Application of a bacterial strain in constructing an animal pneumonia model and its construction method

Abstract

The present invention discloses the application of a strain in constructing an animal pneumonia model and a construction method, and belongs to the technical field of animal model construction. By adopting the infection strain and construction method provided by the present invention, it is not necessary to inject rats with immunosuppressants such as cyclophosphamide before infection. The rats only need to be adaptively raised to be successfully infected and modeled, which simulates the real infection scene to the maximum extent, shortens the experimental cycle, and saves the experimental cost; the constructed rat pneumonia model is stable for a long time, and by the end of the observation, the lung pathological symptoms and blood infection symptoms have always existed, the sick rats do not heal themselves, and the lungs are severely damaged; the infection strain and construction method provided can provide a reference for the establishment of other bacterial pneumonia models, thereby further laying the foundation for the development of pneumonia drugs and the research on their mechanism of action, and providing more basis and inspiration for clinical guidance of medication.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to an application of a bacterial strain in constructing an animal pneumonia model and a construction method thereof. Background Art

[0002] The lung is an important organ for gas exchange in vertebrates. Due to its continuous gas exchange function, the lung is very likely to become a target organ for a variety of pathogens, allergens and other toxins, which in turn cause pneumonia and acute lung injury. Although the nonspecific immune system of the lung can eliminate most pathogens, some of them will escape the immune system and induce a series of diseases such as pneumonia. Pneumonia is one of the major serious and sometimes life-threatening diseases, and its occurrence has seriously affected human health and the development of animal husbandry. Bacteria and viruses are the two most common pathogens that cause pneumonia, of which bacterial pneumonia accounts for about 80% of all types of pathogen pneumonia. According to the National Center for Health Statistics of the United States, bacterial pneumonia and influenza were the eighth leading causes of death in the United States from 2014 to 2018. Studies have shown that viral respiratory infections often lead to bacterial pneumonia, which in turn leads to the death of patients. The prevention and treatment of bacterial pneumonia has become a hot topic of global research. Various Gram-negative bacteria, such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Pasteurella, are the most common causes of clinically acquired pneumonia and are also important causes of pneumonia morbidity and mortality worldwide. At the same time, the increasing level of bacterial resistance has made the treatment of bacterial infections increasingly complicated. Therefore, we urgently need to deepen our understanding of the characteristics and mechanisms of severe pneumonia caused by common Gram-negative bacteria in order to develop therapeutic drugs more quickly and effectively. To achieve the above research goals, animal experiments are an essential step. Therefore, establishing a stable animal bacterial pneumonia model is the basis of all research.

[0003] There are many types of model animals. According to the literature, most animals used for pneumonia research are rodents. Most studies on bacterial pneumonia use SD rats or Wistar rats because of their strong tolerance, low mortality, easy feeding, and economical advantages. However, in the process of establishing many pneumonia models, there are many uncertain factors, which lead to unstable models. Many studies will ensure the occurrence of inflammation by injecting rats with immunosuppressants such as cyclophosphamide before modeling. Cyclophosphamide is a widely used anticancer drug and is also commonly used as an immunosuppressant. Cyclophosphamide can cause hemorrhagic cystitis in animals; it has blood toxicity reactions, which can cause a decrease in animal white blood cells, red blood cells and platelets, and inhibit bone marrow production; it also has digestive system toxicity reactions, such as nausea, vomiting, gastrointestinal discomfort, etc. Therefore, the use of immunosuppressants such as cyclophosphamide leads to an increase in the probability of stress in rats, increases experimental variables, and prolongs the experimental time, affecting the experimental results and increasing the experimental cost. Studies have reported that in some pneumonia models, without any treatment, lung inflammation and tissue pathological changes will gradually decrease over time, indicating that unstable animal pneumonia models have a tendency to heal themselves. Therefore, it is particularly important to establish an animal model of bacterial pneumonia that can simulate clinical conditions as much as possible and is relatively stable.

