Application of protein marker in preparation of detection product for detecting inflammatory bowel disease

Abstract

The invention provides application of a protein marker in preparation of a detection product for detecting inflammatory bowel diseases. Wherein the protein marker is selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA. Experiments prove that the citrulline levels of the 12 proteins are highly associated with the inflammatory bowel disease, so that one or more of the 12 proteins can be used as protein markers to prepare detection products (such as detection or diagnosis kits and the like) for detecting the inflammatory bowel disease. A detection product based on the protein marker is beneficial to early detection and non-invasive detection, and is rapid, convenient and accurate.

Description

Technical Field

[0001] The present application relates to the field of inflammatory bowel disease identification, and more specifically, to the use of a protein marker in the preparation of a detection product for detecting inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of chronic idiopathic intestinal inflammatory disease with unknown etiology, mainly including ulcerative colitis (UC) and Crohn's disease. IBD recurs repeatedly during the course of the disease, leading to frequent hospitalization and severe decline in the quality of life of patients. The resulting interruption of education, sick leave and heavy medical burden also have a huge impact on social development. Nowadays, the diagnosis of IBD mostly relies on clinical manifestations and endoscopic examinations, and patients are often in the active stage of the disease when they are discovered. Dextran sulfatesodium salt (DSS)-induced colitis mice are a classic animal model of IBD and play an important role in the exploration of its pathogenesis, diagnosis and treatment.

[0003] Recently, the study of the interaction mechanism between neutrophils and the intestinal epithelial barrier has gradually become a research hotspot in the field of ulcerative colitis. Activated neutrophils can release depolymerized chromatin to form a DNA mesh scaffold to surround and restrict pathogens. This structure is called neutrophil extracellular traps (NETs), and this process is called neutrophil extracellular trap-osis (NETosis). Although the specific mechanism and functional regulation of NETosis in neutrophils are still unclear, it is generally believed that peptidyl arginine deiminase 4 (PAD4)-mediated citrullination modification is a key link in the formation of NETosis. Many studies have confirmed that PAD4-dependent NETosis is involved in the occurrence and development of ulcerative colitis.

[0004] As a post-translational modification of proteins, citrullination is currently considered to be an irreversible modification process, mainly in which arginine residues on proteins are converted to citrulline by PAD-catalyzed de-imino groups. Citrulline is not one of the 20 main amino acids encoded by DNA, and it only exists in proteins after arginine is deaminated. The chemical mechanism of the deimination reaction is based on the process in which the ketimine group is replaced by a keto group, causing arginine to lose its positive charge and produce ammonia. Unlike positively charged arginine, citrulline is electrically neutral. The loss of positive charge caused by the citrullination process can lead to electrostatic and conformational changes in the modified protein, ultimately changing the protein binding site, protein-protein interaction, and protein degradation susceptibility.

[0005] Currently, there is no solution for molecular markers and related applications of inflammatory bowel disease. Summary of the invention

[0006] The main purpose of the present application is to provide an application of a protein marker in the preparation of a detection product for detecting inflammatory bowel disease, so as to solve the problem that there is no molecular level detection of such disease conditions in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to one aspect of the present application, a protein marker is provided for use in the preparation of a detection product for detecting inflammatory bowel disease, and the protein marker is selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

[0008] Furthermore, the application includes detecting one of the following indicators of one or more protein markers: 1) the citrullination level of the corresponding protein; 2) the expression level of the corresponding protein.

[0009] Furthermore, the citrullination sites of the proteins corresponding to the one or more protein markers are: MRPL43R85, ECHS1 R178, TLN2 R59, ENO3 R372, NCSTN R354, CTSH R315, CKMT1 R243, TUBA4AR221, DYNC1H1 R3158, PDLIM3 R164, GLB1 R592 and PMPCA R267.

[0010] According to the second aspect of the present application, a kit for detecting inflammatory bowel disease is provided, which includes a detection reagent for a protein marker, and the protein marker is selected from any one or more of the following molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

[0011] Furthermore, the detection reagent is a related reagent for detecting one of the following indicators of the protein marker: 1) the corresponding protein expression level; 2) the citrullination level of the corresponding protein; preferably, the detection reagent includes an antibody; preferably, the antibody is arranged on a solid phase carrier; preferably, the solid phase carrier is selected from an ELISA plate, a membrane carrier or a microsphere, and further preferably, the membrane carrier is selected from a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; preferably, the antibody is a monoclonal antibody or a polyclonal antibody; preferably, the kit is an ELISA kit, an immunofluorescence kit or an immunocolloidal gold kit.

