Antibody combined with pemphigus vulgaris specific antigen peptide and application thereof

Abstract

The invention belongs to the field of biological medicine, and provides an antibody combined with pemphigus vulgaris specific antigen peptide and application of the antibody. According to the principle of a phage display technology, a constructed library is subjected to in-vitro affinity screening, phages are combined with a target antigen Dsg3, non-specific binders are eluted, the phages tightly combined with the antigen are reserved, and the pemphigus vulgaris specific antigen peptide is obtained. Then, amplifying and carrying out affinity enrichment for multiple rounds; and carrying out multiple rounds of panning, and after 3-5 rounds of screening, obtaining phage clones with strong binding capacity with Dsg3 and high specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an antibody binding to a specific antigenic peptide of pemphigus vulgaris and application thereof. BACKGROUND

[0002] Autoimmune bullous skin diseases (AIBD) are a group of heterogeneous diseases characterized by organ specificity, which are triggered by autoantibodies against structural proteins of skin and mucosa. This antigen-antibody reaction leads to the destruction of intercellular connections within the epidermis and between the epidermis and the dermis, and then causes the loss of adhesion function of skin and mucosa, resulting in the formation of blisters and erosions. According to the location of the blisters, autoimmune bullous diseases can be divided into two major categories: pemphigus, characterized by intraepidermal blisters; and pemphigoid, characterized by subepidermal blisters. Pemphigus is a chronic autoimmune bullous disease that is life-threatening, and its characteristic is the formation of clefts within the epidermis and adjacent epithelium, accompanied by acantholysis. Pemphigus vulgaris (PV) is the most common type of pemphigus. The pathogenesis of pemphigus mainly involves the attack of autoantibodies on the desmogleins (Dsg) in the skin and mucosa. The main target antigen of pemphigus is desmoglein, especially Dsg3.

[0003] Dsg3 has a molecular weight of 64.95 KD and is a member of the superfamily of cadherins, which plays an important role in maintaining the integrity of the epidermis. Its destruction can cause the basal lamina to loosen, leading to intraepidermal blisters. Dsg3 is the main target antigen of pemphigus and exists both in the intracellular and extracellular hemidesmosomes. The intracellular component of Dsg3 interacts with the cytoskeleton through plakoglobin and forms a connection, while the extracellular region mediates intercellular connections. It is the extracellular region of Dsg3 that provides the epitopes recognized by PV antibodies. These regions that play a major role are called extracellular domains (EC), and the EC of Dsg3 is divided into five domains (EC1-EC5). EC-1 is located at the amino terminal end, and EC-5 is located at the carboxy terminal end. The extracellular domain of the hemidesmosome core glycoprotein contains four cadherin repeat sequences (EC1 to EC4) and an extracellular anchoring domain near the membrane. Among them, the EC1 and EC2 domains at the amino terminal end contain residues that are essential for adhesion interactions. The binding of autoantibodies to DSG3 can directly interfere with cis or trans adhesion interactions and weaken the hemidesmosome adhesion. DSG3 clusters after binding with pemphigus IgG and is internalized through a p38MAPK-dependent pathway. The natural stereochemical conformation of the Dsg3 molecule is important for triggering autoimmune blisters.

[0004] Previous studies have obtained human Dsg3 recombinant protein, including the entire EC of Dsg3, through a baculovirus expression system and a mammalian expression system, and have been confirmed by natural antibodies from human PV patient sera. Therefore, human Dsg3 recombinant protein can not only provide a reliable means for diagnosing PV, but also be used for further research on the pathogenesis of pemphigus. In pemphigus, the calcium signaling pathway is essential for the formation of human epidermal blisters induced by autoantibodies. PV IgG can induce PLC activation and Ca 2+ influx, which requires the participation of Dsg1 (Desmoglein 1), and Dsg3 is not directly involved in the initiation of the Ca 2+ signal pathway. However, the localization and distribution of Dsg3 can be affected by the Ca 2+ signal pathway. When the Ca 2+ signal pathway is activated, the localization of Dsg3 at the cell edge becomes fragmented and partially transferred to the cytoplasm.

[0005] Phage display is a molecular biology method that fuses a gene fragment of foreign protein or peptide to the coat protein of phage, so that the expressed polypeptide or protein is displayed on the surface of phage. This technology was first proposed by George P. Smith in 1985 and has been widely used in antibody screening, vaccine development, drug target discovery, and protein interaction research. The core principle is to insert the gene library to be screened into a phage vector, express and assemble into a complete phage through infection of host cells, and display polypeptide or protein epitopes. Then, through affinity screening (biopanning), specific target molecules are combined, and unbound phages are washed to enrich specific binding. After several rounds of screening and sequencing analysis, polypeptides or antibodies with high affinity to target molecules can be obtained. Compared with traditional methods, phage display technology has the advantages of high throughput, low cost, and short cycle, and is particularly suitable for cases where antibodies are difficult to obtain through in vivo immunization.

[0006] Pemphigus (PV) is an autoimmune bullous disease mainly involving the skin and mucous membranes, which is relatively common in clinical practice. Patients have autoantibodies against the intercellular structural proteins of keratinocytes, leading to impaired cell-cell junction and characteristic lesions such as intraepidermal blisters. Early diagnosis is crucial for developing treatment strategies and predicting prognosis.

[0007] The diagnosis of PV includes the following aspects: 1) clinical manifestations: common features include ① formation of loose and easily broken blisters on the skin; ② persistent erosion after rupture; ③ blisters or erosions on mucosal sites; ④ positive Nikolsky's sign. 2) Histopathology: under the microscope, there is acantholysis between epidermal or epithelial cells, accompanied by blister formation. 3) Immunological detection: direct immunofluorescence (DIF) can observe IgG and / or complement deposition between cells in the lesion or its surrounding normal tissue; indirect immunofluorescence (IIF) detects the presence of anti-epithelial cell antibodies in the serum; enzyme-linked immunosorbent assay (ELISA) detects anti-Dsg3 antibodies. The diagnostic criteria are: 1 item in the clinical manifestations, combined with any of the histopathological or immunological indicators; or 2 items in the clinical manifestations, combined with 2 positive immunological tests.

