Biomarker genes for detecting pathogenic injury of intestinal tracts of piglets as well as screening method and application of biomarker genes

Abstract

The invention discloses a group of biomarker genes for detecting pathogenic injury of intestinal tracts of piglets as well as a screening method and application of the biomarker genes. The biomarker gene comprises at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5, and the biomarker gene comprises at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6 and ENPP7. The corresponding protein expressed by the biomarker gene can be used for diagnosing piglet intestinal pathogenic injury caused by infection of one or more pathogens of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus and enterotoxigenic escherichia coli. The biomarker gene provided by the invention has relatively strong specificity, high sensitivity and good stability, and can be applied to early diagnosis and prognosis analysis of pathogenic injury of intestinal tracts of piglets.

Description

Technical Field

[0001] The present application relates to the field of animal medical technology, and in particular to a group of biomarker genes for detecting pathogenic damage to the intestine of piglets, and a screening method and application thereof. Background Art

[0002] Pathogenic damage to the piglet intestine is a major problem that restricts the healthy breeding of pigs. Common pathogens include epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus and enterotoxigenic Escherichia coli, which pose a major threat to the health of pigs.

[0003] Porcine Epidemic Diarrhea Virus (PEDV) is a highly contagious enteric infectious pathogen. The porcine epidemic diarrhea (PED) caused by it is one of the diseases that seriously threatens the global pig industry. Typical symptoms of PED include diarrhea, vomiting and dehydration in pigs, especially for piglets that have just been born. After infection, the mortality rate of piglets under 7 days old can be as high as 100%, causing huge losses. PEDV infection is mainly transmitted through the digestive tract, and can also be transmitted through contaminated environments, objects and excrement of sick pigs. After the virus enters the pig's body, it mainly infects the epithelial cells of the small intestine, causing damage to the intestinal villi and absorption dysfunction, causing severe diarrhea and dehydration, which leads to rapid weight loss, decreased immunity and even death of piglets. Although adult pigs have milder symptoms, they also have problems such as decreased appetite and reduced litter size, which significantly affect production performance.

[0004] Porcine transmissible gastroenteritis virus (TGEV) is a highly contagious coronavirus that poses a serious threat to the healthy development of the global pig industry. Its typical symptoms are similar to those of PED, including diarrhea, vomiting, and dehydration, and it is also highly lethal to piglets. Especially for piglets under 10 days old, the mortality rate after infection can be as high as over 90%. TGEV infection is mainly transmitted through direct contact, but can also be transmitted through droplets in the air and contaminated feed, drinking water, and objects.

[0005] Porcine rotavirus (PoRV) is one of the main pathogens causing porcine viral diarrhea, posing a long-term and significant threat to the global pig industry, especially in intensive farming areas. PoRV infection usually occurs in piglets aged 1-4 weeks, and its symptoms include watery diarrhea, dehydration and weight loss. In severe cases, it can lead to growth retardation or even death. Since it was first reported in the United States in 1969, PoRV infection has spread throughout the world, and multiple genotypes (such as type A, type B and type C) are prevalent in different regions. PoRV infection not only causes direct death of piglets, but also leads to growth retardation and reduced feed conversion rate, resulting in indirect economic losses.

[0006] Enterotoxigenic Escherichia coli (ETEC) is a common pathogen that mainly causes intestinal diseases such as yellow and white diarrhea in piglets through infection. Typical symptoms of ETEC include watery diarrhea, vomiting, dehydration and rapid weight loss, especially for piglets under 5 days old. The pathogenic mechanism of ETEC is closely related to the enterotoxins it secretes, including heat-stable enterotoxins (such as STa and STb) and heat-labile enterotoxins (LT), which stimulate the intestinal mucosa to secrete a large amount of fluid, thereby causing watery diarrhea. ETEC infection is often accompanied by immunosuppression or viral infection (such as PEDV and TGEV), which aggravates the complexity of the disease.

