Humanized antibodies binding to bp-specific antigenic peptides, methods of making and uses thereof

Abstract

The present disclosure belongs to the field of biological medicine, and relates to a human antibody binding to a BP specific antigen peptide, a preparation method and use. Specifically, the present disclosure relates to a monoclonal antibody, wherein the antibody specifically binds to the NC16A domain of the BP180 antigen protein, a preparation method of the monoclonal antibody, and use of the aforementioned monoclonal antibody in preparation of a drug for diagnosis, prevention or treatment of bullous pemphigoid. The antibody or antigen binding fragment against BP180 screened by the present disclosure specifically binds to the BP180 antigen, can be used as a reference standard for qualitative detection of BP180 positivity, realizes quantitative detection of the level of the anti-BP180 NC16A autoantibody in BP patients, and has important significance for clinical diagnosis, disease monitoring and treatment of the BP patients.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine and relates to a human antibody that binds to a BP-specific antigenic peptide, its preparation method, and its uses. Specifically, this disclosure relates to a monoclonal antibody that specifically binds to the NC16A domain of the BP180 antigen protein, a method for preparing the monoclonal antibody, and its use in the preparation of drugs for the diagnosis, prevention, or treatment of bullous pemphigoid. Background Technology

[0002] Bullous pemphigoid (BP) is an autoimmune disease that occurs when the immune system attacks the skin and causes blisters, most commonly affecting people aged 60-80. The characteristic clinical presentation is tense blisters and intense, widespread itching. Histopathologically, hematoxylin and eosin (HE) staining reveals subepidermal clefts with eosinophilic infiltration; direct immunofluorescence (DIF) shows linear deposits of autoantibodies and / or complement distributed along the basement membrane band; and salt cleft indirect immunofluorescence (SSIIF) shows autoantibodies and / or complement deposits on the epidermal side.

[0003] The target antigens of autoantibodies in the serum of patients with BP are BP180 and BP230, also known as BPAG1 and BPAG2, with molecular weights of 180 kDa and 230 kDa, respectively. BP180 is considered a direct target of autoantibodies. BP180 is a type II transmembrane protein with a cytoplasmic NH2 terminus and an extracellular COOH domain. The N-terminal domain, transmembrane stretching domain, and extracellular C-terminus have 466, 23, and 1008 amino acids, respectively. The extracellular domain contains 15 collagen subdomains (COL1–COL15), interspersed with 16 non-collagenous sequences (NC1–NC16). The NC16A domain is the lateral membrane junction region, which is the core of the collagen-like triple helix formation. The extracellular region contains a helical structure that physiologically detaches from the cell surface via deintegrin metalloproteinases (ADAM). The NC16A domain contains seven antigenic sites, including NC16A1 (aa 490-506), NC16A1-3 (aa 490-534), NC16A1-5 (aa 490-562), NC16A2 (aa 507-520), NC16A2.5 (aa 514-532), NC16A3 (aa 521-534), and NC16A3-4 (aa 522-545). Among these sites, NC16A2 and NC16A2.5 are the major antigenic sites and can be captured by all IgG and IgE antibodies. It has been reported that BP180-NC16A antibody titers are correlated with disease severity in BP patients. Replacing mouse BP180-NC14A with the homologous human BP180-NC16A cluster region and injecting mice with intact IgG or IgG antibodies affinity-purified for BP180-NC16A from BP patients resulted in increased skin fragility. The structure and localization of BP180 indicate that it is a core anchoring protein connecting intracellular and extracellular hemiponectin, playing a crucial role in the pathogenesis of BP. The NC16A domain of BP180 is considered a major pathogenic epitope of BP. Therefore, identifying the target region of BP180 is of great significance for understanding the pathogenesis and clinical characteristics of BP180.

[0004] BP is a spectrum disease; although most patients achieve clinical remission after treatment, there is a significant mortality rate in elderly patients, especially those over 80 years of age, with a mortality rate reaching 25%. Furthermore, immunotherapy in cancer patients can also induce BP. PD-1 / PD-L1 checkpoint inhibitors, widely used to treat various solid and hematologic malignancies, are among the drugs most frequently reported to induce BP in recent years. Most cases develop bullae or vesicles within 6-8 months of starting PD-1 / PD-L1 inhibitor treatment, with a minority of cases showing mucosal involvement. With the increasing prevalence of PD-1 immunotherapy, the demand for BP testing will continue to grow. Currently, there are no effective methods for rapid and accurate diagnosis of this disease.

[0005] IgG is a type of autoantibody that causes BP, with its main target antigen being BP180. After BP-IgG binds to BP180, it activates complement, inducing the internalization of the antigenic peptide within cells, leading to weakened adhesion between keratinocytes and the basement membrane. The serum level of heat shock protein 90 (HSP90) in BP patients is inversely proportional to that of BP180-NC16A IgG antibody; that is, anti-BP180-NC16A IgG antibody indirectly enhances intracellular HSP90 expression through inflammatory responses generated by soluble inflammatory chemokines, while simultaneously inhibiting the release of HSP90 from cells into the peripheral blood. Abnormally high intracellular HSP90 expression releases related chemokines to attract inflammatory cells such as eosinophils and neutrophils, and releases proteolytic enzymes and various inflammatory mediators, participating in the pathological changes and vesicle formation in BP.

[0006] The diagnostic criteria for bullous pemphigoid include clinical manifestations, histopathology, direct immunofluorescence, indirect immunofluorescence, and specific antibody testing. Different treatment regimens are adopted according to the severity of the disease, primarily relying on glucocorticoids, antibiotics, and immunosuppressants. Currently, glucocorticoids are recognized as the first-line drug for treating BP, with significant clinical efficacy; however, long-term use can lead to numerous complications, some of which are so severe that they even prevent certain patients from using the drug clinically. Therefore, there is a need for a reagent for the diagnosis or treatment of bullous pemphigoid.

[0007] Meanwhile, classic hybridoma technology requires significant time and effort to obtain high-affinity antibodies and necessitates subsequent humanization, making it difficult to obtain human monoclonal antibodies and achieve absolute quantification of antibody levels. Therefore, these treatments lack specificity, have poor efficacy, and require long treatment courses. Phage display technology, first established by Smith in 1985, has been widely applied in antigen-antibody library construction, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, and cell signal transduction research after more than 30 years of development and refinement. Phage antibody library technology involves preparing human antibody libraries, expressing proteins or peptides on the surface of phages, and then screening and enriching specific antibodies. It has been proposed that almost all recombinant human monoclonal antibodies that specifically react with antigens can be screened from single-pot antibody library systems. Therefore, when using phage antibody technology, various antibody fragments (Fab or ScFv) applicable to in vivo diagnosis or treatment can be obtained. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] Based on the problems existing in the prior art, this disclosure screens out a human monoclonal antibody against the bullous pemphigoid antigen BP180.