[0008] At present, glucocorticoids are still the first choice for treatment. For moderate to severe cases, early combined use of immunosuppressive agents is recommended, especially when combined with diabetes, hypertension, osteoporosis and other diseases, more attention should be paid to the combined reduction strategy of hormones. Although the traditional hybridoma antibody preparation technology can obtain high affinity antibodies, the steps are complicated, time-consuming, and subsequent humanization process is required, which limits the acquisition of human monoclonal antibodies and the accuracy of antibody quantitative analysis, and also affects the targeting and efficacy in clinical treatment. Phage antibody library technology does not need to go through the steps of immunization and humanization modification, relies on enrichment screening in a completely controllable biochemical environment, and realizes the screening of high-affinity human antibody sequences in a short period.

[0009] Pemphigus is a life-threatening autoimmune bullous skin disease. About 60% of PV patients will develop oral mucosa blisters and bullae, which are prone to spontaneous rupture and form erosions that are difficult to heal, severely affecting patients' eating and daily life, and causing them great physical and psychological burden. In addition, some patients only show persistent oral mucosa erosion without skin damage. Therefore, rapid and accurate diagnostic methods are crucial for the treatment of this disease.

[0010] The pathogenicity of PV is mainly caused by IgG autoantibodies, and the target antigen is Dsg3. After PV-IgG binds to Dsg3, the intercellular connection structure is destroyed, leading to the loss of cell adhesion function, and eventually forming blisters. Therefore, accurately and rapidly detecting the level of anti-Dsg3 antibody in the serum of PV patients is of great significance for in-depth study of the pathogenesis of the disease, as well as for precise diagnosis and treatment. SUMMARY

[0011] In view of the deficiencies in the prior art, the purpose of the present application is to provide an antibody binding to a specific antigenic peptide of pemphigus vulgaris and its application. The present application isolates PBMCs from 10 Dsg3 antibody-positive patients to construct a phage human antibody library, and performs multiple rounds of screening to screen five human anti-Dsg3 monoclonal antibodies. The antibody can quantitatively detect the level of anti-Dsg3 autoantibody in PV patients by specifically binding to Dsg3, thereby monitoring the activity of the disease and being used for clinical diagnosis of PV patients. The research of the present application provides a theoretical and experimental basis for specific treatment of PV, and also provides a new research direction for the treatment of autoimmune diseases mainly caused by autoantibodies.

[0012] To achieve the purpose of the present application, the following technical solutions are adopted:

[0013] In a first aspect, the present application provides an antibody binding to a specific antigen peptide of Pemphigus vulgaris, wherein the CDR1 of the heavy chain variable region of the antibody is shown as SEQ ID NO: 1; the CDR2 is shown as SEQ ID NO: 2, and the CDR3 is shown as SEQ ID NO: 3; the CDR1 of the light chain variable region of the antibody is shown as SEQ ID NO: 6, the amino acid sequence of the CDR2 is AAS, and the CDR3 is shown as SEQ ID NO: 8;

[0014] or, the CDR1 of the heavy chain variable region of the antibody is shown as SEQ ID NO: 11; the CDR2 is shown as SEQ ID NO: 12, and the CDR3 is shown as SEQ ID NO: 13; the CDR1 of the light chain variable region of the antibody is shown as SEQ ID NO: 16, the amino acid sequence of the CDR2 is YDS, and the CDR3 is shown as SEQ ID NO: 18;

[0015] or, the CDR1 of the heavy chain variable region of the antibody is shown as SEQ ID NO: 21; the CDR2 is shown as SEQ ID NO: 22, and the CDR3 is shown as SEQ ID NO: 23; the CDR1 of the light chain variable region of the antibody is shown as SEQ ID NO: 26, the amino acid sequence of the CDR2 is AAS, and the CDR3 is shown as SEQ ID NO: 28;

[0016] or, the CDR1 of the heavy chain variable region of the antibody is shown as SEQ ID NO: 31; the CDR2 is shown as SEQ ID NO: 32, and the CDR3 is shown as SEQ ID NO: 33; the CDR1 of the light chain variable region of the antibody is shown as SEQ ID NO: 36, the amino acid sequence of the CDR2 is SNN, and the CDR3 is shown as SEQ ID NO: 38;

[0017] or, the CDR1 of the heavy chain variable region of the antibody is shown as SEQ ID NO: 41; the CDR2 is shown as SEQ ID NO: 42, and the CDR3 is shown as SEQ ID NO: 43; the CDR1 of the light chain variable region of the antibody is shown as SEQ ID NO: 46, the amino acid sequence of the CDR2 is DNN, and the CDR3 is shown as SEQ ID NO: 48.

[0018] The present application screens a monoclonal antibody binding to a specific antigen peptide of Pemphigus vulgaris. Monoclonal antibodies are one of the most numerous and fastest developing categories of therapeutic proteins. More than 500 therapeutic antibodies and their derivatives are in clinical trials, mainly focusing on the treatment of tumors and autoimmune diseases. Developing antibodies for detecting the level of anti-Dsg3 antibodies in serum is of great significance for the study of the pathogenesis of Pemphigus vulgaris and the realization of precise diagnosis and treatment.

[0019] In order to screen high-affinity human antibodies against pathogenic antigen Dsg3 in pemphigus vulgaris (PV), PBMCs are isolated from peripheral blood of patients positive for Dsg3 antibodies, total RNA is extracted, and quality control is performed. The qualified RNA sample is reversely transcribed into cDNA by reverse transcription PCR, and the VH and VL (including VK and Vλ) gene fragments of the antibody variable region are amplified.

[0020] Subsequently, the light chain variable region fragments obtained by amplification are cloned into the phage display vector pATA-scFv-2 carrying the antibody framework, and are assembled into single-chain antibodies (scFv) together with the heavy chain variable region, thereby constructing an antibody display library with extremely high diversity. The antibody genes in the library are further inserted upstream or N-terminal of the coding region of the M13 phage coat protein gene III (gIII) or gene VIII (gVIII) to ensure that the antibody fragments can be displayed on the phage surface in the form of a fusion protein.

[0021] By infecting E. coli with helper phages, each bacterium produces phage particles carrying a specific antibody, and finally forms a library containing tens of billions of different antibody clones. Based on the principle of phage display technology, we perform in vitro affinity screening on the constructed library: the phages are combined with the target antigen Dsg3, the non-specific binders are eluted, the phages tightly combined with the antigen are retained, and then they are amplified and subjected to multiple rounds of affinity enrichment. After 3-5 rounds of screening, phage clones with strong binding ability and high specificity to Dsg3 can be obtained.