[0012] According to the third aspect of the present application, a device for detecting inflammatory bowel disease is provided, the device having a built-in detection element for protein markers, wherein the protein markers are selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

[0013] Further, the detection element is a related element for detecting one of the following indicators of the protein marker: 1) the citrullination level of the corresponding protein; 2) the corresponding protein expression level; preferably, the detection element includes an antibody; preferably, the antibody is disposed on a solid phase carrier; preferably, the solid phase carrier is selected from an ELISA plate, a membrane carrier or a microsphere, and further preferably, the membrane carrier is selected from a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0014] Furthermore, the device also includes a comparison module and an early warning module. The comparison module is used to compare the numerical value corresponding to the indicator of the protein marker obtained by the detection element with the internal threshold; the early warning module is used to display a warning signal when the comparison result of the comparison module exceeds the threshold; preferably, the internal threshold includes a protein level threshold or a citrullination level threshold.

[0015] According to the fourth aspect of the present application, a method for screening protein markers of inflammatory bowel disease is provided, the screening method comprising: mass spectrometry detection of proteins with significantly increased citrulline levels in the proteome of a wild-type inflammatory bowel disease simulation group compared with a healthy group, to obtain a first differential protein set; mass spectrometry detection of proteins with significantly reduced citrulline levels in a PAD4 gene knockout inflammatory bowel disease simulation group compared with a wild-type inflammatory bowel disease simulation group, to obtain a second differential protein set; taking the intersection of the first differential protein set and the second differential protein set to obtain protein markers of inflammatory bowel disease.

[0016] Furthermore, the wild-type inflammatory bowel disease modeling group is DSS-induced wild-type colitis mice.

[0017] By applying the technical solution of the present application, experiments have proven that the citrulline levels of the above 12 proteins are highly correlated with inflammatory bowel disease, so one or more of them can be used as protein markers to prepare detection products (such as detection or diagnostic kits, etc.) for detecting inflammatory bowel disease. Detection products based on the above protein markers help to achieve early detection and non-invasive detection, which is fast, convenient and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings constituting part of the present application are used to provide a further understanding of the present application. The exemplary embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0019] Figure 1 The results of citrullination levels of 12 differentially expressed proteins in intestinal tissues of normal mice and DSS colitis mice in Example 1 of the present application are shown;

[0020] Figure 2 The protein level results of 12 differentially expressed proteins in the intestinal tissues of normal mice and DSS colitis mice in Example 1 of the present application are shown;

[0021] Figure 3 The results of CKMT1 protein levels in intestinal tissues of normal mice and DSS colitis mice in Example 2 of the present application are shown;

[0022] Figure 4 The colitis phenotype of CKMT1 intestinal epithelial-specific knockout mice according to Example 2 of the present application is shown;

[0023] Figure 5 The CTSH protein levels in the intestinal tissues of normal mice and DSS colitis mice in Example 3 of the present application are shown;

[0024] Figure 6 The colitis phenotype of CTSH knockout mice in Example 3 of the present application is shown;

[0025] Figure 7 The NCSTN protein levels in intestinal tissues of normal mice and DSS colitis mice according to Example 4 of the present application are shown. DETAILED DESCRIPTION

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below in conjunction with the embodiments.

[0027] Terminology explanation:

[0028] Protein marker: This application refers to an isolated marker protein that can specifically mark a disease, the development process or stage of a disease.

[0029] Protein: In this application, protein and protein have the same meaning and can be used interchangeably.

[0030] As mentioned in the background technology, the existing detection of inflammatory bowel disease still relies on clinical manifestations and endoscopic examinations, and cannot be discovered and treated early. Therefore, in this application, the applicant attempts to find relevant molecular markers from the molecular mechanism of the pathogenesis of inflammatory bowel disease, in order to find molecular markers that can mark the occurrence and development of inflammatory bowel disease, and provide a theoretical basis and basis for the early detection of such diseases. Specifically, this application has found 12 protein molecules that are significantly related to inflammatory bowel disease through proteomic analysis, and further confirmed the role of related protein molecules in the onset of inflammatory bowel disease through experiments. Based on this, the applicant has proposed a series of protection schemes for this application.

[0031] In a typical embodiment of the present application, a protein marker is provided for use in the preparation of a detection product for detecting inflammatory bowel disease, and the protein marker is selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

[0032] As mentioned above, the present application has experimentally proved that the citrulline levels of the above 12 proteins are highly correlated with inflammatory bowel disease, so one or more of them can be used as protein markers to prepare detection products (such as detection or diagnostic kits, etc.) for detecting inflammatory bowel disease. Detection products based on the above protein markers help to achieve early detection and non-invasive detection, which is fast, convenient and accurate.

