WO2022075485A1

WO2022075485A1 - Collagen-like modified protein and use thereof

Info

Publication number: WO2022075485A1
Application number: PCT/JP2021/037556
Authority: WO
Inventors: 渉西江; 翔子眞井; 洋輔眞井; 健太郎泉
Original assignee: 国立大学法人北海道大学
Priority date: 2020-10-09
Filing date: 2021-10-11
Publication date: 2022-04-14
Also published as: JPWO2022075485A1

Abstract

The present invention provides: a collagen-like modified protein that contains a trimerization domain corresponding to the coiled-coil sequence of human type II transmembrane collagen and a domain corresponding to NC11-COL7 of human BP180 and/or a domain corresponding to NC7-COL4 thereof, said domain(s) being located on the C-terminal side of the trimerization domain, provided that this collagen-like modified protein does not bind to a human autoantibody targeting NC16A of human BP180; a method and a kit for detecting a human autoantibody using the aforesaid protein; and a method and a kit for diagnosing drug-induced bullous pemphigoid using the aforesaid protein.

Description

Collagen-like modified protein and its use

The present invention comprises a collagen-like modified protein useful for diagnosing drug-induced bullous pemphigoid, a method and kit for detecting human autoantibodies using the protein, and drug-induced bullous pemphigoid using the protein. Regarding methods and kits for making a diagnosis of.

Bullous pemphigoid (BP), which is one of the designated intractable diseases in Japan, occurs frequently in the elderly and is the most common autoimmune pemphigoid. Autoantibodies (anti-BP180 autoantibodies) targeting BP180 (XVII type collagen, also called BPAG2), which is a transmembrane protein existing between the epidermis and dermal of the skin, were detected in the patient's blood, and this was the onset of BP. Is believed to induce. Most anti-BP180 autoantibodies are known to target the 16th non-collagen region ( ^NC16A region, non-collagenous (NC) 16th A region) located in the extracellular domain of BP180.

In recent years, it has been found that a DPP-4 (dipeptidyl peptidase-IV) inhibitor, which is a therapeutic drug for diabetes, contributes to the onset of BP. Autoantibodies that bind to BP180 are also detected in the blood of BP (hereinafter referred to as DPP4i-BP) patients who develop while taking DPP-4 inhibitors, but this autoantibody (DPP4i-BP autoantibody) is It has been clarified that, unlike the anti-BP180 autoantibodies that target NC16A that have been reported so far, they target regions other than NC16A in the extracellular domain of BP180 (Non-Patent Document 1).

Autoantibodies targeting NC16A of BP180 (anti-BP180-NC16A antibody) can be detected by ELISA or CLEIA method (Non-Patent Document 2), and tests using this method are covered by insurance in Japan. However, this method cannot detect anti-BP180 autoantibodies, typically DPP4i-BP autoantibodies, that target regions other than NC16A in the extracellular domain of BP180.

The present inventors succeeded in producing a full-length BP180 recombinant protein by optimizing the method for purifying a membrane protein, and further established an ELISA method using the full-length BP180 (Non-Patent Document 1). Although the DPP4i-BP autoantibody can be detected by using the full-length BP180, the anti-BP180-NC16A antibody is also detected at the same time, and it is difficult to selectively detect the DPP4i-BP autoantibody.

In order to selectively detect DPP4i-BP autoantibodies by immunological methods, it is necessary to prepare an antigenic protein containing a region other than NC16A in the extracellular domain of BP180. However, BP180 is a homotrimer in which three monomer polypeptides having a molecular weight of 180 kDa are associated with each other, and is a giant protein having a molecular weight of 540 kDa. The extracellular domain has a triple helix structure called a collagen spiral structure. It is not easy to make an antigenic protein containing the extracellular domain of BP180.

It is an object of the present invention to provide an antigenic protein that can be utilized for selectively detecting an anti-BP180 autoantibody that targets a region other than NC16A in the extracellular domain of BP180. ..

The present inventors have an amino acid sequence including a specific amino acid sequence involved in the formation of a trimer of type II transmembrane collagen and a partial amino acid sequence of the extracellular domain of BP180 located on the C-terminal side thereof. Collagen-like modified proteins can selectively detect anti-BP180 autoantibodies targeting regions other than NC16A in the extracellular domain of BP180, and further identify non-NC16A BP180 extracellular domains in drug-induced BP patients. We found that self-antibodies targeting the region were detected, but the self-antibodies were hardly or completely detected in BP patients and healthy subjects unrelated to the drug, and the following invention was completed.

Item 1. The trimeric region corresponding to the coiled coil arrangement of human type II transmembrane collagen and the 7th collagen from the 11th non-collagen region (NC11) of human BP180 located on the C-terminal side of the trimeric region. A human that includes a region corresponding to the region (COL7) and / or a region corresponding to the 7th non-collagen region (NC7) to the 4th collagen region (COL4), but targeting the NC16A region of human BP180. Collagen-like modified protein that does not bind to self-antibodies.
Item 2. Item 2. The protein according to Item 1, wherein the human type II transmembrane collagen is human XIII type collagen.
Item 3. Item 2. The protein according to

Item

1 or 2, wherein the amino acid sequence of the trimerization region is the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. ..
Item 4. The amino acid sequence of the region corresponding to NC11 to COL7 of human BP180 is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2, and human BP180. The amino acid sequence of the region corresponding to NC7 to COL4 in No. 1 to Item 1 to 3 is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3. The protein according to any one of the above.
Item 5. Item 6. The protein according to any one of Items 1 to 4, which comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21.
Item 6. Item 6. The protein according to any one of Items 1 to 5, which forms a homotrimer structure.
Item 7. A nucleic acid encoding a protein specified in any one of Items 1 to 5.
Item 8. An expression vector containing the nucleic acid specified in Item 7.
Item 9. A host cell containing the nucleic acid specified in Item 7 or the expression vector specified in Item 8.
Item 10. Human BP180 includes a step of contacting a sample sample collected from a subject with the protein specified in any one of Items 1 to 6, and a step of detecting the binding between the antibody in the sample sample and the protein. A method for detecting a human autoantibody targeting the region from NC11 to COL7 and / or a human autoantibody targeting the region from NC7 to COL4 of human BP180.
Item 11. Item 6. The method according to Item 10, wherein the sample is a blood sample.
Item 12. Target the region from NC11 to COL7 of human BP180 and / or the region from NC7 to COL4 of human BP180, which comprises the protein specified in any one of Items 1 to 6. A kit for detecting human autoantibodies.
Item 13. Drug property, which comprises a step of contacting a sample sample collected from a subject with a protein specified in any one of Items 1 to 6 and a step of detecting a binding between an antibody in the sample sample and the protein. How to collect data for the diagnosis of bullous pemphigoid.
Item 14. Item 3. The method according to Item 13, wherein the sample is a blood sample.
Item 15. A kit for diagnosing drug-induced bullous pemphigoid, which comprises the protein specified in any one of Items 1 to 6.

