Specific biomarker for granulomatous mastitis as well as preparation method and application of specific biomarker

Abstract

The invention relates to the technical field of disease diagnosis, and provides a specific biomarker for granulomatous mastitis and a preparation method and application thereof, the specific biomarker is nicotinamide phosphoribosyltransferase truncated protein, and the amino acid sequence of the specific biomarker is shown as SEQ ID NO. 1. The NAMPT truncated protein is a specific molecule directly related to the GM pathogenesis. The detection result has high directivity, the positive result supports GM diagnosis, and the negative result is helpful for eliminating the GM diagnosis, so that non-iconography objective evidence is provided for differential diagnosis. GM and breast cancer are clearly distinguished in the early stage of diagnosis, and unnecessary anti-cancer treatment or delay of the real illness state is avoided.

Description

Technical Field

[0001] This application belongs to the field of disease diagnosis technology, and in particular relates to a specific biomarker for granulomatous mastitis, its preparation method and application. Background Technology

[0002] Granulomatous mastitis (GM) is a rare but serious chronic inflammatory disease that affects women’s health. It is characterized by the formation of granulomatous structures and non-necrotizing inflammatory infiltration within the breast tissue.

[0003] The etiology of granulomatous mastitis (GM) is not fully understood, but it may be related to autoimmune reactions, infection, hormonal imbalances, or lactation. Currently, the "gold standard" for GM diagnosis relies on histopathological examination, specifically obtaining a breast tissue sample through core needle biopsy or surgical excision. This method has several inherent limitations. Biopsy is an invasive procedure, posing risks of pain, bleeding, infection, and scarring to the patient. Furthermore, the time from sampling to obtaining a pathology report can take several days, failing to meet the need for rapid diagnosis. Needle biopsy can also lead to false negative results due to the inability to obtain representative lesion tissue. Additionally, the medical costs are relatively high due to the surgical procedure and pathological analysis involved.

[0004] Although the exact cause of GM remains unclear, mounting clinical and pathological evidence, such as its granulomatous inflammatory pathological features, good response to glucocorticoid therapy, and frequent coexistence with other autoimmune diseases, strongly suggests that autoimmune mechanisms play a crucial role in its pathogenesis. In stark contrast to the challenges in GM diagnosis, serological diagnostic methods based on specific autoantibody detection have achieved great success in the clinical practice of many other autoimmune diseases, becoming the cornerstone of their diagnosis, monitoring, and prognosis. This diagnostic approach is a well-established technique familiar to those skilled in the art. For example, the diagnosis of systemic lupus erythematosus relies heavily on screening for antinuclear antibodies (ANA) and the identification of specific antibodies such as anti-double-stranded DNA (dsDNA) antibodies and anti-Sm antibodies. Similarly, the diagnosis of rheumatoid arthritis has become more precise and earlier due to the discovery of rheumatoid factor (RF), especially anti-cyclic citrullinated peptide (CCP) antibodies.

[0005] This diagnostic strategy is also widely used in the field of organ-specific autoimmune diseases. For example, Hashimoto's thyroiditis is diagnosed by detecting anti-thyroid peroxidase (TPO) antibodies; Graves' disease is diagnosed by detecting thyroid-stimulating hormone receptor antibody (TRAb); and primary biliary cholangitis (PBC) is diagnosed by detecting anti-mitochondrial antibodies (AMA). These specific autoantibodies have become irreplaceable serological markers for their respective diseases.

[0006] In the field of neurological autoimmune diseases, the detection of acetylcholine receptor (AChR) antibodies is key to the diagnosis of myasthenia gravis.

[0007] The examples above clearly demonstrate that identifying specific autoantigens associated with particular autoimmune diseases and establishing corresponding serological detection methods for autoantibodies is an effective way to achieve rapid, accurate, and non-invasive diagnosis of such diseases. This allows patients to avoid unnecessary invasive examinations and receive appropriate treatment as early as possible. Summary of the Invention

[0008] The purpose of this application is to provide a specific biomarker for granulomatous mastitis, its preparation method, and its application, aiming to solve the above-mentioned problems existing in the diagnostic methods for granulomatous mastitis.

[0009] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0010] In a first aspect, the present invention provides a specific biomarker for granulomatous mastitis, namely nicotinamide phosphoribosyltransferase truncated somatic protein, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0011] As one possible design, the nucleotide sequence of the gene encoding the truncated protein of the nicotinamide phosphoribosyltransferase is shown in SEQ ID NO. 2.

