Construction method and application of a zebrafish sepsis model

Abstract

The present invention relates to a method for constructing a zebrafish sepsis model, and belongs to the technical field of medical animal models. The method for constructing the zebrafish sepsis model comprises the following steps: resuspending and culturing Escherichia coli, one of the common pathogens of sepsis, to prepare a suitable concentration; injecting the prepared Escherichia coli suspension into the zebrafish abdominal cavity, and obtaining a sepsis animal model 48 hours after the injection. Compared with previous methods, the present invention overcomes the shortcomings of the previous LPS water bath method by injecting Escherichia coli into the zebrafish abdominal cavity, avoiding the model defects caused by the physiological differences between zebrafish and mammals and the disadvantage that LPS may not be recognized by zebrafish receptors. At the same time, the zebrafish model prepared by this method is closer to the pathophysiological state of mammalian sepsis; due to the advantages of zebrafish such as fast development, low cost, small size and easy operation, it has significant advantages in high-throughput screening of therapeutic drugs, which is more conducive to the study and exploration of the pathogenesis and treatment of sepsis.

Description

Technical Field

[0001] The present invention relates to the field of animal medical models, in particular to a method for constructing a zebrafish sepsis model, and belongs to the technical field of infection experimental models. Background Art

[0002] Sepsis is an organ dysfunction caused by an dysregulated host response to infection. It is the result of the combined effects of the immune system and pathogens. In severe cases, multiple organ dysfunction syndrome (MODS) may occur. The high morbidity and mortality rates make sepsis a serious challenge facing global health. Currently, clinical treatment options for sepsis are still very limited, relying mainly on traditional methods such as early identification, appropriate antibiotics, and supportive care. However, these methods lack specificity and have poor therapeutic effects. Therefore, exploring the molecular mechanisms of sepsis occurrence and development and finding targeted therapeutic drugs is of great significance in reducing mortality.

[0003] In sepsis research, establishing animal models is a crucial step in exploring the pathogenesis and treatment of sepsis. Researchers have successfully established sepsis models for both research and clinical research in a variety of species, including mice, rats, rabbits, pigs, sheep, and chimpanzees. The most common model is the mouse sepsis model, established through intraperitoneal injection of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP). However, due to physiological differences between species, different animal models have varying limitations. For example, rodents are very resistant to endotoxins and have a stronger resistance to infection, which leads to differences with human pathological conditions. Compared with rodents, rabbits can better reflect the response of the human immune system, but rabbits do not have a significant vomiting reaction and lack a cough reflex. Sheep, like humans, are extremely sensitive to LPS, but for ethical reasons, sheep are rarely used in sepsis survival studies. Non-human primates such as orangutans, baboons, and rhesus monkeys are ideal animal models for studying sepsis because their cardiopulmonary anatomy and physiology, as well as the host's response to infection, are almost identical to those of humans. However, the high cost and complex ethical issues make large-scale application of these animals impossible.

[0004] The zebrafish has recently emerged as a powerful vertebrate model for studying human pathologies. It possesses similar tissues, organs, and systems to humans, with over 85% of its genes and signaling pathways highly conserved with humans. Its physiology, development, and metabolism are highly similar to those of mammals, leading the US National Institutes of Health (NIH) to rank the zebrafish as the third most important vertebrate model organism, after mice and rats. In scientific research, the zebrafish provides a unique and powerful prototype for disease modeling due to: 1. its remarkable physiological similarity and functional conservation with humans in fundamental cellular processes; 2. the relative ease of embryo manipulation and whole-body imaging; 3. its short developmental cycle, high fecundity, low cost, and transparency; and 4. the availability of a wide range of molecular tools for forward and reverse genetics and genome editing. Numerous zebrafish disease models have been established, including those for blood cancers, immune system disorders, and infectious diseases. Using zebrafish disease models to investigate cures for related human diseases has become a global research hotspot in recent years.