[0010] Solution for solving the problem

[0011] In a first aspect, this disclosure provides isolated anti-BP180 antibodies or antigen-binding fragments thereof, comprising a heavy chain variable region, wherein the sequence encoding the heavy chain variable region comprises one or more of the following sequences:

[0012] (a1) The amino acid sequence as shown in SEQ ID NO: 4;

[0013] (a2) Compared with the sequence shown in SEQ ID NO: 4, there are 1, 2 or 3 conserved amino acid sequences;

[0014] (a3) The amino acid sequence as shown in SEQ ID NO: 8;

[0015] (a4) Compared with the sequence shown in SEQ ID NO: 8, there are 1, 2 or 3 conserved amino acid sequences;

[0016] (a5) The amino acid sequence as shown in SEQ ID NO: 12;

[0017] (a6) Compared with the sequence shown in SEQ ID NO: 12, there are 1, 2 or 3 conserved amino acid sequences;

[0018] The heavy chain variable region is encoded according to the IMGT analysis method.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof according to this disclosure comprises a light chain variable region, wherein the sequence encoding the light chain variable region comprises one or more of the following sequences:

[0020] (b1) The amino acid sequence as shown in SEQ ID NO: 3;

[0021] (b2) Compared with the sequence shown in SEQ ID NO: 3, there are 1, 2 or 3 conserved amino acid sequences;

[0022] (b3) The amino acid sequence as shown in SEQ ID NO: 7;

[0023] (b4) Compared with the sequence shown in SEQ ID NO: 7, there are one or two conserved amino acid sequences;

[0024] (b5) The amino acid sequence as shown in SEQ ID NO: 11;

[0025] (b6) Compared with the sequence shown in SEQ ID NO: 11, there are 1, 2 or 3 conserved amino acid sequences;

[0026] The variable region of the light chain is encoded according to the analysis method of IMGT.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof according to this disclosure comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the light chain variable region (VL) comprises VL complementarity-determining region (CDR) 1, VL complementarity-determining region (CDR) 2, and VL complementarity-determining region (CDR) 3, and the heavy chain variable region (VH) comprises VH complementarity-determining region (CDR) 1, VH complementarity-determining region (CDR) 2, and VH complementarity-determining region (CDR) 3; and,

[0028] The VL is encoded by the following amino acids: VLCDR1 contains the amino acid sequence shown in SEQ ID NO: 3, VLCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and VLCDR3 contains the amino acid sequence shown in SEQ ID NO: 11.

[0029] The VH is encoded by the following amino acids: VHCDR1 contains the amino acid sequence shown in SEQ ID NO: 4, VHCDR2 contains the amino acid sequence shown in SEQ ID NO: 8, and VHCDR3 contains the amino acid sequence shown in SEQ ID NO: 12.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof according to this disclosure comprises one or more sequences shown below:

[0031] (i) VH contains the amino acid sequence shown in SEQ ID NO: 16, and VL contains the amino acid sequence shown in SEQ ID NO: 18;

[0032] (ii) Sequences with conserved mutations compared to the sequence shown in (i).

[0033] Secondly, this disclosure provides a polynucleotide, wherein the polynucleotide is selected from any one of (a)-(d):

[0034] (a) Contains a nucleotide sequence as shown in any one or a combination of SEQ ID NO: 15, SEQ ID NO: 17, or the sequence shown in SEQ ID NO: 17;

[0035] (b) A nucleotide sequence comprising the reverse complementary sequence of a nucleotide sequence as shown in any of the sequences of SEQ ID NO: 15, SEQ ID NO: 17 or a combination thereof;

[0036] (c) The reverse complementary sequence of a sequence that can hybridize with any of the nucleotide sequences shown in (a)-(b) under high-strict hybridization conditions or very high-strict hybridization conditions;

[0037] (d) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with any of the nucleotide sequences shown in (a)-(c).

[0038] Thirdly, this disclosure provides a vector comprising the polynucleotide according to the second aspect.

[0039] Fourthly, this disclosure provides an isolated host cell, wherein the host cell comprises a vector as described in the third aspect.

[0040] Fifthly, this disclosure provides a method for preparing host cells that stably express a target protein, wherein the method includes the step of transforming an initial host cell using the vector described in the third aspect.

[0041] In a sixth aspect, this disclosure provides a method for preparing a target protein, the method comprising preparing the target protein using the host cell described in the fourth aspect or by the method described in the fifth aspect.

[0042] In a seventh aspect, this disclosure provides antibodies or their binding fragments prepared according to the method described in the fifth aspect.

[0043] Eighthly, this disclosure provides a method for detecting anti-BP180 antibodies, wherein the method includes the step of detecting a sample to be tested using the antibody or antigen-binding fragment thereof described in the first or seventh aspect;

[0044] Optionally, the method includes the step of quantifying the anti-BP180 antibody in the sample to be tested.

[0045] In a ninth aspect, this disclosure provides a kit comprising an antibody or an antigen-binding fragment thereof as described in the first or seventh aspect.

[0046] In a tenth aspect, this disclosure provides a composition comprising an antibody or an antigen-binding fragment thereof as described in the first or seventh aspect.

[0047] In the eleventh aspect, this disclosure provides for the use of the antibody or antigen-binding fragment thereof according to the first or seventh aspect, or the composition according to the tenth aspect, in at least one of the following (1)-(4):

[0048] (1) Detect anti-BP180 antibody, or prepare reagents or kits for detecting anti-BP180 antibody;

[0049] (2) Prepare reagents or kits for diagnosing bullous pemphigoid;

[0050] (3) Prepare reagents or kits for monitoring the progression of bullous pemphigoid;

[0051] (4) Prepare reagents or kits for studying the pathogenesis of bullous pemphigoid.

[0052] In a twelfth aspect, this disclosure provides a method for preventing or treating bullous pemphigoid, wherein a subject is given an antibody or antigen-binding fragment thereof according to the first or seventh aspect, or a composition according to the tenth aspect.

[0053] The effects of the invention

[0054] This disclosure identifies a humanized monoclonal antibody targeting the bullous pemphigoid antigen BP180. This antibody exhibits high activity, good stability, and strong specificity upon binding to BP180, serving as a qualitative reference standard for BP180 positivity and enabling quantitative detection of anti-BP180 NC16A autoantibody levels in BP patients, thereby monitoring disease activity. It can be used for the clinical diagnosis and treatment of BP patients. Attached Figure Description

[0055] Figure 1 The image shows a monoclonal bacterial PCR agarose gel electrophoresis image of the VL phage library, which corresponds to the VL phage library constructed in this disclosure. Lane M is DL2000, lanes 1-16 are pATA-VK, and lanes 17-32 are pATA-Vλ.

[0056] Figure 2 The image shows a monoclonal bacterial PCR agarose gel electrophoresis image of the KH phage library, which corresponds to the KH phage library constructed in this disclosure. Lane M is DL2000, and lanes 1-48 are pATA-scFv-KH, respectively.

[0057] Figure 3 The image shows a monoclonal bacterial PCR agarose gel electrophoresis image of the λH phage library, which corresponds to the λH phage library constructed in this disclosure. Lane M is DL2000, and lanes 1-48 are pATA-scFv-λH, respectively.

[0058] Figure 4 The results of single-clone sequencing analysis of the phage display library are shown. The left figure shows the light chain analysis results, and the right figure shows the heavy chain analysis results.

[0059] Figure 5 The SDS-PAGE electrophoresis image of the target protein BP180 is shown. Based on the results of the aforementioned electrophoresis image, the protein size is 34 kDa and the purity is greater than 90%.