[0007] Traditionally, the methods for preventing and treating porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus and enterotoxigenic Escherichia coli infections mainly include vaccination and the use of anti-infective drugs. However, vaccination may fail under conditions such as virus mutation or immunosuppression, and long-term use of anti-infective drugs can easily lead to drug resistance and toxic side effects. Therefore, it is urgent to identify biomarker genes for pathogenic damage to the piglet intestine, which can be used for early diagnosis and prognosis analysis of pathogenic damage to the piglet intestine. After the piglet intestine is infected by pathogens, the mechanism of intestinal damage is closely related to pathogen-induced cell damage, inflammatory response, immune disorder and intestinal barrier destruction. In recent years, researchers have explored a variety of biomarkers related to intestinal pathogenic damage through molecular biology and omics technologies, providing an important basis for disease diagnosis and mechanism research. For example, (1) markers of intestinal pathogenic damage in PEDV infection include cell damage markers: apoptosis-related proteins (such as increased Caspase-3 and Bax / Bcl-2 ratio) are significantly upregulated in the early stage of infection, suggesting that programmed cell death is an important mechanism. Inflammatory markers: Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8 were significantly increased, indicating an enhanced local inflammatory response. Barrier function markers: The expression of tight junction proteins (such as Claudin-1, Occludin and ZO-1) decreased, reflecting the damage to the intestinal barrier integrity. (2) Intestinal pathogenic damage markers of TGEV infection include cell damage markers: the expression of small intestinal epithelial cell proliferation factor (Ki67) decreased, indicating a weakened repair capacity; mitochondrial function-related proteins (such as Cytochrome c) leaked, indicating severe mitochondrial damage; inflammatory factors: TGEV infection significantly activated the NF-κB signaling pathway, upregulated TNF-α, IL-1β and IL-18, leading to an imbalance in the intestinal immune microenvironment; oxidative stress markers: The level of reactive oxygen species (ROS) increased, and the activity of antioxidant enzymes (such as SOD, GSH-Px) decreased, which is an important intestinal damage mechanism. (3) Markers of intestinal pathogenic damage caused by PoRV infection include viral replication markers: PoRV VP6 protein is significantly expressed in the small intestine, which is a sign of active viral infection; cell apoptosis and autophagy markers: Caspase-3 and LC3B expression are significantly upregulated, indicating that cell apoptosis and autophagy are simultaneously involved in the pathological process; intestinal permeability markers: damaged intestinal barrier leads to the entry of bacteria and endotoxins into the blood, and elevated plasma D-lactic acid and endotoxin (LPS) levels become indirect markers of systemic damage.(4) Intestinal pathogenic damage markers of ETEC infection include toxin-related markers: heat-stable enterotoxin (STa) and heat-labile enterotoxin (LT) are important virulence factors that can cause excessive intestinal fluid secretion by activating the cAMP / cGMP signaling pathway; inflammatory response markers: IL-8, TNF-α, and IL-22 are significantly upregulated, accompanied by macrophage infiltration, indicating excessive activation of intestinal mucosal immunity; electrolyte disorder markers: after infection, the sodium-potassium pump (Na. + / K + -ATPase) activity decreased, and Na + , Cl - Increased concentrations further exacerbate dehydration.

[0008] However, the discovery of biomarkers related to pathogenic intestinal damage still faces many challenges, such as (1) insufficient specificity and sensitivity of markers: markers of pathogenic intestinal damage may show atypical expressions due to co-infection or secondary infection with multiple pathogens, making it difficult to accurately distinguish the effects of different pathogens. The dynamic changes of markers are complex, and different infection stages may show completely different expression patterns, which brings difficulties to clinical diagnosis and research; (2) limitations of marker detection technology: traditional detection methods (such as ELISA, qPCR) have high requirements for sample processing and equipment, and it is difficult to achieve large-scale and rapid detection; (3) the interaction mechanism between pathogens and hosts is still unclear: the causal relationship between pathogen-induced immune response and host barrier damage has not been fully clarified, and some markers may be the result rather than the direct cause. The diversity and variability of pathogens lead to insufficient universality of markers, especially the large differences in expression between epidemic strains and classical strains; (4) interference from differences in pathological background: differences in age, genetic background and nutritional status among individual piglets have a significant impact on marker expression. The effects of maternal antibodies, environmental stress and feed ingredients may mask changes in markers caused by infection. In short, there is an urgent need to explore the biomarker genes of pathogenic intestinal damage in piglets in depth to provide a theoretical basis for the early diagnosis and prognosis analysis of pathogenic intestinal damage in piglets. Summary of the invention

[0009] In view of this, the purpose of this application is to provide a group of biomarker genes for detecting pathogenic damage to the intestinal tract of piglets, and screening methods and applications thereof. The biomarker genes can be used to diagnose pathogenic damage to the intestinal tract of piglets caused by one or more pathogenic infections of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus and enterotoxigenic Escherichia coli. The biomarker genes have strong specificity, high sensitivity and good stability, and can be used for early diagnosis and prognosis analysis of pathogenic damage to the intestinal tract of piglets.