By utilizing the collagen-like modified protein of the present invention, among the anti-BP180 autoantibodies, the anti-BP180 autoantibodies targeting the region from NC11 to COL7 of BP180 and the anti-BP180 autoantibodies targeting the region from NC7 to COL4 Antibodies can be selectively detected, and drug-induced BP can be distinguished from drug-independent BP.

It is a figure which shows the structure of BP180 and XIII type collagen (collagen 13). It is a figure which shows the structure of the collagen 13-BP180 swap protein of Example 1, which is one Embodiment of this invention. It is a figure which shows the result of the Western blotting about the swap protein 3 of Example 1 using the serum of the DPP4i-BP patient, the BP patient and the healthy person control. It is a figure which shows the result of the Western blotting about the swap protein 4 of Example 1 using the serum of the DPP4i-BP patient, the BP patient and the healthy person control. It is a figure which shows the result of the ELISA about the swap protein 1-5 of Example 1 using the serum of the DPP4i-BP patient, the BP patient and the healthy person control. It is a figure which shows the result of the ELISA about the swap protein 1-5, the BP180 total length and NC16A of Example 1 using the serum of the DPP4i-BP patient, the BP patient and the non-BP patient. The ROC curve drawn with the objective variable as DPP4i-BP and the explanatory variable as the ELISA index of swap protein 4 is shown.

The first aspect of the present invention is a trimeric region corresponding to the coiled coil arrangement of human type II transmembrane collagen, and NC11 to COL7 of human BP180 located on the C-terminal side of the trimeric region. With respect to collagen-like modified proteins comprising corresponding regions and / or regions corresponding to NC7 to COL4, but not binding to human autoantibodies targeting NC16A of human BP180.

Trimerized region type II transmembrane collagen is expressed in many different tissues and cells and protects the host from epithelial and neural cell adhesion and epithelial-mesenchymal interactions during morphogenesis. It is involved in a wide range of biological functions. Type II transmembrane collagen has XIII type collagen, BP180 (XVII type collagen), XXIII type collagen, XXV type collagen, MARCO (macrophage receptor with collagenous structures) which is a class A macrophage scavenger receptor-like protein, and collagen structure. Macrophagee receptor), SRCL (scavenger receptor with C-type lectin), which is a scavenger receptor having C-type lectin, ectodisplacin A and the like.

The coiled coil sequences (called coiled-coil sequences, coiled-coil heptad repeats, coiled-coil oligomerization domain, N-terminal heptad repeats, etc.) existing on the N-terminal side of the type II transmembrane collagen extracellular region are HPPHPPP (H). Is a domain involved in the trimerization of collagen (McAlinden A. et al., J. Biol. Chem. 278: 42200-42207, 2003, Snellman A. et al., J. Biol. Chem. 282: 14898-14905, 2007). The coiled coil arrangement of a typical human type II transmembrane collagen is shown in the table below.

The coiled coil sequence preferably used in the present invention is the coiled coil sequence of human XIII type collagen (SEQ ID NO: 1). The extracellular domain of type XIII collagen includes three collagen regions (COL1 to COL3 from the N-terminal side to the C-terminal side) and four non-collagen regions (NC1 from the N-terminal side to the C-terminal side). ~ NC4) exist alternately, and the coiled coil arrangement exists in NC1. In the case of the amino acid sequence (SEQ ID NO: 9) of human XIII type collagen α1 chain (COL13A1) registered as NP_001123575.1 in the Reference Sequence Database of National Center for Biotechnology Information (NCBI), the coiled coil sequence of XIII type collagen is 65. It is present at the amino acid sequence site at the 85th position.

BP180
BP180 is a transmembrane collagen, also called XVII type collagen, in which three monomers having a molecular weight of 180 kDa are associated to form a homotrimer. The extracellular domain of BP180 includes 15 collagen regions (COL1 to COL15 from the C-terminal side to the N-terminal side) and 16 non-collagen regions (NC1 to NC16 from the C-terminal side to the N-terminal side). ) And alternately exist. The BP180 amino acid sequence and the base sequence of the gene encoding it are known, and are registered as NP_000485.3 (amino acid sequence) and NM_000494.4 (cDNA base sequence) in the Reference Sequence Database of the National Center for Biotechnology Information (NCBI), respectively. ing. The amino acid sequence of BP180 is shown in SEQ ID NO: 10, and the base sequence of the cDNA encoding it is shown in SEQ ID NO: 11.

In the present invention, the region of BP180 from NC11 to COL7 (NC11-COL7) and the region of BP180 from NC7 to COL4 (NC7-COL4) are used. When the amino acid sequence of BP180 is the amino acid sequence shown in SEQ ID NO: 10, the position of NC11-COL7 is 982-1160 and the position of NC7-COL4 is 1161-1279. The amino acid sequences 982 to 1160 of SEQ ID NO: 10 are shown in SEQ ID NO: 2, and the amino acid sequences 1161 to 1279 are shown in SEQ ID NO: 3, respectively.

Collagen-like modified protein The collagen-like modified protein of this embodiment contains a trimerized region, and further corresponds to a region corresponding to NC11-COL7 and / or NC7-COL4 of BP180 on the C-terminal side of the trimerized region. Includes area.

