Application of beta2M as biomarker in animal early weaning stress

Abstract

The invention discloses application of beta2M as a biomarker in early weaning stress of animals, and belongs to the technical field of detection and analysis. According to the application of the beta2M as the biomarker in preparation of a product for detecting the early weaning stress of animals, the product is used for detecting the content of the beta2M in jejunum tissues or serum samples and can be used for judging whether the animals have weaning stress symptoms or not. The invention provides a new biomarker for prevention, diagnosis and treatment of animal weaning stress intestinal injury.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically relating to the application of β2M as a biomarker in early weaning stress in animals. Background Technology

[0002] Early weaning of piglets (usually at 3-4 weeks of age) is a key technology in modern intensive pig farming, but this stage coincides with a critical window for the development of the piglet's gut and immune system. Drastic nutritional and environmental changes constitute complex weaning stress, causing a series of profound and interconnected negative impacts on gut health and immune function, often leading to growth retardation and a high incidence of diarrhea. Weaning stress first directly damages the integrity of the gut. This is mainly manifested in atrophy of small intestinal villi and crypt hyperplasia, resulting in a sharp reduction in digestive and absorptive surface area and decreased function. More fundamentally, the damage lies in the breakdown of the intestinal barrier function. Physical barriers become more permeable due to downregulation of tight junction proteins; chemical barriers, such as reduced mucus secretion, decrease; and biological barriers are characterized by severe dysbiosis, with a decrease in the abundance of beneficial bacteria such as lactobacilli and an increase in the proportion of potentially pathogenic bacteria such as Escherichia coli. This dysbiosis and barrier damage create a vicious cycle, providing conditions for pathogen colonization and endotoxin translocation, and is a major cause of post-weaning diarrhea.

[0003] The gut is not only a digestive organ but also an important site of immune response. Weaning stress leads to dysfunction of the intestinal mucosal immune system, a condition known as "weaning stress syndrome." On the one hand, systemic immune responses are suppressed, manifested as impaired development and decreased indices in immune organs such as the spleen and thymus. On the other hand, local immune responses in the gut become unbalanced. Studies show that weaning stress upregulates the levels of pro-inflammatory cytokines in the intestinal mucosa of piglets, inducing cytokine storms and exacerbating the risk of intestinal infections. However, there are currently no accurate indicators to reflect weaning stress levels, thus lacking guidance for farmers to develop appropriate intervention programs.

[0004] β2-microglobulin (β2M) is a single-chain low-molecular-weight protein (approximately 11.8 kDa) composed of 99 amino acids. As the light chain of the major histocompatibility complex class I (MHC-I) molecule, it has a wide range of crucial biological functions. Under physiological conditions, β2M is non-covalently bound to the MHC-I heavy chain and is an essential structural component for the proper folding, assembly, and stable expression of intact MHC-I molecules on the cell surface. It participates in the core process of antigen presentation: β2M assists the MHC-I heavy chain in loading antigenic peptides in the endoplasmic reticulum and transporting them to the cell membrane, thereby mediating CD8+ antigen presentation. +T cells perform immune surveillance against intracellular pathogens (such as viruses) and mutated cells (such as tumor cells). The absence of β2M leads to unstable expression of MHC-I molecules, severely impairing cellular immune responses. In addition to its classical functions, β2M itself also possesses immunomodulatory activity. Studies have found that free β2M can act as an immune signaling molecule, participating in inflammatory responses. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides the application of β2M as a biomarker in early weaning stress in animals, which is beneficial for timely and accurate judgment of the weaning stress situation in animals.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is to provide β2M as a biomarker for the preparation of products for detecting early weaning stress in animals.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, the product is used to detect the content of β2M in samples.

[0009] Furthermore, the samples used for testing are jejunal tissue or serum.

[0010] Furthermore, the samples were taken from animals three days after weaning.

[0011] Furthermore, the product is a reagent or kit.

[0012] Furthermore, the detection method used to test the β2M content of the product is either ELISA or RT-qPCR.

[0013] Furthermore, the animal is either a pig or a mouse.

[0014] Furthermore, if the β2M content in the jejunal tissue of pigs is greater than 850 ng / g, or the β2M content in the serum is greater than 280 ng / mL, it is judged as the presence of weaning stress symptoms.

[0015] Furthermore, if the β2M content in the mouse jejunal tissue is greater than 2250 ng / g, or the β2M content in the serum is greater than 302 ng / mL, it is considered that weaning stress symptoms have appeared.