[0010] To achieve the above objectives, this application provides at least the following technical solutions:

[0011] In a first aspect, the present application provides a group of biomarker genes for detecting pathogenic damage to the intestine of piglets, including at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5.

[0012] In a second aspect, the present application provides a method for screening the aforementioned biomarker genes, comprising the following steps:

[0013] The jejunum of pathogen-infected and non-infected piglets was used as samples for proteomic sequencing to obtain the protein composition of the two groups of samples;

[0014] Performing differential analysis on the results of the proteomic sequencing, and screening out differentially expressed proteins in the two groups of samples by setting a threshold;

[0015] The String combined with Cytohubba algorithm was used to screen out the top 20 important proteins in the protein composition of the two groups of samples;

[0016] Wayne analysis was performed on the differentially expressed proteins and the top 20 important proteins to screen out potential biomarkers.

[0017] In a third aspect, the present application provides a group of protein markers for detecting pathogenic damage to the intestine of piglets, wherein the protein markers include at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins.

[0018] In a fourth aspect, the present application provides the use of the aforementioned biomarker genes or the aforementioned protein markers in the preparation of reagents for diagnosing pathogenic injuries in piglets or drugs for treating pathogenic injuries in piglets.

[0019] Compared with the prior art, the technical solution provided by this application has at least the following beneficial effects:

[0020] 1. The present application provides a group of biomarker genes for detecting pathogenic damage to the intestine of piglets, which are screened using jejunum as a biological sample, including one or more of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5. Experiments have shown that compared with piglet jejunum samples without pathogenic damage to the intestine in the control group, in piglet jejunum samples with pathogenic damage to the intestine, MMP7, PLA2G3 and AQP5 genes were found to be upregulated, while CYP3A22, HSD17B6, ENPP7 and SDR16C5 genes were downregulated. At the same time, GraphPad Prism V.9 software was used to construct the model, and the ROC curve was obtained. The prediction accuracy of the model was then evaluated by the area under the ROC curve (AUC). The results showed that the AUCs of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins were all greater than 0.94 (95% CI = 1.00-1.00; P < 0.001), indicating that the differentially expressed jejunal proteins MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 can be used as valuable biomarkers for evaluating and diagnosing pathogenic intestinal lesions in piglets.

[0021] 2. This application studies the key targets of intestinal pathogenic damage from piglets, which can be used for the early diagnosis of intestinal pathogenic damage in piglets, and help develop corresponding drugs to intervene and treat intestinal pathogenic damage in piglets in the early stage of the disease, laying the foundation for preventing and controlling the occurrence and development of intestinal pathogenic damage in piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the screening process of biomarker genes for pathogenic damage to the piglet intestine provided in the examples of the present application.

[0023] Figure 2 This is a PCA analysis diagram of the identified proteins provided in the examples of this application.

[0024] Figure 3 Overlapping diagram of the number of proteins identified in the jejunum of piglets in the CT and PEDV groups (Venn diagram).

[0025] Figure 4 The quantitative difference results of jejunum protein provided in the examples of this application.

[0026] Figure 5 A potential pathway analysis diagram based on the significantly different proteins in the jejunum of piglets in the CT group and the PEDV group provided in the examples of the present application.

[0027] Figure 6The significantly differentially expressed proteins (DEPs) in the jejunum of the CT group and the PEDV group of piglets provided in the examples of the present application are combined with the String combined with the Cytohubba algorithm to screen out the top 20 important proteins in the protein composition of the two groups of samples. Wayne analysis diagram.

[0028] Figure 7 The ROC curve of MMP7 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets.

[0029] Figure 8 The ROC curve of PLA2G3 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets.

