A recombinant protein and its use in the preparation of a product for the auxiliary diagnosis of epidermolysis bullosa acquisita

Abstract

The application provides a kind of recombinant protein and its application in the preparation of auxiliary diagnosis of epidermolysis bullosa acquisita product, relate to the field of biotechnology, including the NC1 domain of epidermolysis bullosa acquisita type VII collagen, the amino acid sequence of the NC1 domain of the type VII collagen is as shown in SEQ ID NO.2, the NC1 domain of epidermolysis bullosa acquisita type VII collagen is used as antigen, and the IgG autoantibody of patient is captured, can be used for indirect method magnetic microparticle chemiluminescence detection, significantly improve detection sensitivity and detection accuracy;The kit prepared by it can be matched with full-automatic chemiluminescence immunoassay instrument, realizes full-automatic operation, reaction is fast, and detection is convenient.The method commonly used in the prior art for detecting VII collagen antibody is immunofluorescence method, which needs manual operation, long reaction time, and the technical problem that the interpretation of fluorescence result needs professional personnel.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant protein and its application in the preparation of products for the auxiliary diagnosis of acquired epidermolysis bullosa. Background Technology

[0002] Acquired epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disease of the skin and mucous membranes, characterized by the presence of IgG autoantibodies against type VII collagen. Clinically, EBA is classified into non-inflammatory (classical) and inflammatory subtypes, with varying degrees of inflammatory infiltration on histopathological basis. The inflammatory pattern of EBA is difficult to distinguish from other pemphigoid diseases clinically and histopathologically, requiring indirect immunofluorescence analysis to differentiate EBA from other pemphigoid diseases by identifying the location of antibody deposition. Antibody deposition on the dermal side indicates EBA; antibody deposition in the epidermal layer indicates other pemphigoid diseases. Antibodies deposited on the dermal side are type VII collagen antibodies. Serological detection of anti-type VII collagen antibodies is simpler and aids in the diagnosis of acquired epidermolysis bullosa, while also differentiating it from other pemphigoid diseases.

[0003] Clinical manifestations: Non-inflammatory EBA presents with clinical changes similar to hereditary epidermolysis bullosa or porphyria cutanea tarda, characterized by tense blisters and erosions on the skin of trauma-prone areas such as the extremities, elbows, and knees, leaving milia, scars, and nail deformities and atrophy after healing. Inflammatory EBA lacks specificity and resembles other autoimmune bullous diseases. The same patient may have both non-inflammatory and inflammatory lesions simultaneously or sequentially; in a few cases, mucosal involvement may occur, most commonly in the oral mucosa.

[0004] Auxiliary examinations: ① Histopathology: Subepidermal vesicles with varying degrees of inflammatory infiltration; inflammatory EBA showed dense infiltration of neutrophils, eosinophils, and lymphocytes; ② Direct immunofluorescence (DIF): IgG and C3 were deposited linearly in the basement membrane zone, appearing as a "U"-shaped serrated appearance under a 600x microscope; ③ Salt crack indirect immunofluorescence (IIF): Serum IgG autoantibodies were deposited on the dermal side; ④ Specific antibody detection: ELISA detected elevated levels of anti-type VII collagen antibodies in serum.

[0005] Currently, there are two methods for detecting type VII collagen antibodies: immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA). The IF method typically involves coating human or animal tissue onto a biological slide, reacting it with patient serum, a negative control, and a positive control, followed by washing. Then, FITC-labeled anti-human IgG antibody (fluorescent secondary antibody) is reacted with the biological slide, followed by washing and mounting. The fluorescent pattern is observed under a fluorescence microscope. The IF method has limitations in tissue acquisition, requires specialized personnel for fluorescence interpretation, and has poor specificity. The ELISA method uses recombinant type VII collagen-coated ELISA plates, reacting them with patient serum and calibrators, followed by washing. Then, horseradish peroxidase (HRP)-labeled anti-human IgG antibody is reacted with the plate, followed by washing, addition of substrate, and incubation at room temperature for 20-30 minutes. Finally, stop solution is added, and the OD value is read. The ELISA method requires manual operation and has a longer reaction time.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant protein and its application in the preparation of products for the auxiliary diagnosis of acquired epidermolysis bullosa, in order to solve the problems of the existing method for detecting VII collagen antibodies, which is immunofluorescence, requires manual operation, has a long reaction time, and requires professional technical personnel to interpret the fluorescence results.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a recombinant protein for the auxiliary diagnosis of acquired epidermolysis bullosa, comprising the NC1 domain of type VII collagen of acquired epidermolysis bullosa, the amino acid sequence of the NC1 domain of said type VII collagen being shown in SEQ ID NO.2.