In addition, the collagen-like modified protein of this embodiment does not bind to human autoantibodies targeting NC16A of BP180. In terms of amino acid sequence, the collagen-like modified protein of this embodiment is a sequence recognized by a human autoantibody present in NC16A of BP180 (typically, the amino acid sequence shown in SEQ ID NO: 12; D. Zillikens et al., J Invest Dermatol: 109,573-9) is not included. Preferably, the collagen-like modified protein does not contain the amino acid sequence of NC16A of BP180 (SEQ ID NO: 13).

The fact that BP180 does not bind to a human autoantibody that targets NC16A means that the binding affinity of the antibody to collagen-like modified protein is similar to that of other antigens other than NC16A. , It does not preclude that the antibody non-specifically binds to the collagen-like modified protein.

The trimerization region corresponds to the coiled coil arrangement of type II transmembrane collagen. Here, the equivalent to the coiled-coil sequence of type II transmembrane collagen means that it consists of the amino acid sequence of the coiled-coil sequence of type II transmembrane collagen, or is a functionally equivalent variation in which the amino acid sequence of the coiled-coil sequence is modified. It means that it consists of an amino acid sequence.

Similarly, in the present invention, the equivalent of NC11-COL7 of BP180 consists of the natural amino acid sequence of NC11-COL7 or a functionally equivalent mutant amino acid obtained by modifying the natural amino acid sequence of NC11-COL7. It means that it consists of an array. Further, equivalent to NC7-COL4 means that it consists of a natural amino acid sequence of NC7-COL4 or a functionally equivalent mutant amino acid sequence obtained by modifying the natural amino acid sequence of NC7-COL4. ..

Regarding the coiled coil sequence, the functionally equivalent mutant amino acid sequence is an amino acid sequence obtained by modifying the natural amino acid sequence of the coiled coil sequence, which retains the ability to form a homotrimer in a collagen-like modified protein. An example of a mutant amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97%, 98% or 99 with the natural amino acid sequence of a coiled coil sequence. It is an amino acid sequence having a sequence identity of% or more. Another example is an amino acid sequence in which one or two amino acid residues have been deleted, substituted or added in the natural amino acid sequence of a coiled coil sequence.

With respect to NC11-COL7, functionally equivalent mutant amino acid sequences are humans that retain the ability to form homotrimers when contained in collagen-like modified proteins and that target NC11-COL7 of BP180. It is an amino acid sequence obtained by modifying the natural amino acid sequence of NC11-COL7, which retains the ability to bind to an autoantibody. An example of such an amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97%, 98 with the natural amino acid sequence of NC11-COL7. % Or an amino acid sequence having 99% or more sequence identity. Yet another example is the deletion of 1-18, preferably 1-12, more preferably 1-8, and even more preferably 1-4 amino acid residues in the natural amino acid sequence of NC11-COL7. , Substituted or added amino acid sequence.

With respect to NC7-COL4, functionally equivalent mutant amino acid sequences are humans that retain the ability to form homotrimers when contained in collagen-like modified proteins and that target NC7-COL4 in BP180. A natural amino acid sequence of NC7-COL4 that retains the ability to bind autoantibodies. An example of such an amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97%, 98 with the natural amino acid sequence of NC7-COL4. % Or an amino acid sequence having 99% or more sequence identity. Yet another example is the deletion of 1-12, preferably 1-9, more preferably 1-6, even more preferably 1-3 amino acid residues in the natural amino acid sequence of NC7-COL4. , Substituted or added amino acid sequence.

Substitutions are preferably so-called conservative substitutions, such as glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamine acid ( Amino acids such as Glu) and glutamine (Gln), aspartic acid (Asp) and aspartin (Asn), cysteine (Cys) and threonine (Thr), alanine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg). The substitution between can be mentioned.

Amino acid sequence identity is expressed as the ratio of the number of identical amino acid residues to the alignment length, and the alignment of two amino acid sequences to be compared is performed according to a conventional method so that the number of identical amino acid residues is the largest. Will be. Sequence identity can be determined by any method known to those of skill in the art using a sequence comparison program such as BLAST.

In the collagen-like modified protein, the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 are arranged on the C-terminal side of the trimerization region. This configuration allows collagen-like modified proteins to form trimers. In the collagen-like modified protein, the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 may contain only one or both. When both are included, the order of the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 does not matter as long as they are arranged on the C-terminal side of the trimerization region. Further, one region corresponding to NC11-COL7 and one region corresponding to NC7-COL4 may be included or a plurality of regions may be included.

The collagen-like modified protein of this embodiment retains the ability to form homotrimers and binds to human autoantibodies targeting NC11-COL7 of BP180 and / or targets NC7-COL4 of BP180. Unless it retains the ability to bind to human autoantibodies and does not bind to human autoantibodies that target NC16A of BP180, it is a trimerization region, a region corresponding to NC11-COL7, and a region corresponding to NC7-COL4, respectively. It can contain any additional amino acid sequence other than the amino acid sequence of. The additional amino acid sequence may be located at the N-terminus or C-terminus of the modified protein, or between each region contained in the modified protein (eg, the trimerization region and the region corresponding to NC11-COL7). It may be located between the trimerization region and the region corresponding to NC7-COL4).

Examples of additional amino acid sequences are amino acid sequences derived from type XIII collagen other than the coiled-coil sequence, for example, the amino acid sequences 1 to 64 of SEQ ID NO: 9, the amino acid sequences 86 to 217 of SEQ ID NO: 9. The amino acid sequences from the 700th to the 717th of SEQ ID NO: 9 can be mentioned. Other examples of additional amino acid sequences are amino acid sequences corresponding to restriction enzyme recognition sequences, tag sequences such as His tags, GST tags, HA tags, FLAG tags, and amino acid sequences of GFP and other fluorescent proteins.

Preferred examples of collagen-like modified proteins are the trimerized region corresponding to the coiled coil sequence of human XIII type collagen and the region corresponding to one NC11-COL7 or NC7-COL4 of BP180 located on the C-terminal side thereof. It is a protein containing a region corresponding to. In certain embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 1 or a variant amino acid sequence thereof and the amino acid sequence of SEQ ID NO: 2 or a variant amino acid sequence thereof, or the amino acid sequence of SEQ ID NO: 3 or a variant amino acid sequence thereof.