[0005] Using zebrafish to establish a sepsis model is not only conducive to in-depth exploration of the mechanism of sepsis, but also plays an important role in high-throughput screening of sepsis treatment methods and drugs. According to known research, there are currently two main methods for using zebrafish to model sepsis: (1) LPS water bath. This method is simple and easy to use, and can observe vascular damage and leakage, tail fin edema and immune activation in zebrafish. However, the continuous presence of LPS in the water bath will lead to systemic inflammation starting from the epidermis, which is different from the common cause of sepsis infection in clinical practice. In addition, this method is not conducive to the removal of LPS contact and subsequent inflammation subsidence, which affects the evaluation of treatment effects. (2) Tail vein / yolk sac injection of LPS. By injecting LPS into zebrafish to promote the development of sepsis, this method can observe the signs of sepsis damage, including systemic inflammation, extensive tissue damage, loss of vascular connection integrity, and immune cell mobilization. However, on the one hand, humans lack equivalent tissues of yolk sac; on the other hand, in mammals, LPS is recognized by the TLR4 receptor, triggering a series of subsequent inflammatory responses. However, due to differences in the extracellular domains of zebrafish TLR4, TLR4a, and TLR4b paralogs, LPS may not be recognized. Consequently, this method may not accurately reflect the pathophysiology of mammalian sepsis. Therefore, establishing a zebrafish model that closely resembles mammalian sepsis is an urgent challenge in sepsis treatment and drug development. Summary of the Invention

[0006] The present invention addresses the following technical issues: By intraperitoneally injecting E. coli into zebrafish, the present invention overcomes the shortcomings of the previous LPS waterbath method, avoiding model defects caused by physiological differences between zebrafish and mammals and the potential inability of zebrafish receptors to recognize LPS. Furthermore, the zebrafish model prepared by this method more closely resembles the pathophysiological state of mammalian sepsis, exhibiting high reproducibility and consistency. Due to the advantages of zebrafish, such as rapid development, low cost, small size, and ease of manipulation, zebrafish offer significant advantages in high-throughput screening of therapeutic drugs, further facilitating research and exploration into the pathogenesis and treatment of sepsis. The zebrafish model is relatively easy to manipulate and image whole-body. Due to its transparency, it offers significant advantages over mice in observing disease indicators, such as intestinal peristalsis frequency and changes in specific cell numbers. In particular, in the study of sepsis-related intestinal damage, the extent of intestinal damage can be more intuitively demonstrated than in mice, facilitating assessment of disease progression and subsequent treatment. Furthermore, zebrafish, with their short developmental cycle, high fecundity, and low cost, offer significant advantages over other animal models in drug screening.

[0007] In order to solve the above technical problems, the present invention proposes a technical solution: a method for preparing a sepsis zebrafish model, using 5-day post-fertilization (dpf) transgenic neutrophil green fluorescent MPX strain zebrafish for experiments, characterized by comprising the following steps:

[0008] Step 1: Zebrafish were kept in aquaculture water at 28°C (water quality: 200 mg of instant sea salt per 1 L of reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3).

[0009] Step 2: Randomly distribute zebrafish into 6-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Set up a normal control group and an experimental group, with a total of 180 zebrafish in each group.

[0010] Step 3: Normal control group: 10.0 nL of PBS buffer without E. coli was injected into the abdominal cavity of each zebrafish by microinjection and the fish were cultured at 28°C for 2 days.

[0011] Step 4: Experimental group: 1x10 resuspended E. coli was injected into the abdominal cavity of each zebrafish by microinjection. 4 CFU were added to 10.0 nL of PBS buffer and cultured at 28°C for 2 days.

[0012] Step 5: Thirty zebrafish were randomly selected from each group (5 per well) and placed under a fluorescence microscope to record intestinal peristalsis. The number of intestinal peristalsis was counted based on the video. NIS-Elements D 3.20 advanced image processing software was used to collect data under the fluorescence microscope to analyze the number of neutrophils in the zebrafish intestine.

[0013] Step 6: After observing the frequency of intestinal peristalsis and the number of neutrophils, the zebrafish were killed at low temperature; each group of zebrafish was fixed, dehydrated, embedded, sectioned, and stained with H&E, and intestinal tissue pathology was observed under a microscope.

[0014] The successful construction of the model is marked by a decrease in the number of intestinal peristalsis, an increase in the number of intestinal neutrophils, a decrease in the number of intestinal tissue folds and intestinal villi in pathological sections and a significant decrease in height 48 hours after the zebrafish were injected with E. coli, and the mucosal tissue was destroyed.

[0015] As a preferred technical solution of the construction of the present invention, the zebrafish used in the experiment are 5 dpf transgenic neutrophil green fluorescence MPX strain zebrafish.

[0016] As a preferred technical solution of the construction of the present invention, the Escherichia coli used in step 5 is Escherichia coli GDMCC 801268. The bacteria are cultured in LB medium at 30°C, 5% CO2, 150 rpm shaker, and then resuspended to an appropriate concentration before use.

