A serum marker ITIH4 for detecting feline infectious peritonitis and its application

Abstract

The present invention discloses a serum marker ITIH4 for detecting feline infectious peritonitis and its application, which belongs to the field of biomedical technology. The amino acid sequence of ITIH4 is shown in SEQ ID NO: 1. The present invention also discloses the application of ITIH4 as a serum marker in the preparation of a product for diagnosing feline infectious peritonitis. The present invention uses a data-independent acquisition (DIA) high-throughput proteomics method to analyze and screen out the differentially expressed protein ITIH4 in the serum of host animals before FIPV infection and during the onset of FIP. The experimental results show that ITIH4 is significantly upregulated after cats are infected with FIPV, and has better specificity and sensitivity for the diagnosis of FIP. It can become a serum marker for diagnosing FIP, assist in the diagnosis of FIP, and provide a new direction for the establishment of a FIP diagnostic method.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a serum marker ITIH4 for detecting feline infectious peritonitis and an application thereof. Background Art

[0002] Feline coronavirus (FCoV) causes widespread infection in domestic and wild cats and can be divided into two biotypes with highly similar genomes; one FCoV causes a mild intestinal infection with mild diarrhea (called feline enteric coronavirus [FECV]), and the other FCoV causes a fatal systemic disease called feline infectious peritonitis (FIP). Because FECV is highly contagious and is transmitted horizontally via the fecal-oral route, almost all cats are infected with FECV, but feline infectious peritonitis virus (FIPV) lacks the ability to spread horizontally and does not cause FIP in felines even when the virus is shed in the feces. The first test to detect antibodies to FCoV was developed in 1976, but extensive horizontal transmission of FECV resulted in almost 100% of cat sera being positive for FCoV, but this was not associated with the development of FIP. Therefore, subsequent antigen detection methods including immunohistochemical staining and polymerase chain reaction (PCR) of tissues, effusions, blood, and reverse transcriptase were developed. However, there is currently no method to distinguish FIPV from FECV. In addition, FIP is usually divided into exudative, non-exudative, or a combination of the two types. The exudative type is easier to diagnose based on the accumulation of pleural effusion or ascites; the non-exudative type is characterized by granulomatous symptoms in multiple organs and the central nervous system, so the diagnosis of non-exudative FIP is particularly difficult. Therefore, there is an urgent need to develop a method for the specific diagnosis of FIP.

[0003] At present, the clinical diagnosis of FIP mainly relies on RT-PCR and non-specific inflammatory factors such as SAA and CRP in serum to assist clinical symptoms for diagnosis. In addition, it is impossible to obtain a correct diagnostic result in the latent state after FIPV infection when no clinical symptoms are shown. Therefore, specificity has always been the most important diagnostic value to consider. Therefore, FIP can never be diagnosed based solely on pathogen detection. In order to establish specific diagnostic markers to assist in the diagnosis of FIP, the present invention uses DIA-based quantitative proteomics to study the differences in protein expression in the serum of the same cat before and during FIPV infection in an existing animal model, hoping to screen for potential serum biomarkers that can identify FIP disease. Summary of the invention

[0004] The purpose of the present invention is to provide a serum marker ITIH4 for detecting feline infectious peritonitis and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a more specific diagnostic marker ITIH4 for the clinical diagnosis of FIP, assists in the diagnosis of FIP, helps in the prevention and control of FIP, and promotes the in-depth study of FIP.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a serum marker for detecting feline infectious peritonitis, comprising host protein inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4); the amino acid sequence of the ITIH4 is shown in SEQ ID NO:1.

[0007] The present invention also provides the use of ITIH4 as a serum marker in the preparation of a product for diagnosing feline infectious peritonitis. The amino acid sequence of the ITIH4 is shown in SEQ ID NO: 1.

[0008] Furthermore, the product uses cat blood as a test sample and diagnoses feline infectious peritonitis by detecting the expression level of ITIH4.

[0009] Furthermore, the product includes a reagent for detecting the expression level of ITIH4.

[0010] Furthermore, the feline infectious peritonitis is feline infectious peritonitis caused by FIPV infection.