A more preferred example of a collagen-like modified protein is a protein in which the NC2 to COL3 region of human XIII type collagen (Pro ²¹⁷ -Asp ⁶⁹⁹ shown in the lower part of FIG. 1) is replaced with NC11-COL7 or NC7-COL4 of BP180. Is. In certain embodiments, the protein comprises, in order from the N-terminus, the amino acid sequence from position 2 to 216 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence from position 700 to 717 of SEQ ID NO: 9. Consists of. In yet another embodiment, the protein is composed of the amino acid sequences of SEQ ID NO: 9 from 2 to 216, the amino acid sequence of SEQ ID NO: 3, and the amino acids of SEQ ID NO: 9 from 700 to 717, in order from the N-terminal. It consists of an array.

Another more preferred example of the collagen-like modified protein is that the NC2 to COL3 region of human XIII collagen has one or more ends of either NC11-COL7 or NC7-COL4 of BP180 or both ends. It is a protein that has been replaced with a fragment to which an amino acid has been added. The number of amino acids added is, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 to 2. In certain embodiments, the protein has one or more amino acids added to the N-terminal to the 2nd to 216th amino acid sequences of SEQ ID NO: 9 and the N-terminal and C-terminal of the amino acid sequence of SEQ ID NO: 2. It consists of the amino acid sequence obtained and the amino acid sequence from position 700 to 717 of SEQ ID NO: 9. In yet another embodiment, the protein comprises one or more amino acids at the N-terminal to the N-terminal and C-terminal of the amino acid sequences 2 to 216 of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 3, in order from the N-terminal. It consists of an amino acid sequence to which is added and an amino acid sequence from position 700 to 717 of SEQ ID NO: 9. In a particular embodiment, the protein consists of the amino acid sequence set forth in SEQ ID NO: 19, and in a further specific embodiment, the protein consists of the amino acid sequence set forth in SEQ ID NO: 21.

Collagen-like modified proteins retain the ability to form homotrimers and bind to human autoantibodies targeting NC11-COL7 of BP180 and / or human autoantibodies targeting NC7-COL4 of BP180. It may be chemically modified as long as it retains its ability to bind to and does not bind to human autoantibodies targeting NC16A of BP180. Chemical modifications include, for example, acylation, prenylation, acetylation, phosphorylation, glycosylation, PEGylation.

As shown in later examples, the collagen-like modified protein of this embodiment binds to BP-related autoantibodies resulting from drug administration, particularly DPP4i-BP autoantibodies, but is present in many BP cases. Does not bind to autoantibodies targeting NC16A. Thus, the collagen-like modified protein of this embodiment makes it possible to distinguish drug-induced BP patients, especially DPP4i-BP patients, from BP patients with autoantibodies targeting NC16A of BP180.

Preparation of Collagen-like Modified Protein The collagen-like modified protein can be prepared by a method for producing a recombinant protein using Escherichia coli or other microorganisms, insect cells or animal cells as host cells, or a cell-free protein expression method. Construction of recombinant genes, introduction of expression vectors into host cells, expression of target proteins in host cells and other genetic engineering techniques are based on the instructions in the experimental operation manual that explains various gene recombination operations in detail. Can be done.

For recombinant production of collagen-like modified protein, nucleic acid encoding collagen-like modified protein can be used. When the nucleic acid is DNA, it is preferably used in a form incorporated into an appropriate expression vector. The expression vector carrying the DNA encoding the collagen-like modified protein may be in any form such as circular or linear. Further, the expression vector may have another base sequence in addition to the base sequence encoding the collagen-like modified protein. Examples of other base sequences are enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of amplifying the number of copies, base sequences encoding peptides such as signal peptides and other polypeptides, and poly A addition. A sequence, a splicing sequence, a base sequence of a gene serving as a selection marker, and the like.

Upon gene recombination, a suitable synthetic DNA adapter is used to add translation initiation codons and translation termination codons to the DNA encoding the collagen-like modified protein, or to add or delete appropriate restriction enzyme cleavage sequences in the base sequence. It is also possible. These are within the scope of the work normally performed by those skilled in the art, and those skilled in the art can arbitrarily and easily process the DNA encoding the collagen-like modified protein.

As an expression vector carrying DNA encoding a collagen-like modified protein, an appropriate vector can be selected according to the host to be used, and in addition to the plasmid, bacteriophage, baculovirus, retrovirus, vaccinia virus, etc. can be selected. It is also possible to use various viruses.

The collagen-like modified protein can also be used by linking another appropriate expression promoter upstream of the base sequence encoding the collagen-like modified protein. Such an expression promoter may be appropriately selected depending on the host, for example, if the host is an Escherichia bacterium, preferably Escherichia coli, the T7 promoter, lac promoter, trp promoter, λPL promoter, etc., and the host is Bacillus genus. Bacteria, preferably B. subtilis, include P43 promoter, vegI promoter, xylose-inducible promoter, tetracycline inducible promoter and the like. If the host is yeast, the PHO5 promoter, GAP promoter, ADH promoter, etc., and if the host is animal cells, the SV40-derived promoter, retrovirus promoter, cytomegalovirus (CMV) IE (immediate early). Examples thereof include a gene promoter, a metallothioneine promoter, a heat shock promoter, an SRα promoter and the like.

Examples of host cells include the genera Escherichia, Bacillus, Corynebacterium, Brevibacterium, Serratia, Pseudomonas, and Earthlover. Bacteria such as (Arthrobacter), Erwinia, Methylobacterium and Rhodobacter, fungi such as Streptomyces, Zymomonas and Saccharomyces. Can be mentioned. Insect cells such as silkworm, HEK293 cells, MEF cells, Vero cells, Hela cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG cells and animal cells such as human kidney cell lines. Is also available.

Examples of the transformation method for introducing an expression vector into a host cell include an electroporation method, an alkali metal method, a calcium phosphate precipitation method, a DEAE dextran method, a microinjection method, and a lipofection method.

The collagen-like modified protein can be obtained by culturing a transformed cell into which an expression vector has been introduced, expressing the polypeptide in the cell, recovering the target polypeptide from the cell or medium, and purifying the protein. The transformed cells may be cultured according to a conventional method according to the properties of the host cell such as carbon assimilation and auxotrophy, the selection marker contained in the introduced recombinant gene, the promoter and the like.

