Construction method and application of zebra fish sepsis model

Abstract

The invention relates to a construction method of a zebra fish sepsis model, and belongs to the technical field of medical animal models. The construction method of the zebra fish sepsis model comprises the following steps: performing resuspension culture on escherichia coli, which is one of common sepsis pathogenic bacteria, to prepare escherichia coli with a proper concentration; and injecting the prepared escherichia coli suspension into the abdominal cavity of the zebra fish for 48 hours to obtain the sepsis animal model. Compared with the prior art, the method has the advantages that the escherichia coli is injected into the abdominal cavity of the zebra fish, so that the defects of the conventional LPS water bath method are overcome, and the defects of model defects caused by physiological difference between the zebra fish and mammals and the defect that LPS can not be identified by a zebra fish receptor are avoided. Meanwhile, the zebra fish model prepared by the method is closer to the pathophysiological state of sepsis of mammals; the zebra fish has the advantages of fast development, low cost, small size, convenience in operation and the like, so that the zebra fish has remarkable advantages in high-throughput screening of therapeutic drugs, and is more beneficial to research and exploration of pathogenesis and treatment of sepsis.

Description

Technical Field

[0001] The 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 imbalanced 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 may occur. The high morbidity and mortality make sepsis a severe challenge facing global health. Currently, the clinical treatment of sepsis is still very limited, mainly relying on traditional methods such as early identification, appropriate antibiotics and supportive treatment. However, these methods lack specificity and have poor therapeutic effects. Therefore, exploring the molecular mechanisms of the occurrence and development of sepsis and finding targeted therapeutic drugs is of great significance to reducing mortality.

[0003] In the study of sepsis, establishing an animal model of sepsis is an important part of exploring the mechanism of sepsis and seeking treatment methods. At present, researchers have successfully established animal models of sepsis for scientific research and clinical research in a variety of animals, such as mice, rats, rabbits, pigs, sheep, gorillas, etc. The most common one is the mouse sepsis model constructed by intraperitoneal injection of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP). However, due to the physiological differences between species, different animal models have different limitations. For example, rodents are very resistant to endotoxins and have a stronger resistance to infection, which results in differences with human pathological conditions; rabbits are more able to reflect the response of the human immune system than rodents, but rabbits do not have an obvious vomiting response and lack a cough reflex; sheep, like humans, are extremely sensitive to LPS, but for ethical reasons, sheep are rarely used in survival studies of sepsis; non-human primates such as gorillas, 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, but the high cost and complex ethical issues make it impossible to use these animals on a large scale.

[0004] Zebrafish has recently become a powerful vertebrate model for studying human pathology. It has similar tissues, organs and systems to humans, and more than 85% of its genes and signaling pathways are highly conserved with humans. Its physiology, development and metabolism are highly similar to those of mammals. The National Institutes of Health (NIH) of the United States ranks zebrafish as the third largest vertebrate model organism after mice and rats. In scientific research, zebrafish provides a unique and powerful prototype for disease modeling because: 1. It has significant physiological similarities and functional conservation with humans in basic cell biological processes; 2. Embryonic manipulation and whole-body imaging are relatively easy; 3. The development cycle is short, the fecundity is high, the cost is low and the transparency is transparent; 4. The availability of various molecular tools for forward-reverse genetics and genome editing. Models of many zebrafish diseases (such as blood cancer, immune system diseases, infectious diseases, etc.) have been established. Using zebrafish disease models to study and cure human-related diseases has been a hot research project in the world 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 studies, 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, 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 the differences in the extracellular domains of zebrafish TLR4, TLR4a and TLR4b paralogs, LPS may not be recognized. Therefore, this method may not truly reflect the pathophysiological state of mammalian sepsis. Therefore, how to establish a zebrafish model that is close to the mammalian sepsis state is an urgent problem to be solved in sepsis treatment and drug development. Summary of the invention

