WO1998007848A1

WO1998007848A1 - Recombinant mpo fragment and method for assaying mpo autoantibody reaction site by using the same

Info

Publication number: WO1998007848A1
Application number: PCT/JP1997/002910
Authority: WO
Inventors: Kazuo Suzuki; Masaru Tanokura
Original assignee: Teikoku Hormone Mfg. Co., Ltd.
Priority date: 1996-08-21
Filing date: 1997-08-21
Publication date: 1998-02-26
Also published as: AU3867797A

Abstract

A recombinant MPO fragment reacting with myeloperoxidase-specific antineutrophile cytoplasmic antibody (MPO-ANCA); a process for producing the recombinant MPO fragment which comprises amplifying MPO cDNA by the PCR method, integrating a part of the cDNA into an expression vector to give a recombinant plasmid, transforming Escherichia coli by this plasmid, culturing the E. coli transformant to thereby express the recombinant MPO fragment, and then taking up the recombinant MPO fragment from the E. coli; a method for assaying the MPO-ANCA reaction site and the amount thereof in a specimen by using the recombinant MPO fragment; and reagent kits for assaying the MPO-ANCA reaction site and the amount thereof which contain a panel set of the recombinant MPO fragment and labeled antihuman IgG.

Description

Description Recombinant MPO fragment and method for measuring reactive site of MPO autoantibody using the same

The present invention provides a recombinant MPO fragment that reacts with MPO-ANCA in a body fluid of a patient with an autoimmune disease caused by a myelin peroxidase-specific anti-neutrophil cytoplasmic antibody (PO-ANCA), The present invention relates to a method for producing an MPII fragment, a method for measuring the reaction site of MPO-ANCA, and a kit therefor. Background art

MPO (myeloperoxidase) autoantibody, which is not normally found in the blood, is an autoimmune disease. Tema! ^ One Death (SLE), rheumatoid arthritis, and necrotizing vasculitis at the capillary level.

MPO-ANCA is an autoantibody belonging to the IgG fraction, and its detection methods include indirect fluorescent antibody method (IIF), enzyme antibody method (ELISA), radioimmunoassay method (RIA), and immunoblotting method. (IB) and flow cytometry (FC). Recently, MPO purified from human and neutrophil cytoplasmic α-granules was used as an antigen, alkaline phosphatase-specific anti-human IgG was used as a labeled antibody, and MPO was Kits for measuring ANCA are commercially available. However, even with high MPO-ANCA antibody levels, there are many exceptional cases related to the disease, and there is not always a correlation between the disease and the MPO-ANCA antibody level. There was a problem that there is. Therefore, in the case of autoimmune diseases such as necrotizing crescent-forming inflammation and vasculitis, determining the relationship between the MPO-reactive site of MPO-ANCA and the disease can identify the etiology of those diseases. It will be useful for prevention and treatment of disease.

The amino acid sequence constituting the MP◦ peptide and the nucleic acid sequence of the cDNA encoding it are described in Biochemistry 27, 5906-5914 (1988), and the iopeptide is 14 kDa L chain and a 59 kDa H chain.

The present inventors obtained a recombinant MP0 fragment reactive with MPO-ANCA for the purpose of clarifying the relationship between the reaction site and the disease for the analysis of MPO-ANCA epitope. The panel set of these fragments shown in Fig. 2 examined the reaction with MPO-ANCA, and revealed the difference in recognition sites, thereby making it possible to clarify the relationship with disease. Disclosure of the invention

The present invention relates to a recombinant MPII fragment reactive with MPO-ANCA. The present invention also relates to a recombinant MP0 fragment which is an MPO fragment fused to 6XHis. Furthermore, the present invention relates to a recombinant MPO fragment, wherein the recombinant MPO fragment is a peptide selected from the group consisting of Sequence Listings 1 to 32. In the present invention, the recombinant MPO fragment may be a panel set selected from at least two of the peptide fragments in Sequence Listings 1 to 32. In the method for producing the recombinant MPO fragment of the present invention, a recombinant plasmid is obtained by amplifying MPOc DNA by the PCR method, inserting a part of the cDNA into an expression vector, and obtaining Escherichia coli transformed with this plasmid. Culturing the Escherichia coli to express the recombinant MPO fragment, and collecting the recombinant MP0 fragment from the Escherichia coli. In the production method of the present invention, the expression vector may be a pQE vector, and the recombinant MP0 fragment may be a 6XHis fusion MPO fragment.

The invention further provides:

1) A sample is added to a plate on which the recombinant MPO fragment is immobilized, and an immunoreaction is performed.

2) Next, add immunoglobulin I IgG to make immune reaction.

3) The present invention relates to a method for measuring a reaction site of MPO-ANCA in a sample and the amount thereof by measuring the amount of label bound to the plate.

The present invention further relates to a reagent kit for measuring the PO-ANCA reaction site and the amount thereof, including the panel set of the recombinant MPO fragment and the labeled anti-human IgG. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a structural diagram of a panel set of a recombinant MPO fragment. FIG. 2 is a schematic diagram of a method for producing a recombinant 6 XHίs fusion MPO fragment.

FIG. 3 and FIG. 4 are diagrams showing the reactivity of the anti-MP0 antibody to each fragment by ELISA. BEST MODE FOR CARRYING OUT THE INVENTION

As the recombinant MPO fragment of the present invention, the MPO fragment is fused with a protein or peptide such as 6 histidines (hereinafter, referred to as 6XHis), mul! Those that have been used are used. Among them, the recombinant plasmid can be easily obtained by using the expression vector pQE in which the cDNA corresponding to 6XHis is inserted at the position of restriction enzyme BamHI, in the case of fusion with 6XHis. . 6 XHis fusion MP0 fragment can be absorbed into an affinity column using a Ni-NTA (nickel monotrilotriacetic acid) resin column, and can be produced in large quantities because elution and purification are easy. . This column is also effective if the insolubilized recombinant protein that enters E. coli inclusion bodies is denatured with guanidine hydrochloride-urea. 6 The XHis fusion MP0 fragment has no sugar chain, does not cross over with other proteins, and reacts specifically with ANCA. Can be used for measurement.

Examples of the recombinant MPO fragment of the present invention include those having the amino acid sequences of Sequence Listings 1 to 32 shown in Table 1. By combining at least two of these fragments and using them as a panel set that covers the whole, it is possible to clarify the relationship between the disease state and the MPO-ANCA epitope. table 1

Expression of recombinant MPO fragment Amino acid coordinates SEQ ID NO: kD expression

N P 1.4: AA269—730 1, 2 53.0 +

H 2: A A 279-5 1 13, 4 28.4 + H 3 AA5 1 2-745 5, 6 28.5

H 7 AA279-409, 8 15.7 + H 8 AA341 -479 9, 1 0 16.6 + H 9 AA4 1 0-538 1 1, 1 2 15.5 + H 5 AA 5 1 2- 598 1 3, 1 4 1 1.1 + + + H 6 AA541 -745 1 5, 1 6 23.5 + + H 1 1 AA 598 45 1 7, 1 8 1 7.5 + + 1 AA 1 65- 272 1 9, 20 1