[0004] Pasteurella is an important zoonotic pathogen that can cause infection in livestock, poultry, pets, wild animals and humans. It mainly causes hemorrhagic sepsis or infectious pneumonia in animals, and even causes death. This pathogen is widely prevalent all over the world and can co-infect with a variety of pathogens, thereby increasing the morbidity and mortality of livestock and poultry, causing significant economic losses to the animal husbandry industry. The pathogenicity of Pasteurella is related to various related virulence factors. The key virulence factors of the bacteria have been identified, mainly including lipopolysaccharide and capsule. These virulence factors make it easier for pathogens to invade and colonize in the corresponding target tissues of the host, escape or destroy the host's defense mechanism, and further cause inflammatory responses that are harmful to the host. Studies have shown that with the outbreak of clinical pasteurellosis, one or more antibiotics are used for animal treatment or health care, which accelerates the adaptability of strains to environmental selection pressures, resulting in the enhancement of Pasteurella resistance to multiple antibiotics. Therefore, it is particularly important to find new drugs for the prevention and treatment of pasteurellosis, and then find a practical method to control pasteurellosis infection from a long-term perspective. In order to explore the preventive and therapeutic effects of drugs on pasteurellosis, establishing a stable pasteurellosis model is the cornerstone of all research. However, there is no report on a stable rat pasteurellosis pneumonia model. In 2017, Adhikary et al. reclassified Pasteurella pneumophila and its closely related Pasteurella into eight different species of the genus Rodentibacter. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a strain in constructing an animal pneumonia model and a construction method to fill the gap of the current lack of Pasteurella animal model. The animal model constructed by the provided animal model construction method has high stability and good clinical simulation.

[0006] To achieve the above object, the present invention provides a strain of Bacillus pneumophilus R. pneumotropicus 001, the rodentium pneumophila R. pneumotropicus The taxonomic name of 001 is Rodentibacter pneumotropicus. It was deposited in the General Microbiology Center of China Culture Collection Administration on September 23, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31704.

[0007] A Rodentia pneumophila as described above R. pneumotropicus Application of strain 001 in the construction of animal model of pneumonia.

[0008] A method for constructing an animal model of pneumonia with good stability and no spontaneous healing, using the above-mentioned Bacillus pneumophila R. pneumotropicus 001 strain was used to infect experimental animals, including the following steps:

[0009] S1, take the Rodentia pneumophila R. pneumotropicus 001 strain was cultured in Martin broth at 37°C until the mid-to-late logarithmic period;

[0010] S2. Centrifuge and discard the supernatant. Resuspend and wash the cells 2-3 times with sterile Martin broth medium. Then adjust the bacterial solution concentration to 10 11 CFU / mL;

[0011] S3. After the experimental animals are adaptively raised in an SPF environment for 1 week, the bacterial suspension prepared in step S2 is injected intraperitoneally. Conventional feeding can obtain a stable and non-self-healing pneumonia animal model.

[0012] Preferably, the centrifugation in step S2 refers to centrifugation at 3000 rpm for 5 min, and resuspending and washing the bacteria with sterile Martin broth medium refers to adding sterile Martin broth medium to resuspend the bacteria, centrifuging at 3000 rpm for 5 min, discarding the supernatant, and repeating the process of resuspending the bacteria with sterile Martin broth medium and centrifuging again.

[0013] Preferably, the injection volume of the bacterial suspension in step S3 is 0.5 mL of bacterial suspension / 100 g of experimental animal.

[0014] Preferably, the experimental animals are rats, mice, or rabbits.

[0015] An application of the above method in constructing a stable, non-self-healing pneumonia animal model.

[0016] Therefore, the application of a strain provided by the present invention in constructing an animal pneumonia model and the construction method thereof have the following specific technical effects:

[0017] (1) The infection strain and construction method provided by the present invention can successfully establish a model without immunosuppression. It is not necessary to inject immunosuppressants such as cyclophosphamide into rats before infection. The rats only need to be adaptively raised before artificial infection, which greatly improves the accuracy of modeling, shortens the experimental cycle and saves experimental costs.

