Rabbit hybridoma monoclonal antibody against mouse BP180NC14A, preparation method, application and hybridoma cell line

Abstract

The present invention discloses a rabbit hybridoma monoclonal antibody against mouse BP180NC14A, a preparation method, an application, and a hybridoma cell line. The monoclonal antibody is specific for the polypeptide encoded by nucleotides 499-573 in Q07563-1 (i.e., the amino acid sequence shown in SEQ ID No. 1). The beneficial effects of the present invention are embodied in providing a monoclonal antibody against mouse BP180NC14A, its preparation and application, and a rabbit hybridoma cell line producing the monoclonal antibody. These antibodies provide a basis for further clarifying the pathophysiological research of BP180 in skin tissue, and can be used to induce a mouse bullous pemphigoid (BP) model or detect anti-BP180 IgG titers in mice, thus having important application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a rabbit hybridoma monoclonal antibody against mouse BP180NC14A, a preparation method, an application and a hybridoma cell line. Background Art

[0002] Bullous pemphigoid (BP) is the most common autoimmune bullous skin disease, primarily manifesting as tense or blood-filled blisters affecting the flexor areas of the body, accompanied by varying degrees of pruritus. BP is characterized by subepidermal blister formation and the presence of autoantibodies targeting hemidesmosomes. BP is more common in the elderly, particularly those over 70 years old. Its incidence increases with age, reaching 150-330 cases per million in patients over 80 years old. The annual incidence of BP fluctuates worldwide between 2.5 and 66 cases per million, but the overall trend is upward. BP patients are at significantly increased risk of coexisting conditions. In addition to the most common neurological disorders, they may also suffer from tumors, diabetes, or other immune-mediated skin diseases. Consequently, mortality rates among BP patients are also increasing. BP has become a significant contributor to the burden of disease in the elderly. However, due to the lack of established BP models, researchers have a limited understanding of the pathogenesis of BP, resulting in a lack of disease-specific treatments, and glucocorticoids remain the mainstay of BP treatment. Therefore, establishing a mature BP animal model and deeply exploring the pathogenesis of BP have become urgent tasks in the development of new BP therapies.

[0003] Currently, the autoantibody theory is widely accepted as the pathogenesis of BP. BP patients harbor autoantibodies targeting BP180 and BP230, components of the hemidesmosome at the dermal-epidermal junction. Binding of these pathogenic autoantibodies to antigens triggers a series of inflammatory immune responses, leading to dermal-epidermal separation and blister formation. Numerous studies have identified BP180 as the primary target antigen recognized by pathogenic autoantibodies for BP. Binding to BP180 activates downstream signaling, attracting inflammatory cells such as mast cells, eosinophils, and neutrophils to the lesions, where they release inflammatory mediators, disrupting the dermal-epidermal junction, and ultimately leading to subepidermal blister formation. In this process, the binding of pathological autoantibodies to BP180 is crucial, and the production of anti-BP230 antibodies is often secondary to the production of anti-BP180 antibodies. Therefore, targeting BP180 is crucial for inducing animal models of BP.

[0004] Animal models of BP offer a comprehensive means of investigating the pathogenesis of BP in vivo. However, current BP models primarily utilize neonatal mice induced by subcutaneous injection of purified human anti-BP180-NC16A antibodies. These models are young, inconsistent with the clinical observation that BP primarily occurs in the elderly. The short-lived nature of these models hinders long-term observation of intervention effects. Furthermore, the human anti-BP180-NC16A antibodies are derived from humans, requiring purification from large quantities of serum from BP patients, making them difficult to obtain.

[0005] Therefore, there is currently a lack of effective means to comprehensively observe the pathogenesis of BP. It is imperative to study a new method of targeting BP180 antigen to prepare a mature and convenient BP animal model. Summary of the Invention

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a rabbit hybridoma monoclonal antibody against mouse BP180NC14A, as well as a preparation method, application and hybridoma cell line.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention discloses a rabbit hybridoma monoclonal antibody against mouse BP180 NC14A. The monoclonal antibody has specificity for a polypeptide encoded by nucleotides 499-573 in Q07563-1. The amino acid sequence of the polypeptide is shown in SEQ ID No. 1.