[0033] It should be noted that the above protein marker molecules can be used alone or in combination. Specifically, when used in combination, they can be used in any combination of 2-12 protein markers, such as in a combination of two, three, four, five, six, seven, and so on.

[0034] In a preferred embodiment, the above application includes detecting one of the following indicators of one or more protein markers: 1) the citrullination level of the corresponding protein; 2) the corresponding protein expression level; preferably, it is recommended to detect the citrullination level of the corresponding protein.

[0035] It should be noted that the citrullination level of the protein marker indicates the deimino level of arginine on the corresponding protein, which can more accurately indicate inflammatory bowel disease, especially ulcerative colitis. The higher the citrullination level, the greater the possibility of inflammatory bowel disease. In addition, some experiments in this application also confirmed that the lower the protein expression level of some protein markers, the easier it is to show symptoms of inflammatory bowel disease. It should be noted here that not all protein markers have significant differences in the change levels of protein.

[0036] The above protein markers all contain arginine, and it is the presence of arginine that causes the citrullination of the corresponding proteins. In the application of the present application, in order to further accurately use protein markers for detection, in a preferred embodiment, the citrullination sites of the proteins corresponding to one or more protein markers are: MRPL43 R85, ECHS1 R178, TLN2R59, ENO3 R372, NCSTN R354, CTSH R315, CKMT1 R243, TUBA4A R221, DYNC1H1 R3158, PDLIM3 R164, GLB1 R592 and PMPCA R267.

[0037] It should be noted that not all arginine sites will undergo citrullination, and the sites where citrullination occurs are structurally specific (determined by mass spectrometry), and most proteins are mainly citrullinated at some sites. In view of the fact that there are no citrullinated antibodies targeting specific sites on the market, therefore, in the art, citrullinated pan-antibodies are generally used to prove that the citrullination level of the protein increases by protein immunoblotting or immunohistochemical staining. This application also uses this method to identify changes in citrullination levels.

[0038] In a second typical embodiment of the present application, a kit for detecting inflammatory bowel disease is provided, the kit comprising a detection reagent for a protein marker, wherein the protein marker is selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA. The detection reagent for the above protein markers can predict the presence or absence of inflammatory bowel disease by detecting the corresponding protein level or its citrullination level, so the detection kit of the present application is more convenient and faster than the previous detection means.

[0039] Any reagent capable of detecting the above protein markers is applicable to the present application. Specifically, the detection reagents are different according to the characteristics of the detected protein markers. In a preferred embodiment, the detection reagent is a related reagent for detecting one of the following indicators of the protein marker: 1) the citrullination level of the corresponding protein; 2) the expression level of the corresponding protein.

[0040] It should be noted that the detection reagent for detecting the protein expression level of the protein marker can be all existing related reagents for detecting proteins, such as antibodies, etc. The related reagent for detecting the citrullination level of the protein marker can be an anti-citrullination antibody, such as the Anti-Citrulline antibody of Abcam (Cat. No.: ab40908) or the Anti-peptidyl-citrulline (Cat. No.: 07-377) or Anti-Citrulline Antibody (Cat. No.: MABN328) of Merck, etc.

[0041] It should be noted that the specific components contained in the kit are different depending on the structural form of the specific kit. In a preferred embodiment, the detection reagent includes an antibody; preferably, the antibody is disposed on a solid phase carrier; preferably, the solid phase carrier is selected from an ELISA plate, a membrane carrier or a microsphere, and further preferably, the membrane carrier is selected from a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; preferably, the antibody is a monoclonal antibody or a polyclonal antibody; preferably, the kit is an ELISA kit, an immunofluorescence kit or an immunocolloidal gold kit. The kit of the present application is designed into these conventional forms of kits according to the reagent requirements for ease of use and detection.

[0042] In addition to the aforementioned test products in the form of kits, other forms of test products can also be designed using the protein markers of the present application. In a third typical embodiment of the present application, a device for detecting inflammatory bowel disease is provided, the device having a built-in detection element for a protein marker, the protein marker being selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

[0043] According to the progress of technology or clinical needs, the detection product of the present application can be set as an electronic detection device that can automatically detect or automatically output the status of inflammatory bowel disease, as long as the detection element for detecting the protein marker of the present application is built into the device. The device helps to further improve the convenience of detection.

[0044] In a preferred embodiment, the detection element is a related element for detecting one of the following indicators of the protein marker: 1) the citrullination level of the corresponding protein; 2) the expression level of the corresponding protein. The above preferred detection indicators help to more accurately indicate the onset of inflammatory bowel disease.

[0045] In a preferred embodiment, the detection element includes an antibody; preferably, the antibody is disposed on a solid phase carrier; preferably, the solid phase carrier is selected from an ELISA plate, a membrane carrier or a microsphere, and further preferably, the membrane carrier is selected from a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0046] In the above embodiment, the detection element is set in the above form, which is convenient for design, operation and implementation.