[0011] The present invention also provides a kit for diagnosing feline infectious peritonitis, comprising a reagent for detecting the expression amount of ITIH4.

[0012] Furthermore, the amino acid sequence of ITIH4 is shown in SEQ ID NO: 1.

[0013] The present invention discloses the following technical effects:

[0014] The present invention collected the host animal serum before FIPV infection and during the onset of FIP for the first time through the FIP animal model, and analyzed the differences in protein expression in the serum through the DIA high-throughput proteomics method, and screened out the differentially expressed proteins in the FIP serum samples. The experimental results show that ITIH4 is significantly upregulated after cats are infected with FIPV, and has better specificity and sensitivity for the diagnosis of FIP, and is an effective serum marker for the diagnosis of FIP. Therefore, ITIH4 can become a serum marker for the diagnosis of FIP, assist in the diagnosis of FIP, improve its accuracy in the clinical disease detection process, and provide a new direction for the establishment of FIP diagnostic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 Figure 1 shows the indirect immunofluorescence detection of FIPV on CRFK cells (A) and the growth curve determination (B);

[0017] Figure 2 This is an analysis chart of the temperature changes of experimental cats after infection with FIPV;

[0018] Figure 3 This is an analysis chart of weight changes of experimental cats after infection with FIPV;

[0019] Figure 4 This is a scoring chart of clinical symptoms of experimental cats infected with FIPV;

[0020] Figure 5 This is the survival curve of experimental cats infected with FIPV;

[0021] Figure 6 This is the immunohistochemical analysis of tissues of experimental cats infected with FIPV;

[0022] Figure 7 This is the protein expression status screened by DIA in serum samples;

[0023] Figure 8 This is the result of differentially expressed proteins screened by DIA between serum samples not infected with FIPV and serum samples at the onset of FIP;

[0024] Fig. 9 The top ten proteins with the highest expression changes among the differentially expressed proteins screened by DIA; A: up-regulated expression; B: down-regulated expression;

[0025] Fig.10 Identification of ITIH4 expression levels in experimental cat samples from the control group and FIP group; A: Western Blot test results of ITIH4 expression; B: ITIH4 expression statistical graph; C: SAA expression statistical graph;

[0026] Fig.11 Receiver operating characteristic curves for serum ITIH4 and SAA in predicting feline FIP. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The invention uses a high-throughput proteomics method (DIA) based on mass spectrometry technology to screen and obtain differentially expressed proteins in cat serum before and after FIPV infection, including up-regulated proteins Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), Protein-tyrosine-phosphatase (PTPRC), Serum amyloid A protein (SAA), Tropomyosin 3 (TPM3), CUB domain-containing protein (CDCP), Galectin-3-binding protein (G3BP), Ferritin light chain (FTL), Vimentin (VIM), Prosaposin (PSAP), Golgi membrane protein 1 (GOLM1), etc., and down-regulated proteins Exosome component 7 (EXOSC7), Insulin-like growth factor I (IGF1), Apolipoprotein A-IV (APOA4), Serpinfamily Amember 6 (SERPINA6), Insulin like growth factor binding protein acidlabile subunit (IGFALS), Apolipoprotein C-III (APOC3), Insulin-like growth factor-binding protein 3 (IGFBP3), Retinol binding protein 4 (RBP4), Collectin subfamily member 11 (COLEC11), Alpha-1-B glycoprotein (A1BG), etc., all of which are differentially expressed in FIP cases. Among them, ITIH4 is the most significant among all the upregulated differential proteins, and the expression of ITIH4 protein in FIPV-infected and non-infected serum samples collected clinically was evaluated by laboratory Western Blot method. The results were consistent with the proteomics data, and the expression level was significantly increased after infection with FIPV. It is revealed that ITIH4 has the potential to be a diagnostic marker for FIP. The amino acid sequence of ITIH4 protein is shown in SEQ ID NO: 1:

[0033] MWNSGMGARNDIDIYSLTVDSRVSSRFAHTVVTSRVVNKADVVQEATFQVELPKRAFITNFSMIIDGVTYPGNIKEKAAAQEQYSTAVARGESAGLIKATGRKMEQFQVSVNVAPSAKVTFELVYEELLKRRLEVYELLLKVQPQQLVKHLQMDIHIFEPQGISFLETESTFMTNELADALTISRNKTKAHIRFKPTLSQQKSPGQEDTVLNGNFIIRYDVNRTLSGGSIQIENGYFVHYFAPEGLPTIPKNVIFVIDKSGSMLGRKIQQTREALIKILDDLSPRDQFNLISFSGDATQWKPLLVPASAEDVNQARRYVAGIQAQGGTNINDAMLMAVQLLSNANRQELLPAGSVSLIILLTDGDPTVGETNPARIQKNVQEAIAGQYSLFCLGFGFDVSYAFLEKMALDNGGLARRIYEDSDSALQLQDFYQEVANPLLTAVTFEYPSNAVEEVSQDNFRLLFKGSEIVVAGKLQDQSPDVLSAKVRGHLHMQNITFQTEIRVAEKEEEFQSPKYIFHSFMERLWAYLTIQQLLEKMILASDAEKQALETRALNLSLQYSFVTPLTSMVVTKPEGEERSEVAEKPVETGGCESRTPASLAAQEPSALSTSLSRGGSQSPGWGGGPRHHFREDCVCVNFSKAGPSGKAPGNPGRLGPQGPVHSPSHHLTYHGPALPALGTTPAVPAPIQAPPVILTLPGQSVNPLCVDIKHSQGPMNLLSDPDQGVEVTGQYETEKAQFSWIEVTFKNPQLQVRASPEHVVVTRNRRNSAYKWKETLFSVTPGLNMTMDKAGLLLLSSPDKVTIGLLPWDGPGEGLRLLLRDTDRFSSHVGGTLGQFYQGVLWGPPAAADDSRRTLSVQGRDYSATRLLKLDYQEGSPGTEISCWSVEL*(SEQ ID NO:1)。

[0034] Since the research on cat antibodies is relatively weak, most of the screened data cannot be verified in depth. Therefore, the present invention uses the currently widely recognized FIP detection index SAA as a control to verify the ITIH4 protein with a larger upregulation multiple. The results show that when FIP occurs, the level of ITIH4 in cat serum is significantly higher than that of healthy cats. Therefore, the ITIH4 disclosed in the present invention can be used to establish a FIP diagnostic method, assist in the clinical diagnosis of FIP, and effectively prevent and treat FIP. The specific research is as follows:

[0035] Example 1 Identification and Growth Curve Determination of FIPV Virus

[0036] 1 Test method

[0037] 1.1 Indirect immunofluorescence (IFA) identification: CRFK cells were inoculated into a 24-well culture plate. When the cells grew into a monolayer, the culture medium was discarded and each well was inoculated with FIPV virus solution rQS-79 at 0.01 MOI. At the same time, uninfected CRFK cells were set as negative controls. The culture medium was 2% maintenance solution and the cells were cultured in an incubator for 12 hours. After discarding the supernatant, add 4% paraformaldehyde to fix at room temperature for 30 minutes; discard the waste liquid, wash with PBS three times, add 1 / 1000 Triton to permeabilize for 10 minutes, discard the waste liquid, wash with PBS three times, add 2% BSA blocking solution, and let it stand at 37°C for about 1 hour; after washing with PBS three times, add the primary antibody (FIPV rabbit polyclonal antibody) diluted 1:500, and let it stand at 37°C for 1 hour; after washing with PBS four times, add goat anti-rabbit fluorescent secondary antibody at a dilution of 1:1000, and incubate at 37°C in the dark for 1 hour; discard the secondary antibody, wash with PBS three times, add DAPI dye diluted 1:1000, and let it stand at room temperature for 3 minutes; discard DAPI, wash with PBS three times, add 500μL PBS to each well, observe under a fluorescence microscope and collect pictures.