2 AA 1 65-2 1 4 2 1, 22 6.8 + 3 AA 2 1 5-272 23, 24 + + H 7 a AA279-344 25, 26 6.5 + H 7 b AA 340-41 0 27, 28 7.3 + H 12 AA600-678 29, 308 + + H 13 AA 678-745 31, 32 6.7 + + To prepare the recombinant MPO fragment of the present invention, M POc DNA [Biochemistry 27, 5906-5914 (1988)] The portion of c08 corresponding to the MPO fragment in Table 1 (see the nucleic acid sequences in Sequence Listings 1 to 32) is amplified by PCR. As shown in FIG. 2, the C DNA corresponding to the MPO fragment was incorporated into an expression vector p QE [Nucleic Acids Res., 14, 7617-7631 (1986)] having a cDNA corresponding to the 6XHis tag, Obtain a recombinant plasmid. Escherichia coli is transformed with this plasmid by a conventional method. The recombinant E. coli is cultured to express the MP0 fragment. The Escherichia coli is sonicated to destroy the cells, treated with guanidine hydrochloride, urea, etc. to solubilize, and the denatured protein is absorbed into a Ni-NTA resin chelate column, And elute with Tris buffer containing phosphate. In this way, the recombinant 6XHis-fused MP0 fragment can be purified in one step, so that a large amount of sample can be easily produced. In addition, it is preferable to induce the synthesis of 3-galactosidase (of E. coli) by adding IPTG (Isopropyl 1.1-D-galactoside) during the culturing of recombinant E. coli.

The cultured E. coli is collected by centrifugation and stored at 180 ° C. Thaw the frozen cells at the time of use, hang in a buffer solution, wash, treat with guanidine hydrochloride and urea as described above, and purify the lysate with an Nί-NTA column.

The reaction site of MP 0 -AN C 測定 using the recombinant リ fragment of the present invention can be measured by a known Western plot method or ELISA method. That is, a sample is reacted with an immobilized antigen in which the MP0 fragment is immobilized on a plate, and allowed to bind to the MP0-ANCA antibody in the sample. The labeled anti-human IgG is then bound. As the label, a radioactive label, an enzyme label, a fluorescent label, a luminescent label, or the like is used. For example, in an ELISA method using an enzyme label, a serum of a patient positive for the MP0 antibody is reacted as a primary antibody, and then a horseradish oxidase-like enzyme-like lipase or a 3-galactosidase is used as a secondary antibody. The antibody is allowed to react with an anti-human IgG IgG heron antibody. When the enzyme is alkaline phosphatase III / 3-galactosidase, p-diphenyl phenyl phosphate is used as a substrate, and when the enzyme is horseradish peroxidase, 0-phenylenediamine is used as a substrate.

The present invention also relates to a reagent kit for measuring the amount of ANC and its reaction site by the above immunoassay. The reagent kit can contain a panel set of the above-described recombinant MP0 fragment, labeled anti-human IgG, and, in the case of enzyme labeling, an enzyme substrate and, if necessary, a buffer and other reagents. . Example

Example 1. Expression of MPOc DNA fragment in E. coli

1. Synthesis and purification of ribonucleotides

Oligonucleotides as primers were synthesized using a 0LIG01000 DNA synthesizer (Beckman, USA). 0. 5 ml of AM A reagent (NH ₄ OH and NH ₂ CH ₃ 1 0-20% solution) was placed vial, was coupled column and a syringe. The syringe was aspirated and the sample was adsorbed on the column. After standing for 5 minutes, the adsorbed lignonucleotide was eluted from the column with a syringe together with the AMA solution. The sample containing the AM A reagent was heated at 55 ° C for 10 minutes. After cooling on ice, the AMA reagent was removed with a speed pack.

2. Width of each DNA fragment by PCR

Each cDNA fragment was amplified by PCR based on the primer table below.

Primers consist of the following sequences from 5 'to 3' (left to right): The numbers indicate the amino acid sequence numbers. 5 'primer

165

1 and 2 5 '-GCG CGG ATC CCC GTG ACT TGC CCG GAG CAG

215

3 5'-GCG CGG ATC CCC CCC GGG GTC AAG CGC AAC

279

H 2, H 75 '-GCG GGG ATC CCC GTC AAC TGC GAG ACC AGG

341

H 8 5 '-GCG GGG ATC CCC ATG GTG TAC GGC AGC GAG

410

H 9 5 '-GCG GGG ATC CCC GAG CTC ACC TCC AT

512

H 3, H 55 '-GCG GGG ATC CCC CTG GAC AAT CGG TAC CAG

541

H 6 5 '-GCG CGG ATC CCC GGT GGC ATT GAC CCC ATC

598

H 1 1 5 '-GCG CGG ATC CCC GGA TAC AAT GCC TGG AGG

279

H 7 a 5 'One TCC GGG ATC CCC GTC AAC TGC GAG ACC

340

H 7 b 5 '-C GTG GGA TCC AGC ATG GTG TAC GG

600

H 1 2 5 '-GCA GGA TCC AAT GCC TGG AG

6f 8

H 1 3 5 '-AT GGG GAT CCG TTC TGG TGG GAG AAC G

3 'primer

214

2 5 '-CGC AAG CTT CGT CCA GCC GTA GGG AAG

272

1 and 3 5 '-CGC AAG CTT CCG GGC GGC CGG CTC AGG

409

H 7 5 'I CGC AAG CTT CAT CTC ACT GGA ACG GGT 474

H 8 CGC AAG CTT CTT CCT CAT GGC CGT TGG C

511

H 2 CGC AAG CTT CCC GCG GAA CAT GAA GGG TTG

538

H 9 5 'CGC AAG CTT GAC CCT CCA GGA GGC AAA

598

H 5 CGC AAG CTT TCC TGG GAG GCC GTG GTC

F 45

H 3, H 6, H 1 15 '-CGC AAG CTT CGA GGC TC TTC CCT CCA GGA AGC

344

H 7 a 5 'One CGC AAG CTT GCC GTA CAC CAT GCT GGC

410

H 7 b 5 '-A GGT AAG CTT GGG CAT CTC ACT GG

678

H 1 2 5 '-T CTC AAG CTT AAA CCG ATC ACC ATC CCG

745

H 1 3 'I C TGG AAG CTT GGA GGC TTC ACG CCA GGA AG

The 100 At I reaction contains:

100 ng of MPO cDNA, 8 μ. I of the same amount of ATP, TTP, CTP and GTP nucleotide triphosphates (2.5 mM each), 10 times the concentration of 10 I buffer Solution (500 mM KCI, 200 mM Tris-HCl, pH 8.4), 3 l of 50 mM MgCI2, 0.5 I ( _2.5 U) of Taq DNA polymerase and each fragment Above primer (0.1 ltM).增 The width was measured using a thermal cycler manufactured by Takara. The program repeats〗 minutes at 94 ^, 1 minute at 55 ° C, and 1 minute at 72 ^ 25 times.

The DNA amplified by PCR was digested with HindII and BamHI restriction enzymes and inserted into a pQE expression vector. DN A other than NPI 4 is Hindill and B The am HI site was integrated into plasmid pQE32, and the NP1.4 DNA was integrated into plasmid pQE30 at the SamI and PstI sites.

3. Ligation and Transformation

0.5 I pQE30 or pQE32 vector (5 ng), 0.5 I? 〇 amplified 01 \ 1 fragment (2 0 | ^) and 41 The reaction solution of the shilling kit and the ligase solution of〗 I of the same kit were mixed gently, and the mixture was heated at 16 ° C. for 30 minutes.

Transformation into J109 E. coli was performed as follows.

The reaction solution was ice-cooled for 30 minutes, heated at 42 ° C for 1 minute, and ice-cooled again for 5 minutes. An S OC solution (900 ^ I) was added to the reaction solution, and the mixture was heated at 37 ° C for 2 hours.