[0047] In order to further enhance the user experience of the detection device, in a preferred embodiment, the device also includes a comparison module and an early warning module. The comparison module is used to compare the numerical value corresponding to the indicator of the protein marker obtained by the detection element with the internal threshold value; the early warning module is used to display a warning signal when the comparison result of the comparison module exceeds the threshold value; preferably, the internal threshold value includes a protein level threshold value or a citrullination level threshold value.

[0048] In a fourth typical embodiment of the present application, a method for screening protein markers of inflammatory bowel disease is provided, the screening method comprising: detecting by mass spectrometry the proteins with significantly increased citrulline levels in the proteome of a wild-type inflammatory bowel disease simulation group compared with that in a healthy group, to obtain a first differential protein set; detecting by mass spectrometry the proteins with significantly decreased citrulline levels in a PAD4 gene knockout inflammatory bowel disease simulation group compared with that in a wild-type inflammatory bowel disease simulation group, to obtain a second differential protein set; taking the intersection of the first differential protein set and the second differential protein set to obtain protein markers of inflammatory bowel disease.

[0049] In a preferred embodiment, the wild-type inflammatory bowel disease modeling population is DSS-induced wild-type colitis mice.

[0050] The above screening method of the present application uses wild-type colitis mice induced by DSS as a classic animal model of IBD to study proteins with significantly increased citrulline levels in wild-type colitis mice drinking DSS compared to wild-type mice drinking water. The PAD4 gene in wild-type colitis mice with DSS is further knocked out, even if the activity of citrullinated peptidyl arginine deiminase 4 is reduced or inactivated, and then proteins with significantly increased citrullination levels are screened. Proteins with significant changes in both methods are selected as protein markers of the present application. The experiments of the present application also confirm to a certain extent that these proteins are involved in the formation and characterization of inflammatory bowel disease.

[0051] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0052] It should be noted that the samples in the following examples are mouse colon samples, which are derived from C57 / BL6 mice.

[0053] Embodiment 1:

[0054] (I) Protein sample preparation

[0055] The sample was ground into cell powder with liquid nitrogen and then transferred to a 5 ml centrifuge tube. Four volumes of lysis buffer (8 M urea, 1% protease inhibitor) were added, and then sonicated on ice for three minutes using a high-intensity ultrasonic processor (Scientz). Centrifuged at 12,000 g for 10 minutes at 4°C, the precipitate was removed, and the supernatant was collected.

[0056] Take an equal amount of each sample protein for enzymatic hydrolysis, and adjust the volume to the same with lysis buffer. Slowly add TCA at a final concentration of 20%, vortex to mix, and precipitate at 4°C for 2h. Centrifuge at 4500g for 5min, discard the supernatant, and wash the precipitate with pre-cooled acetone 2-3 times. After drying the precipitate, add TEAB at a final concentration of 200mM, ultrasonically break up the precipitate, add trypsin at a ratio of 1:50 (protease: protein, m / m), and enzymatically hydrolyze overnight. Add dithiothreitol (DTT) to a final concentration of 5mM and reduce at 56°C for 30min. Then add iodoacetamide (IAA) to a final concentration of 11mM and incubate at room temperature in the dark for 15min.

[0057] (II) Mass spectrometry detection

[0058] The digested peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile / water) and directly loaded onto a homemade reverse phase analytical column (25 cm long, 100 μm id). On an EASY-nLC 1200UPLC system (Thermo Fisher Scientific), the peptides were separated in 68 minutes using solvent B (0.1% formic acid, 90% acetonitrile) at a constant flow rate of 550 nL / min, from 6% to 23% in 16 minutes, rising to 80% in 3 minutes, and then kept at 80% for the last 3 minutes.

[0059] The separated peptides were analyzed in an Exploris 480TM (Thermo Fisher Scientific) using a nano-electrospray ion source. The electrospray voltage was 2.3 kV and the compensation voltages were -45 V and -65 V. The full MS scan resolution was set to 60,000 and the scan range was 400-1200 m / z. Up to 25 of the most abundant precursors were then selected for further MS / MS analysis and dynamically excluded for 20 s. HCD fragmentation was performed at a normalized collision energy (NCE) of 27%. These fragments were detected on the Orbitrap at a resolution of 15,000. The fixed first mass was set to 110 m / z. The automatic gain control (AGC) target was set to 100%, the intensity threshold was 5E4 ions / s, and the MS2 maximum injection time was set to Auto.