[0009] In a second aspect, the present invention provides biological materials, including polynucleotides, carriers, or cells; A1. The polynucleotide encodes the recombinant protein described above, and the sequence of the polynucleotide is shown in SEQ ID NO.1; A2. The vector carries the polynucleotide described in A1; A3. The cell carries the polynucleotide described in A1 or the vector described in A2, or expresses the recombinant protein described above.

[0010] Thirdly, the present invention provides a method for preparing the above-mentioned recombinant protein, including culturing the above-mentioned cells; Preferably, the cells are prepared by converting polynucleotides encoding the recombinant protein described above into cells.

[0011] Fourthly, the present invention provides the use of the above-described recombinant protein, the above-described biomaterial, or the recombinant protein prepared by the above-described preparation method in the preparation of products for the auxiliary diagnosis of acquired epidermolysis bullosa.

[0012] Fifthly, the present invention provides an antigen-coated material for assisting in the diagnosis of acquired epidermolysis bullosa, the antigen-coated material comprising magnetic beads and an antigen coated on the magnetic beads, wherein the antigen is the recombinant protein described above or a recombinant protein prepared by the preparation method described above.

[0013] In a sixth aspect, the present invention provides a method for preparing the antigen coating described above, comprising mixing the recombinant protein described above or the recombinant protein prepared by the above method with magnetic beads, and coating the recombinant protein onto the magnetic beads.

[0014] Furthermore, the coating process includes washing and / or activating the magnetic beads prior to the coating. Preferably, the coating process further includes sealing the antigen coating material. Preferably, the washing solution used to wash the magnetic beads before activation is PBS buffer; Preferably, the washing solution used to wash the magnetic beads after activation is MES buffer. Preferably, the buffer solution used in the coating step is MES buffer. Preferably, the pH of the MES buffer solution is 4.75; Preferably, the blocking buffer for blocking the antigen coating is 0.05M Tris buffer with a pH of 7.4; Preferably, the blocking buffer contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300, 0.05-0.2% BRO and 2-5% glycerol; Preferably, the antigen coating is stored in a blocking buffer.

[0015] Furthermore, the magnetic beads include at least one of carboxyl-modified magnetic beads, amino-modified magnetic beads, or toluenesulfonyl-modified magnetic beads, preferably carboxyl-modified magnetic beads; Preferably, during the coating process, 0.2 μg of antigen is used for every 20 μg of magnetic beads.

[0016] In a seventh aspect, the present invention provides a kit for assisting in the diagnosis of acquired epidermolysis bullosa, the kit comprising the antigen coating material described above or reagents for preparing the antigen coating material described above.

[0017] Furthermore, the kit also includes at least one of a sample diluent, an enzyme conjugate, or a calibrator; Preferably, the sample diluent is a 0.05M Tris buffer solution with a pH of 7.4. Preferably, the sample diluent contains 0.85% sodium chloride, 0.02-0.5% EDTA-2K, 1-2% BSA, 0.05-0.5% casein, 0.05-0.2% P300, 0.05-0.2% BRO and 0.05-0.2% Triton X-100.

[0018] Preferably, the enzyme conjugate is a horseradish peroxidase-labeled anti-human IgG antibody; Preferably, the enzyme conjugate dilution solution is 0.05M Tris buffer with a pH of 7.4; Preferably, the enzyme conjugate diluent contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300, 0.05-0.2% BRO and 0.05-0.2% Triton X-100; Preferably, the calibrator diluent is 0.05M Tris buffer with a pH of 7.4; Preferably, the calibrator diluent contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300 and 0.05-0.2% BRO.