[0022] Finally, the positive phage clones are screened and verified by the ELISA method, and full human antibodies with high affinity and high specificity are identified from them. This process not only avoids the limitations of animal immunization and humanization steps in traditional hybridoma methods, but also significantly improves the screening efficiency and the clinical conversion potential of antibodies.

[0023] In the present application, DSG3 is treated with a buffer containing 1 mM Ca 2+ Only DSG3 treated with a buffer containing 1 mM Ca 2+ The antibodies screened under Ca 2+ The antibodies screened under non-Ca 2+ After screening, DSG3 is treated with a buffer containing 1 mM Ca 2+ and a buffer not containing Ca 2+The antibody is good in binding ability and non-pathogenic, and lays a foundation for applying the antibody to treatment of other diseases.

[0024] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0025] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0026] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0027] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0028] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0029] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0030] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0031] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0032] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0033] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0034] In a second aspect, the present application provides a nucleic acid molecule encoding the antibody binding to the specific antigen peptide of pemphigus vulgaris according to the first aspect.

[0035] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0036] In a fourth aspect, the present application provides a host cell comprising at least one copy of the expression vector of the third aspect; or the nucleic acid molecule of the second aspect is integrated into the chromosome of the host cell.

[0037] In a fifth aspect, the present application provides a composition for detecting Dsg3 protein in a sample, which comprises the antibody of the first aspect binding to the pemphigus vulgaris specific antigenic peptide.

[0038] In a sixth aspect, the present application provides a kit for detecting Dsg3 protein in a sample, which comprises the antibody of the first aspect binding to the pemphigus vulgaris specific antigenic peptide.

[0039] In a seventh aspect, the present application provides the use of the antibody of the first aspect binding to the pemphigus vulgaris specific antigenic peptide, the composition of the fifth aspect for detecting Dsg3 protein in a sample, or the kit of the sixth aspect for detecting Dsg3 protein in a sample in the preparation of a product for detecting pemphigus vulgaris.

[0040] In an eighth aspect, the present application provides a method for preparing the antibody of the first aspect binding to the pemphigus vulgaris specific antigenic peptide, which comprises:

[0041] (1) isolating PBMC of a Dsg3 antibody positive patient, extracting total RNA, reverse transcribing into cDNA by reverse transcription PCR, and amplifying VH and VL gene fragments of the antibody variable region to construct a phage display library;

[0042] (2) binding the phage to the target antigen Dsg3, eluting non-specific binders, retaining phage tightly bound to the antigen, and then amplifying and performing affinity enrichment; after several rounds of screening, phage clones with strong binding ability and high specificity to Dsg3 can be obtained.

[0043] Preferably, the buffer used in the screening contains 1 mM CaCl2.

[0044] In the present application, multiple rounds of panning are performed under the condition of 1 mM CaCl2, and after 3-5 rounds of screening, phage clones with strong binding ability and high specificity to Dsg3 can be obtained. Compared with the anti-DSG3 antibody screened under the condition without CaCl2, the anti-DSG3 antibody screened under the condition with CaCl2 has stronger binding ability to the surface protein of HacaT cells and better effect. 2+ The screened antibody has stronger binding force to the surface protein of HacaT cells and better effect.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] The application reverses the RNA of the DSG3 antibody positive patient into cDNA, and amplifies the VH and VL fragments of the DNA. The in vitro amplified VK, Vλ gene fragments are cloned into pATA-scFv-2 vector by cloning technology to form VK library and Vλ library. The plasmid vectors of the VK library and the Vλ library are extracted by using a plasmid extraction kit, the in vitro amplified VH gene fragments are inserted into the plasmid vectors of the VK library and the Vλ library to form KH library and λH library. The artificially synthesized gene of the Dsg3 domain is recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector is transfected into DH10Bac competent cells for culture, and the Dsg3 antigen protein is purified.

[0047] The application screens three rounds of antibody phage library, and the clones with more than 3 times of antigen group than the control group are defined as positive clones. The error antibody sequences and repeated antibody sequences are excluded, and the specific binding ability of the antigen antibody reflected by the ELISA experiment is combined to finally obtain 5 antibodies with high affinity, which are named as DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151) and DSG3-R3P1-B1 (ATGM1152). The above-mentioned antibodies have high activity, good stability and strong specificity, and can be used as a reference standard for qualitative detection of PV positive and quantitative detection of the anti-Dsg3 autoantibody level in PV patients. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a PCR agarose gel electrophoresis map of the monoclonal bacteria of the VL phage library.

[0049] Figure 2 It is a PCR agarose gel electrophoresis map of the monoclonal bacteria of the KH phage library.

[0050] Figure 3 It is a PCR agarose gel electrophoresis map of the monoclonal bacteria of the λH phage library.

[0051] Figure 4 It is a SDS-PAGE electrophoresis map of the target protein Dsg3.

[0052] Figure 5 It is an ELISA result under different concentrations of the antibody.

[0053] Figure 6 It is a WB result of the combination of the antibody and the recombinant DSG3 protein.

[0054] Figure 7 Flow data for antibody affinity to HacaT cell surface antigen.

[0055] Figure 8 IF data for antibody binding to human DSG3 protein. DETAILED DESCRIPTION

[0056] The technical solutions of the present application are further illustrated below by means of specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations to the present application.

[0057] Unless otherwise specified in the embodiments, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0058] Embodiment 1

[0059] This embodiment provides a method for constructing a human ScFv phage display library.

[0060] Table 1 lists the main reagents used in this embodiment and their sources.

[0061] Table 1

[0062]

[0063] 1. Library construction

[0064] 1.1 Assemble the heavy chain variable region (VH) and the light chain variable region (VL). Table 2 is the PCR reaction system, and Table 3 is the PCR reaction steps.

[0065] Table 2

[0066]

[0067] Table 3

[0068]

[0069] Among them, the three steps of denaturation, annealing and extension (1) are repeated for 30 times.