[0012] In a second aspect, the present invention provides the use of specific biomarkers for granulomatous mastitis in the preparation of tools for detecting and / or diagnosing granulomatous mastitis.

[0013] As one possible design, the tool may include a reagent or kit.

[0014] As one possible design, the application involves detecting and / or analyzing antibodies against nicotinamide phosphoribosyltransferase truncated protein in a biological sample from a subject; the biological sample being serum.

[0015] Thirdly, the present invention provides a method for preparing a specific biomarker for granulomatous mastitis, comprising the following steps:

[0016] S1. The gene sequence plasmid encoding the truncated somatic protein of nicotinamide phosphoribosyltransferase was transferred into competent cells by heat shock and cultured overnight;

[0017] S2. Culture the successfully cloned competent cells overnight at 210-230 rpm and 35-38°C.

[0018] S3. Ferment the bacterial culture obtained from overnight culture in step S2 to obtain bacterial cells, and freeze them overnight at -70~-80℃;

[0019] S4. The bacterial cells frozen overnight are subjected to lysis treatment to obtain inclusion bodies, and the inclusion bodies are then washed and denatured sequentially.

[0020] As one possible design, the modification process in step S4 includes the following steps:

[0021] Denaturing buffer was added to the inclusion bodies, and the mixture was incubated overnight. The supernatant was then obtained by centrifugation. The supernatant was purified sequentially by ion exchange chromatography, Ni-NTA affinity chromatography, and dialysis.

[0022] As one possible design, the gene sequence carries a 6×His affinity tag.

[0023] As one possible design, induction is performed in step S3 using an inducer with a concentration of 0.8~1.5mM.

[0024] As one possible design, the denaturing buffer consists of: 45-55 mM tris(hydroxymethyl)aminomethane, 4.5-5.5 mM ethylenediaminetetraacetic acid, 90-110 mM NaCl, and 7-9 M urea.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. NAMPT truncated somatic protein, as a biomarker, can be detected with just a simple blood sample. This non-invasive testing method eliminates all the physical trauma and complication risks associated with biopsies, greatly improving patient safety and the overall experience.

[0027] 2. NAMPT truncated protein is a specific molecule directly related to the pathogenesis of GM. The test results are highly specific: a positive result supports the diagnosis of GM, while a negative result helps to rule it out, thus providing objective, non-imaging evidence for differential diagnosis. Early differentiation of GM from breast cancer can avoid unnecessary anti-cancer treatment or delays in diagnosis. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1This is a mass spectrometry detection result of 40-55kDa protein samples taken in the embodiments of this application;

[0030] Figure 2 This application provides a pull-down experimental result graph using recombinant antibodies in its embodiments;

[0031] Figure 3 This is an expression profile of NAMPT truncated protein in normal, tumor, Para, and GM biopsy tissues, as described in the embodiments of this application.

[0032] Figure 4 This is a graph showing the content of autoantibodies in blood samples from normal individuals, cancer patients, and patients with granulomatous mastitis, as described in the embodiments of this application. Detailed Implementation

[0033] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0034] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0035] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be a single item or multiple items.

[0036] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0037] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0038] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0039] The term "PET" is an abbreviation for "Polyethylene terephthalate," which stands for polyethylene terephthalate; the term "PU" is an abbreviation for "polyurethane," which stands for polyurethane.

[0040] The following description is based on specific embodiments.

[0041] Example 1

[0042] NAMPT truncated protein expression and purification in prokaryotic systems

[0043] I. Reagent Preparation: 1. Cracked bacterial culture: 50mM Tris, 5mM EDTA, 100mM NaCl, 2mM DTT, 5mM β-mercaptoethanol, lysozyme (0.3g lysozyme / kg bacteria), sodium deoxycholate (4g / kg bacteria)

[0044] 2. Buffer A: 50mM Tris, 5mM EDTA, 100mM NaCl, 2mM DTT, 5mM β-mercaptoethanol;

[0045] 3. Buffer B: 50mM Tris, 5mM EDTA, 10mM DTT, 4M Urea, 1% triton-x100;

[0046] 4. Buffer C: 50mM Tris, 100mM NaCl;

[0047] 5. Denaturing Buffer: 50mM Tris, 5mM EDTA, 100mM NaCl, 8M Urea;

[0048] 2. The NAMPT gene sequence encoding the 6*His affinity tag was cloned into the Ecoli expression vector (pET25b); the nucleotide sequence of the NAMPT gene sequence is shown in SEQ ID NO. 2.