[0017] As a preferred technical solution of the present invention, the injection dose of E. coli is 1x10 4 CFUPBS buffer 10.0 nL / zebrafish tail.

[0018] As a preferred technical solution of the construction of the present invention, the time required for modeling after the injection of E. coli is 48 hours.

[0019] As a preferred technical solution of the present invention, the observation indexes are mainly the number of zebrafish intestinal peristalsis and changes in intestinal neutrophils. Pathological analysis mainly focuses on changes in zebrafish intestinal tissue, and hematoxylin-eosin staining is used to observe tissue structure.

[0020] To solve the technical problem of the present invention, another technical solution is proposed: the application of the method for constructing a zebrafish sepsis model, which is used to study the pathogenesis and treatment of sepsis. The zebrafish sepsis model is used to study the pathogenesis and treatment of sepsis. Forty-eight hours after the zebrafish are injected with E. coli, the number of intestinal peristalsis decreases and the number of intestinal neutrophils increases. The zebrafish intestinal peristalsis is recorded under a fluorescence microscope, and the number of intestinal peristalsis is counted based on the video. Furthermore, data is collected under the fluorescence microscope using NIS-Elements D 3.20 advanced image processing software to analyze the number of intestinal neutrophils in the zebrafish.

[0021] The zebrafish sepsis model is used to study the pathogenesis and treatment of sepsis.

[0022] Beneficial effects:

[0023] Compared with the existing technology for constructing zebrafish sepsis models, this method is simple to prepare and simulates the pathological state of sepsis caused by bacterial infection in mammals by intraperitoneal injection of Escherichia coli.

[0024] (1) Compared with the water bath modeling method, this intraperitoneal injection method avoids the impact of continuous exposure to LPS on the evaluation of sepsis treatment effects, which is conducive to evaluating the effectiveness of therapeutic drugs; in addition, compared with the tail vein / yolk sac injection method, on the one hand, it avoids the physiological difference of mammals lacking yolk sacs, and more importantly, it simulates the pathological state of sepsis that originates from intraperitoneal infection in most mammals.

[0025] (2) Compared with the use of LPS modeling, it avoids the possibility that LPS may not be recognized due to the difference between zebrafish TLR4 receptors and mammals. In addition, compared with LPS modeling, bacterial infection is more consistent with the cause of sepsis in clinical conditions. Although LPS is the main endotoxin in Gram-negative bacteria infection, not all pathological changes in sepsis are caused by LPS.

[0026] (3) Compared with the modeling of mice, zebrafish have 1. significant physiological similarities and functional conservation with humans; 2. relatively easy operation and whole-body imaging. Because of the transparency of the whole body, zebrafish have obvious advantages over mice in observing some disease indicators, such as the number of intestinal peristalsis and changes in the number of specific cells. Especially in the study of sepsis-related intestinal damage, zebrafish can more intuitively reflect the degree of intestinal damage than mice, which is more conducive to judging the progression of the disease and carrying out subsequent treatment; 3. short development cycle, high fertility, low cost, and significant advantages over other animals in the process of screening therapeutic drugs.

[0027] (4) In comparative example 1, the intestinal motility of zebrafish modeled by LPS injection was enhanced, which is opposite to the result of Example 1. In clinical and literature studies, it has been proven that sepsis leads to weakened intestinal motility. The results of the comparative example are obviously inconsistent with the actual clinical situation. This may be because LPS cannot be recognized by the TLR4 receptor of zebrafish. In addition, although the number of intestinal neutrophils in the zebrafish in the LPS group increased, it was not as significant as that in the E. coli model group, and the intestinal inflammation was less obvious.

[0028] (5) Figure 1 It showed that 1x10 2 Although the number of neutrophils in the zebrafish intestine in the CFU group increased by about 3.7 times compared with the control group, there was no statistical significance (compared with the control group, p > 0.05); 1x10 3 The number of intestinal neutrophils in the CFU group increased by approximately 5.4 times compared with the control group (** p < 0.01 compared with the control group); 1x10 4 The number of intestinal neutrophils in the CFU group was significantly increased compared with the control group, increasing by about 6.15 times (compared with the control group, *** p < 0.001), indicating significant intestinal inflammation.