[0018] (2) Healthy rats can be made sick by artificially infecting them with the Bacillus pneumophila strain provided by the present invention. The rat pneumonia model is successfully established by observing the clinical symptoms of the rats after infection, performing lung CT examination, measuring hematological parameters, performing pathological autopsy, measuring the lung bacterial load, bacterial types and related inflammatory factors;

[0019] (3) The infection strain and construction method provided by the present invention can be used to construct a long-term stable rat pneumonia model. Pathological symptoms of respiratory moist rales appear on the third day of infection. By the end of the observation, lung pathological symptoms and blood infection symptoms persist. The sick rats do not heal on their own. Detection shows that the lungs are loaded with infection strains, and the lungs are severely damaged but will not cause the death of the rats.

[0020] (4) The infection strain and construction method provided by the present invention can facilitate subsequent research on Pasteurella pneumonia by scientific researchers, and provide a reference for the establishment of other bacterial pneumonia models, thereby laying a foundation for further research on pneumonia drug development and its mechanism of action, and providing more basis and inspiration for clinical guidance of drug use.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0023] Figure 1 These are photos of the purified culture on MacConkey medium (A), blood agar medium containing 5% sheep blood (B), and microscopic photos of Gram staining (C) and Wright staining (D) in Example 1 of the present invention;

[0024] Figure 2 It is the result of comparing the 16srRNA gene sequencing results of the strains on the NCBI website in Example 1 of the present invention;

[0025] Figure 3 CT images of the lungs of the model rat (A) and the healthy rat (B) in Example 2 of the present invention;

[0026] Figure 4 These are photos of lung autopsies of model rats (A) and healthy rats (B) in Example 2 of the present invention;

[0027] Figure 5 This is a HE-stained lung section of the model rat in Example 2 of the present invention, wherein A is a 4-fold visual field, B is a 10-fold visual field, and C is a 50-fold visual field;

[0028] Figure 6 is the bacterial load in the lungs of the model rats on the 8th day of infection in Example 2 of the present invention;

[0029] Figure 7 It is the result of sequence alignment on the NCBI website of the 16srRNA gene sequencing results of the strain isolated from the lungs of the model rats on the 8th day of infection in Example 2 of the present invention;

[0030] Figure 8 This is the result of detecting the expression of IL-6 protein in the lung tissues of the model rats and healthy rats on the 8th day of infection in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0033] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels, and the experimental methods without special explanation were all conventional techniques in the art.

[0034] Embodiment 1

[0035] The specific steps for the isolation and identification of Rodentia pneumophila are as follows:

[0036] A SD rat with wet rales found in the laboratory was autopsied, and the lungs were collected aseptically. An incision was made on the surface of the lungs with a sterile scalpel, and a disposable inoculation loop was used to take samples from the deep tissue of the lungs at 0.5-1.0 cm. The obtained tissue samples were inoculated on MacConkey medium and blood agar medium containing 5% sheep blood, respectively, and the plates were placed in a 37 ℃ constant temperature incubator for 24 hours. The colony morphology was observed and small colonies with droplet-like and non-hemolytic characteristics were selected for further purification. The photos of the purified cultured strains are shown in the figure below. Figure 1 As shown, the isolated strain did not grow on MacConkey medium ( Figure 1 A), while on blood agar medium containing 5% sheep blood, small transparent colonies with dew drop shapes, neat edges, and moistness were formed, and there was no hemolysis ( Figure 1 B). Prepare bacterial smears and observe them under a microscope after Gram staining. Figure 1 As shown in C, the microscopic observation results after Wright staining are as follows Figure 1 As shown in D, blunt-round Gram-negative short rods with dense staining at both ends can be seen. Most bacteria exist individually, and some exist in pairs, chains or clusters. The morphology of these strains conforms to the morphological characteristics of Pasteurella.

[0037] MacConkey medium composition (g / L): peptone 20.0 g, lactose 10.0 g, ox bile salt 5.0 g, sodium chloride 5.0 g, neutral red 0.075 g, agar 12.0 g. pH value 7.4±0.2.

[0038] Blood agar medium containing 5% sheep blood: ingredients (g / L): peptone 15.0 g, liver hydrolysate 2.5 g, yeast extract 5.0 g, sodium chloride 5.0 g, agar 18.0 g. pH 7.0±0.2.