[0038] 1.2 Growth kinetics assay: CRFK cells were subcultured and inoculated in 24-well plates. When the cells grew to a monolayer, the cells were infected with 0.01 MOI virus. At the same time, the culture medium was replaced with 2% maintenance medium and cultured in a 37°C incubator. Supernatant samples were collected at 8, 16, 24, 32, 40, and 48 hours after infection, and the virus TCID was determined using CRFK cells. 50 . The cells were subcultured and inoculated in a 96-well plate. When the cell density reached about 50%, the virus was diluted 10 times with cell maintenance solution, and 100 μL of virus solution was added to each well. Eight replicates were made for each dilution, and the plates were cultured in an incubator. The plates were observed every 12 hours, and the virus dilution and number of wells at which cytopathic effects occurred were recorded. The observation was stopped after 72 hours, and the final dilution and number of wells at which cytopathic effects occurred were recorded. The TCID was calculated according to the Reed-Muench method. 50 .

[0039] 2. Test results

[0040] 2.1 As Figure 1 As shown in A, specific green fluorescence appeared in the cell monolayer infected with the virus rQS-79, while there was no fluorescence in the control group cells.

[0041] 2.2 As Figure 1 As shown in B, CRFK was inoculated with 0.01 MOI virus, and the virus titer reached the highest at 32h after inoculation, which was 10 6.49 TCID 50 / mL.

[0042] Example 2 FIP animal experiment and sample collection

[0043] 1 Test method

[0044] 1.1 Sample collection: Before infection with FIPV virus rQS-79, samples were collected from the test cats. Blood was collected from the forelimb vein, left to stand at room temperature for 1 hour, and then centrifuged at 4°C, 3000×g for 10 minutes. The upper layer of serum was quickly frozen in liquid nitrogen and stored below -70°C.

[0045] 1.2 FIPV challenge: cats in the experimental group were orally administered 1 mL of FIPV virus solution rQS-79 (TCID 50 1×10 6.0 The control group was orally administered with 1 mL of DMEM medium.

[0046] 1.3 After infection with FIPV virus rQS-79, the body temperature, weight, clinical symptoms and survival of the experimental cats were observed and recorded daily. The clinical symptoms were scored according to the established scoring criteria, as follows: 1 point for not eating, 1 point for no defecation, 1 point for no urination, 1 point for extreme depression, and 1 point for jaundice.

[0047] 1.4 After the experimental cats died and were euthanized, they were autopsied and their livers, spleens, and kidneys were collected for immunohistochemical testing.

[0048] 2 Test results

[0049] 2.1 As Figure 2 As shown, after being infected with FIPV, the experimental cats gradually developed elevated body temperatures. On the third day after infection, some cats had fever, with body temperatures even reaching 40°C. During the entire trial, all infected cats showed symptoms of fever.

[0050] 2.2 When recording the weight of the experimental cats, it can be found that all cats with FIP have lost weight, such as Figure 3 shown.

[0051] 2.3 Clinical scoring of FIPV-infected cats revealed that some cats began to show obvious symptoms on the fifth day after infection, and all infected cats gradually developed corresponding symptoms ( Figure 4 ). Blood was collected when the clinical score reached 4 points, and the serum was stored as the FIP group.

[0052] 2.4 After the experimental cats were infected with FIPV, they developed a series of clinical symptoms. As the disease progressed, the experimental cats began to die. Figure 5 As shown, the experimental cats began to die from the 8th day after infection, and by the 12th day after infection, all the infected experimental cats had died.

[0053] 2.5 All experimental cats were autopsied after death to further confirm the credibility of the experiment. Specific immunohistochemical detection of tissues showed obvious positive signals ( Figure 6 ).

[0054] Example 3 Screening of differentially expressed protein indicators in cat serum before and after FIP infection using proteomics analysis

[0055] 1 Test method

[0056] 1.1 Protein enzymatic hydrolysis: Example 2 Take 5 μL of each FIP animal serum sample and add 50 μL 8M Urea for denaturation treatment. Add an appropriate amount of 1M DTT to each sample to a final concentration of 10 mM, incubate at 37°C for 30 minutes, and cool to room temperature. Then add an appropriate amount of IAA to each sample to a final concentration of 50 mM, and incubate at room temperature in the dark for 30 minutes. Add 50 mM NH4HCO3 to each sample to dilute it 6 times, then add 8 μg Trypsin, oscillate at 600 rpm for 1 minute, and 37°C for 20 hours. Add an appropriate amount of TFA to the sample after enzymatic hydrolysis to terminate the enzymatic hydrolysis. Then use C18 Cartridge to desalt the hydrolyzed peptides, and finally use OD 280 Peptide quantification.