[0019] This invention provides a recombinant protein for the auxiliary diagnosis of acquired epidermolysis bullosa. Using the NC1 domain of type VII collagen in acquired epidermolysis bullosa as an antigen, it captures IgG autoantibodies from patients and can be used for indirect magnetic microparticle chemiluminescence detection, significantly improving detection sensitivity and accuracy. The prepared kit for the auxiliary diagnosis of acquired epidermolysis bullosa employs magnetic microparticle chemiluminescence, combining chemiluminescence technology with magnetic microparticle technology. It can be used with fully automated chemiluminescence immunoassay analyzers, achieving fully automated operation, rapid reaction, and convenient detection, enabling antibody detection in a short time. It has the advantages of high sensitivity, strong specificity, and good accuracy, making it highly valuable in clinical applications and suitable for widespread use. It solves the problems of existing methods for detecting type VII collagen antibodies, which commonly use immunofluorescence, requiring manual operation, long reaction time, and technical expertise for interpreting fluorescence results. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 ROC curves for the acquired epidermolysis bullosa kit prepared using different recombinant proteins as antigens, as provided in Example 3 of this invention. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In one aspect, the present invention provides a recombinant protein for the auxiliary diagnosis of acquired epidermolysis bullosa, comprising the NC1 domain of type VII collagen of acquired epidermolysis bullosa, wherein the amino acid sequence of the NC1 domain of type VII collagen is shown in SEQ ID NO.2.

[0026] Using the NC1 domain of type VII collagen in acquired epidermolysis bullosa as an antigen, this method captures patients' IgG autoantibodies and can be used for indirect magnetic microparticle chemiluminescence detection, significantly improving detection sensitivity and accuracy. It solves the problems of existing methods for detecting type VII collagen antibodies, which are mainly immunofluorescence methods, requiring manual operation, long reaction time, and requiring professional technical expertise for interpretation of fluorescence results.

[0027] According to another aspect of the invention, a polynucleotide, a vector, or a cell is also provided; the polynucleotide encodes the recombinant protein described above, and the sequence of the polynucleotide is shown in SEQ ID NO.1; the vector carries the polynucleotide; the cell carries the polynucleotide or the vector, or expresses the recombinant protein described above.

[0028] By linking a polynucleotide encoding a recombinant protein to a vector, the vector is introduced into eukaryotic cells, especially mammalian cells, to construct a cell line capable of expressing the NC1 domain of type VII collagen, thus enabling the expression of the corresponding protein in the cells. Specifically, mammalian cells can be selected from 293 cells or CHO cells.

[0029] According to another aspect of the present invention, a method for preparing the above-mentioned recombinant protein is also provided, comprising culturing the above-mentioned cells.

[0030] In some specific embodiments, the cells are prepared by converting polynucleotides encoding the recombinant protein into cells. In some specific embodiments, the recombinant protein is further purified.

[0031] According to another aspect of the present invention, the use of the above-described recombinant protein, the above-described biomaterial, or the recombinant protein prepared by the above-described preparation method in the preparation of products for the auxiliary diagnosis of acquired epidermolysis bullosa is also provided.

[0032] According to another aspect of the present invention, an antigen-coated material for assisting in the diagnosis of acquired epidermolysis bullosa is also provided, the antigen-coated material comprising magnetic beads and an antigen coated on the magnetic beads, wherein the antigen is the recombinant protein described above or a recombinant protein prepared by the preparation method described above.

[0033] According to another aspect of the present invention, a method for preparing the antigen coating described above is also provided, comprising mixing the recombinant protein described above or the recombinant protein prepared by the above method with magnetic beads, and coating the recombinant protein onto the magnetic beads.

[0034] In some specific embodiments, the coating process includes washing and / or activating the magnetic beads; in some specific embodiments, the washing solution for washing the magnetic beads before activation is PBS buffer; in some specific embodiments, the washing solution for washing the magnetic beads after activation is MES buffer.

[0035] In some specific embodiments, the magnetic beads include at least one of carboxyl-modified, amino-modified, or toluenesulfonyl-modified magnetic beads, preferably carboxyl-modified. In some specific embodiments, the buffer solution in the coating step is a MES buffer. In some specific embodiments, the pH of the MES buffer is 4.75. Ethanolamine is used to terminate the coating process, achieving a highly stable solid-phase interface with low nonspecific binding.