[0070] The primer sequences are as follows:

[0071] Heavy chain variable region upstream primer (F):

[0072] 5' L-VH 1: ACAGGTGCCCACTCCCAGGTGCAG. (SEQ ID NO: 47)

[0073] 5' L-VH 3: AAGGTGTCCAGTGTGARGTGCAG. (SEQ ID NO: 51)

[0074] 5' L-VH 4 / 6: CCCAGATGGGTCCTGTCCCAGGTGCAG. (SEQ ID NO: 52)

[0075] 5' L-VH 5 / 7: CAAGGAGTCTGTTCCGAGGTGCAG. (SEQ ID NO: 53)

[0076] Light chain variable region upstream primer (F):

[0077] 5' L VK 1 / 2: ATGAGGSTCCCYGCTCAGCTGCTGG. (SEQ ID NO: 54)

[0078] 5' L VK 3: CTCTTCCTCCTGCTACTCTGGCTCCCAG. (SEQ ID NO: 55)

[0079] 5' L VK 4 / 5: ATTTCTCTGTTGCTCTGGATCTCTG. (SEQ ID NO: 56)

[0080] 5' L Vl 1: GGTCCTGGGCCCAGTCTGTGCTG. (SEQ ID NO: 57)

[0081] 5' L Vl 2: GGTCCTGGGCCCAGTCTGCCCTG. (SEQ ID NO: 58)

[0082] 5' L Vl 3: GCTCTGTGACCTCCTATGAGCTG. (SEQ ID NO: 59)

[0083] 5' L Vl 4 / 5: GGTCTCTCTCSCAGCYTGTGCTG. (SEQ ID NO: 60)

[0084] 5' L Vl 6: GTTCTTGGGCCAATTTTATGCTG. (SEQ ID NO: 61)

[0085] 5' L Vl 7: GGTCCAATTCYCAGGCTGTGGTG. (SEQ ID NO: 62)

[0086] 5' L Vl 8 / 9 / 10: GAGTGGATTCTCAGACTGTGGTG. (SEQ ID NO: 63)

[0087] Light chain variable region downstream primer (R):

[0088] 3' CK: TGCTGTCCTTGCTGTCCTGCT. (SEQ ID NO: 64)

[0089] 3' Ck: CACCAGTGTGGCCTTGTTGGCTTG. (SEQ ID NO: 65)

[0090] Heavy chain variable region downstream primer (R): 3' VH: ACTCGAGACGGTGACCAGGGTGCC. (SEQ ID NO: 66)

[0091] 1.2 Construction of light chain variable region phage display library

[0092] 1.2.1 Preparation of pATA-scFv-2 vector for library cloning.

[0093] 1.2.2 Digestion of vector and PCR products. Table 4 is the reaction system for digestion of vector and PCR products.

[0094] Table 4

[0095]

[0096] 1.2.3 After digestion, ligation was performed, and Table 5 is the ligation reaction system.

[0097] Table 5

[0098]

[0099] Incubate at 16°C overnight, and heat inactivate at 65°C for 10 min.

[0100] 1.2.4 Electroporation.

[0101] Preparation of TG1 competent cells: 1 mL SOC medium (Sigma, S1797) was pre-warmed at 37°C. An electroporation cuvette (0.1 cm gap) and a microfuge tube were placed on ice (one cuvette and one microfuge tube per transformation reaction). The electrocompetent cells were removed from the -80°C freezer and placed on ice until they were completely thawed (10-15 minutes). After the cells were thawed, they were mixed gently. 50 μL of cells were placed in a chilled microfuge tube on ice. 3 μL of DNA mixture was carefully added to the chilled electroporation cuvette without creating air bubbles. The tube was flicked down quickly with the wrist to deposit the cells at the bottom. The cells were electroporated at 600 Ω, 10 μF, and 1.8 kV. Immediately after the pulse, 1 mL of pre-warmed SOC medium was added to each tube. The cells were shaken at 37°C, 250 rpm for 1 hour. All of the electrotransformation medium was collected. 10 μL of culture was serially diluted into 90 μL of SOC medium and plated on LB / Amp / glucose. The plates were incubated at 37°C overnight. The total number of transformants was calculated by counting the number of colonies, multiplying by the culture volume, and dividing by the plating volume.

[0102] 1.3 Construction of VL-VH phage display library

[0103] 1.3.1 Digestion of vector and PCR products.

[0104] Table 6 is the digestion reaction setup.

[0105] Table 6

[0106]

[0107] 1.3.2 Ligation.

[0108] Table 7 is the ligation reaction setup.

[0109] Table 7

[0110]

[0111] 16°C overnight, 65°C heat inactivation for 10 min.

[0112] 1.3.3 Electroporation.

[0113] Preparation of TG1 competent cells: 4 mL of SOC medium (Sigma, S1797) was pre-warmed at 37°C. The electroporation cuvette (0.2 cm gap) and microfuge tube were placed on ice (one cuvette and one microfuge tube for each transformation reaction). The electrocompetent cells were taken from -80°C freezer and placed on ice until they were completely thawed (10-15 minutes). After the cells were thawed, mix gently. Carefully pipette 6 μL of DNA mixture into the chilled electroporation cuvette without generating air bubbles. Quickly flick the tube down with the wrist to deposit the cells at the bottom. Electroporate at 600 Ω, 10 μF, and 2.5 kV. Immediately after the pulse, add 2 mL of pre-warmed SOC medium into each tube. Shake at 37°C, 250 rpm for 1 hour. Collect all the electrotransformation medium. Serially dilute 10 μL of culture into 90 μL of SOC medium and plate on LB / Amp / glucose. Incubate at 37°C overnight. Count the number of colonies, multiply by the culture volume, and divide by the plating volume to calculate the total number of transformants.

[0114] 1.4 Library evaluation

[0115] 1.4.1 Colony PCR: PCR was performed using the constructed library as template.

[0116] Table 8 is the PCR reaction system, and Table 9 is the PCR reaction condition.

[0117] Table 8

[0118]

[0119] Table 9

[0120]

[0121] Wherein, the three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0122] Primer sequence: pATA-scFv-2 vector identification upstream primer (F): AGCGGATAACAATTTCACACAGGA. (SEQ ID NO: 27) pATA-scFv-2 vector identification downstream primer (R): GCCCCCTTATTAGCGTTTGCCATC. (SEQ ID NO: 37). The results of agarose gel electrophoresis detection after PCR are shown in Figures 1-3

[0123] Figure 1 is the electrophoresis detection result of the constructed VL phage library, wherein lane M: DL2000, lanes 1-16: pATA-VK, lanes 17-32: pATA-Vλ.

[0124] ​Figure 2 is the electrophoresis detection result of the constructed KH phage library, wherein lane M: DL2000, lanes 1-48: pATA-scFv-KH.