The transformed Escherichia coli was spread on an LB medium containing ampicillin (100 μg / ml) and cultured.

4. Transformation of plasmid DNA

The colony generated in the above 3 was inoculated in an LB medium containing 1.2 ml of ampicillin (100 g / ml), shaken at a speed of 350 rpm / min, and cultured at 37 ^ for 6 hours. After collection, 1001 of SET and 10 ^ 1 of lysozyme were added and vigorously shaken. This reaction solution was left at room temperature for 5 minutes, and then boiled for 1 minute to prepare a lysate.

Centrifugation was performed at room temperature at 12,000 OX g for 10 minutes.

To this supernatant was added 100 AiI of isopropanol and vigorously shaken at room temperature for 10 minutes.

Pellet of nucleic acid was obtained by centrifugation at 12,000 X g for 5 minutes 4. The nucleic acid pellet was washed with 200 tI of 70% ethanol. The nucleic acids were dissolved in 50 UL I of TE (pH 8.0). The plasmid DA was digested with a restriction enzyme and subjected to electrophoresis, and the size was confirmed from the mobility. n

Example 2. Preparation of recombinant 6 XHis fusion MPO fragment

(a) Preculture (1 L)

A 900 ml solution containing 12 g of bacto triton and 24 g of bacto yeast extract was used as an A culture solution and autoclaved.

Was prepared as a B _{broth, 0. 1 7 M KH 2 P0} 4 2. 1 00 ml of a solution containing 3 g and _{_{0. 72 MK 2 H Ρ0 4 1}} 2. 54 g were prepared and autoclaved.

Mix solution A and solution B, add ampicillin 50 mg / ml to 100 g / ml, inoculate with E. coli containing cDNA corresponding to the MPO fragment.

The cells were cultured at 7 ° C.

100 mM IPTG was added to this culture solution to a concentration of 1 mM, and the cells were cultured at 37 ^ for 15 hours. The culture was centrifuged at 4,000 OXg for 20 minutes to collect the cells, and the cells were stored at -80 ° C.

Thaw the frozen cells for 5 min and mix with 5 ml Buffer A (6 M guanidine hydrochloride, 0.1 M Na-phosphate, 0.01 M Tris / HC and pH 8 0).

(b) Large scale culture (13.5 L culture)

Add 13 L of tap water to a jar fermenter and bacto tryptone 1 for A culture solution.

80 g and 360 g of bacto yeast extract were added. The volume was adjusted to 13.5 L and autoclaved. Then a predetermined amount of B medium was added. 25 ml of ampicillin (50 mg / ml) was added, a preculture was added, and the cells were cultured at 37 for 14 hours and 30 minutes. IPTG (100 mM) was added to a concentration of 1 mM, and the cells were cultured at 37 for 14 hours and 30 minutes. Cells were centrifuged and the pellet was stored at -8 (TC.

(0 purification Thaw the frozen cells and add buffer A (6 M guanidine hydrochloride, 0.1 M Na-phosphate, 0.0 1 1 ^ 1 Trisuno 1 to 1 at a ratio of 5 ml / g wet weight. The lysate was centrifuged at 10,000 OXg for 15 minutes at 4 ° C. The resulting cell lysate was equilibrated with buffer A 12 Flowed at a rate of 15 ml / hour on the 1-1-1 column of | 711. 10 times column volume of buffer A and 10 times column volume of buffer B (8 M urea, 0. The column was washed with 1 M Na-phosphate, 0.01 M Tris ZHCI, pH 8.0, followed by buffer C (8 M urea, 0.1 M Na-phosphate, 0.1 M 01 M Tris / HC, pH 5.9), Buffer E (8 M Urea, 0.1 M Na-phosphate, 0.01 M Tris ZHCI, pH 4.5), and Buffer The recombinant 6 XHis fusion MPO fragment was eluted from the column in the order of F (6 M guanidine hydrochloride, 0.2 M acetic acid).

Example 3 Measurement of MP0—ANCA Using MPO Fragment by ELISA Method

(1 -a) Preparation of MPO fragment binding plate

Various MPO fragments were dissolved in 0.05 M carbonate buffer (pH 9.6, containing 0.02% sodium azide), diluted to 1 O jag / ml with the same buffer, and then diluted with sodium hydroxide. II (Nunc, Denmark) was dispensed at 100 I per well and incubated overnight at room temperature. After removing the upper part by suction, 100 l of a 1% (W / V) sialbumin PBS solution was dispensed at a time, and a blocking reaction was carried out at room temperature for 1 hour. Each well was washed four times with PBS containing 0.05% Tween 20 (manufactured by Sigma) and used as an MPO fragment binding plate.

(11-b) MPO—measurement of ANCA

The diluted serum sample (diluted with PBS bovine albumin) or a similar sample (standard serum serially diluted from 1Z10 to 1/300) was added to the MP0 fragment binding plate well. After adding 0 μ. I and incubating at room temperature for 2 hours, the wells were washed four times with PBSZ Tween 20. Next, a 1 / 3,000 dilution of a horseradish peroxidase-labeled anti-human IgG IgG heron antibody (Dako, Denmark) was added in 100 / I increments and incubated at room temperature for 2 hours. Then, the plate was washed four times with PB SZ Tween 20 and the substrate solution (0.2 mg / ml o-phenylenediamine, 0.005% hydrogen peroxide, 0.05 M phosphate buffer, pH 5 6) was added in 1 1 Ο μΙ increments. After standing at room temperature for 30 minutes, the reaction was stopped by adding 100 μl of 0.5Μ sulfuric acid. The absorbance at 492 nm can be measured, and the amount of MPO-ANCA in the serum sample can be determined from the standard curve.

FIG. 3 and FIG. 4 show the values measured for the reactivity of the anti-MPO antibody to each fragment by the ELISA method.

The base sequence and amino acid sequence of the recombinant MPO fragment according to the present invention are shown below. Note that H7 was further divided to create H7a and H7b. Also, H11 was divided to create H12 and H13. Industrial applicability

The recombinant MP0 fragment of the present invention can be used for MPO-AN in body fluids of autoimmune disease patients and vasculitis patients caused by myelin peroxidase specific anti-neutrophil cytoplasmic antibody (MPO-ANCA). It is useful for detecting specific sites that react with CA. Further, the present invention provides a method for producing the MP◦ fragment, and further provides a method for measuring a reaction site of MPO-ANCA and a kit used for the method. Sequence Listing SEQ ID NO: 1 (Ν Ρ Ί. 4 = AA269-730)

Sequence length: 462

Sequence type: amino acid

Number of chains: single strand

Topology: Linear

Array:

Pro Ala Ala Arg Ala Ser Phe Val Thr Gly Val Asn Cys Glu Thr

1 5 10 15 Ser Cys Val Gin Gin Pro Pro Cys Phe Pro Leu Lys lie Pro Pro

20 25 30

Asn Asp Pro Arg lie Lys Asn Gin Ala Asp Cys lie Pro Phe Phe

35 40 45

Arg Ser Cys Pro Ala Cys Pro Gly Ser Asn lie Thr lie Arg Asn

50 55 60

Gin lie Asn Ala Leu Thr Ser Phe Val Asp Ala Ser Me Val Tyr

65 70 75

Gly Ser Glu Glu Pro Leu Ala Arg Asn Leu Arg Asn Met Ser Asn

80 85 90 Gin Leu Gly Leu Leu Ala Val Asn Gin Arg Phe Gin Asp Asn Gly

95 100 105

Arg Ala Leu Leu Pro Phe Asp Asn Leu His Asp Asp Pro Cys Leu

110 115 120

Leu Thr Asn Arg Ser Ala Arg lie Pro Cys Phe Leu Ala Gly Asp

125 130 135

Thr Arg Ser Ser Glu Met Pro Glu Leu Thr Ser Met His Thr Leu

140 145 150 Leu Leu Arg Glu His Asn Arg Leu Ala Thr Glu Leu Lys Ser Leu

155 160 165 Asn Pro Arg Trp Asp Gly Glu Arg Leu Tyr Gin Glu Ala Arg Lys

170 175 180 lie Val Gly Ala Met Val Gin lie lie Thr Tyr Arg Asp Tyr Leu

185 190 195 05 ^ ς ίο

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OT6lO / t6, If / 13d 8m0 / 86 ΟΛΛ Met Ser Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys

455 460 SEQ ID NO: 2 (NP 1.4 = NA 9 8 2—2 3 6 7)

Array length: 1 3 8 6

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

CCGGCCGCCC GGGCCTCCTT CGTCACTGGC GTCAACTGCG AGACCAGCTG 50 CGTTCAGCAG CCGCCCTGCT TCCCGCTCAA GATCCCGCCC AATGACCCCC 100 GCATCAAGAA CCAAGCCGAC TGCATCCCGT TCTTCCGCTC CTGCCCGGCT 150

TGCCCCGGGA GCAACATCAC CATCCGCAAC CAGATCAACG CGCTCACTTC 2 00

CTTCGTGGAC GCCAGCATGG TGTACGGCAG CGAGGAGCCC CTGGCCAGGA 2 50 ACCTGCGCAA CATGTCCAAC CAGCTGGGGC TGCTGGCCGT CAACCAGCGC 300

TTCCAAGACA ACGGCCGGGC CCTGCTGCCC TTTGACAACC TGCACGATGA 3 50

CCCCTGTCTC CTCACCAACC GCTCAGCGCG CATCCCCTGC TTCCTGGCAG 400

GGGACACCCG TTCCAGTGAG ATGCCCGAGC TCACCTCCAT GCACACCCTC 4 50

TTACTTCGGG AGCACAACCG GCTGGCCACA GAGCTCAAGA GCCTGAACCC 500 TAGGTGGGAT GGGGAGAGGC TCTACCAGGA AGCCCGGAAG ATCGTGGGGG 550

CCATGGTCCA GATCATCACT TACCGGGACT ACCTGCCCCT GGTGCTGGGG 600

CCAACGGCCA TGAGGAAGTA CCTGCCCACG TACCGTTCCT ACAATGACTC 650

AGTGGACCCA CGCATCGCCA ACGTCTTCAC CAATGCCTTC CGCTACGGCC 700

ACACCCTCAT CCAACCCTTC ATGTTCCGCC TGGACAATCG GTACCAGCCC 750 ATGGAACCCA ACCCCCGTGT CCCCCTCAGC AGGGTCTTTT TTGCCTCCTG 800

GAGGGTCGTG CTGGAAGGTG GCATTGACCC CATCCTCCGG GGCCTCATGG 850

CCACCCCTGC CAAGCTGAAT CGTCAGAACC AAATTGCAGT GGATGAGATC 900

CGGGAGCGAT TGTTTGAGCA GGTCATGAGG ATTGGGCTGG ACCTGCCTGC 950

TCTGAACATG CAGCGCAGCA GGGACCACGG CCTCCCAGGA TACAATGCCT 1000 GGAGGCGCTT CTGTGGGCTC CCGCAGCCTG AAACTGTGGG CCAGCTGGGC 1050

ACGGTGCTGA GGAACCTGAA ATTGGCGAGG AAACTGATGG AGCAGTATGG 1100

CACGCCCAA.C AACATCGACA TCTGGATGGG CGGCGTGTCC GAGCCTCTGA 1150

AGCGCAAAGG CCGCGTGGGC CCACTCCTCG CCTGCATCAT CGGTACCCAG 1200

TTCAGGAAGC TCCGGGATGG TGATCGGTTT TGGTGGGAGA ACGAGGGTGT 1250 GTTCAGCATG CAGCAGCGAC AGGCCCTGGC CCAGATCTCA TTGCCCCGGA 1300

TCATCTGCGA CAACACAGGC ATCACCACCG TGTCTAAGAA CAACATCTTC 1350

ATGTCCAACT CATATCCCCG GGACTTTGTC AACTGC 1386

5 SEQ ID NO: 3 (H 2 = AA 279— 5 1 1)

Array length: 233

Sequence type: amino acid

Number of chains: single strand

Topology: linear

10 arrays:

Val Asn Cys Glu Thr Ser Cys Val Gin Gin Pro Pro Cys Phe Pro

1 5 10 15

Leu Lys lie Pro Pro Asn Asp Pro Arg lie Lys Asn Gin Ala Asp

20 25 30

I 5 Cys lie Pro Phe Phe Arg Ser Cys Pro Ala Cys Pro Gly Ser Asn

35 40 45 lie Thr lie Arg Asn Gin lie Asn Ala Leu Thr Ser Phe Val Asp

50 55 60

Ala Ser Met Val Tyr Gly Ser Glu Glu Pro Leu Ala Arg Asn Leu

20 65 70 75

Arg Asn Met Ser Asn Gin Leu Gly Leu Leu Ala Val Asn Gin Arg

80 85 90

Phe Gin Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn Leu His

95 100 105

^ 5 Asp Asp Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg He Pro Cys

110 115 120

Phe Leu Ala Gly Asp Thr Arg Ser Ser Glu Met Pro Glu Leu Thr

125 130 135

Ser Me His Thr Leu Leu Leu Arg Glu His Asn Arg Leu Ala Thr

30 140 145 150

Glu Leu Lys Ser Leu Asn Pro Arg Trp Asp Gly Glu Arg Leu Tyr

155 160 165

Gin Glu Ala Arg Lys lie Val Gly Ala Met Val Gin lie lie Thr

170 175 180 Tyr Arg Asp Tyr Leu Pro Leu Val Leu Gly Pro Thr Ala Me Arg

185 190 195

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

200 205 210

Arg lie Ala Asn Val Phe Thr Asn Ala Phe Arg Tyr Gly His Thr

215 220 225

Leu lie Gin Pro Phe Met Phe Arg

230 233 SEQ ID NO: 4 (H 2 = NA 1 0 1 2— 1 7 1 0)

Array length: 699

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

GTCAACTGCG AGACCAGCTG CGTTCAGCAG CCGCCCTGCT TCCCGCTCAA 50 GATCCCGCCC AATGACCCCC GCATCAAGAA CCAAGCCGAC TGCATCCCG 100 TCTTCCGCTC CTGCCCGGCT TGCCCCGGGA GCAACATCAC CATCCGCAAC 150