[0030] Fig. 9 The ROC curve of AQP5 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets.

[0031] Fig.10 ROC curve of CYP3A22 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets.

[0032] Fig.11 ROC curve of HSD17B6 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets.

[0033] Fig.12 ROC curve of ENPP7 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets.

[0034] Fig.13 The ROC curve of SDR16C5 protein as a biomarker for diagnosing pathogenic intestinal lesions in piglets. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0036] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0037] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0038] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application may also be used to implement this application.

[0039] The present application example uses pathogen-infected and non-infected piglet jejunum as samples, and screens out potential biomarker genes through proteomic sequencing combined with String and Cytohubba algorithms. The gene expression of these biomarker genes in infected and non-infected pig coelenteric samples shows completely different trends. Compared with healthy piglets without infection, in pathogen-infected piglet jejunum samples, MMP7, PLA2G3 and AQP5 genes are upregulated, while CYP3A22, HSD17B6, ENPP7 and SDR16C5 genes are downregulated, which can be diagnosed as pathogenic intestinal damage. The results of ROC curve analysis have verified that the diagnostic accuracy of the corresponding proteins expressed by these biomarker genes is greater than 94%.

[0040] Based on this, the present application provides a group of biomarker genes for detecting pathogenic intestinal damage in piglets, including at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 genes, wherein the gene ID of the MMP7 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 397411, and the ID of its mRNA is NM_001348795.1; the gene ID of the PLA2G3 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 100511161, and the ID of its mRNA is XM_003132975.4; the gene ID of the AQP5 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 100126278, and the ID of its mRNA is NM_0011104. 24.1; the gene ID of the CYP3A22 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 100144468, and the ID of its mRNA is NM_001195509.1; the gene ID of the HSD17B6 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 100620470, and the ID of its mRNA is XM_003355459.4; the gene ID of the ENPP7 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 100514726, and the ID of its mRNA is XM_013980746.2; the gene ID of the SDR16C5 gene in the gene library of the National Center for Biotechnology Information (NCBI) of the United States is 100157319, and the ID of its mRNA is NM_001195368.1.

[0041] In certain embodiments, the porcine intestinal pathogenic lesions are porcine jejunal lesions.

[0042] Based on this, the present application provides a method for screening the aforementioned biomarker genes, comprising the following steps: (1) using the jejunum of pathogen-infected and non-infected piglets as samples, performing proteomic sequencing to obtain the protein composition of the two groups of samples; (2) performing differential analysis on the results of the proteomic sequencing, and screening the differentially expressed proteins in the two groups of samples by setting a threshold; (3) using String combined with Cytohubba algorithm to screen out the top 20 important proteins in the protein composition of the two groups of samples; (4) performing Wayne analysis on the differentially expressed proteins and the top 20 important proteins to screen out potential biomarkers.

[0043] In certain preferred embodiments, the proteomic sequencing comprises the following steps: lysing and digesting the jejunum sample to obtain peptides, purifying and recombining the peptides, and then performing LC-MS / MS analysis.

[0044] In certain preferred embodiments, the peptide purification comprises desalting and centrifugal concentration.

[0045] In certain preferred embodiments, the difference analysis includes performing a t-test on each group of values, and calculating the difference multiple FC and the difference significance P-value of the comparison group.

[0046] In certain preferred embodiments, the threshold is set to FC>1.5 and P<0.05, or FC<0.67 and P<0.05.

[0047] In certain preferred embodiments, the differential analysis further comprises performing KEGG enrichment analysis on the differentially expressed proteins.

[0048] Based on this, the embodiments of the present application also provide a group of protein markers for detecting pathogenic damage to the intestine of piglets, wherein the protein markers include at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins.

[0049] In the embodiment of the present application, the above 7 genes can express corresponding proteins, wherein protein MMP7 is Matrixmetallopeptidase 7, and its ID on UniProt is F1SV71; protein PLA2G3 is phospholipase A2, and its ID on UniProt is F1RPC3; protein AQP5 is Aquaporin-5, and its ID on UniProt is A8W649; protein CYP3A22 is Cytochrome P450 3A, and its ID on UniProt is I3LEX8; protein HSD17B6 is Hydroxysteroid 17-beta dehydrogenase 6, and its ID on UniProt is A0A4X1W6H0; protein ENPP7 is Ectonucleotide pyrophosphatase / phosphodiesterase 7, and its ID on UniProt is A0A8D1R3X8; protein SDR16C5 is Short chain dehydrogenase / reductase family 16C, its ID on UniProt is A0A4X1ST96.