[0125] Figure 3 is the electrophoresis detection result of the constructed λH phage library, wherein lane M: DL2000, lanes 1-48: pATA-scFv-λH.

[0126] 1.4.2 Sequencing: the positive clones were selected and sent to Wuhan Qikexue Biotechnology Co., Ltd. for sequencing.

[0127] 1.5 Expression of Dsg3 protein

[0128] The Dsg3 gene sequence was artificially synthesized, and the Dsg3 gene was recombined into the expression vector plasmid pFastBacl to obtain the Dsg3-pFastBacl expression vector; the cloning site was BamH1 / Xho1.

[0129] The amino acid sequence of Dsg3 is SEQ ID NO: 7.

[0130] The gene sequence of Dsg3 is SEQ ID NO: 17.

[0131] The Dsg3-pFastBacl expression vector was transfected into DH10Bac competent cells for culture, and the precipitate was collected for GST tag affinity chromatography to obtain Dsg3 protein; the purified Dsg3 also needs to be subjected to SDS-PAGE electrophoresis to verify its purity, and the SDS-PAGE electrophoresis map of the purified Dsg3 is shown in Figure 5 , and the purity is greater than 90%. Figure 4 It is the SDS-PAGE electrophoresis map of the target protein Dsg3, and the protein size is 64.95 KDa, and the purity is greater than 90%.

[0132] Example 2

[0133] Preparation of monoclonal antibody specifically binding to DSG3

[0134] Table 10 is the main reagents used in this example.

[0135] Table 10

[0136]

[0137] 1. Experimental procedure

[0138] 1.1 Panning

[0139] (1) Coating immunotubes: DSG3 50 μg / mL, PBS buffer (1 mM CaCl2), 1 mL per tube, overnight at 4°C.

[0140] (2) Wash the immunotubes three times with 5 mL of PBST.

[0141] (3) Block with 5 mL of 5% milk / PBST (1 mM CaCl2) at 30°C for 1 h.

[0142] (4) Wash once with 5 mL PBS.

[0143] (5) Negative screening of library phages: Add 0.5% skim milk / PBST (1 mM CaCl2) or 0.1% casein / PBST (1 Mm CaCl2) to the library phages (the phages amplified in the previous round), fill with PBS to a total volume of 1 mL, and incubate at room temperature for 0.5 h.

[0144] (6) Add negatively screened phage to each tube and incubate at 30°C for 2 h.

[0145] (7) Wash 6 times with 5 mL PBST.

[0146] (8) Add 1 mL of Gly-HCl (pH = 2.2) to the tube to elute the phage and incubate at room temperature with shaking for about 6-8 minutes. Add 120-130 μL of Tris-HCl (pH = 9.6) to neutralize the solution to pH = 7.0-8.0.

[0147] (9) The eluted phages were diluted and infected with Escherichia coli TG1 in the logarithmic phase, and the titer was determined by plating.

[0148] 1.2 Amplification of eluted phage

[0149] (1) Take the eluted phage and add it to the logarithmic phase Escherichia coli TG1 bacterial solution. After standing at 37°C for 30 minutes, incubate at 220 rpm for 30 minutes to 1 hour.

[0150] (2) Add antibiotic Amp to the culture medium and culture at 37°C and 220 rpm until the bacterial solution OD is about 0.4-0.6.

[0151] (3) Add helper phage to the bacterial solution. After standing at 37°C for 30 min, incubate at 220 rpm for 45 min-1 h.

[0152] (4) Centrifuge the bacterial suspension at 3000-5000 rpm and discard the supernatant. Resuspend the bacteria in an equal volume of 2YT-Amp-Kan medium. Incubate overnight at 30°C and 220 rpm.

[0153] (5) The next day, centrifuge the bacterial solution at 8000 rpm, 4°C for 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of PEG / NaCl solution. After mixing well, place on ice or at 4°C for 1-2 h.

[0154] (6) Centrifuge at 8000 rpm, 4°C for 30 min, and discard the supernatant. Resuspend the precipitate with about 1 mL of PBS. Centrifuge at 8000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube.

[0155] (7) Dilute the amplified phage and infect the logarithmic phase TG1, and plate to determine the titer.

[0156] 1.3 Elutriation

[0157] Table 11 is the elutriation condition of each round.

[0158] Table 11

[0159]

[0160] 1.4 Polyclonal phage ELISA detection

[0161] Experimental group (Ag), control group 1 (NC1), control group 2 (NC2).

[0162] (1) Coat the immunoplate (experimental group: 4 μg / mL DSG3; control group 1: 4 μg / mL NC-His; control group 2: PBS (1 mM CaCl2), 4°C constant temperature overnight.

[0163] (2) Wash 3 times with 300 μL PBST.

[0164] (3) Add 300 μL 5% skim milk (1 mM CaCl2) and block at 37°C for 2 hours.

[0165] (4) Dilute the phage after each round of amplification with PBS, with a 3-fold increase in dilution. The initial concentration is 1×10 12 pfu / mL. Add 100 μL of the diluted amplified phage to each well. Incubate at 32°C for 1 h, and wash 3 times with 300 μL PBST.

[0166] (5) Add 100 μL of secondary antibody (anti-phage M13-HRP) diluent, incubate at 30°C for 1 h, and wash 3 times with 300 μL PBST.

[0167] (6) Add 100 μL of color developing solution TMB and develop color in the dark for 3-8 min, add 50 μL of 2 M HCl to stop the reaction, and read on a microplate reader (450 nm-620 nm).

[0168] 1.5 Monoclonal phage ELISA screening

[0169] (1) Select R3 eluted phage, dilute to the appropriate concentration, infect the logarithmic phase of TG1, and plate.

[0170] (2) The next day, 96 single clones were picked from the plate and inoculated into a 96-deep well plate and cultured at 37°C with 250 rpm shaking until the OD = 0.4-0.6.

[0171] (3) Add helper phage to the 96-deep well plate medium. Incubate at 37°C for 30 min, shake at 250 rpm for 45 min-1 h at 37°C.

[0172] (4) Centrifuge the 96-deep well plate at 4000 rpm for 5 min, discard the supernatant. Resuspend the bacterial solution with 2YT-Amp-Kan medium per well, shake at 250 rpm at 30°C overnight.

[0173] (5) The next day, centrifuge the 96-deep well plate at 4000 rpm for 10-15 min, and take the supernatant for ELISA experiment.