CAGATCAACG CGCTCACTTC CTTCGTGGAC GCCAGCATGG TGTACGGCAG 200

CGAGGAGCCC CTGGCCAGGA ACCTGCGCAA CATGTCCAAC CAGCTGGGGC 250

TGCTGGCCGT CAACCAGCGC TTCCAAGACA ACGGCCGGGC CCTGCTGCCC 300

TTTGACAACC TGCACGATGA CCCCTGTCTC CTCACCAACC GCTCAGCGCG 350

CATCCCCTGC TTCCTGGCAG GGGACACCCG TTCCAGTGAG ATGCCCGAGC 400

TCACCTCCAT GCACACCCTC TTACTTCGGG AGCACAACCG GCTGGCCACA 450

GAGCTCAAGA GCCTGAACCC TAGGTGGGAT GGGGAGAGGC TCTACCAGGA 500

AGCCCGGAAG ATCGTGGGGG CCATGGTCCA GATCATCACT TACCGGGACT 550

ACCTGCCCCT GGTGCTGGGG CCAACGGCCA TGAGGAAGTA CCTGCCCACG 600

TACCGTTCCT ACAA.TGACTC AGTGGACCCA CGCATCGCCA ACGTCTTCAC 650

CAATGCCTTC CGCTACGGCC ACACCCTCAT CCAACCCTTC ATGTTCCGC 699

SEQ ID NO: 5 (H 3 = A A 51 2-745)

Array length: 234

Sequence type: amino acid

Number of chains: single strand Topology: linear

Array:

Leu Asp Asn Arg Tyr Gin Pro Met Glu Pro Asn Pro Arg Val Pro

1 5 10 15 Leu Ser Arg Val Phe Phe Ala Ser Trp Arg Val Val Leu Glu Gly

20 25 30

Gly lie Asp Pro lie Leu Arg Gly Leu Me Ala Thr Pro Ala Lys

35 40 45

Leu Asn Arg Gin Asn Gin lie Ala Val Asp Glu lie Arg Glu Arg

50 55 60

Leu Phe Glu Gin Val Met Arg lie Gly Leu Asp Leu Pro Ala Leu

65 70 75

Asn Met Gin Arg Ser Arg Asp His Gly Leu Pro Gly Tyr Asn Ala

80 85 90 Trp Arg Arg Phe Cys Gly Leu Pro Gin Pro Glu Thr Val Gly Gin

95 100 105

Leu Gly Thr Val Leu Arg Asn Leu Lys Leu Ala Arg Lys Leu Me

110 115 120

Glu Gin Tyr Gly Thr Pro Asn Asn lie Asp lie Trp Met Gly Gly

125 130 135

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

140 145 150

Ala Cys lie lie Gly Thr Gin Phe Arg Lys Leu Arg Asp Gly Asp

155 160 165 Arg Phe Trp Trp Glu Asn Glu Gly Val Phe Ser Met Gin Gin Arg

170 175 180

Gin Ala Leu Ala Gin lie Ser Leu Pro Arg lie lie Cys Asp Asn

185 190 195

Thr Gly lie Thr Thr Val Ser Lys Asn Asn lie Phe Met Ser Asn

200 205 210

Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr Leu Pro Ala Leu

215 220 22 5 Asn Leu Ala Ser Trp Arg Glu Ala Ser

230 234 SEQ ID NO: 6 (H 3 = NA 1 7 1 1-241 2)

Array length: 702

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

CTGGACAATC GGTACCAGCC CATGGAACCC AACCCCCGTG TCCCCCTCAG 50 CAGGGTCTTT TTTGCCTCCT GGAGGGTCGT GCTGGAAGGT GGCATTGACC 100 CCATCCTCCG GGGCCTCATG GCCACCCCTG CCAAGCTGAA TCGTCAGAAC 150 CAAATTGCAG TGGATGGAGTAGGGAGGAGTGAGGATGA

GATTGGGCTG GACCTGCCTG CTCTGAACAT GCAGCGCAGC AGGGACCACG 250

GCCTCCCAGG ATACAATGCC TGGAGGCGCT TCTGTGGGCT CCCGCAGCCT 300

GAAACTGTGG GCCAGCTGGG CACGGTGCTG AGGAACCTGA AATTGGCGAG 350

GAAACTGATG GAGCAGTATG GCACGCCCAA CAACATCGAC ATCTGGATGG 400 GCGGCGTGTC CGAGCCTCTG AAGCGCAAAG GCCGCGTGGG CCCACTCCTC 450

GCCTGCATCA TCGGTACCCA GTTCAGGAAG CTCCGGGAT GGTGATCGGTT 500

TTGGTGGGAG AACGAGGGTG TGTTCAGCAT GCAGCAGCGA CAGGCCCTGG 550

CCCAGATCTC ATTGCCCCGG ATCATCTGCG ACAACACAGG CATCACCACC 600

GTGTCTAAGA ACAACATCTT CATGTCCAAC TCATATCCCC GGGACTTTGT 650 CAACTGCAGT ACACTTCCTG CATTGAACCT GGCTTCCTGG AGGGAAGCCT 700

CC 702 SEQ ID NO: 7 (H 7 = AA 279- 409)

Array length: 1 3 1

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Val Asn Cys Glu Thr Ser Cys Val Gin Gin Pro Pro Cys Phe Pro 1 5 10 15

Leu Lys lie Pro Pro Asn Asp Pro Arg lie Lys Asn Gin Ala Asp

20 25 30 Cys lie Pro Phe Phe Arg Ser Cys Pro Ala Cys Pro Gly Ser Asn

35 40 45 lie Thr lie Arg Asn Gin lie Asn Ala Leu Thr Ser Phe Val Asp

50 55 60

Ala Ser Me Val Tyr Gly Ser Glu Glu Pro Leu Ala Arg Asn Leu

65 70 75

Arg Asn Met Ser Asn Gin Leu Gly Leu Leu Ala Val Asn Gin Arg

80 85 90

Phe Gin Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn Leu His

95 100 105

Asp Asp Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg lie Pro Cys

110 115 120

Phe Leu Ala Gly Asp Thr Arg Ser Ser Glu Met

125 130 SEQ ID NO: 8 (H 7 = NA 1 01 2-1 404)

Array length: 393

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

GTCAACTGCG AGACCAGCTG CGTTCAGCAG CCGCCCTGCT TCCCGCTCAA 50 GATCCCGCCC AATGACCCCC GCATCAAGAA CCAAGCCGAC TGCATCCCGT 100 TCTTCCGCTC CTGCCCGGCT TGCCCCGGGA GCAACATCAC CATCCGCAAC 150 CAGATCAACG CGCTCACTTC CTTCGTGGAC GCCAGCATGG TGTACGGCAG 200 CGAGGAGCCC CTGGCCAGGA ACCTG CGCAA CATGTCCAAC CAGCTGGGGC 250 TGCTGGCCGT CAACCAGCGC TTCCAAGACA ACGGCCGGGC CCTGCTGCCC 300 TTTGACAACC TGCACGATGA CCCCTGTCTC CTCACCAACC GCTCAGCGCG 350 CATCCCCTGC TTCCTGGCAG GGGACACCCG TTCCAGTGAG ATG 393 sequences Number: 9 (H 8 = AA341 -479)

Array Length: 1 39

Sequence type: amino acid

Number of chains: single strand Topology: linear

Array:

Me Val Tyr Gly Ser Glu Glu Pro Leu Ala Arg Asn Leu Arg Asn 1 5 10 15

Met Ser Asn Gin Leu Gly Leu Leu Ala Val Asn Gin Arg Phe Gin

20 25 30

Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn Leu His Asp Asp

35 40 45

Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg lie Pro Cys Phe Leu

50 55 60

Ala Gly Asp Thr Arg Ser Ser Glu Met Pro Glu Leu Thr Ser Met

65 70 75

His Thr Leu Leu Leu Arg Glu His Asn Arg Leu Ala Thr Glu Leu

80 85 90

Lys Ser Leu Asn Pro Arg Trp Asp Gly Glu Arg Leu Tyr Gin Glu

95 100 105

Ala Arg Lys lie Val Gly Ala Met Val Gin lie lie Thr Tyr Arg

110 115 120

Asp Tyr Leu Pro Leu Val Leu Gly Pro Thr Ala Me Arg Lys Tyr

125 130 135

Leu Pro Thr Tyr

139 SEQ ID NO: 1 0 (H8 = NA 1 1 98—1 6 1 4)

Sequence length: 41 7

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

ATGGTGTACG GCAGCGAGGA GCCCCTGGCC AGGAACCTGC GCAACATGTC 50 CAACCAGCTG GGGCTGCTGG CCGTCAACCA GCGCTTCCAA GACAACGGCC 100 GGGCCCTGCT GCCCTTTGAC AACCTGCACG ATGACCCCTG TCTCCTCACC 150 AACCGCTCAG CGCGCATCCCC CTGCGACCCGGC TGAGATGCCC GAGCTCACCT CCATGCACAC CCTCTTACTT CGGGAGCACA 250

ACCGGCTGGC CACAGAGCTC AAGAGCCTGA ACCCTAGGTG GGATGGGGAG 300

AGGCTCTACC AGGAAGCCCG GAAGATCGTG GGGGCCATGG TCCAGATCAT 350

CACTTACCGG GACTACCTGC CCCTGGTGCT GGGGCCAACG GCCATGAGGA 400 AGTACCTGCC CACGTAC 417 SEQ ID NO: 1 1 (H 9 = AA4 10—538)

Array Length: 1 29

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Pro Glu Leu Thr Ser Met His Thr Leu Leu Leu Arg Glu His Asn 1 5 10 15 Arg Leu Ala Thr Glu Leu Lys Ser Leu Asn Pro Arg Trp Asp Glv

20 25 30

Glu Arg Leu Tyr Gin Glu Ala Arg Lys lie Val Gly Ala Met Val

35 40 45

Gin lie lie Thr Tyr Arg Asp Tyr Leu Pro Leu Val Leu Gly Pro

50 55 60

Thr Ala Met Arg Lys Tyr Leu Pro Thr Tyr Arg Ser Tyr Asn Asp

65 70 75

Ser Val Asp Pro Arg lie Ala Asn Val Phe Thr Asn Ala Phe Arg

80 85 90 Tyr Gly His Thr Leu lie Gin Pro Phe Met Phe Arg Leu Asp Asn

95 100 105

Arg Tyr Gin Pro Me Glu Pro Asn Pro Arg Val Pro Leu Ser Arg

110 115 120

Val Phe Phe Ala Ser Trp Arg Val Val SEQ ID NO: 1 2 (H 9 = NA 1 405-1 79 1)

Array length: 387

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

CCCGAGCTCA CCTCCATGCA CACCCTCTTA CTTCGGGAGC ACAACCGGCT 50 GGCCACAGAG CTCAAGAGCC TGAACCCTAG GTGGGATGGG GAGAGGCTCT 100 ACCAGGAAGC CCGGAAGATC GTGGGGGCCA TGGTCCAGAT CATCACTTAC 150 CGGGACTACC TGCCCCTGGT GCTGGGGACAGC

GCCCACGTAC CGTTCCTACA ATGACTCAGT GGACCCACGC ATCGCCAACG 250 TCTTCACCAA TGCCTTCCGC TACGGCCACA CCCTCATCCA ACCCTTCATG 300 TTCCGCCTGG ACAATCGGTA CCAGCCCATG GAACCCAACC CCCGTGTCCC 350 CCTCAGCAGG GTCT 3GT 5CGTCTCGGAG GT CGTCTCGGAG

Array length: 87

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Leu Asp Asn Arg Tyr Gin Pro Met Glu Pro Asn Pro Arg Val Pro 1 5 10 15

Leu Ser Arg Val Phe Phe Ala Ser Trp Arg Val Val Leu Glu Gly

20 25 30

Gly lie Asp Pro lie Leu Arg Gly Leu Met Ala Thr Pro Ala Lys

35 40 45

Leu Asn Arg Gin Asn Gin lie Ala Val Asp Glu lie Arg Glu Arg

50 55 60 Leu Phe Glu Gin Val Met Arg lie Gly Leu Asp Leu Pro Ala Leu

65 70 75

Asn Met Gin Arg Ser Arg Asp His Gly Leu Pro Gly

80 85 SEQ ID NO: Ί 4 (Η 5 = ΝΑ 1 7 1 1 — 1 97 1)

Array length: 26 1

Sequence type: nucleic acid

Number of chains: single strand

5 Topology: Linear

Array:

CTGGACAATC GGTACCAGCC CATGGAACCC AACCCCCGTG TCCCCCTCAG 50 CAGGGTCTTT TTTGCCTCCT GGAGGGTCGT GCTGGAAGGT GGCATTGACC 100 CCATCCTCCG GGGCCTCATG GCCACCCCTG CCAAGCTGAA TCGTCAGAAC 150 10 CAAATTGCAG TGGATGATGTGGGAGA

GATTGGGCTG GACCTGCCTG CTCTGAACAT GCAGCGCAGC AGGGACCACG 250 GCCTCCCAGG A 261 SEQ ID NO: 15 (H 6 = AA54 1-745)

15 Length of array: 205

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

0 Gly Gly lie Asp Pro lie Leu Arg Gly Leu Met Ala Thr Pro Ala

1 5 10 15

Lys Leu Asn Arg Gin Asn Gin lie Ala Val Asp Glu lie Arg Glu

20 25 30

Arg Leu Phe Glu Gin Val Me Arg lie Gly Leu Asp Leu Pro Ala 25 35 40 45

Leu Asn Met Gin Arg Ser Arg Asp His Gly Leu Pro Gly Tyr Asn

50 55 60

Ala Trp Arg Arg Phe Cys Gly Leu Pro Gin Pro Glu Thr Val Gly

65 70 h 5 θ Gin Leu Gly Thr Val Leu Arg Asn Leu Lys Leu Ala Arg Lys Leu

80 85 90

Met Glu Gin Tyr Gly Thr Pro Asn Asn lie Asp lie Trp Met Gly

95 100 105 Gly Val Ser Glu Pro Leu Lys Arg Lys Gly Arg Val Gly Pro Leu

110 115 120

Leu Ala Cys lie lie Gly Thr Gin Phe Arg Lys Leu Arg Asp Gly

125 130 135

Asp Arg Phe Trp Trp Glu Asn Glu Gly Val Phe Ser Met Gin Gin

140 145 150

Arg Gin Ala Leu Ala Gin lie Ser Leu Pro Arg lie lie Cys Asp

155 160 165

Asn Thr Gly lie Thr Thr Val Ser Lys Asn Asn lie Phe Met Ser

1f 0 1f 5 180

Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr Leu Pro Ala

185 190 195

Leu Asn Leu Ala Ser Trp Arg Glu Ala Ser

200 205 SEQ ID NO: 1 6 (H 6 = NA 1 798-24 1 2)

Sequence length: 61 5

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array

GGTGGCATTG ACCCCATCCT CCGGGGCCTC ATGGCCACCC CTGCCAAGCT 50 GAATCGTCAG AACCAAATTG CAGTGGATGA GATCCGGGAG CGATTGTTTG 100 AGCAGGTCAT GAGGATTGGG CTGGACCTGC CTGCTCTGAA CATGCAGCGC 150