[0060] Figure 6 The results of ELISA with different concentrations of 76F-GST-NC16A-R2P1-H2 antibody are shown. Detailed Implementation

[0061] definition

[0062] In the claims and / or specification of this disclosure, the words “a”, “an”, or “the” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0063] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean inclusive or open-ended and do not exclude additional, uncited elements or method steps. At the same time, “comprising,” “having,” “including,” or “containing” can also mean closed-ended, excluding additional, uncited elements or method steps.

[0064] Throughout the application, the term “about” means: a value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0065] While the disclosure supports the definition of the term "or" as merely a substitute and "and / or", the term "or" in the claims means "and / or" unless expressly stated as merely a substitute or as mutually exclusive among substitutes.

[0066] As used in this disclosure, the term "bullous pemphigoid" (BP) is generally considered to be an autoimmune disease in which most patients have anti-basement membrane band autoantibodies in their serum. Antigen-antibody binding leads to damage to the basement membrane band, forming blisters.

[0067] The terms “individual,” “patient,” or “subject” as used in the context of this invention include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0068] As used in this disclosure, the term "BP180 antigen" (abbreviated as BP180) is also referred to as BPAG1 antigen, with a molecular weight of 180 kDa. BP180 is considered a direct target of autoantibodies. BP180 is a type II transmembrane protein with a cytoplasmic NH2 terminus and an extracellular COOH domain. The N-terminal domain, transmembrane stretching domain, and extracellular C-terminus have 466, 23, and 1008 amino acids, respectively. The extracellular domain contains 15 collagen subdomains (COL1–COL15) interspersed with 16 non-collagenous sequences (NC1–NC16).

[0069] As used in this disclosure, the term "conservative mutation" refers to a mutation (e.g., amino acid substitution, insertion, and / or deletion) that maintains the normal function of a protein. For example, a conservative mutation is a conservative substitution.

[0070] As used in this disclosure, a “conservative substitution” generally refers to the exchange of one amino acid at one or more sites in a protein. This substitution can be conserved. Examples of substitutions considered conserved include, specifically, substitutions of Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include: Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. In addition, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin.

[0071] In this disclosure, "sequence identity" and "identity percentage" refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotide or polypeptide and scoring the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residues, comparing this to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides may differ at a position, for example, by containing different nucleotides (i.e., substitution or mutation) or deleted nucleotides (i.e., nucleotide insertion or deletion in one or two polynucleotides). Polypeptides may differ at a position, for example, by containing different amino acids (i.e., substitution or mutation) or deleted amino acids (i.e., amino acid insertion or deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the identity percentage can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0072] The term "phage display technology" in this disclosure refers to a biotechnology that inserts the DNA sequence of a foreign protein or polypeptide into an appropriate position in the structural gene of a phage coat protein, so that the foreign gene is expressed along with the expression of the coat protein, and the foreign protein is displayed on the surface of the phage as the phage is reassembled.

[0073] The term "antibody" as used in this disclosure refers to an immunoglobulin or fragments thereof or derivatives thereof, and includes any polypeptide containing an antigen-binding site, whether or not it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, FV, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments will include antigen-binding fragments.

[0074] The term "single-chain antibody" (scFv) in this disclosure refers to an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide (also known as a linker) of a limited number of amino acids.

[0075] The term "Fab" fragment in this disclosure includes both a heavy chain variable domain and a light chain variable domain, and also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them.

[0076] The term "IMGT numbering scheme" used in this disclosure refers to a new standardized numbering system introduced by Lefranc et al. for all protein sequences in the immunoglobulin superfamily, including variable domains from antibody light and heavy chains and T cell receptor chains from different species. The IMGT numbering method is based on germline V sequence alignment and sequential counting of residues.

[0077] In the technical solutions described in this disclosure, unless otherwise specified, the antibody numbering scheme used for antibodies in this disclosure is the IMGT numbering scheme.

[0078] In some technical solutions, this disclosure relates to the stringency of hybridization conditions used to define the degree of complementarity between two polynucleotides. Optionally, the aforementioned polynucleotide may be selected from DNA. As used in this disclosure, “stringency” refers to the temperature and ionic strength conditions during hybridization and the presence of certain organic solvents. The higher the stringency, the greater the complementarity between the target nucleotide sequence and the labeled polynucleotide sequence. “Stringent conditions” refers to the temperature and ionic conditions under which only nucleotide sequences with high-frequency complementary bases will hybridize. The term “hybridization under high stringency or very high stringency conditions” as used herein describes the conditions used for hybridization and washing. Instructions for performing hybridization reactions can be found in Current Protocols in Molecule & Larbiology, John Wiley and Sons, NY (1989), 6.3.1–6.3.6. The specific hybridization conditions mentioned in this disclosure are as follows: 1) Highly stringent hybridization conditions: in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing once or more with 0.2X SSC and 0.1% SDS at 65°C; 2) Very highly stringent hybridization conditions: in 0.5M sodium phosphate at 65°C with 7% SDS, followed by washing once or more with 0.2X SSC and 1% SDS at 65°C.

[0079] Technical solution

[0080] In the technical solutions disclosed herein, the meanings of the nucleotide and amino acid sequence listing numbers are as follows:

[0081] The sequence shown in SEQ ID NO: 1 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL FR1;

[0082] The sequence shown in SEQ ID NO: 2 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH FR1;

[0083] The sequence shown in SEQ ID NO: 3 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL CDR1;

[0084] The sequence shown in SEQ ID NO: 4 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH CDR1;

[0085] The sequence shown in SEQ ID NO: 5 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL FR2;

[0086] The sequence shown in SEQ ID NO: 6 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH FR2;

[0087] The sequence shown in SEQ ID NO: 7 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL CDR2;

[0088] The sequence shown in SEQ ID NO: 8 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH CDR2;

[0089] The sequence shown in SEQ ID NO: 9 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL FR3;

[0090] The sequence shown in SEQ ID NO: 10 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH FR3;

[0091] The sequence shown in SEQ ID NO: 11 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL CDR3;

[0092] The sequence shown in SEQ ID NO: 12 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH CDR3;

[0093] The sequence shown in SEQ ID NO: 13 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VL FR4;

[0094] The sequence shown in SEQ ID NO: 14 is the amino acid sequence of the 76F-GST-NC16A-R2P1-H2 antibody VH FR4;

[0095] The sequence shown in SEQ ID NO: 15 is the nucleotide sequence encoding the 76F-GST-NC16A-R2P1-H2 antibody VH;

[0096] The sequence shown in SEQ ID NO: 16 is the amino acid sequence of antibody VH of 76F-GST-NC16A-R2P1-H2;

[0097] The sequence shown in SEQ ID NO: 17 is the nucleotide sequence encoding the 76F-GST-NC16A-R2P1-H2 antibody VL;

[0098] The sequence shown in SEQ ID NO: 18 is the amino acid sequence of antibody VL of 76F-GST-NC16A-R2P1-H2;

[0099] The sequences shown in SEQ ID NO: 19-34 are primer sequences;

[0100] The sequence shown in SEQ ID NO: 35 is the amino acid sequence of the BP180 protein;

[0101] The sequence shown in SEQ ID NO: 36 is a nucleotide sequence encoding the BP180 protein.