[0060] The resulting MS / MS data were processed using the Proteome Discoverer search engine (v2.4.0.305). Tandem mass spectra were linked to the reverse library in the Mus_musculus database (17045 entries). Trypsin (Full) was specified as the cleavage enzyme, and up to 2 missed cleavages were allowed. The mass tolerance for precursor ions was 10 ppm, and the mass tolerance for fragment ions was 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and oxidation (M), Citrullination (R), acetylation on protein N-term, Met-loss (M), and Met-loss+acetyl (M) were specified as variable modifications. The quantification method was set to LFQ. The FDR was adjusted to <1%. The minimum peptide length was set to 6. All other parameters in Proteome Discoverer were set to default values.

[0061] (III) Screening of differentially citrullinated proteins:

[0062] Since the chemical mechanism of the deimination reaction is that the ketimine group is replaced by a keto group, causing arginine to lose its positive charge and produce ammonia, in this process, the arginine residue will undergo a mass shift of 0.984016Da. In order to explore the key substrates of PAD4, we further performed bioinformatics analysis by defining citrullination as a mass shift of +0.984016Da at the arginine residue on the secondary mass spectrum based on the results of mass spectrometry analysis. According to the database retrieval MS / MS spectrum in Proteome Discoverer, the static modification was set to carbamoylmethylation (Cys), the dynamic modification was set to citrullination (+0.984016Da) (Arg) + oxidation (Met), and the maximum missing cleavage was 2, and the peptides were further analyzed.

[0063] Compared with wild-type mice with normal drinking water, 42 proteins in DSS-induced wild-type colitis mice had significantly increased citrullination levels, while 40 proteins in DSS-induced PAD4 knockout mice had significantly decreased citrullination levels compared with wild-type colitis mice. After taking the intersection of the two, we obtained 12 proteins whose citrullination levels were increased in DSS wild-type mice and decreased in PAD4 knockout mice (see Table 1 and Figure 1 Fold difference > 1.2-fold, P value < 0.05, where “fold difference” means that citrulline levels were increased by at least 1.2-fold in DSS wild-type mice and decreased by at least 1.2-fold in PAD4 knockout mice).

[0064] Table 1 Differences in citrullination levels of 12 proteins

[0065]

[0066] Among them, the results of citrullination levels of 12 proteins are shown in Figure 1 ,from Figure 1 It can be seen that compared with the colon tissue of normal control group mice, the citrullination levels of these 12 differentially expressed proteins in the colon tissue of DSS group mice were significantly increased.

[0067] The protein level results of 12 differentially expressed proteins are shown in Figure 2 ,from Figure 2 It can be seen that the total protein levels of these 12 proteins are not completely consistent with the changing trends of their citrullination levels. Citrullination of proteins may affect the changes in the levels of some proteins, thereby affecting the functions of the corresponding proteins.

[0068] Example 2

[0069] (I) Test steps:

[0070] 1. DSS-induced acute colitis in mice

[0071] 8-12 week old C57 / BL6 male mice raised in SPF environment were given free drinking water with 3% DSS for 7 days. Their body weight was measured regularly every day, and the feces and bloody stools were recorded for disease activity index scoring. On the 7th day of modeling, samples were collected to measure the colon length, and the colon tissue near the anus was retained for HE staining.

[0072] 2. HE staining

[0073] 1. After sampling, the colon of the mouse was stripped (the mesentery was removed) and soaked in pre-cooled PBS. A 200ul gun tip was placed on the end of a 1000ul long gun tip. The tip of the 200ul gun tip was gently inserted into one end of the colon. The intestinal cavity was rinsed several times with cold PBS. A 0.5cm circular tubular intestinal segment was cut near the anus with ophthalmic scissors and placed in 4% paraformaldehyde for fixation for at least 24 hours. Human colon biopsy samples can be directly placed in 4% paraformaldehyde for fixation.

[0074] 2. Carefully remove the sample from the fixative with tweezers, trim the tissue flat with a scalpel, and rinse the paraformaldehyde with tap water (10 to 20 minutes).

[0075] 3. Dehydrate the tissue in gradient alcohol, alcohol-benzene mixture for 10 min, xylene for 10 min × 2 times, and paraffin melted at 65°C for 1 h × 3 times.

[0076] 4. After the tissue is waxed, mark the embedding frame and place the longitudinal section of the tissue facing up in the embedding frame containing melted wax liquid. After cooling in the refrigerator, trim the wax block.

[0077] 5. Trim the wax block and place it in the microtome, set the thickness to 5um for slicing, transfer the tissue to a glass slide, place it in an oven, set the temperature to 60℃, bake for 2h, and then cool to room temperature.

[0078] 6. Dehydrate the sections in xylene and gradient alcohol (100%, 95%, 75%) and rinse with tap water for 5 minutes.