[0009] Preferably, the monoclonal antibody specifically targets the NC14A dermal-epidermal junction region of mouse BP180, thereby inducing dermal-epidermal separation and subepidermal blister formation, which are pathological features of BP.

[0010] Preferably, the monoclonal antibody is secreted and produced by a hybridoma cell line, and the hybridoma cell line was deposited in the China Center for Type Culture Collection on April 26, 2022, with the deposit number CCTCCNO: C202296.

[0011] The present invention also discloses a method for preparing the above-mentioned rabbit hybridoma monoclonal antibody against mouse BP180 NC14A, comprising the following steps:

[0012] 1) Preparation of antigen

[0013] The nucleotide sequence shown in SEQ ID No. 1 is used to encode the corresponding polypeptide, which is fused with a vector to form an overexpression vector, and the overexpression vector is transfected into human HEK293 cells to promote the production of BP180NC14A protein;

[0014] 2) Immunized animals

[0015] The isolated BP180 NC14A protein was used to immunize Japanese big-eared rabbits to obtain immune Japanese big-eared rabbits;

[0016] 3) Establishment of hybridoma cell lines

[0017] Antibody-producing plasma cells were extracted from the spleen of immunized Japanese large-eared rabbits and fused with myeloma cells from the same animal to prepare hybridoma cells. After fusion, the cells were selectively cultured in HAT medium, positive clones were screened by ELISA, and hybridoma cells from positive wells were cloned and cultured by limiting dilution until the antibody-positive rate of the cloned cells reached 100%. The hybridoma cells were continuously passaged in vitro for more than 3 months and repeatedly frozen and revived until the cell line could stably secrete monoclonal antibodies, thereby obtaining a hybridoma cell line.

[0018] 4) The hybridoma cells established in step 3) are cultured in vitro, the supernatant is collected, and the purified monoclonal antibody is obtained by affinity chromatography.

[0019] Preferably, in step 1), the vector is a pTT5 vector.

[0020] Preferably, in step 2), the specific operation of immunizing Japanese big-eared rabbits is as follows: each rabbit is intraperitoneally injected with 100 μg once every two weeks for a total of three times, with a fourth shock immunization, and after 3 days, the rabbits are allowed to fuse to obtain immune rabbits.

[0021] The present invention also discloses the use of the above rabbit hybridoma monoclonal antibody against mouse BP180 NC14A in preparing a bullous pemphigoid model.

[0022] The present invention also discloses the use of the above-mentioned rabbit hybridoma monoclonal antibody against mouse BP180 NC14A in preparing a kit for detecting the concentration of anti-BP180 antibodies in mice in a mouse bullous pemphigoid model.

[0023] The present invention also discloses a hybridoma cell line that produces the above-mentioned rabbit hybridoma monoclonal antibody against mouse BP180 NC14A. The hybridoma cell line was deposited in the China Center for Type Culture Collection on April 26, 2022, with the deposit number CCTCC NO: C202296.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention discloses a method for preparing a rabbit monoclonal antibody with high affinity and high specificity against mouse BP180 and a rabbit hybridoma cell line producing the rabbit monoclonal antibody. The monoclonal antibody has specificity for the polypeptide encoded by nucleotides 499-573 in Q07563-1 (i.e., the nucleotide sequence shown in SEQ ID No. 1).

[0026] The present invention uses a rabbit hybridoma cell culture method to prepare rabbit anti-mouse BP180-NC14A (corresponding to human BP180-NC16A) IgG antibody, which can be easily and efficiently mass-produced and purified, providing an important basis for constructing a mouse BP model and further exploring the role of BP180 in the pathogenesis of skin diseases.

[0027] Certificate of preservation:

[0028] The hybridoma cell line described in this application was deposited in the China Center for Type Culture Collection on April 26, 2022, with the deposit number CCTCC NO: C202296. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The electrophoresis results of the recombinant protein prepared for the 499-573 nucleotide amino acids are shown;

[0030] Figure 2 The results of immunofluorescence test for the preliminary validation of polyclonal antibodies in the serum of two rabbits immunized for the first time; A is numbered Z1872; B is numbered Z1873;

[0031] Figure 3 These are the three final clones selected from 68 clones; A is No. 60; B is No. 99; and C is No. 117.