[0039] The bacterial suspension was sent to the company for 16s rRNA gene sequencing, and the sequencing results were compared on the NCBI website. The comparison results are as follows: Figure 2 As shown, the isolated strain was determined to be Pneumophilic Pasteurella, also known as Pneumophilic Pasteurella J type, named R. pneumotropicus 001. The taxonomic name of the strain is Rodentibacter pneumotropicus. The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 23, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31704.

[0040] Embodiment 2

[0041] The rodentium pneumophilum isolated in Example 1 was used R. pneumotropicus 001Construct a rat pneumonia model. The specific steps are as follows:

[0042] S21, the rodentium pneumophilum isolated in Example 1 R. pneumotropicus 001 was cultured in Martin broth until the mid-late logarithmic growth period. The bacterial solution was centrifuged at 3000 rpm for 5 min and the supernatant was discarded. The bacteria were resuspended in sterile Martin broth medium and centrifuged again to discard the supernatant. This process was repeated twice. Finally, the concentration of the bacterial solution was adjusted to about 10 11 CFU / mL.

[0043] S22, 9-10 weeks old SPF grade SD rats, weighing 300-400 g, half male and half female, a total of 16 rats, were purchased from Weitong Lihua Co., Ltd. The rats were kept in an SPF environment. The experiment of the present invention was approved by the Animal Ethics Committee of Nanjing Agricultural University (approval number: NJAU.No20210716119). The procedures of killing and sampling were in accordance with the "Standards for the Management and Treatment of Laboratory Animals" (2006) No. 398 of the Ministry of Science and Technology of China, and the "Regulations on the Management and Treatment of Laboratory Animals" (2008) No. 45.

[0044] S23. After 1 week of adaptive feeding, 16 SD rats were randomly divided into two groups. One group was a model group. The rats in the model group were intraperitoneally injected with Bacillus pneumophilus prepared in step S21. R. pneumotropicus 001 strain solution (inoculation volume was 0.5 mL / 100 g, and the concentration of the solution was about 10 11 CFU / mL), recorded as day 0 of bacterial administration. The other group was the control group (healthy rat group), and the rats in the control group were intraperitoneally injected with an equal amount of sterile saline.

[0045] After infection, rats were fed and drank freely. On the 8th day after infection, blood samples were collected from the rats after tail cutting for blood routine test and lung CT examination for CO 2 The mice were killed by asphyxiation and autopsied aseptically. Lung tissue was collected and washed with 4°C precooled saline. A portion of the tissue was cut to detect the lung bacterial load, a portion of the tissue was fixed in 4% paraformaldehyde and then subjected to pathological section examination, and a portion was stored in liquid nitrogen for IL-6 protein level detection.

[0046] It was observed that intraperitoneal injection of Bacillus pneumotropis R. pneumotropicus After the injection of 001 bacterial solution, 8 rats showed pathological symptoms of depression and rough fur on the first day of bacterial administration, and pathological symptoms of respiratory moist rales on the third day of bacterial administration. The pathological symptoms persisted in the subsequent observation. Rats in the group injected with normal saline had no clinical symptoms.

[0047] 1) Blood routine test of rats

[0048] On the first and eighth days after the administration of bacteria, the rats were subjected to tail-cutting method for blood sampling, and blood routine and C-reactive protein indexes were determined. The specific steps are as follows: the awake rats were placed in a fixator, the tails were exposed, alcohol was rubbed or the blood vessels were dilated, the tail tip was cut off, and the tail vein blood was gently squeezed from the base of the tail to the tip of the tail by hand, and the tail vein blood flowed out. 300 μL of whole blood was collected from each rat. After blood collection, cotton balls were used to press and stop bleeding. The collected blood was placed in a 2 mL EDTA anticoagulant tube and a 2 mL EP tube, and slowly turned upside down several times to fully mix the blood and anticoagulant. The blood in the EP tube was centrifuged at 3 000 rpm for 5 min, and the supernatant was aspirated. After the collection was completed, the blood routine and C-reactive protein indexes were immediately determined using a fully automatic animal blood cell analyzer. The results are shown in Table 1. R. pneumotropicus After 001, the white blood cell, lymphocyte, neutrophil, basophil and C-reactive protein index data of the rats were significantly higher than the normal range. On the 8th day of infection, the bacterial infection symptoms of the rats still existed.