[0050] Based on this, the embodiments of the present application also provide the use of the aforementioned biomarker genes or the aforementioned protein markers in the preparation of reagents for diagnosing pathogenic injuries of piglets or drugs for treating pathogenic injuries of piglets.

[0051] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0052] Screening of biomarker genes for pathogenic intestinal lesions in piglets

[0053] 1. Experimental materials and groups

[0054] 1.1 Experimental piglets: PEDV was obtained from the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0055] 1.2 Experimental animals: The piglets used in this experiment were 7-day-old healthy hybrid piglets (Duroc×Landrace×Large White), and all 40 piglets were purchased from Wuhan Jinlong Animal Husbandry Co., Ltd.

[0056] 1.3 Experimental process: During the whole experiment, all experimental piglets were allowed to eat and drink freely at the same temperature (28±2℃) and relative humidity (45±15%). The piglets were randomly divided into 2 groups, 20 in each group, and divided into a control group (CT) and an infection group (PEDV). On the fifth day of the experiment, 3 mL of PEDV virus capsules were administered to the piglets in the PEDV group, and the experiment lasted for 7 days. Figure 1 A schematic diagram of the entire experimental process.

[0057] 2. Sampling and determination methods

[0058] 2.1 Sampling

[0059] On the 7th day of the experiment, 10 piglets were selected from each group and killed by jugular vein bleeding after anesthesia. The jejunal tissue was collected and stored in a -80℃ refrigerator for proteomics research.

[0060] 2.2 Proteomic Sequencing

[0061] 200 mg of cecal samples were treated with 30 μL SDT lysis buffer and protein digestion with trypsin was performed according to the filter-assisted sample preparation (FASP) procedure. The digested peptides of each sample were desalted on a C18 cartridge (EmporeTM SPE cartridge C18 (standard density), bed ID7mm, volume 3mL, Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 μL of 0.1% (v / v) formic acid; after that, these digested peptides were subjected to LC-MS / MS analysis (OE480 mass spectrometry platform, Wuhan University of Technology).

[0062] 3. Results and Analysis

[0063] (1) Observation of intestinal damage: Anatomical and histopathological observations showed that the cecal epithelium of piglets in the PEDV group was severely damaged, with incomplete crypt structure, bleeding, and reduced cecal mucosal thickness. The morphology of the jejunum in the PEDV group was severely damaged.

[0064] (2) Analysis of differentially expressed proteins: All data analyses were performed based on the NR database and the UniProt database. In order to explore the composition of jejunal proteins, LC-MS / MS was used to perform proteomic analysis of jejunal samples. Figure 2 to Figure 6 The results of the study showed that the differentially expressed proteins in the jejunum of piglets with intestinal pathogenic damage. Figure 2 This is the PCA analysis diagram of the identified proteins. It can be seen from the figure that the four biological replicates of the two groups are clustered together in different regions, indicating that there are differences in the proteins of the two groups. Figure 3 The Venn diagram of the number of proteins identified in the jejunum of piglets in the CT group and the PEDV group was shown in Figure 2. Through the Venn diagram, a total of 2516 proteins were identified in the CT group and the PEDV group, and the two groups had 2202 common proteins. The differentially expressed proteins of the two groups were screened by setting the threshold (FC>1.5 and P<0.05, or FC<0.67 and P<0.05). The proteins with significant differences were indicated in red (upward), while the gray dots indicated that the proteins had no significant differences, and the quantitative difference results of jejunal proteins were obtained (such as Figure 4 As shown in the figure, there are 97 differentially expressed proteins in the two groups. At the same time, the differentially expressed proteins in the two groups were annotated with KEGG pathways and the number was counted, forming a potential pathway analysis diagram based on the significantly different proteins in the jejunum of piglets in the CT group and the PEDV group, as shown in the figure. Figure 5 As shown, the size of the bubble indicates the number of significantly differentially enriched proteins in the pathway, and points with different color gradients (from yellow to blue) indicate the range of P values. The larger the bubble, the higher the degree of enrichment, and the lower the P value, the more significant the enrichment. The results show that the differentially expressed proteins are mainly enriched in the PPAR signaling pathway, fatty acid degradation and other pathways. The above results show that the jejunal proteins of infected piglets have changed significantly. Figure 6 The Wayne analysis plot of the significantly differentially expressed proteins (DEPs) in the jejunum of piglets in the CT group and the PEDV group and the top 20 important proteins in the protein composition of the two groups of samples screened out by String combined with Cytohubba algorithm. The overlapping part represents the number of common proteins. It can be seen from the figure that 7 potential biomarkers, namely MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins, were screened out by performing Wayne analysis on all differentially expressed proteins and the top 20 important proteins.