[0016] The beneficial effects of this invention are as follows: Single-cell sequencing data in the embodiments of this invention show that β2M levels in various types of jejunal epithelial cells significantly increased 3 days after early weaning. ELISA kits were used to detect β2M levels in the jejunal tissue and serum of piglets or mice before and after weaning, and the results showed a similar significant upregulation. These data indicate that changes in β2M levels can reflect the occurrence of early weaning stress. Traditional methods for assessing weaning stress, such as behavioral observation, suffer from drawbacks such as high subjectivity, low sensitivity, and poor timeliness, often failing to provide timely intervention. This invention provides the application of β2M as a biomarker in the preparation of products for detecting early weaning stress in animals. By monitoring changes in β2M levels, weaning stress symptoms can be diagnosed quickly and accurately, significantly improving the timeliness of early intervention, thereby effectively preventing digestive system disorders, decreased immunity, and growth retardation in piglets caused by weaning. Furthermore, the application of β2M as a biomarker can promote precision farming practices, optimize weaning management strategies, and provide a scientific basis for the prevention and control of related diseases and vaccine development. Attached Figure Description

[0017] Figure 1 The effect of early weaning on the development of the small intestine in piglets is shown in the figure. A represents body weight, B and C represent the lengths of the duodenum and distal small intestine, and D, E and F represent the unit intestinal weights of the duodenum, jejunum and ileum, respectively. Figure 2 The effects of early weaning on the jejunal structure of piglets are shown in the following figures: A represents the results of HE staining; B and C represent the villus height and crypt depth based on HE staining, respectively; D and E represent the results of Western blot analysis of Occludin protein on day 1 of weaning and statistical analysis; F and G represent the results of Western blot analysis of Occludin protein on day 3 of weaning and statistical analysis; and H and I represent the results of Western blot analysis of Occludin protein on day 7 of weaning and statistical analysis. Figure 3 The experimental results of single-cell sequencing of jejunal epithelial cells of piglets are shown. In the figure, A is the dimension reduction plot of epithelial cell clustering, and B and D are volcano plots of differentially expressed genes in intestinal stem cells, TA cells and absorptive cells before and after weaning, respectively. Figure 4 The results of single-cell sequencing of piglet jejunal epithelial cells, where A and D represent goblet cells and Best4, respectively. + Volcano plot of differentially expressed genes in cells, endocrine cells, and Tuft cells; Figure 5 The effect of early weaning on β2M protein content in piglets is shown in Figure 1. A and B are the results of ELISA kit detection of jejunal tissue and serum, respectively, and C is the result of RT-qPCR detection. Figure 6The effect of early weaning on β2M protein content in mice is shown in Figures A and B, where A and B are the results of ELISA assays of jejunal tissue and serum, and C is the result of RT-qPCR assays. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0019] Example 1 Thirty-six newborn three-way crossbred (Duroc × Landrace × Large White) boars of similar weight and good health were selected and randomly divided into a control group (CON) and a weaning group (EWS). Early weaning was performed on the weaning group piglets at 20 days of age, and samples were collected at three time points: 1 day after weaning, 3 days after weaning, and 7 days after weaning (n=6).

[0020] (1) Record the weight data of piglets at each time point and draw the growth curve; at the same time, measure the length of each segment of the small intestine of piglets and calculate the unit intestinal weight (i.e. the ratio of intestinal weight to length).

[0021] Test results as follows Figure 1 As shown, piglets experienced a significant weight loss 3 days after weaning, and their weight remained lower than the control group 7 days later. Figure 1 (A). Furthermore, only the distal small intestine shortened one day after weaning, while other data showed no significant changes; by three days after weaning, the distal small intestine length and the weight of each segment of the intestine were significantly reduced, and these indicators returned to the control group levels seven days after weaning. Figure 1 (BF). The results showed that the small intestine of piglets suffered the most severe damage 3 days after weaning. Further comparison of the proportion of decreased intestinal weight per unit area in the duodenum, jejunum, and ileum at this time point of 3 days after weaning showed that the jejunum had a weight loss of 30.15%, which was higher than that of the duodenum (11.55%) and ileum (20.05%), suggesting that the jejunum is the main target of early weaning stress injury.

[0022] (2) Jejunal tissue from piglets at each time point was prepared into paraffin sections. The changes in jejunal epithelial structure at each time point after early weaning stress were observed by HE staining. Total protein from jejunal tissue was extracted for Western blot (WB) to detect the expression level of the tight junction protein Occludin.