[0174] (6) Coat the immunoplate with antigen protein (experimental group: 4 μg / mL DSG3; control group: 4 μg / mL NC-His, buffer PBS (1 mM CaCl2), 100 μL, 4°C overnight.

[0175] (7) Wash 3 times with 300 μL PBST.

[0176] (8) Block with 300 μL 5% skim milk / PBST (1 mM CaCl2) at 37°C for 1 h. Wash 3 times with 300 μL PBST.

[0177] (9) Add 100 μL phage supernatant to each well. Incubate at 32°C for 1 h. Wash 3 times with 300 μL PBST.

[0178] (10) Add 100 μL secondary antibody (anti-phage M13) dilution, incubate at 32°C for 1 h, wash 3 times with 300 μL PBST.

[0179] (11) Add 100 μL color developing solution TMB and develop color in the dark for 3-8 min, add 50 μL 2 M HCl to stop the reaction, and read on a microplate reader (450 nm-620 nm).

[0180] 1.6 Secondary verification of positive clones

[0181] (1) Take 50 μL of positive clone and inoculate into 2 mL of 2YT-AG medium, and culture until OD 600 =0.4-0.6.

[0182] (2) Add helper phage to the medium. Incubate at 37°C for 30 min at 250 rpm, and then incubate at 37°C for 45 min to 1 h with shaking.

[0183] (3) Centrifuge the bacterial solution at 4000 rpm for 5 min, and discard the supernatant. Resuspend the bacterial solution with 2 mL of 2YT-Amp-Kan medium per well, and incubate at 30°C with shaking at 250 rpm overnight.

[0184] (4) Centrifuge the overnight culture at 5000 rpm for 5 min, and take the supernatant for Elisa experiment.

[0185] (5) Coat the immunoplate (experimental group: 4 μg / mL DSG3; control group 1: 4 μg / mL NC-His, control group 2: PBS (1 mM CaCl2)) at 32°C for 2 hours. Wash 3 times with 300 μL of PBST.

[0186] (6) Add 300 μL of 5% skim milk (1 mM CaCl2) and block at 37°C for 2 hours.

[0187] (7) Add 100 μL of phage supernatant (5-fold gradient dilution) to each well. Incubate at 32°C for 2 hours. Wash 3 times with 300 μL of PBST.

[0188] (8) Add 100 μL of secondary antibody (anti-phage M13-HRP) dilution, and incubate at 32°C for 1 h. Wash 3 times with 300 μL of PBST.

[0189] (9) Add 100 μL of color developing solution TMB, and develop color in the dark for 3-8 min. Add 50 μL of 2 M HCl to stop the reaction, and read the result with a microplate reader (450 nm-620 nm).

[0190] 2. Experimental results

[0191] (1) Results of panning

[0192] Table 12 shows the results of panning

[0193] Table 12

[0194]

[0195] (2) Results of polyclonal phage ELISA

[0196] Experimental group (Ag): 4 μg / mL DSG3; Control group 1 (NC1): 4 μg / mL NC-His, Control group 2 (NC2): PBS (1 mM CaCl2).

[0197] Table 13 is the polyclonal phage ELISA result.

[0198] Table 13

[0199]

[0200] According to the polyclonal results, we selected the third round for monoclonal screening, and selected 96 clones for antigen group and control group monoclonal ELISA detection.

[0201] (3) Monoclonal phage ELISA results

[0202] Table 14 is the R3P1 monoclonal phage ELISA result (antigen group), Ag: 4 μg / mL DSG3.

[0203] Table 14

[0204]

[0205] Table 15 is the R3P1 monoclonal phage ELISA result (control group) NC: 4 μg / mL NC-His.

[0206] Table 15

[0207]

[0208] According to the experimental results, we selected clones for testing, and after sequencing analysis, we finally obtained 12 different sequences, and the 12 clones were subjected to secondary verification.

[0209] (4) Secondary ELISA detection results

[0210] Experimental group (Ag): 4 μg / mL DSG3.

[0211] Control group 1 (NC1) NC1: 4 μg / mL NC-His.

[0212] Control group 2 (NC2) NC2: PBS.

[0213] Table 16 is the secondary ELISA detection result (without adding 1 mM CaCl2 in the experimental process).

[0214] Table 16

[0215]

[0216] Table 17 is the secondary ELISA detection results (1 mM CaCl2 was added during the experiment process).

[0217] Table 17

[0218]

[0219] 3、Conclusion

[0220] After this selection, 5 positive clones were finally screened out, which could bind with the antigen and did not bind with the control label.

[0221] 4、Sequencing of antibody sequence

[0222] The 5 phage positive clones DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), and DSG3-R3P1-B1 (ATGM1152) screened were subjected to full sequence sequencing, and the corresponding antibody heavy chain and light chain and full sequence were obtained as shown in Table 18 and Table 19.

[0223] Table 18

[0224]

[0225] Table 19

[0226]

[0227] Example 3

[0228] ELISA detection of OD values of the antibody under different dilution concentrations

[0229] Table 20 is the main reagents used in this example

[0230] Table 20

[0231]

[0232] 1、Experimental steps

[0233] (1) Coating: 100 μL of Dsg3 protein (4 μg / mL) (1 mM CaCl2) was coated on the enzyme-labeled plate per well, and incubated at 4°C overnight.

[0234] (2) Washing: Discard the liquid in the enzyme-labeled plate, wash each well with 300 μL of 0.05% PBST for 2 times, and wash with PBS for 1 time.

[0235] (3) Blocking: Add 300 μL of 5% skimmed milk (dissolved in PBS) to each well, and block at 37°C for 2 hours.

[0236] (4) Positive antibody incubation: DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), DSG3-R3P1-B1 (ATGM1152) antibodies were gradiently diluted respectively, 100 μL of the diluted antibody solution was added to each well, and incubated at 30°C for 2 hours.

[0237] (5) Washing: as in step 2.

[0238] (6) Secondary antibody incubation: Goat Anti-Human IgG (H+L) antibody was diluted 10000 times with blocking solution, 100 μL of the diluted secondary antibody was added to each well, and incubated at 37°C for 1 hour.

[0239] (7) Washing: as in step (2).

[0240] (8) Color development: 100 μL of TMB was added to each well, and incubated at 37°C for 10 minutes, and then 50 μL of 2 M HCl was added to each well to stop the reaction.