[0057] 1.2 DIA mass spectrometry data acquisition: 9 μL of peptides were added to 1 μL of 10X iRT peptides for each sample, and 2 μg was injected after mixing. Chromatographic separation was performed using the nanoliter flow rate Easy nLC 1200 chromatography system (Thermo Scientific). Buffer: Liquid A is 0.1% formic acid in water, and liquid B is 0.1% formic acid in acetonitrile (acetonitrile is 85%). The chromatographic column was balanced with 95% liquid A. The sample was injected into the Trap Column (100 μm*20 mm, 5 μm, C18, Dr. Maisch GmbH) and then passed through the chromatographic analysis column (75 μm*150 mm, 3 μm, C18, Dr. Maisch GmbH) for gradient separation at a flow rate of 300 nL / min. The liquid phase gradient was set as follows: 0min-2min, liquid B linear gradient from 5%-8%; 2min-42min, liquid B linear gradient from 8%-23%; 42min-52min, liquid B linear gradient from 23%-40%; 52min-53min, liquid B linear gradient from 40%-100%; 53min-60min, liquid B maintained at 100%. After peptide separation, DIA (data independent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time was 60min, the electrospray voltage was 2.1kV, the detection mode was positive ion, the parent ion scanning range was 350-1200m / z, the primary mass spectrometry resolution was 60,000@m / z 200, AGC target: 3e6, and the primary MaximumIT: 30ms. Secondary mass spectrometry resolution: 15,000@m / z 200, AGC target:1e6, secondary Maximum IT:25ms, MS2Activation Type:HCD, Isolationwindow:20Th, Normalized collision energy:32.

[0058] 1.3 Library-free DIA analysis: All mass spectrometry data were merged by Spectronaut 15 software, and the SLibrary-free DIA (directDIA) method was used to complete the database retrieval of DIA mass spectrometry data and protein DIA quantitative analysis in one step. The database is UniprotFelis catus (Cat)

[9685] _41796_202202.fasta, which comes from the Uniprot protein database, with protein entry: 188359, and download time: 2021-10. The analysis parameter settings of Pulsar Search software are shown in Table 1.

[0059] Table 1 Pulsar Search software analysis parameters

[0060]

[0061] 1.4 Bioinformatics analysis: DEPs between pre-infection and post-infection groups were identified by DEseq2 and the corresponding volcano plots were constructed. DEPs with p < 0.05 and fold change > 1.5 or < 1 / 1.5 identified by Fisher’s exact test were considered significant.

[0062] 2 Test results

[0063] 2.1 DIA protein qualitative and quantitative results data statistics: Each sample in the project was prepared independently, and the protein was digested and then subjected to DIA (data independent acquisition) analysis. The obtained DIA original mass spectrum files were imported into Spectronaut (version 15, Biognosys AG) for directDIA analysis, using Qvalue≤0.01 as the screening parameter. The statistical information of proteins and peptides obtained in this experiment is shown in Table 2, and the quantitative number of each sample is shown in Table 3. Figure 7 This is the protein expression status of serum samples screened by DIA.

[0064] Table 2 Statistics of DIA protein identification results

[0065] Species Precursor Peptides Proteome cat 6099 3876 383

[0066] Table 3 Statistics of protein identification results of each sample

[0067]

[0068]

[0069] 2.2 Differentially expressed proteins: The readouts obtained from the protein expression analysis were used for the analysis of differentially expressed proteins. DEPs were determined based on fold change >1.5 or <1 / 1.5 and p < 0.05. A total of 96 differentially expressed proteins were obtained by comparing the pre-infection and post-infection groups, including 49 downregulated and 47 proteins with increased expression levels ( Figure 8 ).