AGCAGGGACC ACGGCCTCCC AGGATACAAT GCCTGGAGGC GCTTCTGTGG 200

GCTCCCGCAG CCTGAAACTG TGGGCCAGCT GGGCACGGTG CTGAGGAACC 250

TGAAATTGGC GAGGAAACTG ATGGAGCAGT ATGGCACGCC CAACAACATC 300

GACATCTGGA TGGGCGGCGT GTCCGAGCCT CTGAAGCGCA AAGGCCGCGT 350

GGGCCCACTC CTCGCCTGCA TCATCGGTAC CCAGTTCAGG AAGCTCCGGG 400

ATGGTGATCG GTTTTGGTGG GAGAACGAGG GTGTGTTCAG CATGCAGCAG 450

CGACAGGCCC TGGCCCAGAT CTCATTGCCC CGGATCATCT GCGACAACAC 500

AGGCATCACC ACCGTGTCTA AGAACAACAT CTTCATGTCC AACTCATATC 550

CCCGGGACTT TGTCAACTGC AGTACACTTC CTGCATTGAA CCTGGCTTCC 600

TGGAGGGAAG CCTCC 615 SEQ ID NO: 1 7 (H 1 1 = AA 598-745)

Array length: 1 48

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Gly Tyr Asn Ala Trp Arg Arg Phe Cys Gly Leu Pro Gin Pro Glu

1 5 10 15

Thr Val Gly Gin Leu Gly Thr Val Leu Arg Asn Leu Lys Leu Ala

20 25 30

Arg Lys Leu Met Glu Gin Tyr Gly Thr Pro Asn Asn lie Asp lie

35 40 45

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

50 55 60 Gly Pro Leu Leu Ala Cys lie lie Gly Thr Gin Phe Arg Lys Leu

65 70 75

Arg Asp Gly Asp Arg Phe Trp Trp Glu Asn Glu Gly Val Phe Ser

80 85 90

Met Gin Gin Arg Gin Ala Leu Ala Gin lie Ser Leu Pro Arg lie

95 100 105 lie Cys Asp Asn Thr Gly lie Thr Thr Val Ser Lys Asn Asn lie

110 115 120

Phe Met Ser Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr

125 130 135 Leu Pro Ala Leu Asn Leu Ala Ser Trp Arg Glu Ala Ser

140 145 SEQ ID NO: 1 8 (H 1 1 = NA 1 969—241 2)

Array length: 444

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array: GGATACAATG CCTGGAGGCG CTTCTGTGGG CTCCCGCAGC CTGAAACTGT 50 GGGCCAGCTG GGCACGGTGC TGAGGAACCT GAAATTGGCG AGGAAACTGA 100 TGGAGCAGTA TGGCACGCCC AACAACATCG ACATCTGGAT GGGCGGCGTG 150

TCCGAGCCTC TGAAGCGCAA AGGCCGCGTG GGCCCACTCC TCGCCTGCAT 200

CATCGGTACC CAGTTCAGGA AGCTCCGGGA TGGTGATCGG TTTTGGTGGG 2 50

AGAACGAGGG TGTGTTCAGC ATGCAGCAGC GACAGGCCCT GGCCCAGATC 300

TCATTGCCCC GGATCATCTG CGACAACACA GGCATCACCA CCGTGTCTAA 350

GAACAACATC TTCATGTCCA ACTCATATCC CCGGGACTTT GTCAACTGCA 400

GTACACTTCC TGCATTGAAC CTGGCTTCCT GGAGGGAAGC CTCC 444 SEQ ID NO: 19 (L 1 = A A 16 5-2 7 2)

Array length: 0 8

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Val Thr Cys Pro Glu Gin Asp Lys Tyr Arg Thr lie Thr Gly Met 1 5 10 15

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

20 25 30

Phe Val Arg Trp Leu Pro Ala Glu Tyr Glu Asp Gly Phe Ser Leu

35 40 45

Pro Tyr Gly Trp Thr Pro Gly Val Lys Arg Asn Gly Phe Pro Val

50 55 60

Ala Leu Ala Arg Ala Val Ser Asn Glu lie Val Arg Phe Pro Thr

65 70 75

Asp Gin Leu Thr Pro Asp Gin Glu Arg Ser Leu Met Phe Met Gin

80 85 90

Trp Gly Gin Leu Leu Asp His Asp Leu Asp Phe Thr Pro Glu Pro

95 100 105

Ala Ala Arg

108 SEQ ID NO: 20 (L 1 = NA670— 993)

Sequence length: 324

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

GTGACTTGCC CGGAGCAGGA CAAATACCGC ACCATCACCG GGATGTGCAA 50 CAACAGACGC AGCCCCACGC TGGGGGCCTC CAACCGTGCC TTTGTGCGCT 100 GGCTGCCGGC GGAGTATGAG GACGGCTTCT CTCTTCCCTA CGGCTGGACG 150 CCCGGGGTCA AGCGCAACGG CTTCCCGGTG GCTCTGGCTC GCGCGGTCTC 200 CAACGAGATC GTGCGCTTCC CCACTGATCA GCTGACTCCG GACCAGGAGC 250 GCTCACTCAT GTTCATGCAA TGGGGCCAGC TGTTGGACCA CGACCTCGAC 300 TTCACCCCTG AGCCGGCCGC CCGG 324 SEQ ID NO: 2 1 (L 2 = AA 1 65- 2 1 4)

Array length: 50

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Val Thr Cys Pro Glu Gin Asp Lys Tyr Arg Thr lie Thr Gly Met 1 5 10 15

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

20 25 30 Phe Val Arg Trp Leu Pro Ala Glu Tyr Glu Asp Gly Phe Ser Leu

35 40 45

Pro Tyr Gly Trp Thr

50 SEQ ID NO: 22 (L 2 = NA670-81 9)

Array length: 1 50

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear Array:

GTGACTTGCC CGGAGCAGGA CAAATACCGC ACCATCACCG GGATGTGCAA 50 CAACAGACGC AGCCCCACGC TGGGGGCCTC CAACCGTGCC TTTGTGCGCT 100 GGCTGCCGGC GGAGTATGAG GACGGCTTCT CTCTTCCCTA CGGCTGGACG 150 SEQ ID NO: 23 (L 3—2 A 2) 1

Array length: 58

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Pro Gly Val Lys Arg Asn Gly Phe Pro Val Ala Leu Ala Arg Ala 1 5 10 15

Val Ser Asn Glu lie Val Arg Phe Pro Thr Asp Gin Leu Thr Pro

20 25 30

Asp Gin Glu Arg Ser Leu Met Phe Met Gin Trp Gly Gin Leu Leu

35 40 45

Asp His Asp Leu Asp Phe Thr Pro Glu Pro Ala Ala Arg

50 55 SEQ ID NO: 24 (L 3 = N A820-993)

Array Length: 1 74

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

CCCGGGGTCA AGCGCAACGG CTTCCCGGTG GCTCTGGCTC GCGCGGTCTC 50 CAACGAGATC GTGCGCTTCC CCACTGATCA GCTGACTCCG GACCAGGAGC 100 GCTCACTCAT GTTCATGCAA TGGGGCCAGC TGTTGGACCA CGACCTCGAC 150 TTCACCCCTG AGCCGGCCA CCGG 174 A: 344

Array length: 66 Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Val Asn Cys Glu Thr Ser Cys Val Gin Gin Pro Pro Cys Phe Pro