[0241] (10) Plate reading: the values were read using a microplate reader at 450 nm-630 nm, as shown in Figure 5

[0242] Figure 5 The ELISA results of the antibodies at different concentrations. As can be seen from Figure 5 , DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), and DSG3-R3P1-B1 (ATGM1152) can strongly bind to recombinant DSG3 protein, and DSG3-R3P1-C1 (ATGM1151) can weakly bind to recombinant DSG3 protein.

[0243] Example 4

[0244] WB detection of the binding of monoclonal antibodies to target antigen DSG3

[0245] Table 21 is the main reagents used in this example.

[0246] Table 21

[0247]

[0248] 1. Experimental procedure

[0249] ​1.1 Preparation of protein sample

[0250] (1) Wash the collected HacaT cells with 1 mL PBS, centrifuge at 3000 rpm for 5 min, 0.9 g.

[0251] (2) Discard the supernatant, and resuspend with 120 μL lysis solution (strong RIPA + phosphatase inhibitor).

[0252] (3) Lyse on ice for 20 min; after lysis, centrifuge at 12000 rpm for 30 min at 4°C.

[0253] (4) After centrifugation, take 110 μL supernatant to a new label tube.

[0254] (5) Detect the protein concentration by BCA method:

[0255] Prepare BCA working solution: A solution: B solution = 50:1 200 μL / well.

[0256] Prepare Pr sample well: 10-fold dilution of Pr stock solution 3 μL Pr stock solution + 27 μL ddw = 30 μL.

[0257] Add 200 μL / well working solution to the 96-well plate.

[0258] Add 25 μL / well of protein sample and BSA standard.

[0259] Incubate at 37°C for 30 min.

[0260] After incubation, load the machine.

[0261] (6) Calculate the protein concentration and loading volume, control the protein loading amount to be 30 μg, add the total protein extracted from HacaT and ATGE5228 (recombinant DSG3 extracellular protein segment).

[0262] 1.2 Electrophoresis and membrane transfer

[0263] (1) Add the total protein extracted from HacaT and ATGE5228 (recombinant DSG3 extracellular protein segment) to the gel well.

[0264] (2) Pour the electrophoresis solution: concentrate the protein at 80 V for 20 min, separate the protein at 120 V for 40 min.

[0265] (3) Activate the PVDF membrane in advance with methanol, cut the upper left corner to mark, and prepare the membrane transfer sandwich.

[0266] (4) Clasp the membrane transfer clamp, pour new membrane transfer solution into the tank, transfer on ice at 100 V for 120 min.

[0267] 1.3 Blocking, antibody incubation and color development

[0268] (1) After the end of the transfer, add 5% skimmed milk, block at room temperature for 1 h, speed = 0;

[0269] (2) After the end of the blocking, wash the surface blocking solution with 1x TBST;

[0270] (3) Prepare DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), DSG3-R3P1-B1 (ATGM1152) into a primary antibody solution using an antibody diluent at a concentration of 1 μg / mL, and shake overnight at 4°C.

[0271] (4) Wash with 1x TBST for 15 min, wash three times, speed = 80.

[0272] (5) Prepare a HRP-labeled anti-human IgG Fc gamma antibody (Anti-Human IgG, Fc gamma Fragment Specific, HRP-Linked Antibody) secondary antibody solution using an antibody diluent at a concentration of 1:10000, and slowly shake at room temperature for 1 h, speed = 0;

[0273] (6) Wash with 1x TBST for 15 min, wash three times, speed = 80.

[0274] (7) Prepare a color developing solution, A liquid: B liquid = 1:1.

[0275] (8) Expose to the machine. The experimental results are shown in Figure 6 .

[0276] As can be seen from Figure 6 , the human-derived anti-Dsg3 monoclonal antibody has strong specific binding ability with the extracellular segment of the recombinant Dsg3 protein. ATGE5228 is a recombinant DSG3 extracellular segment protein. Among them, DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-B1 (ATGM1152) have strong binding with the recombinant DSG3 protein, DSG3-R3P1-E1 (ATGM1149) has weak binding with the recombinant DSG3 protein, and DSG3-R3P1-C1 (ATGM1151) does not bind to the recombinant DSG3 protein (suggesting that its binding site is a spatial epitope rather than a linear epitope).

[0277] Example 5

[0278] Flow cytometry detection of monoclonal antibody affinity to HacaT cell surface antigen

[0279] Table 22 lists the main reagents used in this example.

[0280] Table 22

[0281]

[0282] 1. Experimental procedure

[0283] (1) Collect 10 6 HacaT cells into 1.5 mL EP tubes, 2% FBS in PBS, 50 μL / tube.

[0284] (2) Blocking: Add 2.5 μL of human Fc blocking solution (Human TruStain FcXTM) to the EP tube, incubate at room temperature for 10 min.

[0285] (3) Directly add primary antibody solution: 4 μg / mL DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), DSG3-R3P1-B1 (ATGM1152), and hIgG1-ISOtype, 2% FBS in PBS, 4°C for 30 min.

[0286] (4) 4°C, 500 g centrifugation for 6 min.

[0287] (5) Wash unbound primary antibody: 2% FBS in PBS, 4°C, 500 g centrifugation for 6 min.

[0288] (6) Add fluorescently labeled secondary antibody: resuspend the cells in 100 μL of 2% FBS in PBS, at this time start to avoid light, 10 6 Prepare an antibody mixture corresponding to 1 μL of antibody for the amount of cells, add 1 μL of PE-labeled goat anti-human IgG Fc end secondary antibody (PE-anti human IgG Fc), 4°C, avoid light for 30 min.

[0289] (7) 4°C, 500 g centrifugation for 6 min.

[0290] (8) Wash unbound secondary antibody: 2% FBS in PBS, 4°C, 500 g centrifugation for 6 min.

[0291] (9) 200 μL PBS resuspended cells, 40 μm sieve to flow tube, machine detection; experimental results as shown in Figure 7

[0292] From Figure 7 It can be seen that human anti-Dsg3 monoclonal antibody has strong specific binding ability with HacaT surface antigen. Blank (Blank): fluorescence negative group, excluding fluorescence background interference; hIgG: isotype control group (ISOtype control group), excluding non-specific binding of antibodies; ATGM1148, ATGM1149, ATGM1150, ATGM1151, ATGM1152 are experimental groups. Among them, DSG3-R3P1-A2 (ATGM1150) has the strongest binding ability with HacaT surface antigen, followed by DSG3-R3P1-E1 (ATGM1149) and DSG3-R3P1-B1 (ATGM1152), and DSG3-R3P1-D1 (ATGM1148) and DSG3-R3P1-B1 (ATGM1152) have relatively weak binding ability with HacaT surface antigen.