[0049] Day 1: Plasmids were transferred into BL21 competent cells using the heat shock method and cultured overnight on LB plates;

[0050] Day 2: Selected clones were incubated overnight in LB liquid medium with gentle shaking at 37°C and 220 rpm.

[0051] Day 3: Inoculate the seed culture at a ratio of 1:50 into the seed bottle and incubate at 37°C and 220 rpm until the OD value reaches 0.5-0.8; inoculate the fermentation culture at a ratio of 1:100 into the fermentation flask and incubate at 37°C and 220 rpm for 3 hours; add IPTG (induction concentration 1 mM) and incubate for 3 hours; centrifuge at 4°C and 13,000 rpm for 5 minutes to collect the cells; freeze at -80°C overnight.

[0052] Day 4: Take the frozen overnight bacterial cells, add the lysing solution at a ratio of 1:10 (bacterial cells: lysing solution) to break the bacteria and stir for 30 minutes. Sonicate at 650W for 3 seconds, stop for 3 seconds, and repeat for 30 minutes. Centrifuge at 13,000 rpm at 4℃ for 15 minutes and discard the supernatant.

[0053] Wash inclusion bodies. Add Buffer A (bacterial cells: Buffer A = 1:10), disperse the precipitate, and wash on a horizontal shaker for 1 hour; centrifuge at 13,000 rpm for 15 minutes at 4°C, and discard the supernatant; add Buffer B (bacterial cells: Buffer B = 1:10), disperse the precipitate, and wash on a horizontal shaker for 1 hour; add Buffer C (bacterial cells: Buffer C = 1:10), disperse the precipitate, and wash on a horizontal shaker for 1 hour; centrifuge at 13,000 rpm for 15 minutes at 4°C, and discard the supernatant;

[0054] Denatured inclusion bodies. Add denaturing buffer (bacterial cells: buffer = 1:10), disperse the precipitate, and incubate overnight on a horizontal shaker for denaturation;

[0055] Day 5: Centrifuge at 13,000 rpm for 15 min at 4℃, remove precipitate, and collect supernatant;

[0056] DEAE column purification. Column equilibration (20 mM Tris, 8 M urea), supernatant diluted 5-fold and loaded onto the column, eluted with a gradient of 100 mM, 250 mM, 500 mM, and 1 M NaCl, and collected the eluents separately.

[0057] Ni column purification. Column equilibration (20 mM Tris, 8 M urea), DEAE-purified 100 mM and 250 mM NaCl eluents were loaded onto the column, followed by gradient elution with 50 mM, 100 mM, 200 mM, and 500 mM imidazole, and the eluents from each step were collected separately.

[0058] Dialysis refolding. Dialyze with 500mM imidazole eluent using a Urea gradient (6M-4M-2M-1M-0.5M-0.25M-0M), changing the dialysate every 4 hours. Finally, dialyze to 20mM Tris, 5% glycerol.

[0059] 12. Ultrafiltration concentration to obtain NAMPT truncated somatic protein concentrate. The amino acid sequence of NAMPT truncated somatic protein is shown in SEQ ID NO. 1.

[0060] Example 2

[0061] Proteins in the 40-55 kDa range were detected by mass spectrometry. Mass spectrometry is a standard and existing technique in this field, and will not be elaborated upon here. The detection results are as follows: Figure 1 As shown, by Figure 1 It is evident that the most significant difference lies in the truncated form of the NAMPT protein.

[0062] Example 3

[0063] The pull-down experiment was performed using the NAMPT truncated protein obtained in Example 1, as detailed below:

[0064] 1. Take 20-50 mg of quick-frozen tissue sample from liquid nitrogen and lyse it in lysis buffer (Millpore, #20-188) containing protease inhibitors and phosphatase inhibitors. Add lysis buffer to the homogenate at a mass-to-volume ratio (mg:μL) of 1:100. Sonicate the homogenate at 64 Hz for 3-5 cycles, 30 seconds each time.

[0065] 2. Incubate the lysis buffer on ice for 30 minutes to promote protein release. Centrifuge at 12,000 rpm for 15 minutes at 4°C to separate the protein-containing supernatant from the tissue fragments.

[0066] 3. Protein concentration was determined by the BCA method;

[0067] 4. 1 mg of protein and 25 μg of NAMPT truncated protein prepared in Example 1 were immunoprecipitated overnight at 4°C.