[0102] Anti-BP180 antibody or its antigen-binding fragment

[0103] Bullous pemphigoid is an autoimmune bullous disease mediated by pathogenic autoantibodies. The severity of BP patients is related to the titer of anti-BP180 antibodies. Therefore, detecting the level of anti-BP180 antibodies is of great significance for the study of the pathogenesis of BP, disease diagnosis and monitoring of the disease.

[0104] In some embodiments, the present disclosure prepares the BP180 protein, the amino acid sequence of which is shown in SEQ ID NO: 35, and the nucleotide sequence encoding the BP180 protein is shown in SEQ ID NO: 36.

[0105] In some specific embodiments, the preparation method of BP180 protein includes the following steps:

[0106] (1) The BP180 gene sequence was artificially synthesized and the BP180 gene was recombined into the expression vector plasmid pGEX-6P-1 to obtain the BP180-pGEX expression vector; the cloning site is BamHI / XhoI.

[0107] (2) The BP180-pGEX expression vector was transfected into BL21(DE3) competent cells and cultured. The precipitate was collected and subjected to GST tag affinity chromatography to obtain BP180 protein.

[0108] In some embodiments, phage display technology is used to screen for antibody or antigen-binding fragments with high affinity for the BP180 protein. Exemplary examples of antibody or antigen-binding fragments with high affinity for the BP180 protein include polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, heterozygous, mutated, and grafted antibodies, or antibody fragments such as Fab, F(ab')2, FV, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function.

[0109] In some embodiments, this disclosure isolates PBMCs from BP180 antibody-positive patients, and constructs a phage display library containing heavy chain variable regions (VH) and light chain variable regions (VL) by amplifying the antibody VH and VL gene fragments therein. By performing biopanning with BP180 protein, human antibodies that can specifically bind to the BP180 antigen are screened.

[0110] In some specific embodiments, the method for screening anti-BP180 antibodies disclosed herein includes the following steps:

[0111] S1. PBMCs from BP180 antibody-positive patients were isolated, and RNA was extracted and quality-checked. The quality-checked RNA was reverse-transcribed into cDNA using RT-PCR, and all VH and VL antibody gene fragments were amplified. The amplified VH and VL gene fragments were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library.

[0112] In S2, an antibody gene conjugate library is inserted immediately downstream of the leader sequence of gene III (g3) encoding a membrane protein of a phage. Through helper phage superinfection, the polypeptide or protein expressed by the exogenous antibody gene can be displayed as a fusion protein at the N-terminus of the phage coat protein pIII. Each phage particle encodes and presents a different antibody, containing billions of individual clones. In these antibody libraries, genes encoding antibodies that bind to the antigen are selected through affinity enrichment-mild elution-phage amplification in vitro. This enrichment and screening process is repeated several times until a phage library with high specificity and affinity is obtained. Positive clones are then screened from this library. Positive clones are identified using ELISA, and finally, fully human antibodies with high specificity and affinity are selected.

[0113] In some more specific embodiments, step S1 includes: using The III 1st StrandcDNA Synthesis Kit (+gDNA wiper) reverse transcription kit reverse transcribed RNA from BP180 antibody-positive patients into cDNA, amplifying the VH and VL fragments of the DNA. The VK and Vλ gene fragments obtained in vitro were cloned into the pATA-scFv-2 vector using cloning technology to form VK and Vλ libraries, respectively. Plasmid vectors for the VK and Vλ libraries were extracted using a plasmid extraction kit, and the in vitro amplified VH gene fragment was inserted into the plasmid vectors of the VK and Vλ libraries, forming KH and λH libraries, respectively.

[0114] This disclosure involves three rounds of screening of antibody phage libraries, identifying clones with an antigen group greater than 3 times that of the control group as positive clones. These single clones were then sequenced and analyzed. Erroneous and duplicate antibody sequences were eliminated, and combined with the antigen-antibody specific binding ability reflected in the ELISA experiment, a high-affinity antibody was finally obtained, named 76F-GST-NC16A-R2P1-H2. This antibody exhibits high activity, good stability, and strong specificity, serving as a reference standard for qualitative detection of BP180 positivity and also for quantitative detection of anti-BP180 autoantibody levels in BP patients. This provides valuable information for the diagnosis, disease progression monitoring, clinical drug treatment, and research on the pathogenesis of bullous pemphigoid.

[0115] Example

[0116] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0117] All reagents used in the examples, unless otherwise emphasized, are commercially available.

[0118] Example 1: Method for constructing a human ScFv phage display library

[0119] The main reagents used in this embodiment are shown in Table 1.

[0120] Table 1. Main reagents used in Example 1

[0121]

[0122] 1. Library Construction

[0123] 1.1 Assemble the heavy chain variable region (VH) and the light chain variable region (VL)

[0124] Table 2 PCR reaction conditions and procedures

[0125]

[0126] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0127] The primer sequences used in this embodiment are as follows:

[0128] Forward(F):

[0129] 5′L-VH 1:ACAGGTGCCCACTCCCAGGTGCAG (SEQ ID NO: 19)

[0130] 5′L-VH 3: AAGGTGTCCAGTGTGARGTGCAG (SEQ ID NO: 20)

[0131] 5′L-VH 4 / 6: CCCAGATGGGTCCTGTCCCAGGTGCAG (SEQ ID NO: 21)

[0132] 5′L-VH 5 / 7: CAAGGAGTCTGTTCCGAGGTGCAG (SEQ ID NO: 22)

[0133] 5′L VK 1 / 2: ATGAGGSTCCCYGCTCAGCTGCTGG (SEQ ID NO: 23)

[0134] 5′L VK 3: CTCTTCCTCCTGCTACTCTGGCTCCCAG (SEQ ID NO: 24)

[0135] 5′L VK 4 / 5: ATTTCTCTGTTGCTCTGGATCTCTG (SEQ ID NO: 25)

[0136] 5'L Vλ1:GGTCCTGGGCCCAGTCTGTGCTG (SEQ ID NO: 26)

[0137] 5'L Vλ2: GGTCCTGGGCCCAGTCTGCCCTG (SEQ ID NO: 27)

[0138] 5'L Vλ3: GCTCTGTGACCTCCTATGAGCTG (SEQ ID NO: 28)

[0139] 5′L Vλ4 / 5: GGTCTCTCTCSCAGCYTGTGCTG (SEQ ID NO: 29)

[0140] 5'L Vλ6: GTTCTTGGGCCAATTTTATGCTG (SEQ ID NO: 30)

[0141] 5'L Vλ7: GGTCCAATTCYCAGGCTGTGGTG (SEQ ID NO: 31)

[0142] 5′L Vλ8 / 9 / 10: GAGTGGATTCTCAGACTGTGGTG (SEQ ID NO: 32)

[0143] Reverse(R):

[0144] 3′CK: TGCTGCCTTGCTGTCCTGCT (SEQ ID NO: 33)

[0145] 3′Cλ: CACCAGTGTGGCCTTGTTGGCTTG (SEQ ID NO: 34)

[0146] 1.2 Construction of a light chain variable region phage display library

[0147] 1.2.1 Preparing the pATA-scFv-2 vector for library cloning

[0148] 1.2.2 Digestion of vectors and PCR products

[0149] Table 3. Reaction system for digesting vector and PCR product

[0150]

[0151] The PCR products were obtained using the technical methods described in Table 3.