Purification of collagen-like modified protein can be performed by appropriately selecting an appropriate method from the methods usually used for purification of protein. That is, various affinity chromatography such as salting out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, hydrophobic chromatography and antibody chromatography, chromatographic focusing method, adsorption. An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and if necessary, purification may be performed in an appropriate order using an HPLC system or the like.

When the collagen-like modified protein contains a tag sequence or other functional protein or an amino acid sequence of a polypeptide other than collagen, it is preferable to adopt a purification method characteristic of the functional protein. Such functional proteins or peptides and corresponding purification methods include histidine tags consisting of about 6 to 10 consecutive histidine residues and nickel-immobilized affinity chromatography, glutathione S-transferase (GST) and glutathione-immobilized affinity. Chromatography, FLAG tags, anti-FLAG antibodies and the like can be mentioned. Furthermore, the collagen-like modified protein can be recovered by cleaving the purified fusion protein with an appropriate protease (thrombin, trypsin, etc.).

Furthermore, a cell-free synthesis method using a nucleic acid encoding a collagen-like modified protein is also one of the genetic engineering production methods. Cell-free protein synthesis systems include systems that use cell extracts such as Escherichia coli, wheat germ, yeast, rabbit reticulocytes, insect cells, and cultured mammalian cells, and reconstituted types that are composed by combining factors necessary for protein synthesis. System can be mentioned.

The collagen-like modified protein is produced by an organic chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) using a suitable commercially available peptide synthesizer. Alternatively, by genetic engineering techniques, the nucleic acids described above, especially the DNA integrated into the expression vector, can be introduced into a suitable expression system using a suitable host cell selected from prokaryotes or eukaryotes. It is preferable to produce it.

The collagen-like modified protein is produced as a homotrimer by the method exemplified above, particularly by culturing and expressing transformed cells into which an expression vector has been introduced, and recovering and purifying the target protein from the cells or medium. can do. Homotrimers can also be converted into monomers and used by placing them in the presence of SDS or other surfactants or high-concentration salts.

As described above, the present invention provides a nucleic acid encoding a collagen-like modified protein, an expression vector containing the nucleic acid, and the nucleic acid or a host cell containing the expression vector, as different embodiments.

Method for detecting autoantibodies Another aspect of the present invention is a step of contacting a sample sample collected from a subject with the collagen-like modified protein of the first aspect, and an antibody in the sample sample and the collagen-like modified protein. The present invention relates to a method for detecting a human autoantibody targeting NC11-COL7 and / or a human autoantibody targeting NC7-COL4 of human BP180, which comprises a step of detecting the binding of. Here, the collagen-like modified protein is as described in the first aspect.

The detection of human autoantibodies can be performed by detecting the binding between the antibody and the collagen-like modified protein in the sample sample, which is caused by contacting the sample sample with the collagen-like modified protein of the first aspect. The sample sample used here is a biological sample derived from humans, and examples thereof include skin tissue, skin cells, and body fluids (eg, blood, lymph, saliva, mucus, bone marrow, urine, semen, and ascitic fluid). Etc.), serum or plasma prepared from blood, etc. can be mentioned. The biological sample may be used as it is collected, or may be used after pretreatment such as pulverization, homogenization, centrifugation, concentration, and dilution. A particularly preferred sample sample is a blood sample, specifically blood, serum or plasma.

Detection of human autoantibodies can be performed by immunoassay utilizing antigen-antibody reaction. Examples of the immunoassay include ELISA, radioimmunoassay, immunoblotting, and immunochromatography.

In the immunoassay, for example, a collagen-like modified protein is immobilized on a carrier, which is brought into contact with a sample sample to react with the collagen-like modified protein and an antibody in the sample sample, and then the antibody bound to the collagen-like modified protein is labeled. It can be carried out by detecting with the obtained human antibody-binding substance.

Examples of human antibody-binding substances include antibody-binding proteins capable of binding to human antibodies, particularly to the Fc region of human IgG, such as antibodies to human antibodies and protein G. ..

Examples of labeling compounds used for labeling human antibody-binding substances include fluorescent substances (eg, FITC, rhodamine, etc.), metal particles such as gold colloid, fluorescent microbeads such as Luminex (registered trademark, Luminex), and dye proteins. (Eg, phycoerythrin, phycocyanin, etc.), radioisotopes (eg, ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I, ¹³¹ I, etc.), enzymes (eg, peroxidase, alkaline phosphatase, etc.), biotin, streptavidin, etc. Can be done.

Carriers and methods for immobilizing collagen-like modified proteins, labeling compounds for labeling human antibody-binding substances and labeling methods using them, and immunoassay operations using these, such as incubation of specimens with carriers. For conditions, washing, blocking treatment, detection of labeled compounds, etc., refer to the description of methods known or well known to those skilled in the art, for example, The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques (4TH), Elsevier. It can be carried out. This document is incorporated herein by reference in its entirety.

Data collection method According to the above-mentioned method for detecting human autoantibodies, human autoantibodies targeting NC11-COL7 and / or NC7-COL4 of human BP180 can be detected. Specifically, when the collagen-like modified protein contains a region corresponding to NC11-COL7 of BP180, a human autoantibody targeting NC11-COL7 can be detected, and the collagen-like modified protein is NC7 of BP180. -Human autoantibodies targeting NC7-COL4 can be detected if they contain a region corresponding to COL4. These human autoantibodies are drug-induced BP-related autoantibodies, especially DPP4i-BP autoantibodies. Therefore, the data collected by performing this method on sample samples taken from BP patients is useful for distinguishing whether the patient's BP is drug-induced or due to other causes. Data. As described above, the present invention comprises a step of contacting a sample sample collected from a subject with a collagen-like modified protein, and a step of detecting binding between an antibody in the sample sample and the collagen-like modified protein. It also provides a method of collecting data for the diagnosis of sex BP, especially for the diagnosis of DPP4i-BP. Further, in the present invention, the sample sample used in this data acquisition method, the collagen-like modified protein, and the method of using these are as described in the above-mentioned method for detecting human autoantibodies.