[0065] Seven biomarkers for the diagnosis of pathogenic intestinal lesions in piglets

[0066] The expression data of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins in the two groups of jejunal samples obtained from the above proteomics study were used to construct a model using GraphPad Prism V.9 software, and the receiver operating characteristic curve (ROC) was obtained. The predictive accuracy of the model was then evaluated using the area under the ROC curve (AUC). P < 0.05 was considered to be significantly different.

[0067] Figure 7 to Figure 13 The ROC curves for evaluating the accuracy of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins as biomarkers for diagnosing pathogenic intestinal lesions in piglets are shown respectively. As can be seen from the figure, the AUC of the ROC curves of the seven protein markers is greater than 0.94 (95% CI = 1.00-1.00; P < 0.001). This indicates that the differentially expressed proteins MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 in the jejunum can be used as valuable biomarkers for evaluating and diagnosing pathogenic intestinal lesions in piglets.

[0068] The above results show that the differentially expressed proteins MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 in the jejunum can be used as valuable biomarkers for evaluating and diagnosing pathogenic damage to the intestines of piglets, and can accurately diagnose pathogenic damage to the intestines of piglets. The screening method of the present invention can accurately obtain valuable biomarkers for diagnosing intestinal damage of piglet intestinal diseases.

[0069] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A set of biomarker genes for detecting pathogenic damage to the intestine of piglets, characterized in that: Including at least one of the MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 genes.

2. The biomarker gene according to claim 1, characterized in that The pathogenic damage to the porcine intestinal tract is porcine jejunal damage.

3. The method for screening biomarker genes according to claim 1, characterized in that: The following steps are involved: (1) Proteomic sequencing was performed on the jejunum of pathogen-infected and non-infected piglets to obtain the protein composition of the two groups of samples; (2) performing differential analysis on the results of the proteomic sequencing, and screening out differentially expressed proteins between the two groups of samples by setting a threshold; (3) String combined with Cytohubba algorithm was used to screen the top 20 important proteins in the protein composition of the two groups of samples; (4) Performing Wayne analysis on the differentially expressed proteins and the top 20 important proteins to screen out potential biomarker genes.

4. The screening method according to claim 3, characterized in that The proteomic sequencing comprises the following steps: lysing and digesting the jejunum sample to obtain peptide segments, purifying and recombining the peptide segments, and then performing LC-MS / MS analysis.

5. The screening method according to claim 4, characterized in that The peptide purification includes desalting and centrifugal concentration.

6. The screening method according to claim 3, characterized in that The difference analysis includes performing a t-test on each group of values, and calculating the difference multiple FC and the difference significance P-value of the comparison group.

7. The screening method according to claim 3, characterized in that The differential analysis also includes performing KEGG enrichment analysis on the differentially expressed proteins.

8. The screening method according to claim 3, characterized in that The threshold was set at FC>1.5 and P<0.05, or FC<0.67 and P<0.

05.

9. A group of protein markers for detecting pathogenic damage to the intestine of piglets, characterized in that: The protein markers include at least one of MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins, and the MMP7, PLA2G3, AQP5, CYP3A22, HSD17B6, ENPP7 and SDR16C5 proteins are the corresponding biomarker gene expressions in claim 1.

10. Use of the biomarker gene according to any one of claims 1 to 2 or the protein marker according to claim 9 in the preparation of a reagent for diagnosing pathogenic damage in piglets or a drug for treating pathogenic damage in piglets.

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