[0070] 2.3 Differential protein analysis: Based on the omics statistical data, the differentially expressed proteins in serum samples before FIP onset and before FIPV infection were obtained, and the top 10 differentially expressed proteins (up-regulated and down-regulated proteins) were screened out ( Fig. 9 ). Fig. 9 A in the middle represents the top 10 up-regulated proteins. Fig. 9B in the figure shows the top 10 down-regulated proteins. As can be seen from the figure, ITIH4 has the most significant differential expression among all up-regulated differentially expressed proteins, suggesting that ITIH4 has the potential to become a diagnostic marker for FIP.

[0071] Example 4 Clinical validation study of ITIH4 as a diagnostic marker for feline infectious peritonitis

[0072] 1 Test method

[0073] 1.1 Materials

[0074] Nine cats diagnosed with feline infectious peritonitis admitted to Wuhan Yihe Animal Hospital from January to April 2024 were selected as the FIP group, and 9 cats with healthy physical examinations and no FIP were selected as the control group. Inclusion criteria: The owner agreed to participate in the clinical study, and the test cats had no other diseases. Cats with FIP symptoms and positive antigens during clinical examination were included in the FIP group; cats without any clinical symptoms and healthy physical examination results were included in the control group.

[0075] 1.2 Methods

[0076] 1.2.1 After the diagnosis, blood was collected from the forelimb vein of the test cat, left to stand at room temperature for 1 hour, and then centrifuged at 4°C, 3000×g for 10 minutes. The upper serum was taken and stored below -70°C. The samples were determined by Western Blot method for ITIH4. First, the serum protein concentration was determined using the BCA protein assay kit. Samples with equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk for 3 hours, they were incubated with antibodies overnight. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour, and the blots were visualized using electrochemiluminescence (ECL) reagents. The grayscale values ​​of protein bands were determined using Image J software.

[0077] 1.2.2 Serum SAA levels were measured using the animal immunofluorescence detector (YG-101) produced by Guangzhou Wondercon Technology Co., Ltd.

[0078] 1.2.3 Observation indicators: compare the levels of ITIH4 and SAA in the serum of the two groups of test cats; evaluate the value of serum ITIH4 level in the clinical diagnosis of FIP in cats.

[0079] 1.3 Data Analysis

[0080] GraphPad Prism 9 statistical software was used to process and analyze all the data obtained in this experiment and draw graphs. The independent sample T test was used for significance analysis between the groups. The measurement data were expressed as mean ± standard deviation (Mean ± SD). p>0.05 was not significantly different, p<0.05 was significantly different, and p<0.01 was extremely significant. The diagnostic value was analyzed by receiver operating characteristic curve analysis to obtain the area under the curve (AUC), confidence interval, sensitivity, specificity and cutoff value.

[0081] 2 Test results

[0082] Serum ITIH4 levels were significantly increased in FIP cats ( Fig.10 A and B), while SAA levels showed an upward trend, but the difference was not significant ( Fig.10 C). The control group was used as a negative sample and the FIP group was used as a positive sample to draw the receiver operating characteristic curve. According to the analysis results of the samples we collected, within the sample range, the AUC of the serum ITIH4 protein level in the tested cats for predicting FIP was 0.9420, the best sensitivity was 88.89%, and the specificity was 88.89%; the AUC of SAA for predicting FIP was 0.8025, the best sensitivity was 88.89%, and the specificity was 77.78% (see Table 4, Fig.11 ), indicating that the ITIH4 screened out by the present invention has better specificity for the diagnosis of FIP and can be used as an effective serum marker for the diagnosis of FIP.

[0083] Table 4 Receiver operating characteristic curves for the prediction of serum ITIH4 and SAA levels for FIP

[0084]

[0085] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of ITIH4 as a serum marker in the preparation of a product for diagnosing feline infectious peritonitis, characterized in that: The amino acid sequence of ITIH4 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The product uses the blood of a cat to be tested as a test sample and diagnoses feline infectious peritonitis by detecting the expression level of ITIH4.

3. The use according to claim 2, characterized in that: The product includes a reagent for detecting the expression level of ITIH4.

4. The use according to claim 1, characterized in that: The feline infectious peritonitis is feline infectious peritonitis caused by FIPV infection.

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