[0036] In some specific embodiments, the coating process further includes sealing the antigen coating.

[0037] In some specific embodiments, the blocking buffer for blocking the antigen coating is 0.05M Tris buffer with a pH of 7.4; in some specific embodiments, the blocking buffer contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300, 0.05-0.2% BRO and 2-5% glycerol.

[0038] In some specific embodiments, the antigen coating is stored in a blocking buffer. The final product is a magnetic microparticle suspension, which can be used for subsequent chemiluminescent immunoassay.

[0039] In some specific implementations, during the coating process, 0.2 μg of antigen is used for every 20 μg of magnetic beads.

[0040] According to another aspect of the present invention, a kit for assisting in the diagnosis of acquired epidermolysis bullosa is also provided, the kit comprising the above-described antigen coating or reagents for preparing the above-described antigen coating.

[0041] This invention's reagent kit employs magnetic microparticle chemiluminescence, combining chemiluminescence technology with magnetic microparticle technology. It can be used with fully automated chemiluminescence immunoassay analyzers, enabling fully automated operation, rapid reaction, and convenient detection. It can detect antibodies in a short time. With advantages of high sensitivity, strong specificity, and good accuracy, it has high clinical value and is suitable for widespread application.

[0042] In some specific embodiments, the kit further includes at least one of a sample diluent, an enzyme conjugate, or a calibrator.

[0043] In some specific embodiments, the sample diluent is a 0.05M Tris buffer solution with a pH of 7.4; in some specific embodiments, the sample diluent contains 0.85% sodium chloride, 0.02-0.5% EDTA-2K, 1-2% BSA, 0.05-0.5% casein, 0.05-0.2% P300, 0.05-0.2% BRO, and 0.05-0.2% Triton X-100.

[0044] The enzyme conjugate may be, but is not limited to, alkaline phosphatase or horseradish peroxidase. In some specific embodiments, the enzyme conjugate is a horseradish peroxidase-labeled anti-human IgG antibody.

[0045] The anti-human IgG antibody is derived from at least one of the following species: mouse, rat, guinea pig, hamster, rabbit, goat, sheep, dairy cow, and pig.

[0046] In some specific embodiments, the enzyme conjugate diluent is a 0.05M Tris buffer at pH 7.4. In some specific embodiments, the enzyme conjugate diluent contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300, 0.05-0.2% BRO, and 0.05-0.2% Triton X-100.

[0047] In some specific embodiments, the calibrator diluent is a 0.05M Tris buffer solution with a pH of 7.4; in some specific embodiments, the calibrator diluent contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300 and 0.05-0.2% BRO.

[0048] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0049] The magnetic microparticle coating general buffer A is a 0.01M PBS buffer: 0.29g sodium dihydrogen phosphate, 2.89g disodium hydrogen phosphate, and 8.5g sodium chloride, dissolved in 1L deionized water, stirred well, and stored at 2~8℃.

[0050] The magnetic microparticle coating universal buffer M is a 0.05M MES buffer with a pH of 4.75: 10.66g of 2-(N-morpholine)ethanesulfonic acid, dissolved in 1L of deionized water, adjusted to pH 4.75 with NaOH, stirred well, and stored at 2~8℃.

[0051] The blocking buffer is a 0.05M Tris buffer with a pH of 7.4: 6.06g of tris(hydroxymethyl)aminomethane and 8.5g of sodium chloride are dissolved in 1L of deionized water and stirred until homogeneous. The pH is then adjusted to 7.4 with concentrated hydrochloric acid. After that, 2% BSA, 0.1% P300, 0.05% BRO, and 5% glycerol are added and stirred until homogeneous. The solution is then stored at 2-8℃.

[0052] The enzyme conjugate dilution solution was a 0.05M Tris buffer solution with a pH of 7.4: 6.06g of tris(hydroxymethyl)aminomethane and 8.5g of sodium chloride were dissolved in 1L of deionized water and stirred until homogeneous. The pH was then adjusted to 7.4 with concentrated hydrochloric acid. After that, 2% BSA, 0.1% P300, 0.05% BRO, and 0.1% Triton X-100 were added and stirred until homogeneous. The solution was then stored at 2-8℃.