[0049] Table 1 Blood routine of rats after infection

[0050] ;

[0051] 2) CT examination of rat lungs

[0052] Eight days after infection, the rats were subjected to lung CT examination to observe their lung lesions. The specific steps are as follows: the rats were anesthetized by intramuscular injection of sulphamide, and the rats were fixed in a prone position with their abdomens attached to the examination bed, with the midsagittal plane perpendicular to the examination bed. After the rats were fixed, the examination site was sent to the scanning frame so that the sagittal laser positioning line coincided with the spinous process. The setting parameters were: tube voltage 120 kV, tube current 100 mA. Representative rat lung CT examination results are shown in the figure. Figure 3 As shown: After infection, the bronchi and bronchioles in the lungs of rats were dilated, there was exudate in the lungs, the cardiac border was blurred, and the lung inflation was poor.

[0053] 3) Rat lung autopsy

[0054] Eight days after infection, all rats were kept in CO 2 The animals were killed by asphyxiation and autopsied. Figure 4 As shown, compared with the lungs of healthy rats ( Figure 4 Compared with the model group, the lungs of rats in the Figure 4 A) Bleeding is obvious, and the arrow points to the area of ​​severe bleeding in the lung.

[0055] 4) Pathological examination of rat lungs

[0056] The lungs of the model rats were fixed with formaldehyde fixative and HE-stained sections were prepared using conventional methods. The results of microscopic observation were as follows: Figure 5As shown, pathological sections of the lungs of rats in the model group showed desquamation of alveolar epithelial cells, congestion of alveolar interstitium and alveolar wall, pink cellulose visible in the alveoli, accompanied by inflammatory cell infiltration. The above results indicate that 8 days after infection, the lungs of rats are severely damaged and cannot heal themselves.

[0057] 5) Detection of bacterial load in rat lung tissue

[0058] Part of the lung tissue of the model group rats was aseptically removed and placed in a 2 mL centrifuge tube, weighed and recorded, and then an appropriate amount of Martin broth was added. The lung tissue was homogenized using a homogenizer. After standing, the supernatant was aspirated and continuously diluted 10 times with Martin broth. The diluted liquid was spread on a blood agar plate and cultured at 37 °C for 24 h. The bacterial load of the rat lung tissue was calculated by counting the number of colonies, and three biological replicates were performed.

[0059] The calculation results are as follows Figure 6 As shown, the bacterial load in the lung tissue of rats was still high 8 days after infection.

[0060] The bacteria collected from the lung tissue were sent to the company for sequencing of the 16s rRNA of the bacteria. The sequencing results were compared on the NCBI website. The results are as follows Figure 7 As shown, the bacteria in the lungs of rats in the model group were Pasteurella pneumophila.

[0061] These results show that the Rodentia pneumophila isolated in Example 1 R. pneumotropicus The 001 strain can be used to construct a rat pneumonia model.

[0062] 6) Detection of IL-6 protein levels in lung tissue of rats in each group

[0063] Western blot was used to detect the protein expression of IL-6 in rat lung tissue. The specific steps are as follows:

[0064] 50 mg of lung tissue was added with 500 μL of RIPA containing protease inhibitors and homogenized in a multi-sample tissue homogenizer at 60 Hz for 20 s, and placed on ice for 5 min to ensure complete lysis. Centrifuge at 4 ℃ and 12000 rpm for 20 min, and take the supernatant to obtain the total protein solution. The total protein concentration was determined according to the instructions of the BCA kit, and the final protein loading amount of each sample was ensured to be the same. A 12% separation gel was prepared and spotted according to the protein loading amount. The concentrated gel was electrophoresed at 80 V for 30 min, and the separation gel was electrophoresed at 120 V. After electrophoresis, the membrane was transferred, and then the NC membrane was blocked in 5% skim milk powder solution at room temperature for 2 h, then transferred to the corresponding primary antibody solution, incubated overnight in a horizontal shaker at 4 ℃ and 50-60 rpm, and then washed 3 times with TBST, each time for 10 min. After washing the membrane, transfer it to the secondary antibody solution and incubate it at room temperature for 1 h on a horizontal shaker at 50-60 rpm, then wash it with TBST for 15 min, for a total of 3 times. Finally, in a dark room, allow the ECL luminescent solution to react with the membrane for 1-2 min, then expose it to observe the expression of the target protein and take pictures, and use ImageJ software for grayscale value analysis. The results are shown in Figure 2. Figure 8 As shown, the expression of IL-6 protein in the lung tissue of rats was significantly increased 8 days after infection.