[0152] 1.2.3 Connection

[0153] Table 4 Connection Reaction System

[0154] pATA-VK pATA-Vλ T4 DNA ligase (Thermo) 3μL 3μL 10×T4 DNA ligase buffer 8μL 8μL Vector (NheI / NotI) 1μg 1μg VK or Vλfragment(NheI / NotI) 0.3μg 0.3μg <![CDATA[H2O]]> Add to a total of 80 μL of the reaction mixture. Add to a total of 80 μL of the reaction mixture.

[0155] The ligation was performed using the technical method described in Table 4. The mixture was incubated overnight at 16°C and then inactivated by heating at 65°C for 10 minutes to obtain the ligation product.

[0156] 1.2.4 Electrical Transfer

[0157] 1.2.4.1 Preparation of TG1 competent cells.

[0158] 1.2.4.2 Preheat 1 mL of SOC medium (Sigma, S1797) to 37 °C. Place the electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0159] 1.2.4.3 Remove Electrocompetent cells from the freezer at -80°C and place them on ice until they are completely thawed (10-15 minutes). After thawing, gently mix the cells. Transfer 50 μL of cells to a frozen microcentrifuge tube placed on ice.

[0160] 1.2.4.4 Carefully add 3 μL of the DNA mixture to a frozen electroporation cuvette, being careful not to create air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom.

[0161] 1.2.4.5 Electroporation was performed at 600 Ω, 10 μF, and 1.8 kV. Within 10 seconds of the pulse, 1 mL of preheated SOC medium was immediately added to each tube. The tubes were incubated at 37°C with shaking at 250 rpm for 1 hour.

[0162] 1.2.4.6 Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / Glucose plates. Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the inoculum volume.

[0163] 1.3 Construction of VL-VH phage display library

[0164] 1.3.1 Digestion of vectors and PCR products

[0165] Table 5 Digestion reaction system

[0166]

[0167] The digested PCR products were obtained using the digestion reaction system and steps shown in Table 5.

[0168] 1.3.2 Connection

[0169] Table 6 Connection Reaction System

[0170]

[0171] The ligation was performed using the technical method described in Table 6. The mixture was incubated overnight at 16°C and then inactivated by heating at 65°C for 10 minutes to obtain the ligation product.

[0172] 1.3.3 Electrical Transfer

[0173] 1.3.3.1 Preparation of TG1 competent cells.

[0174] 1.3.3.2 Preheat 4 mL of SOC medium (Sigma, S1797) to 37 °C. Place the electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0175] 1.3.3.3 Remove Electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed (10-15 minutes). After thawing, gently mix the cells.

[0176] 1.3.3.4 Carefully add 6 μL of the DNA mixture to a frozen electroporation cuvette, being careful not to create air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom.

[0177] 1.3.3.5 Electroporation at 600Ω, 100Ω, and 2.5kV. Immediately within 10 seconds of the pulse, add 2mL of preheated SOC medium to each tube. Incubate at 37°C with shaking at 250rpm for 1 hour.

[0178] 1.3.3.6 Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / Glucose plates. Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the inoculum volume.

[0179] 1.4 Library Evaluation

[0180] 1.4.1 Colony PCR: PCR was performed using the constructed library as a template.

[0181] Table 7 PCR reaction conditions

[0182]

[0183] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0184] The primer sequences in Table 7 are as follows:

[0185] The upstream primer (F) for pATA-scFv-2 vector identification is: AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 35)

[0186] The downstream primer (R) for identifying the pATA-scFv-2 vector is: GCCCCTTATTAGCGTTTGCCATC (SEQ ID NO: 36)

[0187] The results of agarose gel electrophoresis after PCR are as follows: Figures 1-3 As shown.

[0188] 1.4.2 Sequencing

[0189] Positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing quality control results were as follows: Figure 4 As shown.

[0190] 1.5 Expression of BP180 protein

[0191] The BP180 gene sequence was artificially synthesized and recombined into the expression vector plasmid pGEX-6P-1 to obtain the BP180-pGEX expression vector; the cloning site is BamHI / XhoI.

[0192] The amino acid sequence of BP180 is (SEQ ID NO: 35):

[0193] GSEEVRKLKARVDELERIRRSILPYGDSMDRIEKDRLQGMAPAAGADLDKIGLHSDSQEELWMFVRKKLMMEQENGNLR;

[0194] The gene sequence is (SEQ ID NO: 36):

[0195] ggatccGAGGAGGTGAGGAAGCTGAAGGCGCGTGTGGATGAGCTGGAGAGGATCAGGAGGAGCATACTGCCCTATGGGGACAGCATGGATAGAATAGAAAAGGACCGCCTCCAGGGCATGGCA CCCGCGGCGGGAGCAGACCTGGACAAAATTGGGCTGCACAGTGACAGCCAGGAGGAGCTCTGGATGTTCGTGAGGAAGAAGCTAATGATGGAACAGGAAAATGGAAATCTCCGatgactcgag.

[0196] The BP180-pGEX expression vector was transfected into BL21(DE3) competent cells and cultured. The precipitate was collected and subjected to GST tag affinity chromatography to obtain BP180 protein. The purified BP180 was further subjected to SDS-PAGE (polyacrylamide gel electrophoresis) to verify its purity. The SDS-PAGE electrophoresis image of the purified BP180 is shown below. Figure 5 As shown, the purity is greater than 90%.

[0197] Example 2: Preparation of monoclonal antibodies that specifically bind to BP180

[0198] The main reagents used in this Example 1 are shown in Table 8.

[0199] Table 8. Main reagents used in Example 2

[0200] reagents serial number Manufacturer 96-well plate 42592 Costar Tween 20 P2287 Sigma Tris RES3098T-B7 Sigma Glycine G8200 Solarbio PEG 181986 Sigma PBS C10010500BT Life BSA A104912-100g aladdin Skim milk 6342932 BD

[0201] 1. First round

[0202] 1.1 Biological Screening

[0203] 1.1.1 Coating: Coat the immunotubes and incubate overnight at 4°C. Antigen group: 1 mL GST-NC16A transfection solution (50 μg / mL), control group: 500 μL transfection solution (0 μg / mL). GST-NC16A was obtained by recombinant expression of the BP180-pGEX expression vector in Example 1.

[0204] 1.1.2 Washing: Discard the liquid in the immunoassay tube and wash three times with 5 mL of 0.05% PBST.

[0205] 1.1.3 Blocking: Add 5 mL of 5% skim milk (dissolved in PBST) to the tube and incubate at 37°C for 2 hours.

[0206] 1.1.4 Washing: Discard the liquid in the immunoassay tube and wash once with 5 mL of 0.05% PBST.

[0207] 1.1.5 Incubation: Dilute the phage library with 1% skim milk (dissolved in PBST), add 1 mL to an immunoassay tube, and incubate at 32°C for 2 hours.