[0032] Figure 4 This is a photo of the hybridoma cell line culture results of clone 99, which was generated from three clones after two subcloning screenings;

[0033] Figure 5 Immunofluorescence detection of BP180 NC14A expression in mouse salt-cracked skin; A is the control antibody; B is anti-BP180 NC14A IgG;

[0034] Figure 6 The mouse model was induced after subcutaneous injection of anti-BP180 NC14A hybridoma antibody into mice; A shows the changes in the left ear (isotype control IgG antibody) and right ear (anti-BP180 NC14A IgG, see the title) of C57BL / 6 mice at 0, 10, and 20 days after subcutaneous injection; B shows the fluorescence results of subepidermal blisters induced in the model group (right ear) at 0 and 20 days after injection. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0036] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present invention and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or are inherent to these processes, methods, products or apparatuses.

[0037] The present invention is described in further detail below with reference to the accompanying drawings:

[0038] Example 1: BP180 NC14A immunogen design

[0039] a. Preparation of recombinant protein: Based on the mouse BP180 gene sequence (Q07563-1) logged in GenBank, the RNA sequence corresponding to the 499-573 nucleotide amino acids was selected and the recombinant protein was constructed by Shanghai abcam. The prepared recombinant protein was named P0551 with a molecular weight of 10.8KD (see Figure 1 ).

[0040] Among them, the amino acid sequence of 498-573 is:

[0041] AEEVRKLKARVEELEKTKVLYHDVQMDKSNRDRLQAEAPSLGPGLGKAELDGYSQEAIWLFVRNKLMTEQENGNLR, that is, the nucleotide sequence shown in SEQ ID No.1.

[0042] b. Vector Construction: The pTT5 vector was double-digested with EcoRI and HindIII, purified using a PCR product purification kit, and ligated with the BP180 gene at a molar ratio of 1:5 using T4 DNA ligase at 4°C overnight. Competent E. coli DH5α was prepared using the CaCl2 method. The ligation product was transformed into competent E. coli DH5α, plated onto a LB culture plate containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Colonies were picked and added to 10 ml of LB medium containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Plasmids were extracted using a plasmid extraction kit and identified using double-digestion with EcoRI / HindIII. Positive clones were screened and the binding site of the clones was detected by Western blot. The recombinant plasmid with the correct binding site was designated pTT5-BP180NC14A.

[0043] c. Cell transfection: HEK 293 cells were revived and cultured in FreeStyle TM Cells were transferred into 6-well plates in 293 culture medium. When the cell confluency reached 60%, 4 μg each of the purified recombinant plasmid pTT5-BP180NC14A and the empty vector pTT5 were transfected into HEK293 cells according to the instructions of Lipofectamine 2000 transfection reagent. A blank control group (untransfected) was also established.

[0044] d. Detection of pTT5-BP180 NC14A fusion protein expression in transfected cells: 48 hours after transfection, place each group of HEK293 cell 6-well plates on ice, discard the culture medium, wash three times with pre-chilled PBS, scrape the cells, transfer them to a centrifuge tube, add 50 μl of protein lysis buffer containing protease inhibitors, mix well, and place on ice for 30 minutes. Centrifuge at 15,000 g for 10 minutes at 4°C, and collect 5 μl of the supernatant for protein quantification. Forty micrograms of protein were added to 5× SDS-PAGE loading buffer and denatured at 100°C for 5 minutes. Proteins were separated by 10% SDS-PAGE and electrotransferred to a PVDF membrane (4°C, 70V, 3.5 hours). The membrane was washed once with TBST and blocked with 5% skim milk powder for 2 hours at room temperature. Rabbit anti-mouse BP180 monoclonal antibody (1:1000 dilution) and mouse anti-β-actin monoclonal antibody (1:2000 dilution) were added and incubated overnight at 4°C. The membrane was washed three times with PBST, and HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (1:2000 dilution) were added and incubated for 2 hours at room temperature. The membrane was washed three times with TBST. Development was performed with ECL chemiluminescence solution, exposure was 1 minute, and protein expression was detected using an imaging system.