Kit The present invention is for detecting a human autoantibody targeting NC11-COL7 and / or a human autoantibody targeting NC7-COL4 of human BP180, which comprises at least a collagen-like modified protein according to the first aspect. Kits, as well as kits for the diagnosis of drug-induced bullous pemphigoid, are provided as yet another embodiment. In addition to collagen-like modified proteins, the kit includes carriers such as plates used for detecting human autoantibodies that bind to the polypeptide in immunoassays, blocking solutions, washing solutions, human antibody-binding substances, color-developing substrates, and the like. Additional reagents may be included.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Preparation of collagen-like modified protein (1) Preparation of expression vector cDNA encoding five types of collagen-like modified protein (collagen 13-BP180 swap protein 1 to 5) was synthesized using a DNA synthesizer. The cDNA of swap protein 1 consists of FLAG tag (SEQ ID NO: 24), amino acid sequence from 2 to 121 of human XIII collagen isoform COL13A1 (SEQ ID NO: 9), and extracellular domain fragment of BP180 in order from the 5'end side. It encodes the amino acid sequence of Gly ⁵⁶⁷ -Ile ⁸⁰⁸ and the amino acid sequence of human XIII collagen isoform COL13A1 (SEQ ID NO: 9) from position 700 to 717. The cDNAs of swap proteins 2 to 5 are, in order from the 5'end side, the FLAG tag (SEQ ID NO: 24), the amino acid sequences 2 to 216 of the human XIII collagen isoform COL13A1 (SEQ ID NO: 9), and the extracellular of BP180. Amino acid sequence of domain fragment Val ⁸⁰⁹ -Ser ⁹⁸¹ , Glu ⁹⁸² -Ser ¹¹⁶⁰ , Tyr ¹¹⁶¹ -Ser ¹²⁷⁹ or Arg ¹²⁸⁰ -Pro ¹⁴⁹⁷ , and amino acids 700-717 of human XIII collagen isoform COL13A1 (SEQ ID NO: 9). Encoding the array. In each cDNA, EcoRV restriction site and HindIII restriction site were added in-frame to the 5'end and 3'end of the base sequence encoding the BP180 extracellular domain fragment, respectively. In addition, a NheI restriction site was added to the 5'end of each cDNA, and an ApaI restriction site was added to the 3'end. The base sequences of swap proteins 1 to 5 excluding the restriction enzyme sites at the 5'end and 3'end and the sequence corresponding to the FLAG tag sequence are assigned to SEQ ID NOs: 14, 16, 18, 20, and 22 in these base sequences. The corresponding amino acid sequences are shown in 15, 17, 19, 21, 23.

Expression vector A total of 5 types of collagen-like modified protein expression vectors (collagen 13-BP180 swap protein expression vector) by recombining the above cDNAs to the NheI-ApaI sites of pcDNA3.1 / Hyg (Invitrogen) or pcDNA5 / FRT (Invitrogen). Was built.

(2) Preparation of collagen-like modified protein stable expression cells The collagen 13-BP180 swap protein expression vector prepared in (1) alone (when incorporated into pcDNA3.1 / Hyg) or together with pOG44 (Invitrogen) Flp-In 293 cells (Invitrogen) cultured in DMEM medium containing 10% bovine fetal serum were transfected with Avalanche-Omni Transfection Reagent (EZ Biosystems). From 48 hours after gene transfer, Hygromycin (Invitrogen) was added to the medium at a concentration of 50 μg / ml, and the culture was continued for 10 to 14 days, and collagen 13-BP180 swap protein stable expressing cells were selected as drug-resistant clones.

(3) Purification of collagen-like modified protein Collagen 13-BP180 swap protein stable-expressing cells that had been cultured and grown in a petri dish until they became dense were washed twice with PBS (Nakarai), and then cell thawing buffer (25 mM Tris, pH 7.5, 1). % NP-40 (Nakarai), 10 mM EDTA, 1 x Protease inhibitor buffer (Sigma # P8340-1ML)) was added, and the mixture was gently shaken on ice for 30 minutes. The thawed cell-containing solution was collected in an Eppen tube using a cell scraper, centrifuged at 14,000 rpm at 4 ° C. for 20 minutes, and the supernatant was collected. PBS was added to the supernatant and mixed, and then anti-DDDDK antibody magnetic beads (MBL # M185-11) were added, and the mixture was shaken at 4 ° C. for 10 to 12 hours. Then, only the magnetic beads were recovered using a magnetic separator (Invitrogen) and washed with PBS containing 0.1% NP-40. Then, elution buffer (500 μg / ml FLAG peptide (Sigma # F3290-4MG), 0.1% NP-40, PBS) was added, and the mixture was shaken at 700 rpm for 15 minutes at room temperature, and then the eluted protein was recovered using a magnetic separator. Five types of collagen-like modified proteins (collagen 13-BP180 swap proteins 1-5, Fig. 2) were prepared.

Example 2 Immunoassay using human sample (1) Western blotting 17 patients diagnosed with DPP4i-BP (hereinafter referred to as DPP4i-BP patients) by the diagnosis of a dermatologist, no history of oral administration of DPP4i diagnosed with BP Serums were prepared from blood collected from each of 3 patients (hereinafter referred to as BP patients) and 3 healthy subjects, and used as sample samples. It was confirmed in advance that the sera of all DPP4i-BP patients used did not show responsiveness to NC16A.

Swap proteins 1 to 5 prepared in Example 1 are multiplied by 5 times with 5x sample buffer (4M Urea, 0.5M Tris-HCl pH 6.8, 0.0005% bromophenol blue, 10% SDS, 25% Glycerin, 0.05 M DTT). Diluted and subjected to SDS-PAGE on a gel containing 7% acrylamide.

After transcribing the protein in the SDS-PAGE gel after electrophoresis to a nitrocellulose membrane (BioRad), anti-FLAG antibody (M2, Sigma # F1804-1MG, diluted 1: 2,000 with TBS containing 2% skim milk) or sample as the primary antibody. Samples (diluted 1:200 with TBS containing 2% skim milk) were added and reacted at room temperature for 1 hour or at 4 ° C. for 10-12 hours. Then, as a secondary antibody, HPR-labeled anti-mouse IgG (Jackson # 115-036-006) or HRP-labeled anti-human IgG (Dako # P0214) diluted 1: 5,000 with TBS containing 2% skim milk was added, and 30-60 at room temperature. It was reacted for a minute. Color was developed using Clarity Western ECL Substrate (BIO-RAD # 170-5060), and chemiluminescence was identified with LAS-4000mini (FUJIFILM).