[0023] Test results as follows Figure 2 As shown, one day after weaning, the jejunal villi of piglets shortened; three days later, the villi were severely fragmented, with a 55% reduction in height; and seven days later, the villi structure was reconstructed, but still significantly lower than the control group. The crypt depth was significantly higher than the control group at both three and seven days after weaning. Figure 2Western blot results showed that the expression level of the tight junction protein ocludin in the jejunum was significantly inhibited 1 day and 3 days after weaning, and was higher than that in the control group 7 days after weaning. Figure 2 (Middle DI). The above results confirm that the jejunum of piglets is most severely damaged 3 days after early weaning.

[0024] (3) Single-cell sequencing analysis was performed on the jejunal epithelial tissue of piglets collected 3 days after weaning, which was completed by Xunyin Biotechnology Co., Ltd.

[0025] The test and analysis results are as follows Figure 3 and Figure 4 As shown, jejunal epithelial cells were clustered into intestinal stem cells, TA cells, absorptive cells, goblet cells, and Best4 cells. + Cells, endocrine cells and Tuft cells ( Figure 3 (A). Further detailed analysis of each cell type (intestinal stem cells, TA cells, absorptive cells, goblet cells, Best4). + Differentially expressed genes in intestinal epithelial cells (including endocrine cells and Tuft cells) were analyzed. The results showed that, except for Tuft cells, the β2M gene was significantly upregulated in all types of intestinal epithelial cells. Figure 3 Chinese BD and Figure 4 (Chinese AD).

[0026] (4) Jejunal tissue and blood of piglets taken 3 days after weaning were used to detect the β2M content in jejunal tissue and serum using an ELISA kit, and the β2M gene abundance change in jejunal tissue was detected by RT-qPCR. The ELISA kit was a porcine β2 microglobulin (BMG / β2-MG) ELISA kit, purchased from Shanghai Enzyme Research Biotechnology Co., Ltd., catalog number: EK-P40227.

[0027] Test results as follows Figure 5 As shown, the β2M protein content in the jejunal tissue of unweaned piglets was 850 ng / g, and the β2M protein content in serum was 280 ng / mL. Exceeding these levels indicates weaning stress. Three days after weaning, the β2M levels in the jejunal tissue and serum of piglets significantly increased. Figure 5 (AB); further, RT-qPCR results showed a similarly significant upregulation ( Figure 5 (C); The results showed that early weaning stress induced an increase in β2M levels in piglets.

[0028] Example 2 Twelve newborn male C57 mice of similar weight were randomly divided into a control group and a weaning group. Early weaning was performed on 14-day-old C57 mice. Samples were collected 3 days after weaning (n=6). The β2M content in the jejunal tissue and serum of the mice was detected using an ELISA kit (Mouse β2 Microglobulin (BMG / β2-MG) ELISA Quantitative Detection Kit, purchased from Shanghai Enzyme Research Institute Biotechnology Co., Ltd., catalog number: Ek-M20123). Changes in β2M gene abundance in the jejunal tissue were detected using RT-qPCR.

[0029] Test results as follows Figure 6 As shown, the β2M protein content in the jejunal tissue of unweaned mice was 2250 ng / g, and the β2M protein content in serum was 302 ng / mL. Exceeding this standard indicates weaning stress. Three days after early weaning, the β2M levels in jejunal tissue and serum significantly increased. Figure 6 (AB); Furthermore, RT-qPCR detection results also showed a significant upregulation ( Figure 6 (C) The results showed that early weaning stress induced an increase in β2M levels in mice.

[0030] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. Application of β2M as a biomarker in the preparation of products for detecting early weaning stress in animals.

2. The application according to claim 1, characterized in that: The product is used to detect the content of β2M in a sample.

3. The application according to claim 2, characterized in that: The samples used for testing are jejunal tissue or serum.

4. The application according to claim 3, characterized in that: The samples were taken from animals 3 days after weaning.

5. The application according to claim 3, characterized in that: The product is a reagent or kit.

6. The application according to claim 5, characterized in that: The detection method used to determine the β2M content of the product is either ELISA or RT-qPCR.

7. The application according to claim 6, characterized in that: The animal in question is either a pig or a mouse.

8. The application according to claim 7, characterized in that: If the β2M content in the jejunal tissue of pigs is greater than 850 ng / g, or the β2M content in the serum is greater than 280 ng / mL, it is considered that weaning stress symptoms have appeared.

9. The application according to claim 7, characterized in that: If the β2M content in the jejunal tissue of mice is greater than 2250 ng / g, or the β2M content in the serum is greater than 302 ng / mL, it is considered that weaning stress symptoms have appeared.

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