[0006] The technical problem solved by the present invention is that the present invention overcomes the shortcomings of the previous LPS water bath method by injecting Escherichia coli into the peritoneal cavity of zebrafish, avoids 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 pathological and physiological state of mammalian sepsis; it has high repeatability and consistency, because zebrafish has the advantages of fast development, low cost, small size and convenient operation, it has significant advantages in high-throughput screening of therapeutic drugs, and is more conducive to the study and exploration of the pathogenesis and treatment of sepsis. The operation and whole-body imaging of the zebrafish model are relatively easy. Because of the transparency of the whole body, it has obvious advantages over mice in observing some disease indicators, such as the number of intestinal peristalsis, changes in the number of specific cells, etc., especially in the study of sepsis-related intestinal damage, it 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; and zebrafish has a short development cycle, high fertility, and low cost, and has significant advantages over other animals in the process of screening therapeutic drugs.

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

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

[0009] Step 2: Place zebrafish randomly in 6-well plates, with 30 zebrafish in each 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 raised at 28°C for 2 days.

[0011] Step 4: Experimental group: 1x10 resuspended E. coli were injected into the peritoneal cavity of each zebrafish by microinjection. 4 CFU of PBS buffer 10.0 nL, 28 ℃ culture for 2 days.

[0012] Step 5: 30 zebrafish were randomly selected from each group (5 per well) and placed under a fluorescence microscope to record the intestinal peristalsis of the zebrafish. 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 peristalsis frequency and the number of neutrophils in the intestine, the zebrafish were killed at low temperature; each group of zebrafish was fixed, dehydrated, embedded, sliced, H&E stained, and the intestinal tissue pathology was observed under a microscope.

[0014] The signs of successful model construction are that 48 hours after the zebrafish was injected with E. coli, the number of intestinal peristalsis decreased, the number of intestinal neutrophils increased, the number of intestinal tissue folds and intestinal villi decreased and their height decreased significantly in pathological sections, 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 is a 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, and the bacteria are cultured in LB medium at 30°C, 5% CO2, 150 rpm shaking and then resuspended to an appropriate concentration for use.

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

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

[0019] As the preferred technical solution of the invention, the observation index is mainly the change of intestinal peristalsis frequency and intestinal neutrophil granulocytes in zebrafish. Pathological analysis is mainly the change of zebrafish intestinal tissue, and hematoxylin-eosin staining is used to observe the tissue structure.

[0020] In order to solve the technical problem of the present invention, another technical solution is proposed: the application of the method for constructing the zebrafish sepsis model, the zebrafish sepsis model is used to study the pathogenesis and treatment methods of sepsis. The zebrafish sepsis model is used to study the pathogenesis and treatment methods of sepsis. The zebrafish has a reduced number of intestinal peristalsis and an increased number of intestinal neutrophils 48 hours after the injection of Escherichia coli. The zebrafish is placed under a fluorescence microscope to record the intestinal peristalsis of the zebrafish, and the number of intestinal peristalsis is counted according to the video; and the NIS-Elements D 3.20 advanced image processing software is used to collect data under the fluorescence microscope to analyze the number of intestinal neutrophils in the zebrafish.

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

[0022] Beneficial effects:

[0023] Compared with the existing technology for constructing zebrafish sepsis model, 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 effects of therapeutic drugs; in addition, compared with the tail vein / yolk sac injection method, on the one hand, it avoids the physiological difference that mammals lack 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 etiology of sepsis in clinical conditions. Although LPS is the main endotoxin in Gram-negative bacilli 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 to operate and image the whole body. 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, compared with mice, it can more intuitively reflect the degree of intestinal damage, which is more conducive to judging the progression of the disease and carrying out subsequent treatment; 3. short development cycle, high fertility, and low cost, which have significant advantages over other animals in the process of screening therapeutic drugs.

[0027] (4) In comparative example 1, the intestinal motility of zebrafish injected with LPS was enhanced, which is opposite to the result of example 1. In clinical and literature studies, it has been proved 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 modeling group, and intestinal inflammation was less obvious.