[0053] The sample dilution solution was a 0.05M Tris buffer solution with a pH of 7.4: 6.06g tris(hydroxymethyl)aminomethane, 8.5g sodium chloride, and 0.05% EDTA-2K were dissolved in 1L of deionized water and stirred until homogeneous. The pH was then adjusted to 7.4 using concentrated hydrochloric acid. After that, 2% BSA, 0.2% casein, 0.1% P300, 0.05% BRO, and 0.2% Triton X-100 were added and stirred until homogeneous. The solution was then stored at 2–8°C.

[0054] The calibrator diluent is a 0.05M Tris buffer solution with a pH of 7.4: 6.06g of tris(hydroxymethyl)aminomethane and 8.5g of sodium chloride are dissolved in 1L of deionized water. After stirring well, the pH is adjusted to 7.4 with concentrated hydrochloric acid. Then, 2% BSA, 0.1% P300, and 0.05% BRO are added, stirred well, and stored at 2-8℃.

[0055] Example 1: Eukaryotic recombinant VII collagen NC1 domain 1. Construction of eukaryotic expression vectors The amino acid sequence of the VII-NC1 protein is amino acids 17-1253 of type VII collagen (as shown in SEQ ID NO.2), and the nucleotide sequence of its encoding gene is nucleotides 49-3759 of type VII collagen (as shown in SEQ ID NO.1). The synthesized nucleotide sequence was inserted into the pCMV expression vector, resulting in a recombinant vector named pCMV-VII-NC1. Co-expression of the VII-NC1 protein and the HIS tag on the vector was achieved, yielding recombinant VII-NC1-HIS.

[0056] The recombinant vector pCMV-VII-NC1 was transfected into E. coli to obtain recombinant bacteria. The plasmid of the recombinant bacteria was extracted and sent for sequencing. If the sequencing was correct, it was the pCMV-VII-NC1 recombinant plasmid.

[0057] 2. Recombinant VII-NC1 expression HEK293 cells were transiently transfected with the pCMV-VII-NC1 recombinant plasmid and cultured in DMEM containing fetal bovine serum at 37°C in a 5% CO2 / 95% air incubator. Cells were digested with trypsin and collected. Lysis buffer containing protease inhibitors (20 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 7.5) was added, and cells were lysed by sonication. The cells were then centrifuged at 12000 g for 30 min at 4°C, and the supernatant was collected.

[0058] 3. Purify VII-NC1 protein This embodiment employs affinity chromatography to purify the recombinant protein. The supernatant was passed through an equilibrated nickel column (Ni-NTA agarose gel) for affinity chromatography. The recombinant VII-NC1-HIS protein bound to nickel ions and adhered to the column, while other contaminating proteins were washed away. The target protein was eluted using elution buffer containing 200 mM imidazole (50 mM PBS, 150 mM NaCl, pH 8.0, 200 mM imidazole). The eluted protein was then dialyzed to remove the imidazole, and 20% glycerol was added to protect the protein. Protein concentration was determined by UV spectrophotometry. The recombinant VII-NC1-HIS protein was subjected to SDS-PAGE electrophoresis, transferred to a membrane, and incubated with horseradish peroxidase (HRP)-labeled HIS antibody. Immunoblotting showed a band approximately 145 kDa in size.

[0059] 4. Using the same method, construct the VII-NC1-N and VII-NC1-C recombinant expression vectors, express them in eukaryotic HEK293 cells, and obtain the recombinant expressed protein.

[0060] 5. The amino acid sequence of the recombinant VII-NC1-N is amino acid 17-472 of type VII collagen (amino acid 1-456 in SEQ ID NO.2), and the nucleotide sequence of its encoding gene is nucleotide 49-1416 of type VII collagen (nucleotide 1-1368 in SEQ ID NO.1).