[0079] 7. Stain the sections in hematoxylin solution for 4-5 minutes, rinse with tap water for 5 minutes, differentiate with differentiation solution for 1 minute, and rinse again with tap water for 5 minutes.

[0080] 8. Dehydrate with gradient ethanol (85% to 95%) for 5 minutes each, and immerse in eosin solution for staining for 5 minutes.

[0081] 9. Continue gradient dehydration and transparency (anhydrous ethanol 5 min × 3 times → xylene 5 min × 2 times). While the xylene is still wet, drop 1-2 drops of neutral gum on the slices. Gently press down the cover glass from one side to seal the slices. Bake in an oven for 2 hours and observe and photograph under a microscope.

[0082] 10. Colon inflammation score: ① Inflammation degree (0-3 points); ② Depth of inflammatory infiltration (0-3 points); ③ Crypt structure damage (0-4 points); ④ Lesion range (0-4 points); ⑤ Tissue regeneration degree (0-4).

[0083] 3. Western Blot

[0084] 1. Protein

[0085] (1) Tissue sample: Add 3 zirconium oxide grinding beads to a 2 ml round-bottom EP tube. Mix the lysate with protease inhibitor (100×) in a ratio of 100:1 in advance. Add 150 ul of lysate for every 20 mg of colon tissue. Place the EP tube in a low-temperature grinding shaker, oscillate at 55-60 Hz for 50 s, with an interval of 10 s, and repeat 3 times. Place the shaken tissue lysate in an ultrasonic crusher for 6 s, then place the EP tube in a low-temperature high-speed centrifuge at 14000 g for 10 min, discard the precipitate, and transfer the supernatant to a new 1.5 ml EP tube for later use.

[0086] (2) Cell samples: After washing the cells in the 6-well plate with PBS for 2-3 times, add 150ul of lysis buffer to each well and place on ice for 5 min. Gently scrape the bottom of a 200ul sterile pipette tip and pipette 2-3 times. Transfer the lysis buffer to a 1.5ml EP tube and centrifuge at 14000g for 10 min in a low-temperature high-speed centrifuge. Transfer the supernatant to a new EP tube.

[0087] 2. Measure protein concentration

[0088] (1) Dilute the sample to be tested: Take 5ul of the protein supernatant of the tissue or cells in the above step, add 45ul of PBS, dilute 10 times, and transfer to a new EP tube.

[0089] (2) Prepare the color development working solution: The color development working solution uses Yazyme Omni-Rapid TM The working principle of the rapid protein quantification kit is similar to that of the traditional BCA quantitative test. The color development only needs to be incubated at room temperature for 5 minutes before it can be tested on the machine. Two secondary wells are made for the samples to be tested. The total amount of color development working solution A liquid required is calculated according to the number of standard products (8) and the number of samples to be tested (n) (8+2n)×200ul. To avoid sample addition errors, 2 more wells can be configured each time, that is, the total amount of A liquid required is (10+2n)×200ul. Mix A liquid and B liquid in a volume ratio of 50:1, prepare and use immediately, and use it within 1.5h to ensure accuracy.

[0090] (3) Take 20ul of the diluted sample and BSA standard to be tested and add them to a 96-well plate. The concentrations of BSA standards ① to ⑧ are shown in the following table:

[0091] Table 2:

[0092]

[0093] (4) Quantitative detection: After adding the sample to be tested, add 200ul of colorimetric working solution to each well, mix well and incubate at room temperature for 5min. Detect the absorbance value at a wavelength of 480nm on an ELISA reader, draw a standard curve, and calculate the concentration of each sample to be tested.

[0094] 3. Protein denaturation

[0095] The samples with the measured concentration were diluted to 1 mg / ml using 5× Loading Buffer and lysis buffer, and incubated in a metal bath at 100°C for 10 min to complete protein denaturation.

[0096] 4. Electrophoresis

[0097] (1) Prepare 1×MOPS electrophoresis buffer in advance, open the package of GenScript 4-20% gradient precast gel, tear off the plastic seal at the bottom of the gel plate, and place the gel plate in the electrophoresis tank.

[0098] (2) Slowly pour the electrophoresis solution into the inner tank until it overflows. Slowly pull out the comb vertically and gently blow the sample well with a 200ul gun to blow away the precast gel storage solution remaining in the well. Continue to pour MOPS electrophoresis solution into the outer tank of the electrophoresis tank until it reaches more than half the height of the outer tank.

[0099] (3) Add 20ul of sample to each well and 5ul of prestained protein marker on both sides of the sample. After confirming that the positive and negative electrodes are in the correct position, cover the electrophoresis cover and perform electrophoresis at a constant voltage of 150V for 60min.