[0068] 5. After immunoprecipitation, add streptavidin magnetic beads and incubate for 4 hours, then wash with PBST;

[0069] 6. Elute the magnetic beads with loading buffer or acidic glycine, followed by silver staining to observe the antibody's antigen-capturing specificity. Results are as follows: Figure 2 As shown, by Figure 2 It can be seen that the 40-55kDa pull-down protein shows significant differences in GM and perilesional tissues.

[0070] Example 4

[0071] NAMPT truncated protein was displayed in the Ensembl database. The expression profiles of NAMPT truncated protein in normal, tumor, Para, and GM biopsy tissues were analyzed. Results are as follows: Figure 3 As shown, the results indicate that NAMPT-Tr levels in granulomatous mastitis lesions were higher than those in Para tissues, and the levels in Para tissues were significantly higher than those in tumor or normal tissues.

[0072] Example 5

[0073] ELISA detects the binding of antibodies to NAMPT truncated protein in patients' blood.

[0074] 1. Day 1: Dilute NAMPT truncated protein at a concentration of 10 μg / mL in PBS and coat a 96-well plate overnight at 4°C;

[0075] 2. Day 2: Block with 5% BSA-PBST blocking solution at 37°C for 1 hour, then wash the plate;

[0076] 3. Add blood samples from GM patients, healthy individuals, or breast cancer patients, incubate at 37°C for 1 hour, and wash the plate;

[0077] 4. Add HRP-labeled goat anti-human secondary antibody, react at 37°C for 1 hour, and wash the plate;

[0078] 5. Add 100 μL of TMB, incubate in the dark for 5 minutes, then add 2M concentrated sulfuric acid to terminate the reaction; measure the absorbance at 450 nm using a microplate reader. The results are as follows. Figure 4 As shown, by Figure 4 It was found that, using NAMPT truncated protein to detect the levels of autoantibodies in blood samples from normal individuals, cancer patients, and patients with granulomatous mastitis, the levels of NAMPT truncated protein antibodies were significantly increased in patients with granulomatous mastitis.

[0079] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A specific biomarker for granulomatous mastitis, characterized in that, The specific biomarker is nicotinamide phosphoribosyltransferase truncated somatic protein, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The specific biomarker for granulomatous mastitis according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the truncated protein of nicotinamide phosphoribosyltransferase is shown in SEQ ID NO.

2.

3. The use of the specific biomarker for granulomatous mastitis as described in claim 1 or 2 in the preparation of tools for detecting and / or diagnosing granulomatous mastitis.

4. The application according to claim 3, characterized in that, The tools include reagents or kits.

5. The application according to claim 3, characterized in that, The application involves detecting and / or analyzing antibodies against nicotinamide phosphoribosyltransferase truncated protein in biological samples from subjects; the biological sample is serum.

6. A method for preparing a specific biomarker for granulomatous mastitis as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: S1. The gene sequence plasmid encoding the truncated somatic protein of nicotinamide phosphoribosyltransferase was transferred into competent cells by heat shock and cultured overnight; S2. Culture the successfully cloned competent cells overnight at 210-230 rpm and 35-38°C. S3. Ferment the bacterial culture obtained from overnight culture in step S2 to obtain bacterial cells, and freeze them overnight at -70~-80℃; S4. The bacterial cells frozen overnight are subjected to lysis treatment to obtain inclusion bodies, and the inclusion bodies are then washed and denatured sequentially.

7. The method for preparing specific biomarkers for granulomatous mastitis according to claim 6, characterized in that, The denaturation process in step S4 includes the following steps: Denaturing buffer was added to the inclusion bodies, and the mixture was incubated overnight. The supernatant was then obtained by centrifugation. The supernatant was purified sequentially by ion exchange chromatography, Ni-NTA affinity chromatography, and dialysis.

8. The method for preparing specific biomarkers for granulomatous mastitis according to claim 6, characterized in that, The gene sequence carries a 6×His affinity tag.

9. The method for preparing specific biomarkers for granulomatous mastitis according to claim 6, characterized in that, In step S3, an inducer with a concentration of 0.8~1.5mM is used for induction.

10. The method for preparing specific biomarkers for granulomatous mastitis according to claim 7, characterized in that, The denaturing buffer solution consists of: 45-55 mM tris(hydroxymethyl)aminomethane, 4.5-5.5 mM ethylenediaminetetraacetic acid, 90-110 mM NaCl, and 7-9 M urea.

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