[0061] 6. The amino acid sequence of recombinant VII-NC1-C is amino acid 500-1250 of type VII collagen (amino acid 484-1234 in SEQ ID NO.2), and the nucleotide sequence of its encoding gene is nucleotide 1498-3750 of type VII collagen (nucleotide 1450-3702 in SEQ ID NO.1).

[0062] Example 2: Auxiliary Diagnostic Kit for Acquired Epidermolysis Bullosa The kit is an indirect magnetic microparticle chemiluminescence kit, which includes magnetic microparticle suspension, enzyme conjugate, sample diluent and calibrator.

[0063] 1. Preparation of magnetic microparticle suspension The recombinant proteins VII-NC1-HIS, VII-NC1-N, and VII-NC1-C prepared in Example 1 were coupled to magnetic microparticles, specifically according to the following steps: (1) Washing: Take 60mg of magnetic microparticles with carboxyl groups on the surface, use 10ml of magnetic microparticle-coated general buffer A for shaking and washing for 5 minutes, wash twice, use a magnetic rack for separation, and discard the supernatant.

[0064] (2) Activation: Weigh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide, dissolve them in magnetic microparticle coating buffer M, prepare a solution with a final concentration of 20 mg / ml, add 3 ml of each to the washed magnetic microparticles, mix well and shake at room temperature for 1 hour to obtain an activated magnetic microparticle suspension.

[0065] (3) Washing after activation: Use a magnetic rack for separation, discard the supernatant, add 10 ml of magnetic microparticle-coated general buffer M, wash for 10 min, and then use a magnetic rack for separation again, discarding the supernatant.

[0066] (4) Coating: First, add 4.5 ml of magnetic microparticle coating buffer M to the magnetic microparticles, shake the magnetic microparticles, add 1.5 ml of recombinant protein (concentration of 0.4 mg / ml), mix and react at room temperature for 2 hours, and then use a magnetic rack to separate again.

[0067] (5) Termination: Add 10 ml of diluted ethanolamine solution (dilute ethanolamine 16 times with microparticle-coated general buffer A, prepare fresh before use) to the magnetic microparticles, shake at room temperature for 30 minutes, and then separate using a magnetic rack.

[0068] (6) Blocking: Add 10 ml of blocking buffer, shake and mix at room temperature for 10 min, then use a magnetic rack for separation, and block a total of 4 times.

[0069] (7) Volume adjustment: Add 60ml of blocking buffer, shake well the magnetic microparticles, dispense into special magnetic microparticle bottles, and store at 2~8℃ for later use.

[0070] 2. Preparation of enzyme conjugates HRP-labeled mouse anti-human IgG antibody was added to the enzyme conjugate diluent at a ratio of 1 / 5000, stirred evenly, and then dispensed into enzyme conjugate vials and stored at 2-8℃.

[0071] 3. Preparation of sample diluent Dispense the above sample dilution into special bottles and store at 2~8℃.

[0072] 4. Preparation of calibrators The clinically high values ​​of the VII collagen antibody were diluted with calibrator diluent at a certain ratio to make the calibrator concentrations approximately 0 AU / ml, 20 AU / ml, 50 AU / ml, 100 AU / ml, 200 AU / ml and 400 AU / ml; and stored at 2~8℃.

[0073] Example 3: On-machine testing The reagent kit prepared in Example 2 was used for instrumental testing.

[0074] 1. Load the reagents into the matching fully automated chemiluminescence instrument and perform calibration first. After checking the calibration results and the instrument indicating successful calibration, test serum samples from 30 EBA patients and 50 normal volunteers. After the experiment, the results are shown in Table 1.

[0075] 2. Plot ROC curves using SPSS software. like Figure 1 As shown, VII-NC1 (NC1 for short) has an AUC area of ​​0.855 (95% CI: 0.762–0.947), a sensitivity of 63.3% (19 / 30), and a specificity of 98% (49 / 50). VII-NC1-N (NC1-N for short) has an AUC area of ​​0.623 (95% CI: 0.493–0.752), a sensitivity of 26.7% (8 / 30), and a specificity of 100% (50 / 50). VII-NC1-C (NC1-C for short) has an AUC area of ​​0.702 (95% CI: 0.584–0.82), a sensitivity of 30.0% (9 / 30), and a specificity of 100% (50 / 50).