[0100] 5. Transfer

[0101] (1) Prepare 1× transfer buffer in advance and place it in a refrigerator at 4 degrees overnight.

[0102] (2) Cut the PVDF membrane to an appropriate size (8.5×5.5 cm), activate it in methanol for 20 seconds, and then place it in the transfer solution. Prepare the prying board, cutting board, tweezers and roller.

[0103] (3) Rinse the precast gel after electrophoresis with deionized water, carefully pry open the precast gel along the edge, cut the precast gel into appropriate sizes, and place it in ice transfer solution for balance.

[0104] (4) Open the transfer clip and place it on a tray with the black side facing down and the white side facing up. Pour the transfer solution in, place the sponge pad and filter paper in turn to soak, move the gel onto the filter paper, and use a roller to remove the bubbles. Then use tweezers to gently transfer the PVDF membrane onto the gel, use a roller to remove the bubbles between the gel and the filter paper, place the soaked filter paper and sponge pad in turn, and close the transfer clip.

[0105] (5) Place the transfer clips corresponding to the positive and negative electrodes in the transfer tank, place an ice box on the side wall of the transfer tank, fill the entire transfer tank with transfer solution, and cover the lid.

[0106] (6) Place the transfer tank in a foam box filled with crushed ice, turn on the power, and transfer the membrane at a constant current of 250 mA for 70 minutes.

[0107] 6. Closure

[0108] (1) Prepare 50 ml of 5% blocking solution with 2.5 g skim milk powder and 1× TBST for later use.

[0109] (2) After transfer, transfer the PVDF membrane to 1× TBST solution and wash on a shaker for 3 min.

[0110] (3) Pour 25 ml of blocking solution into the sealing box and transfer the PVDF membrane to the sealing box and block it at room temperature for 1 h.

[0111] 7. Primary Antibody Incubation

[0112] (1) After blocking, wash the PVDF membrane three times with 1× TBST, 5 min each time.

[0113] (2) Prepare the primary antibody using WB primary antibody diluent according to the antibody instructions.

[0114] (3) Incubate the PVDF membrane with the prepared primary antibody and shake at 4°C overnight.

[0115] 8. Secondary Antibody Incubation

[0116] (1) After the primary antibody incubation, wash with 1× TBST three times, 5-10 min each time.

[0117] (2) Prepare the secondary antibody (1:3000-1:5000) using WB secondary antibody diluent according to the antibody instructions.

[0118] (3) Incubate the secondary antibody on a shaking table at room temperature for 30 min to 1 h.

[0119] 9. Development and exposure

[0120] (1) After the secondary antibody incubation, wash with 1×TBST three times, 7-10 min each time.

[0121] (2) Prepare the ECL working solution by mixing solution A and solution B in a ratio of 1:1. Use the solution immediately after preparation and keep it away from light.

[0122] (3) Pipette an appropriate amount of luminescent liquid and drop it evenly on the PVDF membrane. Shake it left and right to evenly mix. Allow it to react at room temperature in the dark for 1 minute and then expose it to light in an exposure machine.

[0123] (II) CKMT1 protein verification results

[0124] 1. Protein expression level of CKMT1 in normal drinking water mice and DSS-induced colitis mice

[0125] Results Figure 3 In A and B, in wild-type mice, DSS-induced colitis significantly reduced the protein expression level of CKMT1 in the intestinal tissue of mice.

[0126] 2. DSS-induced colitis phenotype in CKMT1 intestinal epithelial gene-specific knockout mice

[0127] We used CRISPR / Cas9 technology to construct a conditional knockout mouse model of CKMT1. flox / flox Then, the intestinal epithelium-specific knockout mouse CKMT1 was constructed by hybridizing with the Vil-Cre mouse construct that specifically expressed Cre enzyme in the intestinal epithelium. flox / flox,Vil-Cre In DSS-induced colitis, mice with intestinal epithelial-specific knockout of CKMT1 showed more significant weight loss ( Figure 4 Middle A), the colon is shorter ( Figure 4 B and C), HE staining histological damage is more obvious ( Figure 4 (middle D).

[0128] Example 3

[0129] (I) Test steps:

[0130] See Example 2 for details.

[0131] (II) CTSH protein verification results

[0132] 1. Expression levels of CTSH protein in normal drinking water mice and DSS-induced colitis mice

[0133] See also Figure 5 In wild-type mice, DSS-induced colitis significantly reduced the protein expression level of CTSH in the intestinal tissue of mice.

[0134] 2. DSS-induced colitis phenotype in CTSH knockout mice

[0135] We used CRISPR / Cas9 technology to construct CTSH knockout mice. In DSS-induced colitis, CTSH knockout mice showed more significant weight loss ( Figure 6 Middle A), the colon is significantly shortened ( Figure 6 In B and D), the histological destruction of colon tissue was more obvious ( Figure 6 Middle C).