[0029] Figure 2 It showed that 1x10 2 The number of intestinal peristalsis of zebrafish in the CFU group was reduced by about 17% compared with the control group (compared with the control group, *p < 0.05); 1x10 3 The number of intestinal motility in the CFU group was reduced by approximately 38% compared with the control group (** p < 0.01 compared with the control group); 1x10 4 The number of intestinal peristalsis in the zebrafish in the CFU group was significantly reduced compared with that in the control group, by approximately 49.3% (*** p < 0.001 compared with the control group).

[0030] Figure 3 Show injection 1x10 4 The number of intestinal folds and villi in the zebrafish CFU E. coli group decreased and their height dropped significantly, and the mucosal tissue was destroyed; the above results showed that injection of 1x10 4 The zebrafish-related indicators and intestinal inflammation in the CFU Escherichia coli group were the most significant, indicating that the sepsis model was successfully established. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below with reference to the accompanying drawings.

[0032] Figure 1 Schematic diagram of intestinal neutrophil levels in the model animal group prepared by the present invention

[0033] Figure 2 Schematic diagram of the intestinal peristalsis frequency level of the model animal group prepared by the present invention

[0034] Figure 3 Schematic diagram of intestinal pathological tissue sections of zebrafish, a model animal group prepared by the present invention (Normal on the left, E. coli on the right)

[0035] Figure 4 Schematic diagram of the intestinal Nile red fluorescence intensity level of the comparative model animal group prepared by the present invention

[0036] Figure 5 This is a schematic diagram of the intestinal neutrophil level of the comparative model animal group prepared by the present invention

[0037] Figure 6 Schematic diagram of zebrafish mortality during the E. coli modeling dose exploration process DETAILED DESCRIPTION

[0038] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0039] Unless otherwise specified, the materials, reagents, etc. used in the following examples, comparative examples, and effect examples can be obtained from commercial sources.

[0040] The sepsis-causing bacteria used in the following examples were Escherichia coli (lyophilized powder, catalog number GDMCC801268), provided by Guangdong Provincial Microbiological Culture Collection Center; the bacteria were cultured and amplified in LB medium, collected by centrifugation, washed with PBS buffer, and resuspended to an appropriate concentration for later use.

[0041] The endotoxin (LPS) (lyophilized powder, Catalog No. ST1470) used in the following comparative examples was provided by Shanghai Biyuntian Biotechnology Co., Ltd. LPS was resuspended in sterile PBS buffer to prepare a stock solution, which was then diluted to the desired working concentration based on the experiment.

[0042] In the following examples, the amount of E. coli injected into each zebrafish was 1×10 4 CFU in 10.0 nL of PBS buffer.

[0043] In the following comparative example, the sepsis model was established by intraperitoneally injecting 10.0 nL of PBS buffer containing different concentrations of LPS into the zebrafish.

[0044] The Nile Red used in the following comparative examples was provided by Sigma, Germany (product number SLBP9326V) and prepared at a concentration of 10 mg mL -1 The mother solution was stored at -20℃ and prepared to 10 ng·mL-1 solution.

[0045] The zebrafish used in the following examples and comparative examples were transgenic neutrophil green fluorescent MPX strain zebrafish, 5 days post-fertilization (dpf), provided by the Zebrafish Breeding Center of Huante Biotechnology Co., Ltd., with Laboratory Animal Use Permit No. SYXK (Zhejiang) 2022-0004. The experimental animals were housed in a 28°C aquaculture water system (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity of 450-550 μS / cm, pH of 6.5-8.5, and hardness of 50-100 mg / L CaCO₃). The animal husbandry and management met the requirements of AAALAC accreditation (certification number: 001458).

[0046] Example 1

[0047] This embodiment provides a sepsis animal model and a method for constructing the same, the method comprising the following steps:

[0048] Step 1: Zebrafish were maintained in aquaculture water at 28°C (water quality: 200 mg of instant sea salt per 1 L of reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3).

[0049] Step 2: Place zebrafish randomly into 6-well plates, with 30 zebrafish per well and a volume of 3 mL per well; set up a normal control group and an experimental group, with a total of 180 zebrafish in each group.

[0050] Step 3: Normal control group: 10.0 nL of PBS buffer without E. coli was injected into the abdominal cavity of each zebrafish by microinjection and the fish were cultured at 28°C for 2 days.