[0208] 1.1.6 Washing: Discard the liquid in the immunoassay tube, wash three times with 5 mL of 0.05% PBST, and wash twice with PBS.

[0209] 1.1.7 Elution: Elute the phages bound to BP180 with 1 mL of glycine-hydrochloric acid (pH 2.2), and then neutralize to pH 7.0 with Tris-HCl.

[0210] 1.2 Determination of the titer of diluted bacteriophages

[0211] 1.2.1 Culture Escherichia coli TG1 until OD600 = 0.4-0.6.

[0212] 1.2.2 Mix 10 μL of diluted eluted phage with 190 μL of Escherichia coli TG1.

[0213] 1.2.3 Incubate the mixture at 37°C for 15 minutes, then pour it into 2×YT-A (Amp 100μg / mL) medium. Invert the medium and incubate overnight at 37°C.

[0214] 1.3 Phage Library Amplification

[0215] 1.3.1 Add 10 μL of E. coli TG1 to 800 μL of 2YT culture medium and mix and culture at 37℃ until OD600 = 0.4-0.6.

[0216] 1.3.2 Transfer the TG1 cultured to the logarithmic phase to 10 mL of 2YT-G culture medium (final concentration 2% glucose) and culture on a shaker at 37°C until OD600 = 0.4-0.6.

[0217] 1.3.3 Add the eluted product, incubate at 37°C for 30 minutes, and then culture on a shaker at 37°C for 30 minutes.

[0218] 1.3.4 Add 30 mL of 2YT-AG culture medium (final concentration 0.1% Amp, 2% glucose) and incubate at 37°C in a shaker for 1 hour.

[0219] 1.3.5 Add M13KO7 (M13KO7:TG1 = 20:1), incubate at 37°C for 30 minutes, and then culture on a shaker at 37°C for 30 minutes.

[0220] 1.3.6 Centrifuge the bacterial culture at 5000 rpm for 5 minutes. Resuspend in 40 mL of 2YT-AK (final concentrations: Amp 100 μg / mL, Kan 100 μg / mL) and incubate overnight at 30°C on a shaker.

[0221] 1.3.7 Centrifuge at 8000 rpm for 10 minutes, remove the supernatant, resuspend in 1 mL PBS, centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0222] 1.4 Determination of titer of amplified phage library

[0223] The steps are the same as in 1.2.

[0224] 2. Rounds 2 to 3

[0225] 2.1 Biological Screening

[0226] Repeat step 1 twice, using eluted phage from the previous round of amplification for each phage library input.

[0227] Table 9 Results of the biological screening

[0228]

[0229] 3. Polyclonal phage ELISA

[0230] 3.1 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL / well of GST-NC16A protein (4 μg / mL), control group: 100 μL / well of protein dilution buffer (0 μg / mL).

[0231] 3.2 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0232] 3.3 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0233] 3.4 Phage incubation: Add 100 μL of diluted phage to each well as shown in Table 10, and incubate at 32°C for 2 hours.

[0234] 3.5 Washing: Same as step 3.2.

[0235] 3.6 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (1:9000) diluted with blocking buffer to each well and incubate at 32°C for 1 hour.

[0236] 3.7 Washing: Same as step 3.2.

[0237] 3.8 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0238] 3.9 Plate reading: Values ​​were read using a microplate reader at 450nm-630nm. The plate reading results are shown in Table 10.

[0239] Table 10 Results of polyclonal phage ELISA

[0240]

[0241] 4. Monoclonal phage ELISA (based on polyclonal results, the second round of elution products are used for monoclonal ELISA).

[0242] 4.1 Select 96 clones from the culture dish and incubate them at 37°C and 250 rpm until OD600nm = 0.4-0.6.

[0243] 4.2 Infect M13KO7 culture (MOI = 20:1), incubate at 37°C for 30 minutes, then incubate on a shaker at 37°C for 30 minutes. Centrifuge the bacterial culture and resuspend the pellet with an equal volume of 2×YT-AK (final concentrations Amp 100 μg / mL, Kan 100 μg / mL), and incubate overnight at 30°C.

[0244] 4.3 Centrifuge the culture and use the supernatant for ELISA.

[0245] 4.4 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL / well of GST-NC16A protein (4 μg / mL), control group: 100 μL / well of protein dilution buffer (0 μg / mL).

[0246] 4.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0247] 4.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0248] 4.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 hours.

[0249] 4.8 Washing: Same as step 4.5.

[0250] 4.9 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (1:9000) diluted with blocking buffer to each well and incubate at 32°C for 1 hour.

[0251] 4.10 Washing: Same as step 4.5.

[0252] 4.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0253] 4.12 Plate reading: Values ​​were read using a microplate reader at 450nm-630nm, and highly specific clones were sequenced. The plate reading results are shown in Tables 11 and 12.

[0254] Table 11 Results of antigen group monoclonal phage ELISA

[0255] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.16 0.04 0.03 0.05 0.02 0.03 0.05 0.28 0.33 0.41 0.33 0.21 B 0.03 0.02 0.02 0.05 0.03 0.03 0.04 0.05 0.04 0.04 0.06 0.06 C 0.03 0.02 0.02 0.04 0.03 0.20 0.19 0.12 0.17 0.03 3.94 0.05 D 0.03 0.09 0.03 0.03 0.17 0.60 0.03 0.04 0.03 0.03 0.03 0.46 E 0.03 0.03 0.03 0.04 0.03 0.03 0.18 0.04 0.58 0.03 0.03 0.22 F 0.06 0.11 0.05 0.14 0.03 0.03 0.03 0.19 0.03 0.05 0.04 0.03 G 0.03 0.04 0.19 0.11 0.12 0.03 0.03 0.10 0.05 0.16 0.17 0.04 H 0.05 3.38 0.04 0.16 0.04 0.04 0.18 0.04 0.03 0.17 0.04 1.62

[0256] Table 12 Results of monoclonal phage ELISA in the control group

[0257] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.06 0.02 0.02 0.02 0.02 0.02 0.03 0.05 0.05 0.10 0.07 0.25 B 0.03 0.03 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.02 0.02 0.04 C 0.02 0.02 0.02 0.02 0.02 0.04 0.04 0.03 0.05 0.02 3.16 0.02 D 0.02 0.03 0.02 0.02 0.07 0.03 0.02 0.02 0.02 0.02 0.20 0.05 E 0.03 0.02 0.02 0.02 0.02 0.02 0.06 0.02 0.05 0.03 0.09 0.06 F 0.03 0.04 0.03 0.04 0.02 0.02 0.02 0.07 0.05 0.03 0.02 0.02 G 0.03 0.04 0.05 0.02 0.03 0.16 0.02 0.03 0.03 0.08 0.06 0.04 H 0.06 0.03 0.02 0.04 0.02 0.03 0.05 0.03 0.03 0.04 0.05 0.28

[0258] 5. ELISA for positive clone verification

[0259] 5.1 Add 50 μL of positive clones to 2 mL of 2YT-AG medium (final concentration 0.1% Amp, 2% glucose) and culture until OD600 = 0.4-0.6.