[0136] Example 4

[0137] (I) Test steps:

[0138] See Example 2 for details.

[0139] (II) NCSTN protein verification results

[0140] 1. Protein expression level of NCSTN protein in normal drinking water mice and DSS-induced colitis mice

[0141] See Figure 7 In wild-type mice, DSS-induced colitis had no significant effect on the protein expression level of NCSTN in the intestinal tissue of mice. That is, the increase in the protein citrullination level of NCSTN is correlated with colitis in DSS-induced colitis mice, but its total protein level is not necessarily reduced.

[0142] From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects: the present application has found 12 protein molecules significantly related to inflammatory bowel disease through proteomics analysis, and further confirmed the role of related protein molecules in the pathogenesis of inflammatory bowel disease through experiments. Therefore, the detection product for detecting inflammatory bowel disease, especially ulcerative colitis, prepared by using the protein marker of the present application not only provides related detection products for early detection and diagnosis, but also has accurate, convenient and non-invasive detection.

[0143] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a protein marker in the preparation of a detection product for detecting inflammatory bowel disease, characterized in that: The protein marker is selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

2. The use according to claim 1, characterized in that: The application includes detecting one of the following indicators of one or more of the protein markers: 1) the citrullination level of the corresponding protein; 2) the expression level of the corresponding protein.

3. The use according to claim 2, characterized in that: The citrullination sites of the proteins corresponding to one or more of the protein markers are: MRPL43 R85, ECHS1 R178, TLN2 R59, ENO3 R372, NCSTN R354, CTSHR315, CKMT1 R243, TUBA4A R221, DYNC1H1 R3158, PDLIM3 R164, GLB1 R592, and PMPCA R267.

4. A kit for detecting inflammatory bowel disease, characterized in that: The kit includes a detection reagent for a protein marker, and the protein marker is selected from any one or more of the following molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

5. The kit according to claim 4, characterized in that The detection reagent is a related reagent for detecting one of the following indicators of the protein marker: 1) the citrullination level of the corresponding protein; 2) the expression level of the corresponding protein; Preferably, the detection reagent comprises an antibody; Preferably, the antibody is disposed on a solid support; Preferably, the solid phase carrier is selected from ELISA plate, membrane carrier or microsphere, and further preferably, the membrane carrier is selected from nitrocellulose membrane, glass cellulose membrane or nylon membrane; Preferably, the antibody is a monoclonal antibody or a polyclonal antibody; Preferably, the kit is an ELISA kit, an immunofluorescence kit or an immunocolloidal gold kit.

6. A device for detecting inflammatory bowel disease, characterized in that: The device is equipped with a detection element for a protein marker, and the protein marker is selected from any one or more of the following protein molecules: MRPL43, ECHS1, TLN2, ENO3, NCSTN, CTSH, CKMT1, TUBA4A, DYNC1H1, PDLIM3, GLB1 and PMPCA.

7. The device according to claim 6, characterized in that The detection element is a related element for detecting one of the following indicators of the protein marker: 1) the citrullination level of the corresponding protein; 2) the expression level of the corresponding protein; Preferably, the detection element comprises an antibody; Preferably, the antibody is disposed on a solid support; Preferably, the solid phase carrier is selected from ELISA plate, membrane carrier or microsphere, and further preferably, the membrane carrier is selected from nitrocellulose membrane, glass cellulose membrane or nylon membrane; Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

8. The device according to claim 6, characterized in that The device also includes a comparison module and an early warning module. The comparison module is used to compare the numerical value corresponding to the index of the protein marker obtained by the detection element with the internal threshold value; The early warning module is used to display a warning signal when the comparison result of the comparison module exceeds a threshold value; Preferably, the built-in threshold comprises a protein level threshold or a citrullination level threshold.

9. A method for screening protein markers for inflammatory bowel disease, characterized in that: The screening method comprises: Mass spectrometry was used to detect proteins with significantly increased citrulline levels in the proteome of the wild-type inflammatory bowel disease simulation group compared with the healthy group, and the first differential protein set was obtained; Mass spectrometry was used to detect proteins with significantly lower citrulline levels in the PAD4 gene knockout inflammatory bowel disease simulation group than in the wild-type inflammatory bowel disease simulation group, to obtain the second differential protein set; The intersection of the first differential protein set and the second differential protein set is taken to obtain the protein marker of inflammatory bowel disease.

10. The screening method according to claim 9, characterized in that The wild-type inflammatory bowel disease simulation group is wild-type colitis mice induced by DSS.

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