[0051] Step 4: Experimental group: 1x10 resuspended E. coli was injected into the abdominal cavity of each zebrafish by microinjection. 2 , 1x10 3 , 1x10 4 CFU PBS buffer 10.0 nL, 28 ℃ culture for 2 days. (During the exploration of E. coli injection dose, it was found that when the E. coli dose exceeded 1x10 4 After CFU, the mortality rate of zebrafish can exceed 73%, which does not meet the requirements. Figure 6 )

[0052] Step 5: After modeling, 30 zebrafish were randomly selected from each group (5 per well) and placed under a fluorescence microscope to record intestinal peristalsis. The number of intestinal peristalsis was counted based on the video. NIS-Elements D 3.20 advanced image processing software was used to collect data under the fluorescence microscope to analyze the number of neutrophils in the zebrafish intestine.

[0053] Step 6: After observing the frequency of intestinal peristalsis and the number of neutrophils, the zebrafish were killed at low temperature; each group of zebrafish was fixed, dehydrated, embedded, sectioned, and stained with H&E, and intestinal tissue pathology was observed under a microscope.

[0054] 1. Changes in intestinal motility and intestinal neutrophils

[0055] After the model was established, randomly selected zebrafish were placed under a fluorescence microscope to record intestinal peristalsis videos of the zebrafish, and the number of intestinal peristalsis was counted based on the videos. NIS-Elements D 3.20 advanced image processing software was used to collect data under the fluorescence microscope to analyze the number of neutrophils in the zebrafish intestine.

[0056] 2. Pathological observation of intestinal tissue

[0057] After observing the number of intestinal peristalsis and the number of neutrophils, the zebrafish were killed at low temperature; the zebrafish in each group were fixed, dehydrated, embedded, sectioned, and stained with H&E, and intestinal tissue pathology was observed under a microscope.

[0058] Figure 1 It showed that 1x10 2 Although the number of neutrophils in the zebrafish intestine in the CFU group increased by about 3.7 times compared with the control group, there was no statistical significance (compared with the control group, p > 0.05); 1x10 3 The number of intestinal neutrophils in the CFU group increased by approximately 5.4 times compared with the control group (** p < 0.01 compared with the control group); 1x10 4 The number of intestinal neutrophils in the CFU group increased significantly compared with the control group, increasing by about 6.15 times (compared with the control group, *** p < 0.001), indicating significant intestinal inflammation;

[0059] Figure 2 It showed that 1x10 2 The number of intestinal peristalsis of zebrafish in the CFU group was reduced by about 17% compared with the control group (compared with the control group, *p < 0.05); 1x10 3 The number of intestinal motility in the CFU group was reduced by approximately 38% compared with the control group (** p < 0.01 compared with the control group); 1x10 4The number of intestinal peristalsis in zebrafish in the CFU group was significantly reduced compared with the control group, by approximately 49.3% (compared with the control group, *** p < 0.001);

[0060] Figure 3 Show injection 1x10 4 The number of intestinal folds and villi in the zebrafish CFU E. coli group decreased and their height dropped significantly, and the mucosal tissue was destroyed; the above results showed that injection of 1x10 4 The zebrafish-related indicators and intestinal inflammation in the CFU Escherichia coli group were the most significant, indicating that the sepsis model was successfully established.

[0061] Comparative Example 1

[0062] This comparative example provides a sepsis animal model using LPS injection and a method for constructing the same, the method comprising the following steps:

[0063] 1. Expose the zebrafish intestine to a solution of Nile red (final concentration: 10 ng mL⁻¹) in water in a dark incubator at 28°C for 4 hours, until the zebrafish intestine is completely filled with Nile red dye. After exposure, wash the stained zebrafish three times.

[0064] 2. Randomly distribute zebrafish into 6-well plates, with 30 zebrafish per well and a volume of 3 mL per well. Set up a normal control group and an experimental group, with a total of 180 zebrafish in each group.

[0065] 3. Normal control group: 10.0 nL of PBS buffer without LPS was injected into the peritoneal cavity of each zebrafish by microinjection and the fish were cultured at 28°C for 2 days.

[0066] 4. Experimental Groups: Zebrafish were microinjected with 10.0 nL of PBS buffer containing different LPS concentrations into the peritoneal cavity of each zebrafish and cultured at 28°C for 2 days. The LPS concentrations were 62.5, 125, 250, and 500 ng / zebrafish, respectively.

[0067] 5. After modeling, 30 zebrafish were randomly selected from each group (5 per well) and observed and photographed using a fluorescence microscope. Images were processed using ImageJ software, and the intensity of intestinal Nile red fluorescence was calculated. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the number of neutrophils in the zebrafish intestine was analyzed.