The results of Western blotting of swap protein 3 are shown in FIG. 3, and the results of Western blotting of swap protein 4 are shown in FIG. 4, respectively. When the anti-FLAG antibody was used as the primary antibody, a protein band was detected at the position corresponding to the molecular weight of the monomer of each swap protein and the position corresponding to the molecular weight of the homotrimer (FIGS. 3 and 3). Image on the left side of 4).

As shown in FIG. 3, all 17 DPP4i-BP patients responded to both the monomer and trimer to the swap protein 3 containing NC11-COL7 of BP180, and 2 BP patients. Reacted with both monomers and trimers. Healthy subject controls did not respond to either monomers or trimers.

In addition, as shown in FIG. 4, 4 DPP4i-BP patients responded to the monomer and 7 DPP4i-BP patients became trimers to the swap protein 4 containing NC7-COL4 of BP180. It reacted. On the other hand, BP patients and healthy control did not respond to either monomers or trimers.

For

swap proteins

1, 2 and 5, bands were detected at positions corresponding to the molecular weight of the monomer and at positions corresponding to the molecular weight of the homotrimer by Western blotting using the anti-FLAG antibody as the primary antibody. However, none of the DPP4i-BP patients, BP patients and healthy person control sample samples showed a response to monomers and trimers.

(2) ELISA
Serum was prepared from blood collected from each of 15 DPP4i-BP patients, 5 BP patients, and 3 healthy subjects, and used as sample samples. It was confirmed in advance that the sera of all DPP4i-BP patients used did not show responsiveness to NC16A.

Swap proteins 1 to 5 prepared in Example 1 were diluted with Carbonate buffer (50 mM CB pH 9.6) to a concentration of 0.4 μg / ml, and added to a 96-well plate (Nunc # 442404) at a concentration of 50 μl / well. After the reaction at 4 ° C. for 10 to 12 hours, the cells were washed 3 times with 200 μl of PBS, and Blocking buffer (Roche # 11112589001) was added at 90 μl / well. After reacting at room temperature for 2 hours, the cells were washed twice with 200 μl of PBS to prepare 5 types of 96-well plates for ELISA in which swap proteins 1 to 5 were immobilized.

As the primary antibody, a sample sample diluted 101-fold with PBS or an anti-FLAG antibody (M2) diluted 1: 2,000 was added at a rate of 100 μl / well and reacted at room temperature for 1 hour. After washing 4 times with PBS containing 0.05% Tween and 200 μl / well, HRP-labeled anti-human and anti-mouse IgG diluted 1: 10,000 with PBS were added at 100 μl / well each and reacted at room temperature for 1 hour. .. After washing 4 times with PBS containing 0.05% Tween and 200 μl / well, a color reaction was carried out at room temperature for 12 minutes using TMB (NOVEX). The color reaction was stopped with Stop solution (0.5N sulfuric acid), and the absorbance at wavelengths of 450 nm and 620 nm was measured with an absorptiometer (Sunrise, TECAN), and these differences were used as an index of the antigen-antibody reaction.

The result of ELISA is shown in Fig. 5. Specimens of DPP4i-BP patients showed high reactivity among swap proteins 1 to 5, especially swap protein 4 containing NC7-COL4 of BP-180.

(3) ELISA with increased sample size
In the same manner as in (2) above, five types of 96-well plates for ELISA in which swap proteins 1 to 5 were fixed were prepared. Also, a 98-well plate for ELISA with NC16A recombinant protein immobilized was used for ELISA with full-length BP180 recombinant protein immobilized by the method described in Kobayashi M, et al. J Dermatol. Sci., 30: 224-232, 2002. 98-well plates were made by the method described in Izumi K, et al. J Invest. Dermatol., 136: 2201-10, 2016.

Patients with skin diseases other than BP (eczema dermatitis group, etc.) shown in Table 3 (hereinafter, non-BP patients), BP patients and DPP4i-BP patients' sera are used as sample samples, and one healthy person's serum is used as a sample. As a negative control, ELISA using the above 7 types of plates was carried out in the same manner as in (2) above.

The difference between the absorbance at a wavelength of 450 nm and the absorbance at a wavelength of 620 nm measured by ELISA was used as the OD value, and the ELISA index was calculated by the following formula.
Formula: (OD value of each sample sample-OD value of healthy person sample) / (OD value of anti-FLAG antibody-OD value of healthy person sample) x 100

The result of ELISA is shown in FIG. The sera of DPP4i-BP patients were significantly more responsive to swap protein 3 containing NC11-COL7 and swap protein 4 containing NC7-COL4 of BP180 than the sera of non-BP and BP patients. Indicated. The sera of all DPP4i-BP patients used did not show reactivity to NC16A.

In addition, using the ELISA index for swap protein 4, non-BP and DPP4i-BP patient datasets, BP and DPP4i-BP patient datasets, and non-DPP4i-BP patients (non-BP patients + BP patients) ROC analysis was performed for each of the DPP4i-BP patient data sets, with the objective variable being DPP4i-BP and the explanatory variable being the ELISA index. The analysis was performed using GraphPad Prism 8 (MDF), and the AUC was calculated from the prepared ROC curve. The AUC was 0.8 or higher regardless of which data set was used, and it was confirmed that the determination of DPP4i-BP by ELISA for swap protein 4 showed a good accuracy.

Comparative example Preparation of BP180 extracellular domain fragment Expression vector pGEX-6P-1 (Amersham # 27-4597-01) and human BP180 COL15, NC15-COL9, NC9-NC4, NC4-NC1 region amino acids (SEQ ID NO:) DNA encoding 26, 28, 30, 32) (SEQ ID NOs: 25, 27, 29, 31) was introduced into the BamHI-NotI site to construct a total of 5 types of BP180 extracellular domain dividing region Escherichia coli expression vectors. Ampicillin drug-resistant clones were selected after transformation of BL21 Escherichia coli strain (GE Healthcare # 27-1542-01) using these expression vectors. Cell lysates were recovered from Escherichia coli whose expression was induced by Overnight Expression Autoinduction System 1 (Novagen # 71300) by B-PERII (Thermo Fisher Scientific # 78243). When Western blotting was performed, a sufficient amount of target protein that could be purified could not be identified from either the soluble fraction or the insoluble fraction, and it was difficult for E. coli to express the divided region of the BP180 extracellular domain. It turned out to be.