1 5 10 15

Leu Lys lie Pro Pro Asn Asp Pro Arg lie Lys Asn Gin Ala Asp

20 25 30

Cys lie Pro Phe Phe Arg Ser Cys Pro Ala Cys Pro Gly Ser Asn

35 40 45 lie Thr lie Arg Asn Gin lie Asn Ala Leu Thr Ser Phe Val Asp

50 55 60

Ala Ser Met Val yr Gly

65 SEQ ID NO: 26 (H 7 a = NA 1 0 1 2-1 209)

Array length: 1 98

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

GTCAACTGCG AGACCAGCTG CGTTCAGCAG CCGCCCTGCT TCCCGCTCAA 50 GATCCCGCCC AATGACCCCC GCATCAAGAA CCAAGCCGAC TGCATCCCGT 100 TCTTCCGCTC CTGCCCGGCT TGCCCCGGGA GCAACATCAC CATCCGCAAC 150 CAGATCAACG CGCTCAGTC CGTCTCTCGCGGTC CGTCTCTCGGAC CGTCTCTCGGAC

Array length: 7 1

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array: Ser Met Val yr Gly Ser Glu Glu Pro Leu Ala Arg Asn Leu Arg

1 5 10 15

Asn Met Ser Asn Gin Leu Gly Leu Leu Ala Val Asn Gin Arg Phe

20 25 30

Gin Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn Leu His Asp

35 40 45

Asp Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg lie Pro Cys Phe

50 55 60

Leu Ala Gly Asp Thr Arg Ser Ser Glu Met Pro

65 70 SEQ ID NO: 28 (H 7 b = N A 1 1 95— 1 407)

Array length: 2 1 3

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

AGCATGGTGT ACGGCAGCGA GGAGCCCCTG GCCAGGAACC TGCGCAACAT 50 GTCCAACCAG CTGGGGCTGC TGGCCGTCAA CCAGCGCTTC CAAGACAACG 100 GCCGGGCCCT GCTGCCCTTT GACAACCTGC ACGATGACCC CTGTCTCCTC 150 ACCAACCGCT CAGCGCGTC CCGCTGCCCCTCCAGGCCCCTCTCGCGG

Array length: 79

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array

Asn Ala Trp Arg Arg Phe Cys Gly Leu Pro Gin Pro Glu Thr Val 1 5 10 15

Gly Gin Leu Gly Thr Val Leu Arg Asn Leu Lys Leu Ala Arg Lys

20 25 30 Leu Met Glu Gin Tyr Gly Thr Pro Asn Asn lie Asp lie Trp Met

35 40 45

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

50 55 60

Leu Leu Ala Cys lie lie Gly Thr Gin Phe Arg Lys Leu Arg Asp

65 70 75

Gly Asp Arg Phe

F 9 SEQ ID NO: 30 (H 1 2 = N A 1 975-22 1 1)

Sequence length: 237

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array

AATGCCTGGA GGCGCTTCTG TGGGCTCCCG CAGCCTGAAA CTGTGGGCCA 50 GCTGGGCACG GTGCTGAGGA ACCTGAAATT GGCGAGGAAACTGATGGAGC 100 AGTATGGCAC GCCCAACAAC ATCGACATCT GGATGGGCGG CGTGTCCGAG 150

CCTCTGAAGC GCAAAGGCCG CGTGGGCCCA CTCCTCGCCT GCATCATCGG 200

TACCCAGTTC AGGAAGCTCC GGGATGGTGA TCGGTTT 237 SEQ ID NO: 3 1 (H 13 = AA 678-745)

Array length: 68

Sequence type: amino acid

Number of chains: single strand

Topology: linear

Array:

Phe Trp Trp Glu Asn Glu Gly Val Phe Ser Met Gin Gin Arg Gin 1 5 10 15

Ala Leu Ala Gin lie Ser Leu Pro Arg lie lie Cys Asp Asn Thr

20 25 30

Gly lie Thr Thr Val Ser Lys Asn Asn lie Phe Met Ser Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr Leu Pro Ala Leu Asn

50 55 60

Leu Ala Ser Trp Arg Glu Ala Ser

65 68 SEQ ID NO: 32 (H 13 = NA 2209-24 1 2)

Array length: 204

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Array:

TTTTGGTGGG AGAACGAGGG TGTGTTCAGC ATGCAGCAGC GACAGGCCCT 50 GGCCCAGATC TCATTGCCCC GGATCATCTG CGACAACACA GGCATCACCA 100 CCGTGTCTA AGAACAACATC TTCATGTCCA ACTCATATCC CCGGCACTT 150 GTCAACTGTC CTGCTCCTGCTCGCTCTCGCTCACTG

Claims

The scope of the claims

1. Recombinant myelin peroxidase (MPO) fragment that reacts with myelinated peroxidase-specific anti-neutrophil cytoplasmic antibody (1 ^ 1urethane).

2. The fragment according to claim 1, wherein the recombinant MPO fragment is an MPO fragment fused with six histidines.

3. The fragment according to claim 1, wherein the recombinant MPII fragment is an MPO fragment fused to a maltose binding protein.

4. The fragment according to claim 1, wherein the recombinant MPO fragment is an MP0 fragment fused with Daryuthion-S-transferase.

5. The fragment according to claim 2, wherein the recombinant MPO fragment is a peptide selected from the group consisting of Sequence Listings 1 to 32.

6. Recombinant MP. 6. The fragment according to claim 5, wherein the fragment is a panel set selected from at least two of the peptide fragments in Sequence Listings 1 to 32.

7. Myelin peroxidase-complementary DNA (MPO cDNA) is amplified by polymerase chain reaction (PCR), and a portion of the cDNA is incorporated into an expression vector to obtain a recombinant plasmid. E. coli transformed with this plasmid was obtained. 2. The method for producing a recombinant MPO fragment according to claim 1, wherein the recombinant MPO fragment is expressed by culturing enterobacteria, and the recombinant MP0 fragment is collected from the Escherichia coli.

8. The production method according to claim 7, wherein the expression vector is a pQE vector.

9. The production method according to claim 7, wherein the recombinant MPO fragment is an MP0 fragment in which six histidines are fused.

10. The method according to claim 7, wherein the expression vector is a pQE vector, and the recombinant MPO fragment is a MPO fragment comprising six histidines.

1 1. The production method according to claim 7, wherein isopropyl 3-hydroxy-galactoside is added during the culturing of Escherichia coli.

12.1) A sample is added to a plate on which the recombinant MPO fragment according to claim 5 is immobilized, and an immunoreaction is performed.

2) Then, a labeled anti-human IgG was added to carry out an immunoreaction,

3) A method of measuring the amount of reaction bound to myelin peroxidase specific anti-neutrophil cytoplasmic antibody (MP0-ANCA) in a sample and the amount thereof by measuring the amount bound to the plate.

1 3.After reacting MPO antibody-positive patient's serum as primary antibody, add horseradish peroxidase-labeled, alkaline phosphatase-labeled or) 3_galactosidase-labeled anti-human IgG antibody The method according to claim 12, wherein the reaction is carried out.

14. The method according to claim 12, wherein alkaline phosphatase or / 3-galactosidase is used as an enzyme label, and P-two-port phenyl phosphate is used as a substrate.

1 5. The method according to claim 12, wherein horseradish peroxidase is used as an enzyme label and 0-phenylenediamine is used as a substrate.

1 6. A panel set of recombinant MP0 fragment according to claim 6 and a myelin periostease-specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) comprising a labeled anti-human IgG. )) A reagent kit for measuring the reaction site and the amount thereof.