[0068] In the comparative example, Nile red dye was used to determine the intestinal motility of zebrafish. The zebrafish intestine was pre-filled with Nile red dye. The intensity of the zebrafish's intestinal motility was inversely proportional to the remaining Nile red in the intestine. The stronger the intestinal motility, the more Nile red dye was excreted, and the lower the intestinal Nile red fluorescence intensity.

[0069] Figure 4 The results showed that in the zebrafish sepsis model established by intraperitoneal injection of LPS, the intestinal fluorescence intensity of the zebrafish was reduced compared with the normal group, with an average decrease of 56% (compared with the control group, *** p < 0.001), indicating that the intestinal peristalsis of the zebrafish in all control cases was enhanced; Figure 5 The results showed that in the LPS model group, the number of neutrophils in the zebrafish intestine increased compared with the control group, with an average increase of 67.3% (compared with the control group, *** p < 0.001).

[0070] In this comparative example, the intestinal motility of the zebrafish modeled with LPS was enhanced, which is contrary to the results of Example 1. In clinical and literature studies, it has been shown that sepsis leads to weakened intestinal motility. The results of the comparative example are obviously inconsistent with the actual clinical situation. This may be because LPS cannot be recognized by the TLR4 receptor of zebrafish. In addition, although the number of intestinal neutrophils in the zebrafish in the LPS group increased, it was not as significant as that in the E. coli model group, and intestinal inflammation was less obvious.

[0071] In summary, the zebrafish model constructed by the method of Example 1 showed symptoms of sepsis, indicating that the sepsis model was successfully established; and the sepsis model preparation method is simple. Compared with the existing sepsis zebrafish model preparation method, (1) it avoids the defects caused by physiological differences with mammals (2) it avoids the possibility that LPS cannot be recognized by the TLR4 receptor of zebrafish (3) intraperitoneal injection of Escherichia coli is more consistent with the pathophysiological state of mammalian sepsis (4) the two indicators of intestinal peristalsis frequency and intestinal neutrophil number can be used to simply, quickly and reliably judge the pathological state of zebrafish sepsis, which is particularly suitable for studying intestinal damage caused by sepsis; (5) compared with mice, the sepsis model constructed using zebrafish has the advantages of low cost and high efficiency in high-throughput screening of sepsis-related therapeutic drugs and methods.

[0072] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A method for constructing a zebrafish sepsis model, characterized in that : Includes the following steps: Step 1: Five-day-post-fertilization (DPF) transgenic neutrophil green fluorescent MPX zebrafish were used. Zebrafish were maintained at 28°C in aquaculture water containing 200 mg of instant sea salt per liter of reverse osmosis water, with a conductivity of 450-550 μS / cm, a pH of 6.5-8.5, and a hardness of 50-100 mg / L CaCO3. Step 2: Randomly distribute zebrafish into 6-well plates, with 30 zebrafish per well and a volume of 3 mL per well; a total of 180 zebrafish per group; Step 3: Inject 1x10 resuspended E. coli into the abdominal cavity of each zebrafish. 4 The zebrafish sepsis model was established by adding 10.0 nL of PBS buffer containing CFU and raising the zebrafish at 28°C for 2 days. The successful establishment of the model was indicated by a decrease in intestinal peristalsis and an increase in the number of intestinal neutrophils 48 hours after injection of E. coli. The product number of the E. coli is GDMCC 801268.

2. The method for constructing a zebrafish sepsis model according to claim 1, wherein: The E. coli was cultured and amplified in LB medium, collected by centrifugation, washed with PBS buffer, and then resuspended to an appropriate concentration for later use.

3. The method for constructing a zebrafish sepsis model according to claim 1, wherein The time required for modeling after injection of E. coli is 48 hours.

4. The use of the method for constructing a zebrafish sepsis model according to claim 1, characterized in that: The zebrafish sepsis model is used to study the pathogenesis and treatment of sepsis. Forty-eight hours after the zebrafish were injected with E. coli, the number of intestinal peristalsis decreased and the number of intestinal neutrophils increased. The zebrafish intestinal peristalsis was recorded under a fluorescence microscope, and the number of intestinal peristalsis was counted based on the video. Advanced image processing software was also used to collect data under the fluorescence microscope to analyze the number of neutrophils in the zebrafish intestine.

5. The use of the method for constructing a zebrafish sepsis model according to claim 1, characterized in that: The zebrafish sepsis model is used to screen drugs or vaccines for the prevention or treatment of sepsis.

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