[0260] 5.2 Infect M13KO7 culture (MOI = 20:1), incubate at 37°C for 30 minutes, then incubate on a shaker at 37°C for 30 minutes. Centrifuge the bacterial culture and resuspend the pellet with an equal volume of 2×YT-AK (final concentrations: Amp 100 μg / mL, Kan 100 μg / mL), and incubate overnight at 30°C.

[0261] 5.3 Centrifuge the culture and use the supernatant for ELISA.

[0262] 5.4 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL / well of GST-NC16A protein (4 μg / mL), control group: 100 μL / well of protein dilution buffer (0 μg / mL).

[0263] 5.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0264] 5.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0265] 5.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 hours.

[0266] 5.8 Washing: Same as step 4.5.

[0267] 5.9 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (1:9000) diluted with blocking buffer to each well and incubate at 32°C for 1 hour.

[0268] 5.10 Washing: Same as step 4.5.

[0269] 5.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0270] 5.12 Plate reading: Values ​​were read using a microplate reader at 450nm-630nm, and highly specific clones were sequenced. The plate reading results are shown in Table 13.

[0271] Table 13 Results of ELISA for positive monoclonal phages

[0272]

[0273] 6. Sequencing of antibody sequences

[0274] The phage-positive clones obtained through screening were subjected to full-sequence sequencing to obtain the corresponding antibody heavy and light chains, as well as the full sequences, as shown in Table 14.

[0275] Table 14 76F-GST-NC16A-R2P1-H2 Monoclonal Antibody Sequence

[0276]

[0277] The heavy chain base sequence of the 76F-GST-NC16A-R2P1-H2 antibody is as follows (SEQ ID NO: 15):

[0278]

[0279] The amino acid sequence of the heavy chain of the 76F-GST-NC16A-R2P1-H2 antibody is as follows (SEQ ID NO: 16):

[0280] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGLGLEWMGWISGYNGNTHYAQKLQDRVTMTTDTSTSTAYMEMRSLGSDDTAVYYCARDYLPGYCSSTSCPHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0281] The nucleotide sequence of the light chain of the 76F-GST-NC16A-R2P1-H2 antibody is as follows (SEQ ID NO: 17):

[0282] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGCGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTACGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGA;

[0283] The amino acid sequence of the light chain of the 76F-GST-NC16A-R2P1-H2 antibody is as follows (SEQ ID NO: 18):

[0284] QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKVTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0285] Example 3: ELISA detection of OD values ​​of antibodies at different dilution concentrations

[0286] Enzyme-linked immunosorbent assay (ELISA) experimental procedures:

[0287] 1. Coating: Coat the microplate with 100 μL of GST-NC16A protein (4 μg / mL) per well and incubate overnight at 4°C.

[0288] 2. Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0289] 3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0290] 4. Positive antibody incubation: The 76F-GST-NC16A-R2P1-H2 antibody was serially diluted, and 100 μL of the diluted antibody solution was added to each well. The mixture was incubated at 37°C for 1 hour.

[0291] 5. Washing: Same as step 4.5.

[0292] 6. Secondary antibody incubation: Dilute Goat Anti-Human IgG (H+L) antibody (Jackson, code: 109-035-088) 10000 times with blocking buffer, add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 30 minutes.

[0293] 7. Washing: Same as step 4.5.

[0294] 8. Color development: Add 100 μL TMB to each well, incubate at 37°C for 10 minutes, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0295] 9. Plate reading: Use an ELISA reader to read values ​​at 450nm-630nm, such as... Figure 6 As shown.

[0296] from Figure 6 The results demonstrate that 76F-GST-NC16A-R2P1-H2 has a strong ability to specifically bind to BP180.