[0028] (5) Figure 1 It showed that 1x10 2 Although the number of neutrophils in the intestine of zebrafish in the CFU group increased by about 3.7 times compared with the control group, it was not statistically significant (compared with the control group, p > 0.05); 1x10 3 The number of intestinal neutrophils in the CFU group increased by about 5.4 times compared with the control group (compared with the control group, ** p < 0.01); 1x10 4 The number of intestinal neutrophils in the CFU group was significantly increased compared with that in 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 peristalsis in the CFU group was reduced by about 38% compared with the control group (compared with the control group, ** p < 0.01); 1x10 4 The number of intestinal peristalsis of zebrafish in the CFU group was significantly lower than that in the control group, by about 49.3% (compared with the control group, *** p < 0.001).

[0030] Figure 3 Display Injection 1x10 4 The number of intestinal folds and intestinal villi in the zebrafish group with CFU E. coli 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 in conjunction with the accompanying drawings.

[0032] Figure 1 Schematic diagram of intestinal neutrophil level 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 This is a schematic diagram of the intestinal pathological tissue sections of the model animal group zebrafish 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 Schematic diagram of intestinal neutrophil level in the comparative model animal group prepared by the present invention

[0037] Figure 6 This is a 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 pathogen used in the following examples is Escherichia coli (lyophilized powder, item number GDMCC801268), provided by Guangdong 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 use.

[0041] The endotoxin (LPS) (lyophilized powder, catalog number ST1470) used in the following comparative examples was provided by Shanghai Bio-Tech Biotechnology Co., Ltd.; LPS was resuspended in sterile PBS buffer to prepare a storage solution, and diluted to the required working concentration according to the experiment.

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

[0043] The method for creating the sepsis model in the following comparative example is to inject 10.0 nL of PBS buffer containing different concentrations of LPS into the peritoneal cavity of zebrafish.

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

[0045] The zebrafish used in the following examples and comparative examples are transgenic neutrophil green fluorescent MPX strain zebrafish 5 days after fertilization (5 dpf), provided by the Zebrafish Breeding Center of Huante Biotechnology Co., Ltd., and the experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The experimental animal breeding environment and conditions are 28°C fish farming water (water quality: 200 mg instant sea salt is added to 1 L of reverse osmosis water, the conductivity is 450~550 μS / cm; pH is 6.5~8.5; hardness is 50~100 mg / L CaCO3), and the breeding management meets the requirements of the international AAALAC certification (certification number: 001458).

[0046] Example 1

[0047] This embodiment provides a sepsis animal model and a construction method thereof, wherein the construction method comprises the following steps:

[0048] Step 1: Zebrafish were kept in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450~550 μS / cm; pH was 6.5~8.5; hardness was 50~100 mg / L CaCO3).

[0049] Step 2: Place zebrafish randomly in 6-well plates, with 30 zebrafish in each 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 raised at 28°C for 2 days.

[0051] Step 4: Experimental group: 1x10 resuspended E. coli were injected into the peritoneal cavity of each zebrafish by microinjection. 2 , 1x10 3 , 1x10 4 CFU PBS buffer 10.0 nL, 28 ℃ culture for 2 days. (In 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 the modeling was completed, 30 zebrafish were randomly selected from each group (5 per well) and placed under a fluorescence microscope to record the intestinal peristalsis of the zebrafish. 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 a fluorescence microscope to analyze the number of neutrophils in the zebrafish intestine.

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

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

[0055] After the modeling was completed, randomly selected zebrafish were placed under a fluorescence microscope to record the intestinal peristalsis video of the zebrafish, and the number of intestinal peristalsis was counted based on the video; and 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 peristalsis frequency and the number of neutrophils in the intestine, the zebrafish were killed at low temperature; the zebrafish in each group were fixed, dehydrated, embedded, sliced, H&E stained, and the intestinal tissue pathology was observed under a microscope.

