of described patient being used the claim 12 of significant quantity.

25. in conjunction with the compound of mpl acceptor, it comprises following formula:

(V1) _v--(TMP1) _a--(V2) _w

Wherein V1 and V2 respectively are IgG1 Fc districts, are 1 if condition is v, and then w is zero, if v is 0, then w is 1, and TMP1 is the peptide of SEQ ID NO:2 to 30, and a is 1 to 20 integer.

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