[0045] Example 2: Immunization of Animals

[0046] A mixture containing 50 mg of purified BP180 NC14A-his protein was thoroughly mixed with an equal volume of complete Freund's adjuvant until emulsified, with a water-in-oil mixture considered successful. Primary immunizations were performed by subcutaneous injection into 4-5 sites on the back of 3.5-4 year old female hybrid Japanese rabbits. Two weeks later, booster immunizations were performed with an equal volume of incomplete Freund's adjuvant emulsified with 50 mg of BP180 NC14A-his protein. Thereafter, booster immunizations were repeated every two weeks. After three booster immunizations, intraperitoneal blood was collected from the rabbits and serum was isolated. The titer was determined by indirect ELISA. When the titer reached 1 CT4, the rabbits were ready for fusion. A booster immunization was performed three days before fusion.

[0047] Example 3: Blood collection and antiserum purification

[0048] Two 3.5-4 month old Japanese white rabbits were anesthetized before antigen immunization, and at 8, 10, and 11 weeks after immunization. 20-25 ml of serum was collected. Antiserum was purified using a protein affinity column. The purified sample was analyzed by immunoblotting, and concentration and antigen binding assays were performed simultaneously. This produced purified polyclonal antibodies. Immunofluorescence analysis was used to initially verify the binding site and efficacy of the antibodies produced by the positive rabbits. Two clones were then selected for subsequent hybridoma cell production. Figure 2 ,from Figure 2 It can be seen that the polyclonal antibodies produced by the two clones Z1872 and Z1873 screened both bind to the dermal-epidermal junction and have strong fluorescence intensity.

[0049] Example 4: Preparation of hybridoma cells by electrofusion

[0050] Resuspend rabbit myeloma cells and immune rabbit spleen cells in 1640 incomplete medium, count them separately using a hemocytometer, and mix them in a 50-ml sterile centrifuge tube at a ratio of 1:3 spleen cells:myeloma cells. After thorough mixing, add 1640 incomplete medium to 40 ml of the mixture and centrifuge at 4°C at 1,500 rpm for 5 minutes. Discard the supernatant and the liquid from the tube wall (use sterile absorbent paper strips to blot dry). Gently tap the mixed pellet of myeloma cells and immune mouse cells to loosen it, and immerse the bottom of the centrifuge tube in 37°C warm water. Wash the suspended cells twice with fusion medium (FM) and then resuspend them in FM. After washing, centrifuge the cells at 4°C at 1,500 rpm for 5 minutes to obtain a cell pellet. Discard the supernatant and resuspend the cells in fresh FM medium. After thorough mixing, transfer the suspended cells to the appropriate fusion chamber. Start the dielectrophoresis field (amplitude <200 V / cm, frequency <2 MHz). Check with a microscope to see if the cells are aligned in a chain. Adjust the amplitude and frequency for optimal results. Turn the dielectrophoresis field on for about 1 minute and then turn it off, and immediately apply a fusion pulse with an amplitude on the order of 1 kV / cm. The pulse width is less than 1 ms. Immediately after the fusion pulse, start the dielectrophoresis field again and keep it on for about 2 minutes as usual. Turn off the dielectrophoresis field and let the cells sit in the sample pool for 10 minutes. Remove the FM, resuspend the cell pellet with 200 ml of HAT complete culture medium solution, and culture in a 37°C, 5% CO2 incubator. When the cells grow to a suitable density, collect the supernatant of the hybridoma cells, use standard ELISA to screen 45 ELISA-positive polyclones from a plate of 120 clones, transfer them to a 24-well plate, and then collect the supernatant. Standard ELISA determines the concentration of the 45 clones screened and the antibodies in the supernatant. Screen suitable clones for primary screening. The screened clone numbers are shown in Table 1 below:

[0051] Table 1 45 clones screened in the second round and their 1:20 diluted ELISA concentrations

[0052]