[0058] Figure 1 It showed that 1x10 2 Although the number of neutrophils in the intestine of zebrafish in the CFU group increased by about 3.7 times compared with the control group, it was not statistically significant (compared with the control group, p > 0.05); 1x10 3 The number of intestinal neutrophils in the CFU group increased by about 5.4 times compared with the control group (compared with the control group, ** p < 0.01); 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 peristalsis in the CFU group was reduced by about 38% compared with the control group (compared with the control group, ** p < 0.01); 1x10 4The number of intestinal peristalsis of zebrafish in the CFU group was significantly lower than that in the control group, by about 49.3% (compared with the control group, *** p < 0.001);

[0060] Figure 3 Display Injection 1x10 4 The number of intestinal folds and intestinal villi in the zebrafish group with CFU E. coli 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 construction method thereof, wherein the construction method comprises the following steps:

[0063] 1. Water-soluble Nile red (final mass concentration of 10 ng·mL-1) was exposed to light in a 28 ℃ incubator for 4 h to completely fill the zebrafish intestine with Nile red dye. After the Nile red exposure, the stained zebrafish was washed 3 times.

[0064] 2. The zebrafish were randomly placed in 6-well plates, with 30 zebrafish in each well and a volume of 3 mL per well. A normal control group and an experimental group were set up, 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 kept at 28℃ for 2 days.

[0066] 4. Experimental group: 10.0 nL of PBS buffer containing different LPS contents was injected into the peritoneal cavity of each zebrafish by microinjection and the fish were kept at 28°C for 2 days; the LPS contents 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. The images were processed using ImageJ software and the fluorescence intensity of Nile red in the intestine was calculated. NIS-Elements D 3.20 advanced image processing software was used to collect data and analyze the number of neutrophils in the zebrafish intestine.

[0068] In the comparative example, Nile red dye was used to judge the intestinal peristalsis of zebrafish. The zebrafish intestine was filled with Nile red dye in advance. The intensity of zebrafish intestinal peristalsis was inversely proportional to the remaining Nile red in the intestine. The stronger the intestinal peristalsis, the more Nile red dye was discharged, and the lower the Nile red fluorescence intensity of the intestine.

[0069] Figure 4 The results showed that in the zebrafish sepsis model established by intraperitoneal injection of LPS, the intestinal fluorescence intensity of 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 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 zebrafish modeled with LPS was enhanced, which is opposite to the result of Example 1. In clinical and literature studies, it has been proved that sepsis leads to weakened intestinal motility. The results of the comparative example are obviously not in line 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 modeling group, and intestinal inflammation was less obvious.

[0071] In summary, the zebrafish model constructed by the method of Example 1 exhibited 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 pathological physiological 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: Transgenic neutrophil green fluorescent MPX zebrafish were used 5 days after fertilization. The zebrafish were kept in 28°C fish farming water with the following water quality: 200 mg instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3; Step 2: Place zebrafish randomly in 6-well plates, with 30 zebrafish in each well and a volume of 3 mL per well; a total of 180 zebrafish in each group; Step 3: Inject 1x10 resuspended E. coli into the abdominal cavity of each zebrafish. 4 The zebrafish sepsis model was obtained by adding 10.0 nL of PBS buffer containing CFU and raising the fish at 28°C for 2 days.

2. The method for constructing a zebrafish sepsis model according to claim 1, characterized in that: The successful construction of the model is marked by a decrease in the number of intestinal peristalsis and an increase in the number of intestinal neutrophils in zebrafish 48 hours after injection of E. coli.

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

4. The method for constructing a zebrafish sepsis model according to claim 1, characterized in that: The injection dose of E. coli is 1x104 CFU / tail zebrafish, and the product number of E. coli is GDMCC 801268.

5. The method for constructing a zebrafish sepsis model according to claim 1, characterized in that: The time required for modeling after injection of E. coli is 48 hours.

6. 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. 48 hours after the injection of Escherichia coli, the number of intestinal peristalsis of zebrafish decreased and the number of intestinal neutrophils increased. The intestinal peristalsis of zebrafish was recorded under a fluorescence microscope, and 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 intestinal neutrophils in zebrafish.

7. 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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