[0297] This disclosure is not intended to limit itself to the specific embodiments disclosed herein, but is provided to illustrate aspects of this disclosure, for example. Various modifications to the compositions and methods will become apparent from the description and teaching herein. Such changes may be practiced without departing from the true scope and spirit of this disclosure, and are intended to fall within the scope of this disclosure. SEQUENCE LISTING <110> Suzhou Fangke Biotechnology Co., Ltd. Suzhou Institute of Systems Medicine Chinese Academy of Medical Sciences Dermatology Hospital (Institute of Dermatology, Chinese Academy of Medical Sciences) <120> Human antibodies containing BP-specific antigenic peptides, preparation methods and applications <130> 6A59-2103407I <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL FR1 <400> 1 Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser 20 25 <210> 2 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH FR1 <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL CDR1 <400> 3 Ser Ser Asn Ile Gly Ser Asn Tyr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH CDR1 <400> 4 Gly Tyr Thr Phe Thr Asn Tyr Gly 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL FR2 <400> 5 Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH FR2 <400> 6 Ile Ser Trp Val Arg Gln Ala Pro Gly Leu Gly Leu Glu Trp Met Gly 1 5 10 15 Trp <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL CDR2 <400> 7 Arg Asn Asn 1 3 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH CDR2 <400> 8 Ile Ser Gly Tyr Asn Gly Asn Thr 1 5 <210> 9 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL FR3 <400> 9 Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1 5 10 15 Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 10 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH FR3 <400> 10 His Tyr Ala Gln Lys Leu Gln Asp Arg Val Thr Met Thr Thr Asp Thr 1 5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Met Arg Ser Leu Gly Ser Asp Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL CDR3 <400> 11 Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val 1 5 10 <210> 12 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH CDR3 <400> 12 Ala Arg Asp Tyr Leu Pro Gly Tyr Cys Ser Ser Thr Ser Cys Pro His 1 5 10 15 Phe Asp Tyr <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL FR4 <400> 13 Phe Gly Gly Gly Thr Lys Val Thr Val Leu 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH FR4 <400> 14 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 15 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> sequence of VH <400> 15 caggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cctctggtta cacctttacc aactatggta tcagctgggt gcgacaggcc 120 cctggactag ggcttgagtg gatgggatgg atcagcggtt acaatggtaa cacacactat 180 gcacagaagc tccaggacag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagatga ggagcctggg atctgacgac acggccgtgt attactgtgc gagagattac 300 ctgcccggat attgtagtag taccagctgc cctcactttg actactgggg ccagggcacc 360 ctggtcaccg tctcgagtgc tagcaccaag ggaccttctg tgttccctct ggctccttct 420 tctaagtcca cttccggtgg tacagcagct ctgggttgtc tggtgaagga ttacttccca 480 gaaccagtga ctgtgtcctg gaactccgga gctctgactt ctggagtgca tactttccca 540 gcagtgctgc aatctagcgg actgtactct ctgtcttccg tggtgactgt gccttcttct 600 tccctgggga ctcaaactta catctgcaac gtgaaccaca agccctccaa caccaaggtg 660 gacaagaagg tggagccaaa gagctgcgat aagacccaca cctgtccacc ttgtccagct 720 ccagaactgc tgggtgggcc ttctgtgttt ctgttcccac ctaagccaaa ggataccctg 780 atgatctcta ggaccccaga agtgacctgt gtggtcgtcg atgtgtctca tgaagaccct 840 gaagtgaagt tcaactggta cgtggacggg gtggaagtgc ataacgcaaa gaccaagccc 900 agggaagagc aatacaactc cacctacagg gtggtctccg tcctgacagt cctgcatcag 960 gattggctga acggcaagga gtacaagtgc aaggtctcca ataaagccct gcctgcccct 1020 atcgagaaaa ccattagcaa agccaaaggc cagcccaggg agccccaggt ctatacactg 1080 ccccccagca gggaggagat gawaaaaat caggtcagcc tgacatgcct ggtcaaggc 1140 ttttatccca gcgacattgc cgtcgagtgg gagtccaatg gccagcccga gaataattat 1200 aaaaacacac cccccgtcct ggacagcgac ggcagcttt ttctgtatag caaactgaca 1260 gtcgataaaa gcaggtggca gcaggggcaat gtctttcct gcagcgtcat gcacgaggcc 1320 ctgcacaatc actatactca gaaagcctg agcctgtccc ccgggaatg a 1371 <210> 16 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> sequence of VH <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Ile Trp Ser Val Arg Gln Ala Pro Gly Leu Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Contains Gly Tyr and Gly Asn Thr His Tyr Ala Gln Lys Leu 50 55 60 Gln Asp Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Met Arg Ser Leu Gly Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Leu Pro Gly Tyr Cys Ser Ser Thr Ser Cys Pro His 100 105 110 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp Has Only Glu Val More Than Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys 450,455 <210> 17 <211> 654 <212> DNA <213> Artificial Sequence <220> <223> sequence of VL <400> 17 cagcctgtgc tgactcagcc accctcagcg tctgggaccc ggggcagcg ggtcaccatc 60 tcttgttctg gaagcagctc siacatcgga agtataatg tatactggta ccaccagctc 120 ccaggaacgg ccccaacct cctcatctat aggaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctgcacc tcagcctccc tggccatcag tggctccgg 240 tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag tggttgggtg 300 ttcggcggag ggaccaaggt caccgtccta cgtacggtgg ctgcaccttc tgtgttcatc 360 ttccctccat ctgatgagca gctgaagtct ggaaccgcat ctgtcgtctg tctgctgaac 420 aacttttacc ccagggaggc taaggtccaa tggaaggtgg acaacgccct gcagtctggt 480 aatagccagg aaagcgtgac cgaacaggat tccaaggact ccacctactc cctgtcctcc 540 acactgacac tgagcaaagc cgactatgaa aagcacaaag tgtatgcctg cgaggtcact 600 catcaggggcc tgtccagccc cgtgactaaa agctttaata ggggggagtg ctga 654 <210> 18 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> sequence of VL <400> 18 Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 19 acaggtgccc actcccaggt gcag 24 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 20 aaggtgtcca gtgtgargtg cag 23 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 21 cccagatggg tcctgtccca ggtgcag 27 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 22 caaggagtct gttccgaggt gcag 24 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 23 atgaggstcc cygctcagct gctgg 25 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 24 ctcttcctcc tgctactctg gctcccag 28 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 25 atttctctgt tgctctggat ctctg 25 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 26 ggtcctgggc ccagtctgtg ctg 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 27 ggtcctgggc ccagtctgcc ctg 23 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 28 gctctgtgac ctcctatgag ctg 23 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 29 ggtctctctc scagcytgtg ctg 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 30 gttcttgggc caattttatg ctg 23 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 31 ggtccaattc ycaggctgtg gtg 23 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 32 gagtggattc tcagactgtg gtg 23 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 33 tgctgtcctt gctgtcctgc t 21 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer sequence <400> 34 caccagtgtg gccttgttgg cttg 24 <210> 35 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> sequence of BP180 <400> 35 Gly Ser Glu Glu Val Arg Lys Leu Lys Ala Arg Val Asp Glu Leu Glu 1 5 10 15 Arg Ile Arg Arg Ser Ile Leu Pro Tyr Gly Asp Ser Met Asp Arg Ile 20 25 30 Glu Lys Asp Arg Leu Gln Gly Met Ala Pro Ala Ala Ala Gly Ala Asp Leu 35 40 45 Asp Lys Ile Gly Leu His Ser Asp Ser Gln Glu Glu Leu Trp Met Phe 50 55 60 Val Arg Lys Lys Leu Met Met Glu Gln Glu Asn Gly Asn Leu Arg 65 70 75 <210> 36 <211> 246 <212> DNA <213> Artificial Sequence <220> <223> sequence of BP180 <400> 36 ggatccgagg aggtgaggaa gctgaaggcg cgtgtggatg agctggagag gatcaggagg 60 agcatactgc cctatgggga cagcatggat agaatagaaa aggaccgcct ccagggcatg 120 gcacccgcgg cgggagcaga cctggacaaa attgggctgc acagtgacag ccaggaggag 180 ctctggatgt tcgtgaggaa gaagctaatg atggaacagg aaaatggaaa tctccgatga 240 ctcgag 246

Claims

1. An isolated anti-BP180 antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the light chain variable region (VL) comprises VL complementarity-determining region (CDR) 1, VL complementarity-determining region (CDR) 2, and VL complementarity-determining region (CDR) 3, and the heavy chain variable region (VH) comprises VH complementarity-determining region (CDR) 1, VH complementarity-determining region (CDR) 2, and VH complementarity-determining region (CDR) 3; and, The VL includes: VLCDR1 as shown in SEQ ID NO: 3, VLCDR2 as shown in SEQ ID NO: 7, and VLCDR3 as shown in SEQ ID NO: 11; The VH includes: VHCDR1 as shown in SEQ ID NO: 4, VHCDR2 as shown in SEQ ID NO: 8, and VHCDR3 as shown in SEQ ID NO: 12; The heavy chain variable region and the light chain variable region are coded according to the IMGT analysis method.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises the VH sequence as shown in SEQ ID NO: 16 and the VL sequence as shown in SEQ ID NO:

18.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in claim 1 or 2, said polynucleotide comprising (a) or (b): (a) A nucleotide sequence as shown in any one of SEQ ID NO: 15, SEQ ID NO: 17, or a combination thereof; (b) The nucleotide sequence of the reverse complementary sequence of the nucleotide sequence shown in any of the sequences of SEQ ID NO: 15, SEQ ID NO: 17 or a combination thereof.

4. A carrier, wherein, The vector comprises the polynucleotide according to claim 3.

5. An isolated host cell, wherein, The host cell comprises the vector as described in claim 4.

6. A method for preparing host cells that stably express a target protein, wherein, The method includes the step of transforming an initial host cell using the vector of claim 4.

7. A method for preparing a target protein, the method comprising preparing the target protein using the host cell of claim 5 or by the method of claim 6.

8. A reagent kit, wherein, The kit contains the antibody or its antigen-binding fragment as described in claim 1 or 2.

9. A composition wherein, The composition contains the antibody or its antigen-binding fragment as described in claim 1 or 2.

10. Use of the antibody or antigen-binding fragment thereof according to claim 1 or 2, or the composition according to claim 9, in at least one of (1)-(3) below: (1) Detection of anti-BP180 antibodies for non-disease diagnosis, or preparation of reagents or kits for detecting anti-BP180 antibodies; (2) Prepare reagents or kits for diagnosing bullous pemphigoid; (3) Prepare reagents or kits for monitoring the progression of bullous pemphigoid.

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