SEQ ID NO: 1 Amino acid sequence of the coiled coil sequence of human XIII type collagen SEQ ID NO: 2 Amino acid sequence of NC11-COL7 of human BP180 SEQ ID NO: 3 Amino acid sequence of NC7-COL4 of human BP180 SEQ ID NO: 4 Amino acid sequence of coiled coil sequence of human XXIII type collagen SEQ ID NO: 5 Amino acid sequence of human XXV type collagen coiled coil sequence SEQ ID NO: 6 Amino acid sequence of human MARCO coiled coil sequence SEQ ID NO: 7 Amino acid sequence of human SRCL coiled coil sequence SEQ ID NO: 8 Amino acid sequence of human ectodisplacin A coiled coil sequence No. 9 Amino acid sequence of human XIII type collagen α1 chain (COL13A1) SEQ ID NO: 10 Amino acid sequence of human BP180 SEQ ID NO: 11 Base sequence of cDNA encoding human BP180 SEQ ID NO: 12 Human autoantibodies present in NC16A of human BP180 are recognized. Amino Acid SEQ ID NO: 13 Amino Acid SEQ ID NO: 14 of NC16A region of human BP180 Base sequence of DNA encoding collagen-like modified protein (collagen 13-BP180 swap protein 1) containing COL15 of human BP180 SEQ ID NO: 15 COL15 of human BP180 Amino acid sequence number of collagen-like modified protein (collagen 13-BP180 swap protein 1) containing NC15-COL11-containing DNA base sequence number of DNA encoding collagen-like modified protein (collagen 13-BP180 swap protein 2) 17 Amino acid sequence of human BP180 NC15-COL11-containing collagen-like modified protein (collagen 13-BP180 swap protein 2) Amino acid sequence number 18 Human BP180 NC11-COL7-containing collagen-like modified protein (collagen 13-BP180 swap protein 3) Base sequence of encoding DNA SEQ ID NO: 19 Amino acid sequence of collagen-like modified protein containing NC11-COL7 of human BP180 (Collagen 13-BP180 swap protein 3) SEQ ID NO: 20 Collagen-like modified protein containing NC7-COL4 of human BP180 (collagen) 13-Basic sequence of DNA encoding BP180 swap protein 4) SEQ ID NO: 21 Amino of collagen-like modified protein (collagen 13-BP180 swap protein 4) containing NC7-COL4 of human BP180 Acid sequence SEQ ID NO: 22 Nucleotide sequence of DNA encoding NC4-NC1-containing collagen-like modified protein (collagen 13-BP180 swap protein 5) of human BP180 SEQ ID NO: 23 Collagen-like modified protein containing NC4-NC1 of human BP180 (collagen) 13-BP180 Swap protein 5) Amino acid sequence SEQ ID NO: 24 FLAG tag amino acid sequence SEQ ID NO: 25 Nucleotide sequence of artificially synthesized DNA encoding COL15 of human BP180 SEQ ID NO: 26 Artificial synthetic amino acid sequence of human BP180 COL15 SEQ ID NO: 27 Human Nucleotide sequence of artificial DNA encoding NC15-COL9 of BP180 SEQ ID NO: 28 Nucleotide sequence of human BP180 Nucleotide sequence of NC15-COL9 Nucleotide sequence of human BP180 Nucleotide sequence of human BP180 Nucleotide sequence of NC9-NC4 artificially synthesized amino acid sequence of BP180 SEQ ID NO: 31 Nucleotide sequence of artificially synthesized DNA encoding NC4-NC1 of human BP180 SEQ ID NO: 32 Nucleotide sequence of NC4-NC1 of human BP180

Claims

The trimeric region corresponding to the coiled coil arrangement of human type II transmembrane collagen and the 7th collagen from the 11th non-collagen region (NC11) of human BP180 located on the C-terminal side of the trimeric region. A human that includes a region corresponding to the region (COL7) and / or a region corresponding to the 7th non-collagen region (NC7) to the 4th collagen region (COL4), but targeting the NC16A region of human BP180. Collagen-like modified protein that does not bind to self-antibodies.
The protein according to claim 1, wherein the human type II transmembrane collagen is human XIII type collagen.
The amino acid sequence of claim 1 or 2, wherein the amino acid sequence of the trimerization region is the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. protein.
The amino acid sequence of the region corresponding to NC11 to COL7 of human BP180 is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2, and human BP180. 1 to claim 1, wherein the amino acid sequence of the region corresponding to NC7 to COL4 of the above is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3. The protein according to any one of 3.
The protein according to any one of claims 1 to 4, which comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21.
The protein according to any one of claims 1 to 5, which forms a homotrimer structure.
Nucleic acid encoding the protein specified in any one of claims 1 to 5.
An expression vector containing the nucleic acid specified in claim 7.
A host cell containing the nucleic acid according to claim 7 or the expression vector according to claim 8.
A human including a step of contacting a sample sample collected from a subject with the protein specified in any one of claims 1 to 6, and a step of detecting the binding between the antibody in the sample sample and the protein. A method for detecting a human autoantibody targeting the region from NC11 to COL7 of BP180 and / or a human autoantibody targeting the region from NC7 to COL4 of human BP180.
The method according to claim 10, wherein the sample is a blood sample.
Targeting a human autoantibody targeting the region NC11 to COL7 of human BP180 and / or the region NC7 to COL4 of human BP180 containing the protein specified in any one of claims 1 to 6. A kit for detecting human autoantibodies.
A drug comprising a step of contacting a sample sample collected from a subject with the protein specified in any one of claims 1 to 6 and a step of detecting the binding of the antibody in the sample sample to the protein. How to collect data for the diagnosis of bullous pemphigoid.
The method according to claim 13, wherein the sample is a blood sample.
A kit for diagnosing drug-induced bullous pemphigoid, which comprises the protein specified in any one of claims 1 to 6.