[0053] Example 5: Subcloning and ELISA screening

[0054] 45 subcloned cells were diluted appropriately to allow plating at three cell density levels: 100 cells / well, 10 cells / well, and 1 cell / well. Cells were grown to approximately 80% confluence, then gently resuspended and viable cells counted. 5,000 viable cells were transferred to a polypropylene conical tube containing 5 ml of complete DMEM medium containing HT, resulting in a cell suspension at a concentration of 1,000 cells / ml. This cell suspension was then serially diluted 10-fold to obtain cell suspensions at concentrations of 100 cells / ml and 10 cells / ml, respectively. 100 μl of this 1,000 cell / ml cell suspension was added to the top row (row A) of two 96-well cell culture plates supplemented with feeder cells or supplements (100 μl / well), resulting in 100 viable hybridoma cells per well. At this cell density, cells should reach confluence in approximately 10 days. In the second row (row B) of each cell culture plate, 100ul of cell suspension at a concentration of 100 cells / ml was added so that each well contained 10 viable hybridoma cells. In the other rows (rows CH) of the cell culture plate, 100ul of cell suspension at a concentration of 10 cells / ml was added so that each well contained 1 viable hybridoma cell. After the hybridoma cells grew to an appropriate density, the culture supernatant was collected, and the antibody concentration was tested by standard ELISA. The binding and intensity of the selected subclones were tested by immunofluorescence. Thus, three suitable subclones (No. 60, 99 and 117) were screened. Figure 3 ).

[0055] Example 6: Secondary subcloning and ELISA screening

[0056] The three subclones screened from Example 6 were again subjected to limiting dilution and the supernatant was collected. The concentration of the supernatant was detected by ELSIA and the binding site and fluorescence intensity of the antibody were detected by immunofluorescence. The final hybridoma cell clone 99 cells were screened and cultured ( Figure 4 ).from Figure 4 It can be seen that the expression intensity of 99 is higher than that of 117, and the expression specificity is better than that of 60, and it is more concentrated in the dermis-epidermis junction.

[0057] Example 8: Hybridoma cell-secreted antibodies induce BP mouse model

[0058] The supernatant from hybridoma cells 2021-XJTU-50 clone No: 99 was purified and concentrated to obtain rabbit anti-mouse BP180 NC14A monoclonal antibody. The antibody was then initially subjected to salt-split experiments to verify binding. It was found that the rabbit isotype control antibody had no significant binding to the salt-split mouse skin ( Figure 5 A), while anti-BP180NC14A antibody clearly bound to the epidermal side of the salt-split site ( Figure 5 B), which is consistent with the current BP salt splitting experimental results.

[0059] To further investigate the ability of the hybridoma monoclonal antibody to induce BP, BALB / c mice were subcutaneously injected into the ears to create a model. Rabbit isotype control antibody was injected into the left ear, and rabbit anti-mouse BP180NC14A monoclonal antibody was injected into the right ear. The injections were repeated every other day for a total of 10 times, with 50 μl injected each time. Obvious erythema, erosion, and scabs were observed on the monoclonal antibody-injected ear, which was significantly thickened and occasionally had small vesicles ( Figure 6 Immunofluorescence detection revealed that small subepidermal blisters appeared on the skin of the anti-BP180 NC14A monoclonal antibody-injected side, accompanied by a decrease in the expression of BP180 antigen protein ( Figure 6 (B) The clinical and pathological manifestations are consistent with BP, indicating that the antibody can successfully induce a BP model in mice.

[0060] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A rabbit hybridoma monoclonal antibody against mouse BP180 NC14A, characterized in that: The monoclonal antibody is specific for the polypeptide encoded by nucleotides 499-573 in Q07563-1, the amino acid sequence of which is shown in SEQ ID No. 1; This monoclonal antibody is secreted and produced by a hybridoma cell line. The hybridoma cell line was deposited in the China Center for Type Culture Collection on April 26, 2022, with the deposit number CCTCC NO: C202296.

2. Use of the rabbit hybridoma monoclonal antibody against mouse BP180 NC14A according to claim 1 in preparing a bullous pemphigoid model.

3. A hybridoma cell line producing the rabbit hybridoma monoclonal antibody against mouse BP180 NC14A according to claim 1, characterized in that: The hybridoma cell line was deposited in the China Center for Type Culture Collection on April 26, 2022, with the deposit number CCTCC NO: C202296.

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