[0076] Data shows that VII-NC1 has the highest sensitivity and the largest AUC area. Therefore, VII-NC1 is used as a biomarker for the detection of acquired epidermolysis bullosa.

[0077] Table 1

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant protein for the auxiliary diagnosis of acquired epidermolysis bullosa, characterized in that, The invention includes the NC1 domain of type VII collagen in acquired epidermolysis bullosa, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A biomaterial, characterized in that, Including polynucleotides, carriers, or cells; A1. The polynucleotide encodes the recombinant protein of claim 1, and the sequence of the polynucleotide is shown in SEQ ID NO. 1; A2. The vector carries the polynucleotide described in A1; A3. The cell carries the polynucleotide described in A1 or the vector described in A2, or expresses the recombinant protein described in claim 1.

3. The method for preparing the recombinant protein according to claim 1, characterized in that, Includes culturing the cells as described in claim 2; Preferably, the cells are prepared by converting a polynucleotide encoding the recombinant protein of claim 1 into cells.

4. The use of the recombinant protein of claim 1, the biomaterial of claim 2, or the recombinant protein prepared by the preparation method of claim 3 in the preparation of products for the auxiliary diagnosis of acquired epidermolysis bullosa.

5. An antigen-coated material for the auxiliary diagnosis of acquired epidermolysis bullosa, characterized in that, The antigen coating comprises magnetic beads and antigen coated on the magnetic beads, wherein the antigen is the recombinant protein of claim 1 or the recombinant protein prepared by the preparation method of claim 3.

6. The method for preparing the antigen coating material according to claim 5, characterized in that, This includes mixing the recombinant protein of claim 1 or the recombinant protein prepared by the preparation method of claim 3 with magnetic beads, thereby coating the recombinant protein onto the magnetic beads.

7. The preparation method according to claim 6, characterized in that, The coating process also includes washing and / or activating the magnetic beads; Preferably, the coating process further includes sealing the antigen coating. Preferably, the washing solution used to wash the magnetic beads before activation is PBS buffer; Preferably, the washing solution used to wash the magnetic beads after activation is MES buffer. Preferably, the buffer solution used in the coating step is MES buffer. Preferably, the pH of the MES buffer solution is 4.75; Preferably, the blocking buffer for blocking the antigen coating is 0.05M Tris buffer with a pH of 7.4; Preferably, the blocking buffer contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300, 0.05-0.2% BRO and 2-5% glycerol; Preferably, the antigen coating is stored in a blocking buffer.

8. The preparation method according to claim 6, characterized in that, The magnetic beads include at least one of carboxyl-modified magnetic beads, amino-modified magnetic beads, or toluenesulfonyl-modified magnetic beads, preferably carboxyl-modified magnetic beads. Preferably, during the coating process, 0.2 μg of antigen is used for every 20 μg of magnetic beads.

9. A kit for the auxiliary diagnosis of acquired epidermolysis bullosa, characterized in that, The kit contains the antigen coating as described in claim 5 or a reagent for preparing the antigen coating as described in claim 5.

10. The reagent kit according to claim 9, characterized in that, The kit also includes at least one of a sample diluent, an enzyme conjugate, or a calibrator; Preferably, the sample diluent is a 0.05M Tris buffer solution with a pH of 7.

4. Preferably, the sample diluent contains 0.85% sodium chloride, 0.02-0.5% EDTA-2K, 1-2% BSA, 0.05-0.5% casein, 0.05-0.2% P300, 0.05-0.2% BRO, and 0.05-0.2% Triton X-100; Preferably, the enzyme conjugate is a horseradish peroxidase-labeled anti-human IgG antibody; Preferably, the enzyme conjugate dilution solution is 0.05M Tris buffer with a pH of 7.4; Preferably, the enzyme conjugate diluent contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300, 0.05-0.2% BRO and 0.05-0.2% Triton X-100; Preferably, the calibrator diluent is 0.05M Tris buffer with a pH of 7.4; Preferably, the calibrator diluent contains 0.85% sodium chloride, 1-2% BSA, 0.05-0.2% P300 and 0.05-0.2% BRO.

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