[0065] The above test results show that the Rodentia pneumophila isolated in Example 1 R. pneumotropicus The 001 strain can establish a stable rat pneumonia model, which can cause pathological symptoms such as wet rales in the rat's breathing, rough fur, and severe lung damage, but will not cause the rat's death. At the same time, the expression of the inflammatory factor IL-6 in the rat's lungs was significantly increased. The above results all indicate that the rat pneumonia model was successfully established.

[0066] Therefore, by adopting the infection strain and construction method provided by the present invention, it is not necessary to inject immunosuppressants such as cyclophosphamide into rats before infection. The rats only need to be adaptively raised before artificial infection and successful modeling can be achieved, which greatly improves the accuracy of modeling, shortens the experimental period, and saves experimental costs; the constructed rat pneumonia model is stable for a long time, and pathological symptoms of respiratory moist rales appear on the third day of infection. By the end of the observation, lung pathological symptoms and blood infection symptoms have always existed, the sick rats do not heal themselves, and the lungs are severely damaged but the rats will not die; the provided infection strain and construction method can provide a reference for the establishment of other bacterial pneumonia models, thereby further laying a foundation for the development of pneumonia drugs and research on their mechanism of action, and providing more basis and inspiration for clinical guidance of medication.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Bacillus pneumotropis R. pneumotropicus The application of 001 in the construction of a pneumonia animal model is characterized by: Rodentia pneumophila R. pneumotropicus The taxonomic name of 001 is: Rodentibacter pneumotropicus, which was deposited in the General Microbiology Center of China Culture Collection Administration on September 23, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31704; the application is for constructing a rat pneumonia model.

2. A method for constructing a stable, non-self-healing pneumonia animal model, characterized in that: Utilizing the Rodentia pneumophila described in claim 1 R. pneumotropicus 001 strain was used to infect experimental animals, including the following steps: S1, take the described Bacillus pneumophila strain R. pneumotropicus 001, cultured in Martin broth at 37°C to mid-to-late logarithmic period; S2. Centrifuge and discard the supernatant. Resuspend and wash the cells 2-3 times with sterile Martin broth medium. Then adjust the bacterial solution concentration to 10 11 CFU / mL; S3. After the experimental animals are adaptively raised in an SPF environment for 1 week, the bacterial suspension prepared in step S2 is injected intraperitoneally. Conventional feeding can obtain a stable and non-self-healing pneumonia animal model.

3. The method for constructing a stable, non-self-healing pneumonia animal model according to claim 2, characterized in that: The centrifugation in step S2 refers to centrifugation at 3,000 rpm for 5 min, and resuspending and washing the bacteria with sterile Martin broth medium refers to adding sterile Martin broth medium to resuspend the bacteria, centrifuging at 3,000 rpm for 5 min, discarding the supernatant, and repeating the process of resuspending the bacteria with sterile Martin broth medium and centrifuging again.

4. The method for constructing a stable, non-self-healing pneumonia animal model according to claim 2, characterized in that: The injection volume of the bacterial suspension in step S3 is 0.5 mL of bacterial suspension / 100 g of experimental animal.

5. The method for constructing a stable, non-self-healing pneumonia animal model according to claim 2, characterized in that: The experimental animals are rats, mice and rabbits.

6. Use of the method according to any one of claims 2 to 5 in constructing a stable, non-self-healing pneumonia animal model.

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