[0293] Example 6

[0294] IF method for detecting monoclonal antibody binding to human DSG3

[0295] Table 23 is the main reagent used in this example

[0296] Table 23

[0297]

[0298] 1. Experimental procedure

[0299] 1.1 Plasmid transfection

[0300] (1) 293T cells were plated in a confocal dish, and when the cell density was 70-80%, 2 μg DSG3-pTT5 plasmid was transfected.

[0301] Plasmid transfection MIX system:

[0302] a. Add 2 μg DSG3-pTT5 plasmid to Opti-MEM, vortex well, and stand at room temperature for 5 min.

[0303] b. Add PEI to the prepared Opti-MEM mixture according to the ratio of all plasmids: PEI = 1:2, mix well, vortex well, and stand at RT for 20 min.

[0304] c. The prepared plasmid suspension was directly added to the culture dish.

[0305] ​d After 5 h, change the medium and continue to culture for 24 h.

[0306] (2) After 24 h, when the cell density in the confocal dish is appropriate, pour off the culture medium and wash with PBS three times. The washing condition is: standing at room temperature / light shaking for 5 min.

[0307] (3) Add 1 mL of 4% paraformaldehyde and stand at room temperature for 15 min.

[0308] (4) Pour off the 4% paraformaldehyde and wash with PBS three times.

[0309] (5) Add 500 μL of PBS containing 10% FBS for blocking for 1 h.

[0310] (6) After pouring off the blocking solution, wash with PBS three times.

[0311] (7) Add 4 μg / mL of DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), DSG3-R3P1-B1 (ATGM1152) and hIgG1-ISOtype solution, and stand at 4°C overnight.

[0312] (8) Take out the next day and wash with PBS three times.

[0313] (9) Add the prepared AF568 fluorescently labeled anti-human secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568) (1:1000 dilution in PBS) and stand at room temperature in the dark for 1 h.

[0314] (10) After aspirating the secondary antibody, wash with PBS three times, add 1 mL of prepared diphenylmethanimine (Hoechst) (1:10000 dilution in PBS) for staining the cell nucleus, and stand at room temperature in the dark for 15 min.

[0315] (11) After pouring off the diphenylmethanimine (Hoechst), wash with PBS three times, and then take pictures under the Lecia laser confocal microscope. The experimental results are shown in Figure 8 .

[0316] From Figure 8It can be seen that the human anti-Dsg3 monoclonal antibody specifically binds to 293t cells overexpressing human DSG3 protein. hIgG: isotype control group, to exclude non-specific binding of the antibody; Flag: protein tag, to show protein expression; ATGM1148, ATGM1149, ATGM1150, ATGM1151, ATGM1152 are experimental groups. Among them, ATGM1148, ATGM1150, ATGM1152 have stronger binding ability with human DSG3 protein, and ATGM1149, ATGM1151 have relatively weaker binding ability with human DSG3.

[0317] The above merely provides a specific implementation of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily conceived by those skilled in the art, and all such changes and replacements fall within the protection scope and disclosure scope of the present application.

Claims

1. An antibody that binds to a pemphigus vulgaris-specific antigenic peptide, characterized in that: The heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO: 1; CDR2 is shown in SEQ ID NO: 2, and CDR3 is shown in SEQ ID NO: 3; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO: 6, the amino acid sequence of CDR2 is AAS, and CDR3 is shown in SEQ ID NO: 8; Or, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO: 11; CDR2 is shown in SEQ ID NO: 12, and CDR3 is shown in SEQ ID NO: 13; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO: 16, the amino acid sequence of CDR2 is YDS, and CDR3 is shown in SEQ ID NO: 18; Or, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO: 21; CDR2 is shown in SEQ ID NO: 22, and CDR3 is shown in SEQ ID NO: 23; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO: 26, the amino acid sequence of CDR2 is AAS, and CDR3 is shown in SEQ ID NO: 28; Or, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO: 31; CDR2 is shown in SEQ ID NO: 32, and CDR3 is shown in SEQ ID NO: 33; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO: 36, the amino acid sequence of CDR2 is SNN, and CDR3 is shown in SEQ ID NO: 38; Alternatively, the heavy chain variable region CDR1 of the antibody is shown as SEQ ID NO:41; CDR2 is shown as SEQ ID NO:42, and CDR3 is shown as SEQ ID NO:43; the light chain variable region CDR1 of the antibody is shown as SEQ ID NO:46, the amino acid sequence of CDR2 is DNN, and CDR3 is shown as SEQ ID NO:

48.

2. The antibody binding to pemphigus vulgaris-specific antigenic peptide according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9; Or, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 14; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; Or, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29; Or, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 34; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39; Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 44; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

49.

3. The antibody binding to pemphigus vulgaris-specific antigenic peptide according to claim 1, characterized in that The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 5; the light chain amino acid sequence is shown in SEQ ID NO: 10; Or, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 15; the light chain amino acid sequence is shown in SEQ ID NO: 20; Or, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 25; the light chain amino acid sequence is shown in SEQ ID NO: 30; Or, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 35; the light chain amino acid sequence is shown in SEQ ID NO: 40; Alternatively, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 45; and the light chain amino acid sequence is shown in SEQ ID NO:

50.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody that binds to the pemphigus vulgaris-specific antigenic peptide according to any one of claims 1 to 3.

5. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 5; or the nucleic acid molecule according to claim 4 is integrated into the chromosome of the host cell.

7. A composition for detecting Dsg3 protein in a sample, characterized in that: The composition comprises the antibody binding to the pemphigus vulgaris-specific antigenic peptide according to any one of claims 1 to 3.

8. A kit for detecting Dsg3 protein in a sample, characterized in that: The kit comprises the antibody binding to the pemphigus vulgaris-specific antigenic peptide according to any one of claims 1 to 3.

9. Use of the antibody binding to a pemphigus vulgaris-specific antigenic peptide according to any one of claims 1 to 3, the composition for detecting Dsg3 protein in a sample according to claim 7, and the kit for detecting Dsg3 protein in a sample according to claim 8 in the preparation of a product for detecting pemphigus vulgaris.

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